Exploiting whole genome sequence variants in cattle breeding : Unraveling the distribution of genetic variants and role of rare variants in genomic evaluation
Zhang, Qianqian - \ 2017
Wageningen University. Promotor(en): H. Bovenhuis; M.S. Lund, co-promotor(en): G. Sahana; M. Calus; B. Guldbrandtsen. - Wageningen : Wageningen University - ISBN 9788793643147 - 249
cattle - genomes - genetic variation - inbreeding - homozygosity - longevity - quantitative traits - animal breeding - animal genetics - rundvee - genomen - genetische variatie - inteelt - homozygotie - gebruiksduur - kwantitatieve kenmerken - dierveredeling - diergenetica
The availability of whole genome sequence data enables to better explore the genetic mechanisms underlying different quantitative traits that are targeted in animal breeding. This thesis presents different strategies and perspectives on utilization of whole genome sequence variants in cattle breeding. Using whole genome sequence variants, I show the genetic variation, recent and ancient inbreeding, and genome-wide pattern of introgression across the demographic and breeding history in different cattle populations. Using the latest genomic tools, I demonstrate that recent inbreeding can accurately be estimated by runs of homozygosity (ROH). This can further be utilized in breeding programs to control inbreeding in breeding programs. In chapter 2 and 4, by in-depth genomic analysis on whole genome sequence data, I demonstrate that the distribution of functional genetic variants in ROH regions and introgressed haplotypes was shaped by recent selective breeding in cattle populations. The contribution of whole genome sequence variants to the phenotypic variation partly depends on their allele frequencies. Common variants associated with different traits have been identified and explain a considerable proportion of the genetic variance. For example, common variants from whole genome sequence associated with longevity have been identified in chapter 5. However, the identified common variants cannot explain the full genetic variance, and rare variants might play an important role here. Rare variants may account for a large proportion of the whole genome sequence variants, but are often ignored in genomic evaluation, partly because of difficulty to identify associations between rare variants and phenotypes. I compared the powers of different gene-based association mapping methods that combine the rare variants within a gene using a simulation study. Those gene- based methods had a higher power for mapping rare variants compared with mixed linear models applying single marker tests that are commonly used for common variants. Moreover, I explored the role of rare and low-frequency variants in the variation of different complex traits and their impact on genomic prediction reliability. Rare and low-frequency variants contributed relatively more to variation for health-related traits than production traits, reflecting the potential of improving prediction reliability using rare and low-frequency variants for health-related traits. However, in practice, only marginal improvement was observed using selected rare and low-frequency variants when combined with 50k SNP genotype data on the reliability of genomic prediction for fertility, longevity and health traits. A simulation study did show that reliability of genomic prediction could be improved provided that causal rare and low-frequency variants affecting a trait are known.
Comparative genomics and trait evolution in Cleomaceae, a model family for ancient polyploidy
Bergh, Erik van den - \ 2017
Wageningen University. Promotor(en): M.E. Schranz; Y. van de Peer. - Wageningen : Wageningen University - ISBN 9789463431705 - 106
capparaceae - genomics - polyploidy - evolution - genomes - reproductive traits - flowers - colour - glucosinolates - genetic variation - biosystematics - taxonomy - identification - capparaceae - genomica - polyploïdie - evolutie - genomen - voortplantingskenmerken - bloemen - kleur - glucosinolaten - genetische variatie - biosystematiek - taxonomie - identificatie
As more and more species have been sequenced, evidence has been piling up for a fascinating phenomenon that seems to occur in all plant lineages: paleopolyploidy. Polyploidy has historically been a much observed and studied trait, but until recently it was assumed that polyploids were evolutionary dead-ends due to their sterility. However, many studies since the 1990’s have challenged this notion by finding evidence for ancient genome duplications in many genomes of current species. This lead to the observation that all seed plants share at least one ancestral polyploidy event. Another polyploidy event has been proven to lie at the base of all angiosperms, further signifying the notion that ancient polyploidy is widespread and common. These findings have led to questions regarding the apparent disadvantages that can be observed in a first generation polyploid. If these disadvantages can be overcome however, duplication of a genome also presents an enormous potential for evolutionary novelty. Duplicated copies of genes are able to acquire changes that can lead to specialization of the duplicated pair into two functions (subfunctionalization) or the development of one copy towards an entirely new function (neofunctionalization).
Currently, most research towards polyploidy has focused on the economically and scientifically important Brassicaceae family containing the model plant Arabidopsis thaliana and many crops such as cabbage, rapeseed, broccoli and turnip. In this thesis, I lay the foundations for the expansion of this scope to the Cleomaceae, a widespread cosmopolitan plant family and a sister family of Brassicaceae. The species within Cleomaceae are diverse and exhibit many scientifically interesting traits. They are also in a perfect position phylogenetically to draw comparisons with the much more studied Brassicaceae. I describe the Cleomaceae and their relevance to polyploid research in more detail in the Introduction. I then describe the important first step towards setting up the genetic framework of this family with the sequencing of Tarenaya hassleriana in Chapter 1.
In Chapter 2, I have studied the effects of polyploidy on the development of C4 photosynthesis by comparing the transcriptome of C3 photosynthesis based species Tarenaya hassleriana with the C4 based Gynandropsis gynandra. C4 photosynthesis is an elaboration of the more common C3 form of photosynthesis that concentrates CO2 in specific cells leading to decreased photorespiration by the RuBisCO and higher photosynthetic efficieny in low CO2 environments. I find that polyploidy has not led to sub- or neofunctionalization towards the development of this trait, but instead find evidence for another important phenomenon in postpolyploid evolution: the dosage balance hypothesis. This hypothesis states that genes which are dependent on specific dosage levels of their products will be maintained in duplicate; any change in their function would lead to dosage imbalance which would have deleterious effects on their pathway. We show that most genes involved in photosynthesis have returned to single copy in G. gynandra and that the changes leading to C4 have mostly taken place at the expression level confirming current assumptions on the development of this trait.
In Chapter 3, I have studied the effects of polyploidy on an important class of plant defence compounds: glucosinolates. These compounds, sometimes referred to as ‘mustard oils’, play an important role in the defence against herbivores and have radiated widely in Brassicaceae to form many different ‘flavors’ to deter specific herbivores. I show that in Cleomaceae many genes responsible for these compounds have benefited from the three rounds of polyploidy that T. hassleriana has undergone and that many duplicated genes have been retained. We also show that more than 75% is actively expressed in the plant, proving that the majority of these duplications has an active function in the plant.
Finally, in Chapter 4 I investigate a simple observation made during experiments with T. hassleriana in the greenhouse regarding the variation in flower colour between different individuals: some had pink flowers and some purple. Using LC-PDA mass spectrometry we find that the two colours are caused by different levels of two anthocyanin pigments, with cyanidin dominating in the purple flowers and pelargonidin being more abundant in pink flowers. Through sequence comparison and synteny analysis between A. thaliana and T. hassleriana we find the orthologs of the genes involved in this pathway. Using a Genotyping by Sequencing method on a cross between these two flower colours, we produce a collection of SNP markers on the reference genome. With these SNPs, we find two significant binary trait loci, one of which corresponds to the location of the F3’H ortholog which performs the conversion of a pelargonidin precursor to a cyanidin precursor.
In the General Conclusion, I combine all findings of the previous chapters and explain how they establish part of a larger species framework to study ancient polyploidy in angiosperms. I then put forth what these findings can mean for possible future research and the directions that are worth to be explored further.
From species to trait evolution in Aethionema (Brassicaceae)
Mohammadin, Setareh - \ 2017
Wageningen University. Promotor(en): M.E. Schranz. - Wageningen : Wageningen University - ISBN 9789463431385 - 125
brassicaceae - evolution - rna - genomes - genetic diversity - phytogeography - glucosinolates - quantitative trait loci - next generation sequencing - brassicaceae - evolutie - rna - genomen - genetische diversiteit - plantengeografie - glucosinolaten - loci voor kwantitatief kenmerk - next generation sequencing
The plant family Brassicaceae (or crucifers) is an economically important group that includes many food crops (e.g. cabbages and radishes), horticultural species (e.g. Draba, Iberis, Lunaria), and model plant species (particularly Arabidopsis thaliana). Because of the fundamental importance of A. thaliana to plant biology, it makes the Brassicaceae an ideal system for comparative genomics and to test wider evolutionary, ecological and speciation hypotheses. One such hypothesis is the ‘Whole Genome Duplication Radiation Lag Time’ (WGD-RLT) model for the role of polyploidy on the evolution of important plant families such as the Brassicaceae. The WGD-RLT model indicates a higher rate of diversification of a core-group compared to its sister group, due to a lag time after a whole genome duplication event that made it possible for novel traits or geo- or ecological events to increase the core groups diversification rate.
Aethionema is the species-poor sister genus of the core Brassicaceae and hence is at an important comparative position to analyse trait and genomic evolution of the species-rich core group. Aethionema species occur mainly in the western Irano-Turanian region, which is concordantly the biodiversity hotspot of the Brassicaceae family. Moreover comparing Aethionema to the Brassicaceae core group can help us to understand and test the ‘WGD-RLT’ model. However to be able to do so we first need to know more about Aethionema. In this thesis, I investigated various levels of evolutionary change (from macro, to micro to trait evolution) within the genus Aethionema, with a major focus the emerging model species Aethionema arabicum.
Next generation sequencing has made it possible to use the genomes of many species in a comparative framework. However, the formation of proteins and enzymes, and in the end the phenotype of the whole plant, relies on transcription from particular regions of the genome including genes. Hence, the transcriptome makes it possible to assess the functional parts of the genome. However, the functional part of the genome not only relies on the protein coding genes. Gene regulatory elements like promoters and long non-coding RNAs function as regulators of gene expression and hence are involved in increasing or decreasing transcription. In Chapter 2 I used the transcriptome of four different Aethionema species to understand the lineage specificity of these long non-coding RNAs. Moreover in a comparison with the Brassicaceae core group and Brassicaceae’s sister family the Cleomaceae I show that although the position of long non-coding RNAs can be conserved, their sequences do not have to be.
Most of the Aethionema species occur in the Irano-Turanian region, a politically instable region, making it hard for scientist to collect from. However the natural history collections made throughout the last centuries are a great resource. Combing these collections with the newest sequencing techniques, e.g. next generation sequencing, have allowed me to infer the phylogeny of ~75% of the known Aethionema species in a time calibrated and historical biogeographical framework. Hence, I was able to establish that Aethionema species likely originated from the Anatolian Diagonal and that major geological events like the uplift of the Turkish and Iranian plateau have had a hand in their speciation (Chapter 3).
To examine species-level processes I sequenced and analysed transcriptomes of eight Ae. arabicum accessions coming from Cyprus, Iran and Turkey to investigate population structure, genetic diversity and local adaptation (Chapter 4). The most prominent finding was a ploidy difference between the Iranian and Turkish/Cypriotic lines, whereby the former were (allo)tetraploid and the latter diploid. The tetraploid Iranian lines seem to have one set of alleles from the Turkish/Cypriotic gene-pool. However we do not know where the other alleles come from. In addition to the differences in ploidy level there are also differences in glucosinolate defence compounds between these two populations (Iranian vs Turkish/Cypriotic), with the Iranian lines lacking the diversity and concentration of indolic glucosinolates that the Turkish/Cypriotic lines have. This chapter serves as a good resource and starting point for future research in the region, maybe by using the natural history collections that are at hand.
Glucosinolates (i.e. mustard oils) are mainly made by Brassicales species, with their highest structural diversity in the Brassicaceae. In Chapter 5, I examined two Ae. arabicum lines (CYP and TUR) and their recombinant inbred lines to assess glucosinolate composition in different tissues and throughout the plants development. The levels of glucosinolates in the leaves changed when Ae. arabicum went from vegetative to a reproductive state. Moreover, a major difference in glucosinolate content (up to 10-fold) between CYP and TUR indicates a likely regulatory pathway outside of the main glucosinolate biosynthesis pathway. Multi-trait and multi-environment QTL analyses based on leaves, reproductive tissues and seeds identified a single major QTL. Fine mapping this region reduced the interval to only fifteen protein coding genes, including the two most intriguing candidates: FLOWERING LOCUS C (FLC) and the sulphate transporter SULTR2;1. These findings show an interesting correlation between development and defence.
Finally, Chapter 6 gives a final discussion of this thesis and its results. It brings the different topics together, put them in a bigger picture and look forward to new research possibilities.
The transcriptome as early marker of diet-related health : evidence in energy restriction studies in humans
Bussel, Inge P.G. van - \ 2017
Wageningen University. Promotor(en): Sander Kersten, co-promotor(en): Lydia Afman. - Wageningen : Wageningen University - ISBN 9789463430678 - 194
energy restricted diets - energy intake - gene expression - genomes - proteins - endurance - food composition - human nutrition research - energiearme diëten - energieopname - genexpressie - genomen - eiwitten - uithoudingsvermogen - voedselsamenstelling - voedingsonderzoek bij de mens
Background: Nutrition research is facing several challenges with respect to finding diet related health effects. The effects of nutrition on health are subtle, show high interindividual variations in response, and can take long before they become visual. Recently, the definition of health has been redefined as an organism’s ability to adapt to challenges and ‘this definition’ can be extended to metabolic health. In the metabolic context the ability to adapt has been named ‘phenotypic flexibility’. A potential new tool to magnify the effects of diet on health is the application of challenge tests. Combined with a comprehensive tool such as transcriptomics, the study of challenge tests before and after an intervention might be able to test a change in phenotypic flexibility. A dietary intervention well-known to improve health through weight loss is energy restriction (ER). ER can be used as a model to examine the potential of challenge tests in combination with transcriptomics to magnify diet-induced effects on health. As opposed to ER, caloric restriction (CR) is a reduction in energy intake aimed at improving health and life span in non-obese subjects and not directly aimed at weight loss. In this thesis, we aimed to investigate the use of the transcriptome as an early and sensitive marker of diet-related health.
Methods: First we studied the consequences of age on the effects of CR on the peripheral blood mononuclear cells (PBMCs) transcriptome. For that purpose, we compared the changes in gene expression in PBMCs from old men with the changes in gene expression in PBMCs from young men upon three weeks of 30% CR. To study the effect of a change in dietary composition during ER, we compared the changes in gene expression upon a 12 weeks high protein 25% ER diet with the changes in gene expression upon a 12 weeks normal protein 25% ER diet in white adipose tissue (WAT). Next, we investigated the added value of measuring the PBMC transcriptome during challenge tests compared to measuring the PBMC transcriptome in the fasted state to magnify the effects of ER on health. This was investigated by measuring the changes in gene expression upon an oral glucose tolerance test (OGTT) and upon a mixed meal test (MMT), both before and after 12 weeks of 20% ER. Finally, we determined the differences between a challenge test consisting of glucose alone, the OGTT, or consisting of glucose plus other macronutrients, the MMT, on the PBMC transcriptome in diet-related health.
Results: We observed that the transcriptome of PBMCs of healthy young men had a higher responsiveness in immune response pathways compared to the transcriptome of PBMCs of aged men upon CR (chapter 2). Also, we showed that upon a normal protein-ER diet the transcriptome of WAT showed a decrease in pathways involved in immune response and inflammasome, whereas no such effect was found upon a high protein-ER diet. These effect were observed while parameters such as weight loss, glucose, and waist circumference did not change due to the different protein quantities (chapter 3). 12 weeks of 20% ER was shown to increase phenotypic flexibility as reflected by a faster and more pronounced downregulation of OXPHOS, cell adhesion, and DNA replication during the OGTT compared to the control diet (chapter 4). Finally, two challenge tests consisting of either glucose (OGTT) or glucose plus fat and protein (MMT), were shown to result in a larger overlap than difference in the changes in gene expression of PBMCs (chapter 5).
Conclusions: Based on the differential changes in gene expression upon CR at different ages, we concluded that age is an important modulator in the response to CR. As a high protein ER diet induced transcriptional changes seemed to reflect less beneficial health effects than a normal protein ER diet we concluded that the diet composition is important in the health-effect of ER as measured by the transcriptome. Based on the faster PBMCs changes in gene expression during an OGTT upon 12 weeks of 20% ER, we concluded that the PBMC transcriptome combined with a challenge test can reflect changes in phenotypic flexibility. This makes challenge tests a suitable tool to study diet-related health effects. Finally, based on the changes in gene expression of the MMT and OGTT, we conclude that glucose in a challenge test is the main denominator of the postprandial changes in gene expression in the first two hours. Overall, these results lead to the conclusion that the transcriptome, especially in combination with challenges test, can be used as an early marker of diet-related health. The direct relation to health still needs to be investigated, but the possibility to use the transcriptome as an early marker of diet-related health gives rise to a better understanding of the effects of nutrition on health.
Selection for pure- and crossbred performance in Charolais
Vallée-Dassonneville, Amélie - \ 2017
Wageningen University. Promotor(en): Johan van Arendonk; Henk Bovenhuis. - Wageningen : Wageningen University - ISBN 9789463430180 - 151
charolais - cattle - animal breeding - crossbreeding - crossbreds - selection - beef cattle - genomes - genetic parameters - charolais - rundvee - dierveredeling - kruisingsfokkerij - kruising - selectie - vleesvee - genomen - genetische parameters
Two categories of beef production exist; i.e. (i) purebred animals from a beef sire and a beef dam and (ii) crossbred animals from a beef sire and a dairy dam.
For the purebred beef production, there is a growing interest to include behavior and type traits in the breeding goal. Heritabilities for behavior traits, estimated using subjective data scored by farmers, range from 0.02 to 0.19. Heritabilities for type traits range from 0.02 to 0.35. Results show that there are good opportunities to implement selection for behavior traits using a simple on-farm recording system to allow collection of large data set, and for type traits in Charolais. A genome-wide association study detected 16 genomic regions with small effect on behavior and type traits. This suggests that behavior and type traits are influenced by many genes each explaining a small part of the genetic variance.
The two main dairy breeds mated to Charolais sires for crossbred beef production in France are Montbéliard and Holstein. The genetic correlation between the same trait measured on Montbéliard x Charolais and on Holstein x Charolais was 0.99 for muscular development, 0.96 for birth weight; and 0.91 for calving difficulty, 0.80 for height, and 0.70 for bone thinness. Thus, for these last three traits, results show evidence for re-ranking of Charolais sires depending on whether they are mated to Montbéliard or Holstein cows. When using genomic prediction, the Montbéliard x Charolais and Holstein x Charolais populations could be combined into a single reference population to increase size and accuracy of genomic prediction. Results indicate that the higher the genetic correlation is between the two crossbred populations, the higher the gain in accuracy is achieved when combining the two populations into a single reference.
The selection of Charolais sires to produce purebred or crossbred animals is made through distinct breeding programs. An alternative could be to combine selection into one breeding program. Decision for combining or keeping breeding programs separate is determined by the correlation between the breeding objectives, the selection intensity, the difference in level of genetic merit, the accuracy of selection, and the recent implementation of genomic evaluation. Considering all parameters and based on estimations for selection on birth weight, I recommend combining both breeding programs because this will lead to higher genetic gain, and might simplify operating organization and reduce associated costs.
Utilization of complete chloroplast genomes for phylogenetic studies
Ramlee, Shairul Izan Binti - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Rene Smulders; Theo Borm. - Wageningen : Wageningen University - ISBN 9789462579354 - 186
phylogenetics - genomes - chloroplasts - models - solanum - orchidaceae - phylogenomics - dna sequencing - fylogenetica - genomen - chloroplasten - modellen - solanum - orchidaceae - phylogenomica - dna-sequencing
Chloroplast DNA sequence polymorphisms are a primary source of data in many plant phylogenetic studies. The chloroplast genome is relatively conserved in its evolution making it an ideal molecule to retain phylogenetic signals. The chloroplast genome is also largely, but not completely, free from other evolutionary processes such as gene duplication, concerted evolution, pseudogene formation and genome rearrangements. The conservation of the chloroplast genome sequence allows designing primers targeting regions conserved well beyond species boundaries, and amplification of these targets.
The small size together with their high copy number in leaf cells greatly facilitates chloroplast genome sequencing. In this thesis, chloroplast phylogenomics was conducted using complete chloroplast DNA genomes obtained by a newly developed method of de novo assembly. The method was not only cost-effective but also has the potential to extract a wealth of useful information of thousands of chloroplast genomes from Whole Genome Shotgun (WGS) data. We used k-mer frequency tables to identify and extract the chloroplast reads from the WGS reads and assemble these using a highly integrated and automated custom pipeline. This pipeline includes steps aimed at optimizing assemblies and filling gaps that are left due to coverage variation in the WGS dataset. The pipeline enabled successful de novo assembly across a range of nuclear genome sizes, from Solanum lycopersicon (tomato, 0.9 Gb) to Paphiopedilum heryanum (slipper orchid, 35 Gb).
The pipeline is suitable for studying structural variation in the chloroplast genome, as opposed to the common procedure of read mapping against a reference genome. To support the putative rearrangements, a flexible assembly quality comparison tool was created that combines and visualizes read mapping and alignment results in a two-dimensional plot. We have evaluated the ability of this tool using the de novo assemblies of S. lycopersicon and P. henryanum chloroplasts. The results show that not only we can immediately select the best of two options, but also determine the location of specific artefacts.
In order to explore and evaluate the utility of complete chloroplast phylogenomics, tomato and Paphiopedilum spp were used to conduct phylogenetic inferences based on the complete chloroplast genome. In total 84 tomato chloroplast genomes within the section Lycopersicon were assembled and phylogenetic trees produced. The analyses revealed that next to the chloroplast regions and spacers traditionally used for phylogenetics, additional regions of protein coding and non-coding DNA may be exploited for intraspecific phylogenetic studies. In particular, more than 50% of all phylogenetically relevant information could be included by just using four genes (ycf1, ndhF, ndhA, and ndhH), of which 34% in ycf1 alone. The topology of the phylogenetic tree inferred from ycf1 was the same as that of trees based on all other protein coding genes, although with lower bootstrap values. The phylogenetic analyses based on 32 complete Paphiopedilum spp. chloroplast genomes confirmed the division of the genus into three subgenera Parvisepalum, Brachypetalum and Paphiopedilum. The division of five sections of subgenus Paphiopedilum was also recovered. The de novo assemblies revealed several structural rearrangements including gene loss and inversion. In addition, the chloroplast genome of Paphiopedilum has experienced extreme IR expansion that has included part of or the entire SSC region, resulting in larger IR regions than commonly observed among monocots.
In conclusion, WGS data offer opportunities to generate partial or entire chloroplast genomes for phylogenetic studies. Species discrimination can be achieved already with partial data (subsets of genes), but evolutionarily young lineages may require more informative characters. Therefore, it is expected that many complete chloroplast genomes will be produced in the years to come. While generating these genomes, the urge for de novo assembly of chloroplast genomes rather than mapping against reference genomes is adamant in order to also uncover structural rearrangements in chloroplast genome.
Antibodies and longevity of dairy cattle : genetic analysis
Klerk, B. de - \ 2016
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Jan van der Poel; Bart Ducro. - Wageningen : Wageningen University - ISBN 9789462577589 - 134
dairy cattle - dairy cows - antibodies - longevity - genetic analysis - breeding value - genomes - genetic improvement - animal genetics - melkvee - melkkoeien - antilichamen - gebruiksduur - genetische analyse - fokwaarde - genomen - genetische verbetering - diergenetica
The dairy sector has a big impact on food production for the growing world population and contributes substantially to the world economy. In order to produce food in a sustainable way, dairy cows need to be able to produce milk without problems and as long as possible. Therefore, breeding programs focuses on improvement of important traits for dairy cows. In order to improve desirable traits and obtain genetic gain there is a constant need for optimization of breeding programs and search for useful parameters to include within breeding programs. Over the last several decades, breeding in dairy cattle mainly focused on production and fertility traits, with less emphasis on health traits. Health problems, however, can cause substantial economic losses to the dairy industry. The economic losses, together with the rising awareness of animal welfare, increased herd size, and less attention for individual animals, have led to an increased need to focus more on health traits. Longevity is strongly related to disease resistance, since a more healthy cow will live a longer productive life (longevity). The identification of biomarkers and the detection of genes controlling health and longevity, would not only greatly enhance the understanding of such traits but also offer the opportunity to improve breeding schemes. The objectives of this thesis therefore were 1) to find an easy measurable disease resistance related biomarker in dairy cows, 2) identify the relation between antibodies and longevity, 3) identify genomic regions that are involved with antibody production/expression. In this thesis antibodies are investigated as parameter for longevity. Antibodies might be a novel parameter that enables selection of cows with an improved ability to stay healthy and to remain productive over a longer period of time. In this thesis antibodies bindiging the naive antigen keyhole limpet hemocyanin (KLH) were assumed to be natural antibodies. Antibodies binding bacteria-derived antigens lipoteichoic acid (LTA), lipopolysaccharide (LPS) and peptidoglycan (PGN) were assumed to be specific antibodies. In chapter 2 it was shown that levels of antibodies are heritable (up to h2 = 0.23). Additionally, antibody levels measured in milk and blood are genetically highly correlated (± 0.80) for the two studied isotypes (IgG and IgM). On the other hand, phenotypically, natural antibodies (from both IgG and IgM isotype) measured in milk cannot be interpreted as the same trait (phenotypic correlation = ± 0.40). In chapter 3 and 4 it was shown that levels of antibodies (both natural-and specific antibodies) showed a negative relation with longevity: first lactation cows with low IgM or IgG levels were found to have a longer productive life. When using estimated breeding values for longevity, only a significant relation was found between natural antibody level (IgM binding KLH) and longevity. Lastly chapter 5 reports on a genome-wide-association study (GWAS), to detect genes contributing to genetic variation in natural antibody level. For natural antibody isotype IgG, genomic regions with a significant association were found on chromosome 21 (BTA). These regions included genes have impact on in isotype class switching (from IgM to IgG). The gained knowledge on relations between antibodies and longevity and the gained insight on genes responsible for natural antibodies level make antibodies potential interesting biomarkers for longevity.
Conservation genetics of the frankincense tree
Bekele, A.A. - \ 2016
Wageningen University. Promotor(en): Frans Bongers, co-promotor(en): Rene Smulders; K. Tesfaye Geletu. - Wageningen : Wageningen University - ISBN 9789462576865 - 158
boswellia - genomes - dna sequencing - tropical forests - genetic diversity - genetic variation - genetics - forest management - plant breeding - boswellia - genomen - dna-sequencing - tropische bossen - genetische diversiteit - genetische variatie - genetica - bosbedrijfsvoering - plantenveredeling
Boswellia papyrifera is an important tree species of the extensive Combretum-Terminalia dry tropical forests and woodlands in Africa. The species produces a frankincense which is internationally traded because of its value as ingredient in cosmetic, detergent, food flavor and perfumes productions, and because of its extensive use as incense during religious and cultural ceremonies in many parts of the world. The forests in which B. papyrifera grows are increasingly overexploited at the expense of the economic benefit and the wealth of ecological services they provide. Populations of B. papyrifera have declined in size and are increasingly fragmented. Regeneration has been blocked for the last 50 years in most areas and adult productive trees are dying. Projections showed a 90% loss of B. papyrifera trees in the coming 50 years and a 50% loss of frankincense production in 15 years time.
This study addressed the conservation genetics of B. papyrifera. Forty six microsatellite (SSR) markers were developed for this species, and these genetic markers were applied to characterize the genetic diversity pattern of 12 B. papyrifera populations in Ethiopia. Next to this, also the generational change in genetic diversity and the within-population genetic structure (FSGS) of two cohort groups (adults and seedlings) were studied in two populations from Western Ethiopia. In these populations seedlings and saplings were found and natural regeneration still takes place, a discovery that is important for the conservation of the species.
Despite the threats the populations are experiencing, ample genetic variation was present in the adult trees of the populations, including the most degraded populations. Low levels of population differentiation and isolation-by-distance patterns were detected. Populations could be grouped into four genetic clusters: the North eastern (NE), Western (W), North western (NW) and Northern (N) part of Ethiopia. The clusters corresponded to environmentally different conditions in terms of temperature, rainfall and soil conditions. We detected a low FSGS and found that individuals are significantly related up to a distance of 60-130 m.
Conservation of the B. papyrifera populations is urgently needed. The regeneration bottlenecks in most existing populations are an urgent prevailing problem that needs to be solved to ensure the continuity of the genetic diversity, species survival and sustainable production of frankincense. Local communities living in and around the forests should be involved in the use and management of the forests. In situ conservation activities will promote gene flow among fragmented populations and scattered remnant trees, so that the existing level of genetic diversity may be preserved. Geographical distance among populations is the main factor to be considered in sampling for ex situ conservation. A minimum of four conservation sites for B. papyrifera is recommended, representing each of the genetic clusters. Based on the findings of FSGS analyses, seed collection for ex situ conservation and plantation programmes should come from trees at least 100 m, but preferably 150 m apart.
Mining microbiota signatures in human intestinal tract metagenomes
Tims, S. - \ 2016
Wageningen University. Promotor(en): Michiel Kleerebezem; Willem de Vos, co-promotor(en): Erwin Zoetendal. - Wageningen : Wageningen University - ISBN 9789462576933 - 264
gastrointestinal microbiota - intestines - genomes - man - hosts - host guest relations - dna microarrays - gastrointestinal diseases - inflammatory bowel diseases - irritable colon - prebiotics - body mass index - oligosaccharides - microbiota van het spijsverteringskanaal - darmen - genomen - mens - gastheren (dieren, mensen, planten) - relaties tussen gastheer en gast - dna microarrays - maagdarmziekten - chronische darmontstekingen - prikkelbaar colon - prebiotica - quetelet index - oligosacchariden
Genetic diversity and evolution in Lactuca L. (Asteraceae) : from phylogeny to molecular breeding
Wei, Z. - \ 2016
Wageningen University. Promotor(en): Eric Schranz. - Wageningen : Wageningen University - ISBN 9789462576148 - 210
lactuca sativa - leafy vegetables - phylogeny - genetic diversity - domestication - molecular breeding - genomes - dna - quantitative trait loci - evolution - lactuca sativa - bladgroenten - fylogenie - genetische diversiteit - domesticatie - moleculaire veredeling - genomen - dna - loci voor kwantitatief kenmerk - evolutie
Cultivated lettuce (Lactuca sativa L.) is an important leafy vegetable worldwide. However, the phylogenetic relationships between domesticated lettuce and its wild relatives are still not clear. In this thesis, I focus on the phylogenetic relationships within Lactuca L., including an analysis of the wild Lactuca species that are endemic to Africa for the first time. The genetic variation of responses to salinity in a recombinant inbred line population, derived from a cross between the lettuce crop (L. sativa ‘Salinas’) and wild species (L. serriola), was investigated and the candidate gene in the identified QTL regions was further studied.
In Chapter 1, I introduce and discuss topics related to genetic diversity and evolution in Lactuca, including an overview of lettuce cultivars and uses, its hypothesized domestication history, the taxonomic position of Lactuca, current status of molecular breeding in lettuce and mechanisms of salinity tolerance in plants, especially the High-affinity K+ Transporter (HKT) gene family.
In Chapter 2, the most extensive molecular phylogenetic analysis of Lactuca was constructed based on two chloroplast genes (ndhF and trnL-F), including endemic African species for the first time. This taxon sampling covers nearly 40% of the total Lactuca species endemic to Africa and 34% of all Lactuca species. DNA sequences from all the subfamilies of Asteraceae in Genbank and those generated from Lactuca herbarium samples were used to elucidate the monophyly of Lactuca and the affiliation of Lactuca within Asteracaeae. Based on the subfamily tree, 33 ndhF sequences from 30 species and 79 trnL-F sequences from 48 species were selected to infer phylogenetic relationships within Lactuca using Randomized Axelerated Maximum Likelihood (RAxML) and Bayesian Inference (BI) analyses. In addition, biogeographical, chromosomal and morphological character states were analysed based on the Bayesian tree topology. The results showed that Lactuca contains two distinct phylogenetic clades - the crop clade and the Pterocypsela clade. Other North American, Asian and widespread species either form smaller clades or mix with the Melanoseris species in an unresolved polytomy. The newly sampled African endemic species probably should be excluded from Lactuca and treated as a new genus.
In Chapter 3, twenty-seven wild Lactuca species and four outgroup species were sequenced using next generation sequencing (NGS) technology. The sampling covers 36% of total Lactuca species and all the important geographical groups in the genus. Thirty chloroplast genomes, including one complete (partial) large single copy region (LSC), one small single copy region (SSC), one inverted repeat (IR) region, and twenty-nine nuclear ribosomal DNA sequences (containing the internal transcribed spacer region ) were successfully assembled and analysed. A methodology paper for which I am co-author, but is not included in this thesis, of the sequencing pipeline was published: ‘Herbarium genomics: plastome sequence assembly from a range of herbarium specimens using an Iterative Organelle Genome Assembly (IOGA) pipeline’. These NGS data helped resolve deeper nodes in the phylogeny within Lactuca and resolved the polytomy from Chapter 2. The results showed that there are at least four main groups within Lactuca: the crop group, the Pterocypsela group, the North American group and the group containing widely-distributed species. I also confirmed that the endemic African species should be removed and treated as a new genus.
In Chapter 4, quantitative trait loci (QTLs) related to salt-induced changes in Root System Architecture (RSA) and ion accumulation were determined using a recombinant inbred line population derived from a cross between cultivated lettuce and wild lettuce. I measured the components of RSA by replicated lettuce seedlings grown on vertical agar plates with different NaCl concentrations in a controlled growth chamber environment. I also quantified the concentration of sodium and potassium in replicates of greenhouse-grown plants watered with 100 mM NaCl. The results identified a total of fourteen QTLs using multi-trait linkage analysis, including three major QTLs associated with general root development (qRC9.1), root growth in salt stress condition (qRS2.1), and ion accumulation (qLS7.2).
In Chapter 5, one of the identified QTL regions (qLS7.2) reported in Chapter 4 was found to contain a homolog of the HKT1 from Arabidopsis thaliana. I did a phylogenetic analysis of Lactuca HKT1-like protein sequences with other published HKT protein sequences and determined transmembrane and pore segments of lettuce HKT1;1 alleles, according to the model proposed for AtHKT1;1. Gene expression pattern and level of LsaHKT1;1 (L. sativa ‘Salinas’) and LseHKT1;1 (L. serriola) in root and shoot were investigated in plants growing hydroponically over a time-course. The measurements of Na+ and K+ contents were sampled at the same time as the samples used for gene expression test. In addition, I examined the 5’ promoter regions of the two genotypes. The results showed low expression levels of both HKT1;1 alleles in Lactuca root and relatively higher expression in shoot, probably due to the negative cis-regulatory elements of HKT1 alleles found in Lactuca promoter regions. Significant allelic differences were found in HKT1;1 expression in early stage (0-24 hours) shoots in and in late stage (2-6 days) roots. shoot HKT1;1 expression/root HKT1;1 expression was generally consistent with the ratios of Na+/K+ balance in the relevant tissues (shoot Na+/K+ divided by root Na+/K+).
In Chapter 6, I summarize and discuss the results from previous chapters briefly. The implications of Chapter 2 and 3 for Lactuca phylogenetics are discussed, including some key characters for the diagnosis of species within Lactuca, the use of herbarium DNA for NGS technology, and perspectives into Lactuca phylogeny. Future perspectives of genome-wide association mapping for lettuce breeding were also discussed. Lastly, I propose to integrate phylogenetic approaches into investigations of allelic differences in lettuce, not just associated with salinity stress but also with other stressed and beneficial characters, both within and between species.
Natural genetic variation in Arabidopsis thaliana photosynthesis
Flood, P.J. - \ 2015
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts; Jeremy Harbinson. - Wageningen : Wageningen University - ISBN 9789462575004 - 278
arabidopsis thaliana - genetische variatie - fotosynthese - genomen - chlorofyl - fenotypen - arabidopsis thaliana - genetic variation - photosynthesis - genomes - chlorophyll - phenotypes
Oxygenic photosynthesis is the gateway of the sun’s energy into the biosphere, it is where light becomes life. Genetic variation is the fuel of evolution, without it natural selection is powerless and adaptation impossible. In this thesis I have set out to study a relatively unexplored field which sits at the intersection of these two topics, namely natural genetic variation in plant photosynthesis. To begin I reviewed the available literature (Chapter 2), from this it became clear that the main bottleneck restricting progress was the lack of high-throughput phenotyping platforms for photosynthesis. To address this an automated high-throughput chlorophyll fluorescence phenotyping system was developed, which could measure 1440 plants in less than an hour for ΦPSII, a measure of photosynthetic efficiency (Chapter 3). Using this phenotyping platform I screened five populations of Arabidopsis thaliana. Three of these populations resulted from bi-parental crosses and segregated for only two genomes, using these I conducted family mapping (Chapter 4). The final two populations were composed of natural, field collected, accessions and were analysed using a genome wide association approach (Chapter 5). The family mapping approach had greater statistical power due to within population replication and the genome wide association approach had higher mapping resolution due to historical recombination. Both approaches were used to identify genomic regions (loci) which were responsible for some of the variation in photosynthesis observed. The number and average effect of these loci was used to infer the genetic architecture of photosynthesis as a highly complex polygenic trait for which there are many loci of very small effect. In addition to screening these large populations a smaller subset of 18 lines was assayed for natural variation in phosphorylation of photosystem II (PSII) proteins in response to changing light (Chapter 6). This exploratory study indicated that this process shows considerable variation and may be important for adaptation of the photosynthetic apparatus to photosynthetic extremes. The genetic mapping studies just described, focus exclusively on genetic variation in the nuclear genome, whilst this contains the majority of the plants genetic information there is also a store of genetic information in the chloroplast and mitochondria. These genetic repositories contain genes which are essential for photosynthesis and energy metabolism. Any variation in these genes could have a large impact on photosynthesis. To study natural variation in these genomes I developed a new population of reciprocal nuclear-organellar hybrids (cybrids) which could be used to study the effect of genetic variation in organelles whilst controlling for nuclear genetic variation (Chapter 7). Preliminary results indicate that this resource will be of great use in disentangling natural genetic variation in nucleo-organelle interactions. Finally I looked at one chloroplast encoded photosynthetic mutation in more detail (Chapter 8). This mutation had evolved in response to herbicide application and had spread along British railways. When studying this population of resistant plants I found empirical evidence for organelle mediated nuclear genetic hitchhiking. This is a previously undescribed evolutionary phenomenon and is likely to be quite common. In conclusion there is an abundance of genetic variation in photosynthesis which can be used to improve the trait for agriculture and provide insights into novel evolutionary phenomena in the field.
Using natural variation to unravel the dynamic regulation of plant performance in diverse environments
Molenaar, J.A. - \ 2015
Wageningen University. Promotor(en): Harro Bouwmeester; Joost Keurentjes, co-promotor(en): Dick Vreugdenhil. - Wageningen : Wageningen University - ISBN 9789462573444 - 186
planten - genomen - loci voor kwantitatief kenmerk - warmtestress - genetische kartering - groei - droogte - plantengenetica - plantenfysiologie - plants - genomes - quantitative trait loci - heat stress - genetic mapping - growth - drought - plant genetics - plant physiology
All plants are able to respond to changes in their environment by adjusting their morphology and metabolism, but large differences are observed in the effectiveness of these responses in the light of plant fitness. Between and within species large differences are observed in plant responses to drought, heat and other abiotic stresses. This natural variation is partly due to variation in the genetic composition of individuals. Within-species variation can be used to identify and study genes involved in the genetic regulation of plant performance.
Growth of the world population will, in the coming years, lead to an increased demand for food, feed and other natural products. In addition, extreme weather conditions with, amongst others, more and prolonged periods of drought and heat are expected to occur due to climate change. Therefore breeders are challenged to produce stress tolerant cultivars with improved yield under sub-optimal conditions. Knowledge about the mechanisms and genes that underlie tolerance to drought, heat and other abiotic stresses will ease this challenge.
The aim of this thesis was to identify and study the role of genes that are underlying natural variation in plant performance under drought, salt and heat stress. To reach this goal a genome wide association (GWA) mapping approach was taken in the model species Arabidopsis thaliana. A population of 350 natural accessions of Arabidopsis, genotyped with 215k SNPs, was grown under control and several stress conditions and plant performance was evaluated by phenotyping one or several plant traits per environment. Genes located in the genomic regions that were significantly associated with plant performance, were studied in more detail.
Plant performance was first evaluated upon osmotic stress (Chapter 2). This treatment resulted not only in a reduced plant size, but also caused the colour of the rosette leaves to change from green to purple-red due to anthocyanin accumulation. The latter was visually quantified and subsequent GWA mapping revealed that a large part of the variation in anthocyanin accumulation could be explained by a small genomic region on chromosome 1. The analysis of re-sequence data allowed us to associate the second most frequent allele of MYB90 with higher anthocyanin accumulation and to identify the causal SNP. Interestingly MYB75, a close relative of MYB90, was not identified by GWA mapping, although causal sequence variation of this gene for anthocyanin accumulation was identified in the Cvi x Ler and Ler x Eri-1 RIL populations. Re-sequence data revealed that one allele of MYB75 was dominating the population and that the MYB75 alleles of Cvi and Ler were both rare, explaining the lack of association at this locus in GWA mapping. For MYB90, two alleles were present in a substantial part of the population, suggesting balancing selection between them.
Next, the natural population was exposed to short-term heat stress during flowering (Chapter 3). This short-term stress has a large impact on seed set, while it hardly affects the vegetative tissues. Natural variation for tolerance against the effect of heat on seed set was evaluated by measuring the length of all siliques along the inflorescence in both heat-treated and control plants. Because the flower that opened during the treatment was tagged, we could analyse the heat response for several developmental stages separately. GWA mapping revealed that the heat response before and after anthesis involved different genes. For the heat response before anthesis strong evidence was gained that FLC, a flowering time regulator and QUL2, a gene suggested to play a role in vascular tissue development, were causal for two strong associations.
Furthermore, the impact of moderate drought on plant performance was evaluated in the plant phenotyping platform PHENOPSIS. Homogeneous drought was assured by tight regulation of climate cell conditions and the robotic weighing and watering of the pots twice a day. Because plant growth is a dynamic trait it was monitored over time by top-view imaging under both moderate drought and control conditions (Chapter 4 and 5). To characterise growth it was modelled with an exponential function. GWA mapping of temporal growth data resulted in the detection of time-dependent QTLs whereas mapping of model parameters resulted in another set of QTLs related to the entire growth period. Most of these QTLs would not have been identified if plant size had only been determined on a single day. For the QTLs detected under control conditions eight candidate genes with a growth-related mutant or overexpression phenotype were identified (Chapter 4). Genes in the support window of the drought-QTLs were prioritized based on previously reported gene expression data (Chapter 5). Additional validation experiments are needed to confirm causality of the candidate genes.
Next, to search for genes that determine plant size across many environments, biomass accumulation in the natural population was determined in 25 different environments (Chapter 6). Joint analysis of these data by multi-environment GWA mapping resulted in the detection of 106 strongly associated SNPs with significant effects in 7 to 16 environments. Several genes involved in starch metabolism, leaf size control and flowering time determination were located in close proximity of the associated SNPs. Two genes, RPM1 and ACD6, were located in close proximity of SNPs with significant GxE effects. For both genes, alleles have been identified that increase resistance to bacterial infection, but that reduce biomass accumulation. The sign of the allelic effect is therefore dependent on the environmental conditions. Whole genome predictions revealed that most of the GxE interactions observed at the phenotypic level were not the consequence of strong associations with strong QxE effects, but of moderate and weak associations with weak QxE effects.
Finally, in Chapter 7 I discuss the usefulness of GWA mapping in the identification of genes underlying natural variation in plant performance under drought, heat stress and a number of other environments. Strong associations were observed for both environment-specific as well as common plant performance regulators. Some choices in phenotyping and experimental design were crucial for our success, like evaluation of plant performance over time and simplification of the quantification of the phenotype. It is suggested that follow-up work should focus on the functional characterization of the causal genes, because such analyses would be helpful to identify pathways in which the causal genes are involved and to understand why sequence variation results in changes at the phenotype level. Although translation of the findings to applications in crops is challenging, this thesis contributes to the understanding of the genetic regulation of stress response and therefore will likely contribute to the development of stress tolerant and stable yielding crops.
Breeding program for indigenous chicken in Kenya
Ngeno, K. - \ 2015
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Liesbeth van der Waaij; A.K. Kahi. - Wageningen : Wageningen University - ISBN 9789462572775 - 154
kippen - pluimvee - inheems vee - dierveredeling - veredelingsprogramma's - genetische diversiteit - ecotypen - genomen - genetische verbetering - kenya - fowls - poultry - native livestock - animal breeding - breeding programmes - genetic diversity - ecotypes - genomes - genetic improvement - kenya
Ngeno, K. (2015). Breeding program for indigenous chicken in Kenya. Analysis of diversity in indigenous chicken populations. PhD thesis, Wageningen University, the Netherlands
The objective of this research was to generate knowledge required for the development of an indigenous chicken (IC) breeding program for enhanced productivity and improved human livelihood in Kenya. The initial step was to review five questions; what, why and how should we conserve IC in an effective and sustainable way, who are the stakeholders and what are their roles in the IC breeding program. The next step of the research focused on detecting distinctive IC ecotypes through morphological and genomic characterization. Indigenous chicken ecotypes were found to be populations with huge variability in the morphological features. Molecular characterization was carried out using microsatellite markers and whole genome re-sequenced data. The studied IC ecotypes are genetically distinct groups. The MHC-linked microsatellite markers divided the eight IC ecotypes studied into three mixed clusters, composing of individuals from the different ecotypes whereas non-MHC markers grouped ICs into two groups. Analysis revealed high genetic variation within the ecotype with highly diverse MHC-linked alleles which are known to be involved in disease resistance. Whole genome re-sequencing revealed genomic variability, regions affected by selection, candidate genes and mutations that can explain partially the phenotypic divergence between IC and commercial layers. Unlike commercial chickens, IC preserved a high genomic variability that may be important in addressing present and future challenges associated with environmental adaptation and farmers’ breeding goals. Lastly, this study showed that there is an opportunity to improve IC through selection within the population. Genetic improvement utilizing within IC selection requires setting up a breeding program. The study described the systematic and logical steps in designing a breeding program by focusing on farmers’ need, how to improve IC to fit the farming conditions, and management regimes.
The hybrid nature of pig genomes : unraveling the mosaic haplotype structure in wild and commercial Sus scrofa populations
Bosse, M. - \ 2015
Wageningen University. Promotor(en): Martien Groenen, co-promotor(en): Hendrik-Jan Megens; Ole Madsen. - Wageningen : Wageningen University - ISBN 9789462573000 - 253
dieren - varkens - dierveredeling - genomen - hybridisatie - sus scrofa - haplotypen - genomica - populaties - genetische variatie - animals - pigs - animal breeding - genomes - hybridization - sus scrofa - haplotypes - genomics - populations - genetic variation - cum laude
cum laude graduation
Genomics 4.0 : syntenic gene and genome duplication drives diversification of plant secondary metabolism and innate immunity in flowering plants : advanced pattern analytics in duplicate genomes
Hofberger, J.A. - \ 2015
Wageningen University. Promotor(en): Eric Schranz. - Wageningen : Wageningen University - ISBN 9789462573147 - 142
genomica - planten - metabolisme - bloeiende planten - genomen - genen - next generation sequencing - genomics - plants - metabolism - flowering plants - genomes - genes - next generation sequencing
Genomics 4.0 - Syntenic Gene and Genome Duplication Drives Diversification of Plant Secondary Metabolism and Innate Immunity in Flowering Plants
Johannes A. Hofberger1, 2, 3
1 Biosystematics Group, Wageningen University & Research Center, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands (August 2012 – December 2013)
2 Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands (December 2010 – July 2012)
3 Chinese Academy of Sciences/Max Planck Partner Institute for Computational Biology, 320 Yueyang Road,
Shanghai 200031, PR China (January 2014 – December 2014)TWO-SENTENCE SUMMARY
Large-scale comparative analysis of Big Data from next generation sequencing provides powerful means to exploit the potential of nature in context of plant breeding and biotechnology. In this thesis, we combine various computational methods for genome-wide identification of gene families involved in (a) plant innate immunity and (a) biosynthesis of defense-related plant secondary metabolites across 21 species, assess dynamics that affected evolution of underlying traits during 250 Million Years of flowering plant radiation and provide data on more than 4500 loci that can underpin crop improvement for future food and live quality.
As sessile organisms, plants are permanently exposed to a plethora of potentially harmful microbes and other pests. The surprising resilience to infections observed in successful lineages is due to a complex defense network fighting off invading pathogens. Within this network, a sophisticated plant innate immune system is accompanied by a multitude of specialized biosynthetic pathways that generate more than 200,000 secondary metabolites with ecological, agricultural, energy and medicinal importance. The rapid diversification of associated genes was accompanied by a series of duplication events in virtually all plant species, including local duplication of short sequences as well as multiplication of all chromosomes due to meiotic errors (plant polyploidy). In a comparative genomics approach, we combined several bioinformatics techniques for large-scale identification of multi-domain and multi-gene families that are involved in plant innate immunity or defense-related secondary metabolite pathways across 21 representative flowering plant genomes. We introduced a framework to trace back duplicate gene copies to distinct ancient duplication events, thereby unravelling a differential impact of gene and genome duplication to molecular evolution of target genes. Comparing the genomic context among homologs within and between species in a phylogenomics perspective, we discovered orthologs conserved within genomic regions that remained structurally immobile during flowering plant radiation. In summary, we described a complex interplay of gene and genome duplication that increased genetic versatility of disease resistance and secondary metabolite pathways, thereby expanding the playground for functional diversification and thus plant trait innovation and success. Our findings give fascinating insights to evolution across lineages and can underpin crop improvement for food, fiber and biofuels production
Structural variations in pig genomes
Paudel, Y. - \ 2015
Wageningen University. Promotor(en): Martien Groenen, co-promotor(en): Ole Madsen; Hendrik-Jan Megens. - Wageningen : Wageningen University - ISBN 9789462572171 - 204
varkens - dierveredeling - genomen - genomica - single nucleotide polymorphism - dna-sequencing - fenotypische variatie - chromosoomafwijkingen - evolutie - soortvorming - pigs - animal breeding - genomes - genomics - single nucleotide polymorphism - dna sequencing - phenotypic variation - chromosome aberrations - evolution - speciation
Paudel, Y. (2015). Structural variations in pig genomes. PhD thesis, Wageningen University, the Netherlands
Structural variations are chromosomal rearrangements such as insertions-deletions (INDELs), duplications, inversions, translocations, and copy number variations (CNVs). It has been shown that structural variations are as important as single nucleotide polymorphisms (SNPs) in regards to phenotypic variations. The general aim of this thesis was to use next generation sequencing data to improve our understanding of the evolution of structural variations such as CNVs, and INDELs in pigs. We found that: 1) the frequency of copy number variable regions did not change during pig domestications but rather reflected the demographic history of pigs. 2) CNV of olfactory receptor genes seems to play a role in the on-going speciation of the genus Sus. 3) Variation in copy number of olfactory receptor genes in pigs (Sus scrofa) seems to be shaped by a combination of selection and genetic drift, where the clustering of ORs in the genome is the major source of variation in copy number. 4) Analysis on short INDELs in the pig genome shows that the level of purifying selection of INDELs positively correlates with the functional importance of a genomic region, i.e. strongest purifying selection was observed in gene coding regions. This thesis provides a highly valuable resource for copy number variable regions, INDELs, and SNPs, for future pig genetics and breeding research. Furthermore, this thesis discusses the limitations and improvements of the available tools to conduct structural variation analysis and insights into the future trends in the detection of structural variations.
Sociable swine: prospects of indirect genetic effects for the improvement of productivity, welfare and quality
Duijvesteijn, N. - \ 2014
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Piter Bijma; E.F. Knol. - Wageningen : Wageningen University - ISBN 9789462571525 - 202
varkens - sociaal gedrag - groepsinteractie - genetische effecten - prestatieniveau - dierenwelzijn - androsteron - genomen - genetische correlatie - varkensfokkerij - stakeholders - beoordeling - dierlijke productie - pigs - social behaviour - group interaction - genetic effects - performance - animal welfare - androsterone - genomes - genetic correlation - pig breeding - stakeholders - assessment - animal production
Towards Healthy Diets for parents: efectiveness of a counselling intervention
Eveline J.C. Hooft van Huysduynen
Introduction and Objective: As parents’ modelling of dietary behaviour is one of the factors influencing children’s diets, improving parents’ diets is expected to result in improved dietary intake of their children. This thesis describes research that was conducted to develop and evaluate a counselling intervention to improve parental adherence to the Dutch dietary guidelines.
Methods: A counselling intervention was developed, which was underpinned with the theory of planned behaviour and the transtheoretical model. In 20 weeks, five face-to-face counselling sessions were provided by a registered dietician who used motivational interviewing to improve parental adherence to the Dutch dietary guidelines. In addition, parents received three individually tailored email messages. During the counselling, the dietary guidelines and additional eating behaviours, that were hypothesized to affect diet quality, were addressed. The intervention was evaluated in a randomised controlled trial with 92 parents receiving the counselling and 94 parents as controls. Effects on dietary intake, biomarkers, intermediate markers of health and children’s dietary intake were evaluated. With mediation analyses, it was investigated if changes in dietary intake were established via changes in behavioural determinants. Thereby, it was also examined if spot urine samples could be used to replace 24 h urine samples for evaluating changes in sodium and potassium intake.
Results: The intervention group increased their adherence to the dietary guidelines, as assessed with the Dutch Healthy Diet-index (ranging from 0 to 100 points), by 6.7 points more than the control group did. This improvement was achieved by small increases in the scores of seven out of ten index components. The most substantial changes were shown in fruit and fish intakes of which increases in fish intake were reflected in changes in fatty acid profiles derived from blood plasma. Also a small decrease in waist circumference was observed. Based on parental reports, the children in the intervention group increased their intakes of fruit, vegetables and fish more than the children in the control group. Improvements in parental fruit intake were mediated by changes in the behavioural determinants attitude and habit strength. Decreases in snack intake were mediated by changes in self-identity as a healthy eater. Although the results of a study in young Caucasian women showed that spot urine can be used to rank individuals for their ratios of sodium to potassium, no intervention effects on these ratios were observed.
Conclusion: This thesis provides empirical knowledge on potential effective elements for counselling interventions aiming at improving the dietary pattern as a whole of parents and provides knowledge on methods to evaluate changes in dietary intake.
Filling the gap between sequence and function: a bioinformatics approach
Bargsten, J.W. - \ 2014
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Jan-Peter (Jp) Nap. - Wageningen : Wageningen University - ISBN 9789462570764 - 170
bio-informatica - planten - genomica - nucleotidenvolgordes - functionele genomica - vergelijkende genomica - vergelijkende genetische kartering - genomen - genetische kartering - plantenveredeling - methodologie - bioinformatics - plants - genomics - nucleotide sequences - functional genomics - comparative genomics - comparative mapping - genomes - genetic mapping - plant breeding - methodology
The research presented in this thesis focuses on deriving function from sequence information, with the emphasis on plant sequence data. Unravelling the impact of genomic elements, in most cases genes, on the phenotype of an organism is a major challenge in biological research and modern plant breeding. An important part of this challenge is the (functional) annotation of such genomic elements. Currently, wet lab experiments may provide high quality, but they are laborious and costly. With the advent of next generation sequencing platforms, vast amounts of sequence data are generated. This data are used in connection with the available experimental data to derive function from a bioinformatics perspective.
The connection between sequence information and function was approached on the level of chromosome structure (chapter 2) and of gene families (chapter 3) using combinations of existing bioinformatics tools. The applicability of using interaction networks for function prediction was demonstrated by first markedly improving an existing method (chapter 4) and by exploring the role of network topology in function prediction (chapter 5). Taken together, the combination of methods and results presented indicate the potential as well as the current state-of-the-art of function prediction in (plant) bioinformatics.
Chapter 1 introduces the basis for the approaches used and developed in this thesis. This includes the concepts of genome annotation, comparative genomics, gene function prediction and the analysis of network topology for gene function prediction. A requirement for the study of any new organism is the sequencing and annotation of its genome. Current genome annotation is divided into structural identification and functional categorization of genomic elements. The de facto standard for categorizing functional annotation is provided by the Gene Ontology. The Gene Ontology is divided into three domains, molecular function, biological process and cellular component. Approaches to predict molecular function and biological process are outlined. Accurate function prediction generally relies on existing input data, often of experimental origin, that can be transferred to unannotated genomic elements. Plants often lack such input data, which poses a big challenge for current function prediction algorithms. In unravelling the function of genomic elements, comparative genomics is an important approach. Via the comparison of multiple genomes it gives insights into evolution, function as well as genomic structure and variation. Comparative genomics has become an essential toolkit for the analysis of newly sequenced organisms. Often bioinformatics methods need to be adapted to the specific needs of plant genome research. With a focus on the commercially important crop plants tomato and potato, specific requirements of plant bioinformatics, such as the high amount of repetitive elements and the lack of experimental data, are outlined.
In chapter 2, the structural homology of the long arm of chromosome 2 (2L) of tomato, potato and pepper is analyzed. Molecular organization and collinear junctions are delineated using multi-color BAC FISH analysis and comparative sequence alignment. We identify several large-scale rearrangements including inversions and segmental translocations that were not reported in previous comparative studies. Some of the structural rearrangements are specific for the tomato clade, and differentiate tomato from potato, pepper and other solanaceous species. There are many small-scale synteny perturbations, but local gene vicinity is largely preserved. The data suggests that long distance intra-chromosomal rearrangements and local gene rearrangements have evolved frequently during speciation in the Solanum genus, and that small changes are more prevalent than large-scale differences. The occurrence of transposable elements and other repeats near or at junction breaks may indicate repeat-mediated rearrangements. The ancestral 2L topology is reconstructed and the evolutionary events leading to the current topology are discussed.
In chapter 3, we analyze the Snf2 gene family. As part of large protein complexes, Snf2 family ATPases are responsible for energy supply during chromatin remodeling, but the precise mechanism of action of many of these proteins is largely unknown. They influence many processes in plants, such as the response to environmental stress. The analysis is the first comprehensive study of Snf2 family ATPases in plants. Some subfamilies of the Snf2 gene family are remarkably stable in number of genes per genome, whereas others show expansion and contraction in several plants. One of these subfamilies, the plant-specific DRD1 subfamily, is non-existent in lower eukaryote genomes, yet it developed into the largest Snf2 subfamily in plant genomes. It shows the occurrence of a complex series of evolutionary events. Its expansion, notably in tomato, suggests novel functionality in processes connected to chromatin remodeling. The results underpin and extend the Snf2 subfamily classification, which could help to determine the various functional roles of Snf2 ATPases and to target environmental stress tolerance and yield in future breeding with these genes.
In chapter 4, a new approach to improve the prediction of protein function in terms of biological processes is developed that is particularly attractive for sparsely annotated plant genomes. The combination of the network-based prediction method Bayesian Markov Random Field (BMRF) with the sequence-based prediction method Argot2 shows significantly improved performance compared to each of the methods separately, as well as compared to Blast2GO. The approach was applied to predict biological processes for the proteomes of rice, barrel clover, poplar, soybean and tomato. Analysis of the relationships between sequence similarity and predicted function similarity identifies numerous cases of divergence of biological processes in which proteins are involved, in spite of sequence similarity. Examples of potential divergence are identified for various biological processes, notably for processes related to cell development, regulation, and response to chemical stimulus. Such divergence in biological process annotation for proteins with similar sequences should be taken into account when analyzing plant gene and genome evolution. This way, the integration of network-based and sequence-based function prediction will strengthen the analysis of evolutionary relationships of plant genomes.
In chapter 5 the influence of network topology on network-based function prediction algorithms is investigated. The analysis of biological networks using algorithms such as Bayesian Markov Random Field (BMRF) is a valuable predictor of the biological processes that proteins are involved in. The topological properties and constraints that determine prediction performance in such networks are however largely unknown. This chapter presents analyses based on network centrality measures, such as node degree, to evaluate the performance of BMRF upon progressive removal of highly connected hub nodes (pruning). Three different protein-protein interaction networks with data from Arabidopsis, human and yeast were analyzed. All three show that the average prediction performance can improve significantly. The chapter paves the way for further improvement of network-based function prediction methods based on node pruning.
Chapter 6 discusses the results and methods developed in this thesis in the context of the vast amount of generated sequencing data. Sequencing or re-sequencing a (plant) genome has become fairly straightforward and affordable, but the interpretation for subsequent use of this sequence data is far from trivial. The topics addressed in this thesis, annotation of function, analysis of genome structure and identifying genomic variation, focus on this main bottleneck of biological research. Issues discussed in connection with this work and its future are data accuracy, error propagation, possible improvements and future implications for biological research in crop plants. In particular the shift of costs from sequencing to downstream analyses, with functional genome annotation as essential step, is covered. One of the biggest challenges biology and bioinformatics will face is the integration of results from such downstream analyses and other sources into a complete picture. Only this will allow understanding of complex biological systems.
Natural variation in memory formation among Nasonia parasitic wasps : from genes to behaviour
Hoedjes, K.M. - \ 2014
Wageningen University. Promotor(en): Louise Vet; Marcel Dicke, co-promotor(en): Hans Smid. - Wageningen : Wageningen University - ISBN 9789461739483 - 191
nasonia - hymenoptera - geheugen - leervermogen - genetische factoren - dierecologie - diergedrag - genomen - nasonia - hymenoptera - memory - learning ability - genetic factors - animal ecology - animal behaviour - genomes
The ability to learn and form memory has been demonstrated in various animal species, ranging from relatively simple invertebrates, such as snails and insects, to more complex vertebrate species, including birds and mammals. The opportunity to acquire new skills or to adapt behaviour through learning is an obvious benefit. However, memory formation is also costly: it can be maladaptive when unreliable associations are formed and the process of memory formation can be energetically costly. The balance between costs and benefits determines if learning and memory formation are beneficial to an animal or not. Variation in learning abilities and memory formation between species is thought to reflect species-specific differences in ecology.
This thesis focused on variation in the number of trials required to form long-term memory (LTM). LTM is considered the most stable and durable type of memory, but also the most costly, because it requires protein synthesis. Many animal species require multiple learning experiences, which are spaced in time, to form LTM. This allows re-evaluation of information before an animal invests in costly LTM. There is, however, variation in the number of trials that animal species require to induce LTM formation. A number of insect species, including a number of parasitic wasp species, form LTM after only a single learning experience. Parasitic wasps can learn odours that guide them towards suitable hosts for their offspring, so-called oviposition learning. Substantial differences in LTM formation are observed among closely related species of parasitic wasps, which provides excellent opportunities for comparative studies. Both ecological and genetic factors involved in variation in LTM formation have been studied in this project. A multidisciplinary approach is essential to understand the evolution of variation in LTM formation, because the interaction between genes and environment shapes learning and memory formation.
LTM formation was studied in closely related species of the genus Nasonia. These small parasitic wasps (~2 mm in length) lay their eggs in various species of fly pupae and differences in the ecology of the four known species of this genus have been described. A high-throughput method for olfactory conditioning was developed in which the wasps associated an odour, either chocolate or vanilla, with the reward of a host. A T-maze olfactometer was designed for high-throughput testing of memory retention. Using these methods, variation in memory retention was observed between three Nasonia species. Both N. vitripennis and N. longicornis form a long-lasting memory after a single conditioning trial, which lasts at least 5 days. Nasonia giraulti, on the other hand, lost its memory after 1 to 2 days after a single conditioning trial. Further studies focused on the difference between N. vitripennis and N. giraulti, which was most pronounced. By inhibiting LTM with transcription and translation inhibitors, it was confirmed that N. vitripennis forms this type of memory after a single conditioning trial. LTM is visible 4 days after conditioning in N. vitripennis. Nasonia giraulti does not form LTM after a single conditioning trial. Long-lasting memory is only formed after two trials, with a 4-hour interval between them. This difference in LTM formation makes N. vitripennis and N. giraulti excellent model species to study both ecological and genetic factors involved in this difference.
Ecological factors such as the value of the reward and the reliability of the learned association have been shown to affect memory formation in a number of animal species. A recent study on oviposition learning in two parasitic wasp species demonstrated that LTM formation depends on the host species, i.e. the reward offered during conditioning. LTM was formed when a host with a higher quality was offered, but not when a host of lower quality was offered. The effect of host quality on memory retention of N. vitripennis and N. giraulti was tested. Either a large host, Calliphora vomitoria, a medium-sized host, Lucilia sericata, or a small host, Musca domestica, was offered during conditioning. These hosts were observed to differ significantly in their quality, i.e. in the number of parasitoid offspring that emerged and the size of the offspring. There was, however, no effect of host species on memory retention in either Nasonia species. These results suggest that host quality is not important for LTM formation in N. vitripennis and N. giraulti. This observation shows that ecological factors that are important for memory formation in one species may not be important for another species.
The genetic basis of memory formation is highly conserved among distant animal phyla. A large number of genes involved in LTM formation have been identified in genetic model organisms, including fruit flies, honeybees, the California sea hare, mice and rats, and the zebra finch.Genetic factors responsible for natural variation in LTM formation between species are currently unknown, however. Two approaches were used to study genetic factors responsible for the difference in LTM formation between N. vitripennis and N. giraulti. The first approach took advantage of the unique possibility to interbreed Nasonia species. Hybrid offspring of N. vitripennis and N. giraulti did not form LTM after a single conditioning trial, similar to N. giraulti. The dominant LTM phenotype of N. giraulti was then backcrossed into the genetic background of N. vitripennis for up to 5 generations. Using a genotyping microarray analysis and subsequent confirmation experiments, we detected two genomic regions (quantitative trait loci – QTLs) that both reduce long-lasting memory, but not completely remove this memory. These results indicate that multiple QTLs regulate the difference in LTM formation between the two Nasonia species. Concluding, our approach has provided insights in the genomic basis of a naturally occurring difference in LTM formation between two species. Excellent opportunities for fine-scale QTL mapping are available for the genus Nasonia. This will allow identification of decisive regulatory mechanisms involved in LTM formation that are located in the two genomic regions detected in this study.
The second approach took advantage of next-generation sequencing techniques that allow transcriptome-wide studies of gene expression levels. RNA from heads of N. vitripennis and N. giraulti was collected before conditioning and immediately, 4 hours, or 24 hours after conditioning. This RNA was sequenced strand-specifically using HiSeq technology, which allows detection of sense and antisense transcripts. Various genes, from a number of different signalling pathways known to be involved in LTM formation, were uniquely differentially expressed after conditioning in N. vitripennis. These genes are likely involved in the ongoing process of LTM formation in this species. A number of other genes with a known role in LTM formation,including genes involved in dopamine synthesis and in the Ras-MAPK and PI3K signalling pathways, were uniquely differentially expressed in N. giraulti. These genes may have a role in a LTM inhibitory mechanism in this species. Antisense transcripts were detected for a number of known memory genes, which may indicate a role inregulation of transcription, alternative splicing, or translation. This study is the first to compare gene expression patterns after conditioning between two species that differ in LTM formation. The results provide promising candidate genes for future studies in which the regulation of these genes, the function of specific splice variants, and spatial expression patterns in the brain should be studied to understand how these genes are involved in the regulation of LTM formation.
Learning and memory formation have an important role in animal and human behaviour.Novel and valuable insights on both ecological and genetic factors responsible for variation in LTM formation have been revealed by the research presented in this thesis. Integrating ecological factors and genetic factors is essential, as genes are the level on which ecological factors can drive the evolution of variation in learning and memory formation. The genus Nasonia has offered excellent opportunities for ecological research as well as unique opportunities for studies on genomic and genetic factors, which were addressed by comparing closely related species that differ in memory formation. This thesis provides the basis for the identification of genomic differences responsible for the difference in memory formation between Nasonia species, but it also characterized the consequences of these genomic differences on gene expression. The genetic basis of learning and memory formation are highly conserved among distant animal species and insights from this thesis are likely applicable to other animal species and humans, as well.Altogether, these small parasitic wasps allow us to understand and value differences in memory formation.
Comparative genomics of Dothideomycete fungi
Burgt, A. van der - \ 2014
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jerome Collemare. - Wageningen : Wageningen University - ISBN 9789461739056 - 176
dothideomycetes - plantenziekteverwekkende schimmels - passalora fulva - dothistroma - genomen - vergelijkende genomica - dothideomycetes - plant pathogenic fungi - passalora fulva - dothistroma - genomes - comparative genomics
Fungi are a diverse group of eukaryotic micro-organisms particularly suited for comparative genomics analyses. Fungi are important to industry, fundamental science and many of them are notorious pathogens of crops, thereby endangering global food supply. Dozens of fungi have been sequenced in the last decade and with the advances of the next generation sequencing, thousands of new genome sequences will become available in coming years. In this thesis I have used bioinformatics tools to study different biological and evolutionary processes in various genomes with a focus on the genomes of the Dothideomycetefungi Cladosporium fulvum, Dothistroma septosporumand Zymoseptoria tritici.
Chapter 1introduces the scientific disciplines of mycology and bioinformatics from a historical perspective. It exemplifies a typical whole-genome sequence analysis of a fungal genome, and focusses in particular on structural gene annotation and detection of transposable elements. In addition it shortly reviews the microRNA pathway as known in animal and plants in the context of the putative existence of similar yet subtle different small RNA pathways in other branches of the eukaryotic tree of life.
Chapter 2addresses the novel sequenced genomes of the closely related Dothideomyceteplant pathogenic fungi Cladosporium fulvumand Dothistroma septosporum. Remarkably, it revealed occurrence of a surprisingly high similarity at the protein level combined with striking differences at the DNA level, gene repertoire and gene expression. Most noticeably, the genome of C. fulvumappears to be at least twice as large, which is solely attributable to a much larger content in repetitive sequences.
Chapter 3describes a novel alignment-based fungal gene prediction method (ABFGP) that is particularly suitable for plastic genomes like those of fungi. It shows excellent performance benchmarked on a dataset of 7,000 unigene-supported gene models from ten different fungi. Applicability of the method was shown by revisiting the annotations of C. fulvumand D. septosporumand of various other fungal genomes from the first-generation sequencing era. Thousands of gene models were revised in each of the gene catalogues, indeed revealing a correlation to the quality of the genome assembly, and to sequencing strategies used in the sequencing centres, highlighting different types of errors in different annotation pipelines.
Chapter 4focusses on the unexpected high number of gene models that were identified by ABFGP that align nicely to informant genes, but only upon toleration of frame shifts and in-frame stop-codons. These discordances could represent sequence errors (SEs) and/or disruptive mutations (DMs) that caused these truncated and erroneous gene models. We revisited the same fungal gene catalogues as in chapter 3, confirmed SEs by resequencing and successively removed those, yielding a high-confidence and large dataset of nearly 1,000 pseudogenes caused by DMs. This dataset of fungal pseudogenes, containing genes listed as bona fide genes in current gene catalogues, does not correspond to various observations previously done on fungal pseudogenes. Moreover, the degree of pseudogenization showing up to a ten-fold variation for the lowest versus the highest affected species, is generally higher in species that reproduce asexually compared to those that in addition reproduce sexually.
Chapter 5describes explorative genomics and comparative genomics analyses revealing the presence of introner-like elements (ILEs) in various Dothideomycetefungi including Zymoseptoria triticiin which they had not identified yet, although its genome sequence is already publicly available for several years. ILEs combine hallmark intron properties with the apparent capability of multiplying themselves as repetitive sequence. ILEs strongly associate with events of intron gain, thereby delivering in silico proof of their mobility. Phylogenetic analyses at the intra- and inter-species level showed that most ILEs are related and likely share common ancestry.
Chapter 6provides additional evidence that ILE multiplication strongly dominates over other types of intron duplication in fungi. The observed high rate of ILE multiplication followed by rapid sequence degeneration led us to hypothesize that multiplication of ILEs has been the major cause and mechanism of intron gain in fungi, and we speculate that this could be generalized to all eukaryotes.
Chapter 7describes a new strategy for miRNA hairpin prediction using statistical distributions of observed biological variation of properties (descriptors) of known miRNA hairpins. We show that the method outperforms miRNA prediction by previous, conventional methods that usually apply threshold filtering. Using this method, several novel candidate miRNAs were assigned in the genomes of Caenorhabditis elegansand two human viruses. Although this chapter is not applied on fungi, the study does provide a flexible method to find evidence for existence of a putative miRNA-like pathway in fungi.
Chapter 8provides a general discussion on the advent of bioinformatics in mycological research and its implications. It highlights the necessity of a prioriplanning and integration of functional analysis and bioinformatics in order to achieve scientific excellence, and describes possible scenarios for the near future of fungal (comparative) genomics research. Moreover, it discusses the intrinsic error rate in large-scale, automatically inferred datasets and the implications of using and comparing those.
‘Speciale technieken alleen voor topfokkerij': Gary Hennip voorspelt het einde van KI als geavanceerde technieken worden gecombineerd
Hogenkamp, W. ; Veerkamp, R.F. - \ 2014
Boerderij 99 (2014)27. - ISSN 0006-5617 - p. 38 - 38.
veehouderij - dierveredeling - fokwaarde - kunstmatige inseminatie - genetica - genomen - technieken - nieuwe combinatie - toekomst - livestock farming - animal breeding - breeding value - artificial insemination - genetics - genomes - techniques - new combination - future
Gary Hennip, onderzoeker bij Penn State University (VS), denkt dat ki overbodig is bij slim combineren van technieken als ovum pick-up, ivf, genomics en sperma seksen op het bedrijf. Boerderij legt de stelling voor aan professor generieke genetcia Roel Veerkamp.
Physiology and biochemistry of aromatic hydrocarbon-degrading bacteria that use chlorate and/or nitrate as electron acceptor
Oosterkamp, M.J. - \ 2013
Wageningen University. Promotor(en): Fons Stams, co-promotor(en): Caroline Plugge; Peter Schaap. - Wageningen : Wageningen University - ISBN 9789461737779 - 191
bacteriën - aromatische koolwaterstoffen - fysiologie - biochemie - elektronen - genomen - nitraten - chloraten - microbiologie - bacteria - aromatic hydrocarbons - physiology - biochemistry - electrons - genomes - nitrates - chlorates - microbiology
Optimizing genomic selection for scarcely recorded traits
Pszczola, M.J. - \ 2013
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Mario Calus; T. Strabel. - Wageningen : Wageningen UR - ISBN 9789461737663 - 158
melkvee - genomen - selectief fokken - genetische verbetering - fokwaarde - fenotypen - genotypen - kenmerken - voeropname - dierveredeling - dairy cattle - genomes - selective breeding - genetic improvement - breeding value - phenotypes - genotypes - traits - feed intake - animal breeding
Animal breeding aims to genetically improve animal populations by selecting the best individuals as parents of the next generation. New traits are being introduced to breeding goals to satisfy new demands faced by livestock production. Selecting for novel traits is especially challenging when recording is laborious and expensive and large scale recording is not possible. Genetic improvement of novel traits may be thus limited due to the small number of observations. New breeding tools, such as genomic selection, are therefore needed to enable the genetic improvement of novel traits. Using the limited available data optimally may, however, require alternative approaches and methodologies than currently used for conventional breeding goal traits. The overall objective of this thesis was to investigate different options for optimizing genomic selection for scarcely recorded novel traits. The investigated options were: (1) genotype imputation for ungenotyped but phenotyped animals to be used to enlarge the reference population; (2) optimization of the design of the reference population with respect to the relationships among the animals included in it; (3) prioritizing genotyping of the reference population or the selection candidates; and (4) using easily recordable predictor traits to improve the accuracy of breeding values for scarcely recorded traits. Results showed that: (1) including ungenotyped animals to the reference population can lead to a limited increase in the breeding values accuracy; (2) the reference population is designed optimally when the relationship within the reference are minimized and between reference population and potential selection candidates maximized; (3) the main gain in accuracy when moving from traditional to genomic selection is due to genotyping the selection candidates, but preferably both reference population and selection candidates should be genotyped; and (4) including the predictor traits in the analysis when it is recorded on both reference population and selection candidates can lead to a significant increase in the selection accuracy. The key factors for successful implementation of selection for a novel trait in a breeding scheme are: (1) maximizing accuracy of genotype prediction for ungenotyped animals to be used for updating the reference population; (2) optimizing the design of the reference population; (3) determining easy to record indicator traits that are also available on the selection candidates (4) developing large scale phenotyping techniques; and (5) establishing strategies and policies for increasing the engagement of farmers in the recording of novel traits.
De tomatenkaart is klaar, wat nu?
Finkers, H.J. ; Visser, R.G.F. - \ 2013
Kennis Online 10 (2013)mei. - p. 3 - 5.
moleculaire genetica - plantengenetica - dna-sequencing - tomaten - rassen (planten) - plantenveredeling - genetische bronnen van plantensoorten - genomen - molecular genetics - plant genetics - dna sequencing - tomatoes - varieties - plant breeding - plant genetic resources - genomes
In 2012 publiceerde Nature de genomische sequentie van de tomaat. Maar daarmee is het werk niet af, zegt Richard Finkers. Hij bepaalde de basenvolgorde van nog eens 150 verwanten van de modeltomaat, om plantenveredelaars in staat te stellen op zoek te gaan naar nieuwe genen in oude rassen.
Genomics in de fokkerij: in tien jaar van topwetenschap naar big business
Arendonk, J.A.M. van; Groenen, M. - \ 2013
Kennis Online 10 (2013)mei. - p. 7 - 8.
dierveredeling - genomica - fokkerijmethoden - moleculaire technieken - single nucleotide polymorphism - selectiemethoden - melkvee - varkens - genomen - animal breeding - genomics - animal breeding methods - molecular techniques - single nucleotide polymorphism - selection methods - dairy cattle - pigs - genomes
Genomics was tien jaar geleden topwetenschap die alleen bedreven werd in dure laboratoria van universiteiten en onderzoeksinstituten. Nu gebruiken fokbedrijven DNA-chips om al op jonge leeftijd te kunnen voorspellen welke stier het beste sperma levert, en om te zien of berengeur bij varkens door een slim fokprogramma kan worden voorkomen.
'Ui is een puzzel met 150 duizend stukjes'
Smulders, M.J.M. - \ 2013
Kennis Online 10 (2013)mei. - p. 12 - 12.
moleculaire genetica - dna-sequencing - genomen - plantenveredeling - onderzoek - molecular genetics - dna sequencing - genomes - plant breeding - research
Om zeer grote genomen in kaart te kunnen brengen, moeten eerst fundamentele vragen worden opgelost. Zonder hulp van de overheid komen die antwoord er niet, zegt René Smulders. Terwijl grondige sequencing veredeling aantoonbaar versnelt.
Use of SNP markers to conserve genome-wide genetic diversity in livestock
Engelsma, K.A. - \ 2012
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Jack Windig. - S.l. : s.n. - ISBN 9789461733863 - 178
dierveredeling - rundvee - single nucleotide polymorphism - genetische diversiteit - genomen - genomica - conservering - animal breeding - cattle - single nucleotide polymorphism - genetic diversity - genomes - genomics - conservation
Conservation of genetic diversity in livestock breeds is important since it is, both within and between breeds, under threat. The availability of large numbers of SNP markers has resulted in new opportunities to estimate genetic diversity in more detail, and to improve prioritization of animals for conservation of genetic diversity. The aim of this thesis was to further explore the potential of SNP markers for estimation and conservation of genetic diversity within livestock breeds. This was evaluated analyzing Holstein cattle populations, genotyped with a commonly used 50k SNP chip. Genetic diversity was estimated with SNP markers and compared to genetic diversity estimated with pedigree information. Both methods could detect differences in overall genetic diversity, even between two closely related populations. With SNP markers, differences in genetic diversity at the chromosomal level could be identified as well. Subsequently, SNP markers and pedigree information were used to prioritize animals for conservation in a gene bank using optimal contributions. SNP based prioritization was slightly more effective than pedigree based information, both over the whole genome and at specific regions of the genome. We extended the optimal contribution method to simultaneously conserve a single allele at a specific frequency and maximize the overall genetic diversity conserved in a gene bank. The loss of overall genetic diversity was larger when the target frequency for animals conserved in the gene bank differed more from the original frequency in the population. It can be concluded that dense SNP data form a powerful tool for estimation and conservation of genetic diversity in livestock breeds. Although pedigree information gives a good representation of the overall genetic diversity, SNP markers can provide more detailed information about the genetic diversity over the genome. Especially for small populations, SNP markers can play an important role in conservation of unique alleles, while simultaneously minimizing the loss of genetic diversity at the rest of the genome.
The role and evolution of fungal effectors in plant pathogenesis
Jonge, R. de - \ 2012
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Bart Thomma. - S.l. : s.n. - ISBN 9789461733917 - 148
plantenziekteverwekkende schimmels - moleculaire plantenziektekunde - evolutie - gastheer parasiet relaties - pathogenese - genomica - immuniteit - genomen - plant pathogenic fungi - molecular plant pathology - evolution - host parasite relationships - pathogenesis - genomics - immunity - genomes - cum laude
cum laude graduation (with distinction)
Co-option of pre-existing pathways during Rhizobium-legume symbiosis evolution
Lillo, A. - \ 2012
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - S.l. : s.n. - ISBN 9789461733443 - 151
rhizobium - fabaceae - symbiose - evolutie - stikstoffixatie - wortels - fylogenetica - genomen - medicago - eerste wortels - rhizobium - fabaceae - symbiosis - evolution - nitrogen fixation - roots - phylogenetics - genomes - medicago - root primordia
Fixed nitrogen is one of the most limiting factors for plant growth. One of the most important nitrogen-fixing systems is the rhizobium root nodule symbiosis. In this Thesis I have studied the legume-rhizobium symbiosis, starting from the idea that part of pre-existing signalling pathways have been co-opted during evolution of this mutualistic interaction. Gene duplications -of which a whole genome duplication (WGD) is the most dramatic variant- are known as important driving forces in evolution of new traits. 56 to 65 million years ago an ancestral legume species within the Papilionoidae subfamily (Papilionoids) experienced a WGD event and subsequently gave rise to several major phylogenetic crowns. I hypothesize that among the orthologous gene pairs maintained are genes that are essential for nodulation. I adopted a phylogenetic strategy to identify new candidate genes involved in the legume-Rhizobium symbiosis
Waarom maakt aardappelplant knollen?
Bachem, C.W.B. - \ 2012
Kennis Online 9 (2012)mei. - p. 8 - 8.
aardappelen - genomica - moleculaire genetica - genomen - genen - genexpressie - potatoes - genomics - molecular genetics - genomes - genes - gene expression
Nu het aardappelgenoom in kaart is gebracht, kan het werk eigenlijk pas goed beginnen. ‘We kunnen doelgerichter zoeken naar genen die betrokken zijn bij resistenties en kwaliteit.’ En naar waarom de aardappel knollen vormt en zijn zusje de tomaat niet.
Een kaart van het gigantische leliegenoom en de genetische hutspot van prei
Arens, P. ; Scholten, O.E. - \ 2012
Kennis Online 9 (2012)april. - p. 10 - 11.
moleculaire genetica - dna-sequencing - genomen - lilium - allium porrum - chromosoomkaarten - molecular genetics - dna sequencing - genomes - lilium - allium porrum - chromosome maps
Van allerlei organismen is de complete DNA-volgorde bekend. Lelieveredelaars moeten het echter nog doen met een kaart van niks. Dat komt door de onwaarschijnlijke omvang van het genoom van de bloem. Een lelie heeft tien keer meer DNA dan een mens. Niet alleen de lelie, ook prei laat zich lastig in kaart brengen. Dat komt niet alleen door de omvang van het DNA, maar vooral door de organisatie ervan. Waar mensen en veel planten van elk chromosoom twee kopieën hebben, heeft prei er vier.
Evolution of rhizobium symbiosis
Camp, R.H.M. Op den - \ 2012
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Rene Geurts. - S.l. : s.n. - ISBN 9789461731982 - 136
papilionoideae - rhizobium - symbiose - genomen - parasponia - mycorrhizae - evolutie - moleculaire biologie - papilionoideae - rhizobium - symbiosis - genomes - parasponia - mycorrhizas - evolution - molecular biology
The evolution of rhizobium symbiosis is studied from several points of view in this thesis. The ultimate goal of the combined approaches is to unravel the genetic constrains of the symbiotic interaction. To this end the legume rhizobium symbiosis is studied in model plant species from the Papilionoideae subfamily such as Medicago truncatula and Lotus japonicus. In these model plants the genetic signaling cascade used for rhizobium symbiosis has been largely unraveled. The cascade is triggered by lipo-chitooligosaccharade-based signal molecules excreted by rhizobia, called Nod factors.
In chapter 2 we make use of a whole genome duplication that has occurred at the root of the legume Papilionoideae subfamily to identify maintained paralogous gene pairs. We hypothesized that a substantial fraction of gene pairs which are maintained in distinct Papilionoideae lineages that split roughly 54 million years ago fulfill legume specific functions, among which is rhizobium symbiosis. Furthermore we argue that such approach could identify novel genes as it can also identify genes pairs that are (partially) redundant in function. With applying this approach specifically to the cytokinin phosphorelay pathway we identified a pair of type-A cytokinin Response Regulators that are involved in rhizobium symbiosis. This study provides a proof-of-principle for this strategy.
It is known for over fifty years that cytokinin plays an important role in the symbiotic interaction between rhizobia and legume hosts. External application of cytokinin can even result in nodule formation. Only, never had cytokinin levels been quantified in legume root extracts upon symbiotic interaction. In chapter 3 we describe a method for extraction of both cytokinins and auxin from Medicago truncatula roots. We show that cytokinins accumulate in the root zone susceptible to symbiotic interaction upon Nod factor exposure and that this response is dependent on CCaMK; a key gene of the Nod factor signaling cascade. Furthermore, it was found that ethylene signaling has a negative effect on Nod factor induced cytokinin accumulation. The method set up to measure cytokinin as well as auxin provides a tool to further study hormone interactions in rhizobium symbiosis.
Parasponia,the only non-legume that can engage the rhizobium symbiosis is also subject of study in this thesis. The genetics of the Parasponia-rhizobium symbiosis had not been studied before. It was therefore unknown whether this independently evolved rhizobium symbiosis makes use of the same symbiotic signaling cascade as legumes. In chapter 4 we provide first evidence that Parasponia indeed makes use of the same signaling cascade as found in legumes. Furthermore, we show that in Parasponia a single Nod factor-like receptor is indispensable for two symbiotic interactions; rhizobium and mycorrhiza, respectively. Therefore we conclude that the rhizobium Nod factor perception mechanism is recruited from the widespread endomycorrhizal symbiosis.
Parallel to our studies in Parasponia (Chapter 4), the research team of Jean Dénarié of the French National Institute for Agricultural Research (INRA) published the structure of the signal molecule of the arbuscular endomycorrhizae; theMyc factor (Maillet et al., 2011, Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature, 469, 58-63). It appeared that Myc factors and Nod factors are structurally very similar. In chapter 5 we discuss these findings and present a more thorough phylogenetic analysis of the NFP-like LysM-type receptor kinases. Together, these results suggest that non-legumes that can engage an arbuscular endomycorrhizaesymbiosis can recognize Nod factor-like molecules as well.
The last chapter is about a study on the promiscuity and effectiveness of the Parasponia-rhizobium symbiosis. Parasponia uses a single receptor to control entry of rhizobium as well as arbuscular endomycorrhizal fungi and has evolved the rhizobium symbiosis only recently. This made us to hypothesize that Parasponia Nod factor receptors did not coevolve yet with rhizobia and therefore did not diverge from mycorrhizal recognition to develop specificity for the Nod factor. This implies that Parasponia could be a very promiscuous host for rhizobium species. In chapter 6 we describe that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to 4 different genera, and therefore it is concluded that Parasponia is highly promiscuous for rhizobial engagement. There is a drawback to this high symbiotic promiscuity. Among the strains identified to nodulate Parasponia, a very inefficient rhizobium species, Rhizobium tropici WUR1, was characterized. As this species is able to make effective nodules on two different legume species it suggests that the ineffectiveness of Parasponia andersonii nodules is the result of the incompatibility between both partners. In Parasponia andersonii nodules rhizobia of the ineffective strain become embedded in a dense matrix, but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia. Therefore we argue that the Parasponia-Rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization.
Parasponiahas been given little attention in the rhizobium symbiosis field over the past two decades but with our efforts renewed interest has been established. We believe that in the end, the comparison of Parasponia to its closest related non-symbiotic sister species Trema, will result in the determination of the genetic constrains of rhizobium symbiosis.
Genome bioinformatics of tomato and potato
Datema, E. - \ 2011
Wageningen University. Promotor(en): W. Stiekema, co-promotor(en): Roeland van Ham. - [S.l.] : S.n. - ISBN 9789461730473 - 139
gewassen - solanum lycopersicum - solanum tuberosum - genomica - bio-informatica - nucleotidenvolgordes - genomen - genen - crops - solanum lycopersicum - solanum tuberosum - genomics - bioinformatics - nucleotide sequences - genomes - genes
In the past two decades genome sequencing has developed from a laborious and costly technology employed by large international consortia to a widely used, automated and affordable tool used worldwide by many individual research groups. Genome sequences of many food animals and crop plants have been deciphered and are being exploited for fundamental research and applied to improve their breeding programs. The developments in sequencing technologies have also impacted the associated bioinformatics strategies and tools, both those that are required for data processing, management, and quality control, and those used for interpretation of the data.
This thesis focuses on the application of genome sequencing, assembly and annotation to two members of the Solanaceae family, tomato and potato. Potato is the economically most important species within the Solanaceae, and its tubers contribute to dietary intake of starch, protein, antioxidants, and vitamins. Tomato fruits are the second most consumed vegetable after potato, and are a globally important dietary source of lycopene, beta-carotene, vitamin C, and fiber. The chapters in this thesis document the generation, exploitation and interpretation of genomic sequence resources for these two species and shed light on the contents, structure and evolution of their genomes.
Chapter 1introduces the concepts of genome sequencing, assembly and annotation, and explains the novel genome sequencing technologies that have been developed in the past decade. These so-called Next Generation Sequencing platforms display considerable variation in chemistry and workflow, and as a consequence the throughput and data quality differs by orders of magnitude between the platforms. The currently available sequencing platforms produce a vast variety of read lengths and facilitate the generation of paired sequences with an approximately fixed distance between them. The choice of sequencing chemistry and platform combined with the type of sequencing template demands specifically adapted bioinformatics for data processing and interpretation. Irrespective of the sequencing and assembly strategy that is chosen, the resulting genome sequence, often represented by a collection of long linear strings of nucleotides, is of limited interest by itself. Interpretation of the genome can only be achieved through sequence annotation – that is, identification and classification of all functional elements in a genome sequence. Once these elements have been annotated, sequence alignments between multiple genomes of related accessions or species can be utilized to reveal the genetic variation on both the nucleotide and the structural level that underlies the difference between these species or accessions.
Chapter 2describes BlastIf, a novel software tool that exploits sequence similarity searches with BLAST to provide a straightforward annotation of long nucleotide sequences. Generally, two problems are associated with the alignment of a long nucleotide sequence to a database of short gene or protein sequences: (i) the large number of similar hits that can be generated due to database redundancy; and (ii) the relationships implied between aligned segments within a hit that in fact correspond to distinct elements on the sequence such as genes. BlastIf generates a comprehensible BLAST output for long nucleotide sequences by reducing the number of similar hits while revealing most of the variation present between hits. It is a valuable tool for molecular biologists who wish to get a quick overview of the genetic elements present in a newly sequenced segment of DNA, prior to more elaborate efforts of gene structure prediction and annotation.
In Chapter 3 a first genome-wide comparison between the emerging genomic sequence resources of tomato and potato is presented. Large collections of BAC end sequences from both species were annotated through repeat searches, transcript alignments and protein domain identification. In-depth comparisons of the annotated sequences revealed remarkable differences in both gene and repeat content between these closely related genomes. The tomato genome was found to be more repetitive than the potato genome, and substantial differences in the distribution of Gypsy and Copia retrotransposable elements as well as microsatellites were observed between the two genomes. A higher gene content was identified in the potato sequences, and in particular several large gene families including cytochrome P450 mono-oxygenases and serine-threonine protein kinases were significantly overrepresented in potato compared to tomato. Moreover, the cytochrome P450 gene family was found to be expanded in both tomato and potato when compared to Arabidopsis thaliana, suggesting an expanded network of secondary metabolic pathways in the Solanaceae. Together these findings present a first glimpse into the evolution of Solanaceous genomes, both within the family and relative to other plant species.
Chapter 4explores the physical and genetic organization of tomato chromosome 6 through integration of BAC sequence analysis, High Information Content Fingerprinting, genetic analysis, and BAC-FISH mapping data. A collection of BACs spanning substantial parts of the short and long arm euchromatin and several dispersed regions of the pericentrometric heterochromatin were sequenced and assembled into several tiling paths spanning approximately 11 Mb. Overall, the cytogenetic order of BACs was in agreement with the order of BACs anchored to the Tomato EXPEN 2000 genetic map, although a few striking discrepancies were observed. The integration of BAC-FISH, sequence and genetic mapping data furthermore provided a clear picture of the borders between eu- and heterochromatin on chromosome 6. Annotation of the BAC sequences revealed that, although the majority of protein-coding genes were located in the euchromatin, the highly repetitive pericentromeric heterochromatin displayed an unexpectedly high gene content. Moreover, the short arm euchromatin was relatively rich in repeats, but the ratio of Gypsy and Copia retrotransposons across the different domains of the chromosome clearly distinguished euchromatin from heterochromatin. The ongoing whole-genome sequencing effort will reveal if these properties are unique for tomato chromosome 6, or a more general property of the tomato genome.
Chapter 5presents the potato genome, the first genome sequence of an Asterid. To overcome the problems associated with genome assembly due tothe high level of heterozygosity that is observed in commercial tetraploid potato varieties, a homozygous doubled-monoploid potato clone was exploited to sequence and assemble 86% of the 844 Mb genome. This potato reference genome sequence was complemented with re-sequencing of aheterozygous diploid clone, revealing the form and extent of sequence polymorphism both between different genotypes and within a single heterozygous genotype. Gene presence/absence variants and other potentially deleterious mutations were found to occur frequently in potato and are a likely cause of inbreeding depression. Annotation of the genome was supported by deep transcriptome sequencing of both the doubled-monoploid and the heterozygous potato, resulting in the prediction of more than 39,000 protein coding genes. Transcriptome analysis provided evidence for the contribution of gene family expansion, tissue specific expression, and recruitment of genes to new pathways to the evolution of tuber development. The sequence of the potato genome has provided new insights into Eudicot genome evolution and has provided a solid basis for the elucidation of the evolution of tuberisation. Many traits of interest to plant breeders are quantitative in nature and the potato sequence will simplify both their characterization and deployment to generate novel cultivars.
The outstanding challenges in plant genome sequencing are addressed in Chapter 6. The high concentration of repetitive elements and the heterozygosity and polyploidy of many interesting crop plant species currently pose a barrier for the efficient reconstruction of their genome sequences. Nonetheless, the completion of a large number of new genome sequences in recent years and the ongoing advances in sequencing technology provide many excitingopportunities for plant breeding and genome research. Current sequencing platforms are being continuously updated and improved, and novel technologies are being developed and implemented in third-generation sequencing platforms that sequence individual molecules without need for amplification. While these technologies create exciting opportunities for new sequencing applications, they also require robust software tools to process the data produced through them efficiently. The ever increasing amount of available genome sequences creates the need for an intuitive platform for the automated and reproducible interrogation of these data in order to formulate new biologically relevant questions on datasets spanning hundreds or thousands of genome sequences.
Genome structure and pathogenicity of the fungal wheat pathogen Mycosphaerella graminicola
M'Barek, S. Ben - \ 2011
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Gert Kema. - [S.l.] : S.n. - ISBN 9789085859970 - 229
triticum aestivum - triticum turgidum - tarwe - mycosphaerella graminicola - plantenziekteverwekkende schimmels - genoomstructuur - eiwitexpressieanalyse - pathogeniteit - pathogenesis-gerelateerde eiwitten - genomen - plasticiteit - plant-microbe interacties - triticum aestivum - triticum turgidum - wheat - mycosphaerella graminicola - plant pathogenic fungi - genomic structure - proteomics - pathogenicity - pathogenesis-related proteins - genomes - plasticity - plant-microbe interactions
The phytopathogenic fungus Mycosphaerella graminicola (Fuckel) J. Schröt. in Cohn (asexual stage: Zymoseptoria tritici (Desm.) Quaedvlieg & Crous) causes septoria tritici leaf blotch (STB) in wheat and is one of the most important diseases of this crop worldwide. However, STB control, mainly based on the use of resistant cultivars and fungicides, is significantly hampered by the limited understanding of the genetic and biochemical bases of pathogenicity, and mechanisms of infection and resistance in the host. M. graminicola has a very active sexual cycle under field conditions, which is an important driver of STB epidemics. Moreover, it results in high genetic diversity of field populations that causes a major challenge for the development and sustainable management of resistant cultivars and the discovery of new antifungal compounds. Understanding the role of the sexual and asexual life cycles on genome composition of this versatile pathogen and its infection strategy is crucial in order to develop novel control methods.
Chapter 1 is an introduction to the biology and pathogenicity of M. graminicola. In addition, it shortly describes the impact of improved and novel technologies on the speed, scope and scale of comparative genomics research.
Chapter 2 provides detailed genetic analyses of two M. graminicola mapping populations, using mainly DArT markers, and the analysis of the meiotic transmission of unequal chromosome numbers. Polymorphisms in chromosome length and number were frequently observed in progeny isolates, of which 15–20% lacked one or more chromosomes despite their presence in one or both parents, but these had no apparent effect on sexual and pathogenic fitness. M. graminicola has up to eight so called dispensable chromosomes that can be easily lost - collectively called the dispensome - which is, so far, the highest number of dispensable chromosomes reported in filamentous fungi. They represent small-sized chromosomes and make up 38% of the chromosome complement of this pathogen. Much of the observed genome plasticity is generated during meiosis and could explain the high adaptability of M. graminicola in the field. The generated linkage map was crucial for finishing the M. graminicola genome sequence.
Chapter 3 describes the M. graminicola genome sequence with highlights on genome structure and organization including the eight dispensable chromosomes. The genome comprises a core set of 13 chromosomes and a dispensome, consisting of eight chromosomes that are distinct from the core chromosomes in structure, gene and repeat content. The dispensome contains a higher frequency of transposons and the genes have a different codon use. Most of the genes present one the dispensome are also present on the core chromosomes but little synteny is observed neither between the M. graminicola dispensome and the core chromosomes nor with the chromosomes of other related Dothideomycetes. The dispensome likely originates from ancient horizontal transfer(s) from (an) unknown donor(s).
Chapter 4 shows a global analysis of proteins secreted by M. graminicola in apoplastic fluids during infection. It focuses mainly on fungal proteins secreted in a compatible interaction. The study showed that many of the annotated secreted proteins have putative functions in fungal pathogenicity, such as cell wall degrading enzymes and proteases, but the function of a substantial number of the identified proteins is unknown. During compatible interactions proteins are primarily secreted during the later stages. However, many pathogenesis-related host proteins, such as PR-2, PR-3 and PR-9, accumulated earlier and at higher concentrations during incompatible interactions, indicating that fungal effectors are recognized by resistant plants and trigger resistant gene-mediated defence responses, though without a visible hypersensitive response.
Chapter 5 further details the initial identification and characterization of necrosis-inducing proteins that are produced in culture filtrates (CFs) of M. graminicola. The necrosis-inducing activity of CFs is light dependent and inactivated by proteinase K and heat treatment (100C). This is reminiscent of the necrosis-inducing properties of host selective toxins of other Dothideomycete pathogens such as Stagonospora nodorum and Pyrenophora tritici-repentis. Subsequent purifications of CFs and mass spectrometry identified several candidate proteins with necrosis-inducing activity. Heterologous expression of the two most prominent proteins in Pichia pastoris produced sufficient quantities for infiltration assays in a panel of wheat cultivars that showed differential responses, suggesting specific recognition.
Chapter 6 provides a general discussion of the thesis and puts the results obtained in a broader perspective with a focus on the genome structure of M. graminicola and its function. In addition, aspects of the hemi-biotrophic lifestyle, the relevance of secreted proteins for the wheat-M. graminicola pathosystem in relation to gene-for-gene models and the potential implications for resistance breeding strategies are discussed.
Genome transcription/translation of segmented, negative-strand RNA viruses
Geerts-Dimitriadou, C. - \ 2011
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Richard Kormelink. - [s.l.] : S.n. - ISBN 9789085859314 - 151
rna-virussen - tomatenbronsvlekkenvirus - genomen - transcriptie - translatie - luzernemozaïekvirus - nucleotidenvolgordes - genexpressie - genexpressieanalyse - rna viruses - tomato spotted wilt virus - genomes - transcription - translation - alfalfa mosaic virus - nucleotide sequences - gene expression - genomics
The requirements for alignment of capped RNA leader sequences along the viral genome during influenza transcription initiation (“cap-snatching”) have long been an enigma. Previous work on Tomato spotted wilt virus (TSWV) transcription initiation has revealed that this virus displays a preference for leaders with increasing base complementarity to the 3'-ultimate residues of the viral RNA template. Assuming that cap-snatching is a highly conserved mechanism, it is tempting to speculate that the findings for TSWV apply to all segmented negative RNA viruses. The research in this thesis aimed to analyze whether similar cap donor requirements applied for Influenza A virus transcription initiation as compared to what has been found for TSWV. Indeed, in vitro studies demonstrated that influenza transcriptase prefers multiple base-pairing capped leaders. Additionally, the occurrence of “prime-and-realign” during influenza transcription initiation was observed, as well as internal priming at the 3'-penultimate viral residue. The in vitro findings were confirmed by similar studies performed during influenza infection of cell cultures. Whereas transcription initiation of TSWV has been relatively well studied, transcription termination has not. It is postulated that transcription termination/translation is triggered by the formation of a hairpin structure. In cell experiments support a role of the TSWV hairpin structure in translation.
Genetic variation in the chicken genome: insights in selection
Elferink, M.G. - \ 2011
Wageningen University. Promotor(en): Martien Groenen, co-promotor(en): Richard Crooijmans. - [S.l.] : S.n. - ISBN 9789085859208 - 162
pluimvee - dierveredeling - genomen - populatiegenetica - genen - rassen (dieren) - genetische kartering - genetische variatie - selectie - poultry - animal breeding - genomes - population genetics - genes - breeds - genetic mapping - genetic variation - selection
The chicken currently provides more than a quarter of the meat and nearly all eggs produced worldwide. For future improvements in production traits and animal welfare as well as to address future consumer demands it is necessary to understand the etiology and biology underlying production traits and diseases. The primary aim of the research described in this thesis was to investigate the utility of several molecular approaches to identify causative variants underlying a variety of traits in the chicken.
Efficiënte koe stoot minder methaan uit : fokken op voerefficiëntie in mogelijk
Haas, Y. de; Haan, M.H.A. de - \ 2011
V-focus 8 (2011)2. - ISSN 1574-1575 - p. 22 - 23.
melkveehouderij - rundveevoeding - efficiëntie - dierveredeling - genomen - selectie - fokkerijmethoden - dairy farming - cattle feeding - efficiency - animal breeding - genomes - selection - animal breeding methods
Fokken op een hoge voerefficiëntie zal de voerkosten drukken en het milieu sparen. Dat er nog geen fokprogramma is om op voerefficiëntie van melkkoeien te fokken, komt doordat de dataverzameling van voeropname erg lastig en kostbaar is. Met genomic selection lijken er nieuwe mogelijkheden te komen.
The role of non-specific interactions in nuclear organization
Nooijer, S. de - \ 2010
Wageningen University. Promotor(en): Ton Bisseling; Bela Mulder, co-promotor(en): Joan Wellink. - [S.l. : S.n. - ISBN 9789085857921 - 117
arabidopsis - celkernen - chromosomen - genomen - chromatine - interacties - cytogenetica - moleculaire biologie - arabidopsis - nuclei - chromosomes - genomes - chromatin - interactions - cytogenetics - molecular biology
The most important organelle in eukaryotic cells is the nucleus. Many processes occurring within the nucleus depend on spatial organization of the nucleus. The spatial organization of the eukaryotic nucleus derives from interactions between its constituents. Both specific interactions, for instance the interactions between a DNA binding protein and its target DNA sequence, and non-specific interactions occur. Non-specific interactions stem from physical encounters between molecules or particles, which can favour particular organizations, i.e. the ones that have the lowest entropy. The role of non-specific interactions in nuclear organization is so far not extensively studied. Here, we investigate the effects of non-specific interactions on nuclear organization, using molecular dynamics simulation techniques. Chromatin folding models can be implemented in these simulations as chains of monomers, which can form loops, branches or networks. Through a comparison of simulation results with experimental data, these models can be verified or falsified.
We used MD simulations of models for Arabidopsis chromatin organisation to show that non-specific interactions can explain the in vivo localisation of nucleoli and chromocenters. Also, we quantitatively demonstrate that chromatin looping contributes to the formation of chromosome territories. Focussing on the forces driving nuclear organization in the rosette model, we derive effective interaction potentials for rosette-loop interactions. These potentials are weak, but nevertheless drive chromocenters and nucleoli to the nuclear periphery and away from each other.
We also study the folding of a single human chromosome within its territory. The results of our simulations are analysed using a virtual confocal microscope algorithm which has the same limitations as a real confocal microscope. Thus we show that chromatin looping increases the volume occupied by a 10Mbp chromosomal sub-domain, but decreases the overlap between two neighbouring sub-domains. Our results furthermore show that the measured amount of overlap is highly dependent on both spatial resolution and signal detection threshold of the confocal microscope, and that in typical fluorescence in situ hybridisation experiments these two factors contribute to a gross underestimation of the real overlap. Zooming out to whole nucleus organization, we show that an interplay between interactions between heterochromatin and nuclear lamina generates a wide variety of nuclear organizations, with those occurring in nature requiring a fine balance between both interactions.
The differences between chromosome folding in human and Arabidopsis can be explained through differences in genomic structure and chromosome loop formation, but the underlying mechanisms and forces that organize the nucleus are very similar. The insight how specific and non-specific forces cooperate to shape nuclear organization, is therefore the most important contribution of this thesis to scientific progress.
Bioinformatics' approaches to detect genetic variation in whole genome sequencing data
Kerstens, H.H.D. - \ 2010
Wageningen University. Promotor(en): Martien Groenen; Mari Smits. - [S.l. : S.n. - ISBN 9789085857808 - 182
bio-informatica - genomen - nucleotidenvolgordes - genetische variatie - varkens - kalkoenen - kippen - anas platyrhynchos - dierveredeling - genexpressieanalyse - single nucleotide polymorphism - marker assisted breeding - bioinformatics - genomes - nucleotide sequences - genetic variation - pigs - turkeys - fowls - anas platyrhynchos - animal breeding - genomics - single nucleotide polymorphism - marker assisted breeding
Current genetic marker repositories are not sufficient or even are completely lacking for most farm animals. However, genetic markers are essential for the development of a research tool facilitating discovery of genetic factors that contribute to resistance to disease and the overall welfare and performance in farm animals.
By large scale identification of Single Nucleotide Polymorphisms (SNPs) and Structural Variants (SVs) we aimed to contribute to the development of a repository of genetic variants for farm animals. For this purpose bioinformatics data pipelines were designed and validated to address the challenge of the cost effective identification of genetic markers in DNA sequencing data even in absence of a fully sequenced reference genome.
To find SNPs in pig, we analysed publicly available whole genome shotgun sequencing datasets by sequence alignment and clustering. Sequence clusters were assigned to genomic locations using publicly available BAC sequencing and BAC mapping data. Within the sequence clusters thousands of SNPs were detected of which the genomic location is roughly known.
For turkey and duck, species that both were lacking a sufficient sequence data repository for variant discovery, we applied next-generation sequencing (NGS) on a reduced genome representation of a pooled DNA sample. For turkey a genome reference was reconstructed from our sequencing data and available public sequencing data whereas in duck the reference genome constructed by a (NGS) project was used. SNPs obtained by our cost-effective SNP detection procedure still turned out to cover, at intervals, the whole turkey and duck genomes and are of sufficient quality to be used in genotyping studies. Allele frequencies, obtained by genotyping animal panels with a subset our SNPs, correlated well with those observed during SNP detection. The availability of two external duck SNP datasets allowed for the construction of a subset of SNPs which we had in common with these sets. Genotyping turned out that this subset was of outstanding quality and can be used for benchmarking other SNPs that we identified within duck.
Ongoing developments in (NGS) allowed for paired end sequencing which is an extension on sequencing analysis that provides information about which pair of reads are coming from the outer ends of one sequenced DNA fragment. We applied this technique on a reduced genome representation of four chicken breeds to detect SVs. Paired end reads were mapped to the chicken reference genome and SVs were identified as abnormally aligned read pairs that have orientation or span sizes discordant from the reference genome. SV detection parameters, to distinguish true structural variants from false positives, were designed and optimized by validation of a small representative sample of SVs using PCR and traditional capillary sequencing.
To conclude: we developed SNP repositories which fulfils a requirement for SNPs to perform linkage analysis, comparative genomics QTL studies and ultimately GWA studies in a range of farm animals. We also set the first step in developing a repository for SVs in chicken, a relatively new genetic marker in animal sciences.
The interplay between a Phytophthora RXLR effector and an Arabidopsis lectin receptor kinase
Bouwmeester, K. - \ 2010
Wageningen University. Promotor(en): Francine Govers; Pierre de Wit. - [S.l. : S.n. - ISBN 9789085856474 - 223
phytophthora infestans - arabidopsis - solanum tuberosum - genen - genomen - virulentie - kinasen - lectinen - receptoren - genexpressie - plant-microbe interacties - phytophthora infestans - arabidopsis - solanum tuberosum - genes - genomes - virulence - kinases - lectins - receptors - gene expression - plant-microbe interactions
Phytophthora infestans – the causal agent of potato late blight – secretes a plethora of effector proteins to facilitate plant infection. The central subject of this thesis is ipiO, one of the first cloned Phytophthora genes with a putative function in pathogenicity as was anticipated based on its in planta induced (ipi) expression, in particular during early stages of host infection. IPI-O contains two striking motifs: RXLR-dEER and RGD. RGD is a cell adhesion motif and was shown to be involved in binding to the extracellular lectin domain of LecRK-I.9, a lectin receptor kinase of Arabidopsis. The RXLR-dEER motif plays a role in effector trafficking into host cells and is shared by several secreted oomycete effector proteins which are known to function as race-specific avirulence (Avr) factors. In a previous study, that was aimed at identifying novel pairs of P. infestans Avr and host plant resistance (R) genes, a high-throughput effector genomics screen identified ipiO as Avr-blb1, the counterpart of the late blight R gene Rpi-blb1 which originates from the nightshade Solanum bulbocastanum. Often R genes exploited in late blight resistance breeding become rapidly ineffective as a result of adaptation of P. infestans. However, unlike most late blight R genes that interact in a gene-for-gene manner with Avr genes, Rpi-blb1 seemed to have the potential to remain its effectiveness. In section 2 we monitored the genetic variation and distribution of the ipiO family in an extensive isolate collection of P. infestans and closely related species. This resulted in the identification of 16 IPI-O variants that could be sub-divided in three distinct classes. Variants from class I and class II were shown to induce cell death when co-infiltrated with Rpi-blb1 in Nicotiana benthamiana. Class III consists solely of the highly divergent variant IPI-O4, that is not able to trigger Rpi-blb1-mediated cell death. Class I is highly diverse and represented in all P. infestans isolates analyzed so far, except in two Mexican P. infestans isolates. The latter two are capable to infect Rpi-blb1 plants, suggesting that the lack of class I variants in the genome of these strains allows them to escape recognition by Rpi-blb1 plants. We propose that profiling of the ipiO variants within P. infestans populations can predict the effectiveness of Rpi-blb1-mediated resistance in potato and, as such, can facilitate integrated disease management.
Section 3 of this thesis deals with legume-like lectin receptor kinases (LecRKs), membrane-spanning proteins with potential roles in adaptive responses and cell wall integrity. We present an inventory and a phylogenetic analysis of the Arabidopsis LecRK gene family. The rationale behind this study was to gain better insight into the diversity of LecRKs and their potential roles in plant defense. A comprehensive expression analysis based on exploration of existing databases revealed that several LecRK genes are induced upon treatment with elicitors or during pathogen infection. Based on the phylogenetic analysis we have reclassified the LecRK genes and proposed a new nomenclature.
LecRK-I.9, one of the clade I Arabidopsis LecRKs which binds the RGD cell adhesion motif of IPI-O, was shown to mediate adhesion between the cell wall (CW) and plasma membrane (PM). In contrast, IPI-O disrupts these adhesions by virtue of its RGD motif. We analyzed Arabidopsis LecRK-I.9 knock-out lines (lecrk-I.9) for their response to pathogen infection, in particular to Phytophthora brassicae. We also analyzed transgenic Arabidopsis lines expressing ipiO, and observed that both the ipiO-expressing lines and lecrk-I.9 lines are impaired in their resistance to oomycete pathogens. To unravel the mechanisms underlying this phenomenon we analysed callose deposition upon MAMP (i.e. flg22) treatment and investigated the strength of CW-PM adhesions under plasmolysis-inducing conditions. The results indicated that LecRK-I.9 is not only important for the maintenance of the CW-PM continuum, but also in MAMP-triggered immunity. Also here, both the ipiO-expressing lines and the lecrk-I.9 knock-outs displayed a destabilized CW-PM continuum and impaired callose deposition, and hence, they can be regarded as phenocopies. Arabidopsis plants that constitutively express LecRK-I.9 were smaller in size, and displayed increased levels of anthocyanin and lignin. Additionally, these lines were shown to exhibit enhanced resistance to P. brassicae. Furthermore, we studied transgenic potatoes that constitutively Arabidopsis LecRK-I.9. In comparison to the parental control potato line the transgenic lines were less susceptible to mild and moderately aggressive P. infestans isolates, but the increased tolerance was not sufficient to provide resistance to aggressive isolates. These results strongly suggest that LecRK-I.9 is a novel resistance component that plays a role in defense against Phytophthora.
In Section 4 we describe a novel method for propagating P. brassicae zoospores on an intermediate host plant. This resulted in the production of high numbers of zoospores thereby facilitating highly reproducible small and large scale inoculation experiments.
This thesis is completed with a general discussion (Section 5) addressing the current understanding of effector uptake by host cells, the subsequent recognition by cognate R proteins mediating effector-triggered immunity, and RXLR-dEER effector diversity. We also discuss the role of the RGD motif in effectors of both animal and plant pathogens, and the potential functions of LecRKs. Finally, we high-light the advantages of Arabidopsis-Phytophthora pathosystems as research object.
Nucleotide variation and footprints of selection in the porcine and chicken genomes
Amaral, A.J. - \ 2010
Wageningen University. Promotor(en): Martien Groenen, co-promotor(en): Hendrik-Jan Megens; Henri Heuven. - [S.l. : S.n. - ISBN 9789085856559 - 160
varkens - kippen - genomen - nucleotiden - genetische diversiteit - kunstmatige selectie - selectief fokken - rassen (dieren) - genetica - pigs - fowls - genomes - nucleotides - genetic diversity - artificial selection - selective breeding - breeds - genetics
Jubileum voor de zandraket
Sikkema, A. - \ 2010
Resource: weekblad voor Wageningen UR 4 (2010)8. - ISSN 1874-3625 - p. 18 - 20.
arabidopsis thaliana - mutanten - genomen - genetische bronnen - genetische modellen - plantenfysiologie - wetenschappelijk onderzoek - arabidopsis thaliana - mutants - genomes - genetic resources - genetic models - plant physiology - scientific research
Je kunt ’m in Nederland op elke straathoek tegenkomen tussen de trottoirtegels: de zandraket of Arabidopsis thaliana. Dit jaar viert dit ‘onkruid’ zijn zilveren jubileum als modelplant van de plantenwetenschappers. Onderzoek aan dit plantje heeft geleid tot detailkennis van vrijwel alle moleculaire processen in planten.
Genome-wide gene expression surveys and a transcriptome map in chicken
Nie, H. - \ 2010
Wageningen University. Promotor(en): Martien Groenen; Mari Smits, co-promotor(en): Richard Crooijmans. - [S.l.] : S.n. - ISBN 9789085856221 - 164
kippen - pluimvee - dierveredeling - genomen - genexpressie - transcriptie - genetische kartering - genexpressieanalyse - microarrays - dna microarrays - transcriptomics - marker assisted breeding - moleculaire veredeling - fowls - poultry - animal breeding - genomes - gene expression - transcription - genetic mapping - genomics - microarrays - dna microarrays - transcriptomics - marker assisted breeding - molecular breeding
The chicken (Gallus gallus) is an important model organism in genetics, developmental biology, immunology, evolutionary research, and agricultural science. The completeness of the draft chicken genome sequence provided new possibilities to study genomic changes during evolution by comparing the chicken genome to that of other species. The development of long oligonucleotide microarrays based on the genome sequence made it possible to survey genome-wide gene expression in chicken. This thesis describes two gene expression surveys across a range of healthy chicken tissues in both adult and embryonic stages. Specifically, we focus on the mechanisms of regulation of gene transcription and their evolution in the vertebrate genome.
Chapter 1 provides a brief history of the chicken as a model organism in biological and genomics research. In particular a brief overview is presented about expression profiling experiments, followed by an introduction to gene transcription regulation in general. Finally, the aim and outline of this thesis is presented.
An important aim of this thesis is to generate surveys of genome-wide gene expression data in chicken using microarrays. In chapter 2, we introduce microarray data normalization including background correction, within-array normalization and between-array normalization. Based on these results an analysis approach is recommended for the analysis of two-color microarray data as performed in the experiments described in this thesis. We also briefly explain the relevant methodology for the identification of differentially expressed genes and how to translate resulting gene lists into biological knowledge. Finally, specific issues related to updating microarray probe annotation in farm animals, is discussed. For the analysis of the microarray data in this thesis re-annotation of the probes on the chicken 20K oligoarray was done using the oligoRAP, analysis pipeline.
The vast amount of data generated from a single transcriptomics study makes it impossible to extract meaningful biological knowledge by manually going through individual genes from a list with hundreds and thousands of differentially expressed genes. In chapter 3, we present a practical approach using a collection of R/Bioconductor packages to extract biological knowledge from a microarray experiment in farm animals. Furthermore, a locally adaptive statistical procedure (LAP) analysis approach is used to identify differentially expressed chromosomal regions in a microarray experiment.
Chapter 4 presents a genome-wide gene expression survey across eight different tissues (brain, bursa of Fabricius, kidney, liver, lung, small intestine, spleen, and thymus from 10-week old chickens) in adult birds using a chicken 20K microarray. To a certain extent, most genes show some tissue-specific pattern of expression. Housekeeping and tissue-specific genes are identified based on gene expression patterns across the eight different tissues. The results show that housekeeping genes are more compact, i.e. are smaller, with shorter, coding sequence length, intron length, and smaller length of the intergenic regions. This observed compactness of housekeeping genes may be a result of selection on economy of transcription during evolution. Furthermore, a comparative analysis of gene expression among mouse, chicken, and frog showed that the expression patterns of orthologous genes are conserved during evolution between mammals, birds, and amphibians.
The chicken embryo has been a very popular model for developmental biology. To study the overall gene expression pattern in whole chicken embryos at different developmental stages and/or embryonic tissues, a genome-wide gene expression survey across different developmental and embryonic stages was performed (chapter 5). The study included four different developmental stages (HH stage 3, 10, 15, 22) and eight different embryonic tissues (brain, bursa of Fabricius, heart, kidney, liver, lung, small intestine, and spleen from HH stage 36). We were able to identify several embryonic stage- and tissue-specific genes in our analysis. Genomic features of genes widely expressed under these 12 conditions suggest that widely expressed genes are more compact than tissue-specific genes, confirming the findings described in chapter 4. The analysis of the differentially expressed genes during the different developmental stages of whole embryo indicates a gradual change in gene expression during embryo development. A comparison of the gene expression profiles between the same organs, of adults and embryos reveals both striking similarities as well as differences.
The overall goal of this thesis was to improve our understanding of the mechanisms of transcriptional regulation in the chicken. In chapter 6, a transcriptome map for all chicken chromosomes is presented based on the expression data described in chapter 4. The results reveal the presence of two distinct types of chromosomal regions characterized by clusters of highly or lowly expressed genes respectively. Furthermore, these regions show a high correlation with a number of genome characteristics, like gene density, gene length, intron length, and GC content. A comparative analysis between the chicken and human transcriptome maps suggests that the regions with clusters of highly expressed genes are relatively conserved between the two genomes. Our results revealed the presence of a higher order organization of the chicken genome that affects gene expression, confirming similar observations in other species.
Finally, in chapter 7 I summarize the main findings and discuss some of the limitations of the analyses described in this thesis. I also discuss the different merits and shortcomings of studying gene expression using either microarrays or next-generation sequencing technology and propose directions for future research. The rapid developments in new-generation sequencing technology will facilitate better coverage and depth of the chicken genome. This will provide a better genome assembly and an improved genome annotation. The sequence-based approaches for studying gene expression will reduce noise levels compared to hybridization-based approaches. Overall, next-generation sequencing is already providing greatly enhance tools to further improve our understanding of the chicken transcriptome and its regulation.
On some surprising statistical properties of a DNA fingerprinting technique called AFLP
Gort, G. - \ 2010
Wageningen University. Promotor(en): A. Stein; Fred van Eeuwijk. - [S.l. : S.n. - ISBN 9789085855378 - 154
planten - statistische analyse - dna-fingerprinting - genomen - biometrie - moleculaire genetica - dna - aflp - biostatistiek - toegepaste statistiek - plants - statistical analysis - dna fingerprinting - genomes - biometry - molecular genetics - dna - amplified fragment length polymorphism - biostatistics - applied statistics
AFLP is a widely used DNA fingerprinting technique, resulting in band absence - presence profiles, like a bar code. Bands represent DNA fragments, sampled from the genome of an individual plant or other organism. The DNA fragments travel through a lane of an electrophoretic gel or microcapillary system, and are separated by length, with shorter fragments traveling further. Multiple individuals are simultaneously fingerprinted on a gel. One of the applications of AFLP is the estimation of genetic similarity between individuals, e.g. in diversity and phylogenetic studies. In that case, profiles of two individuals are compared, and the fraction of shared (comigrating) bands is calculated, e.g. using the Dice similarity coefficient. Two comigrating bands may share the same fragment, but band sharing could also be due to chance, if two equally sized, but different fragments are amplified. This is called homoplasy. Homoplasy biases similarity coefficients. Homoplasy could also occur within a lane, if two different fragments of equal length are amplified, resulting in a single band. We call this collision. The main objective of this thesis is the study of collision and homoplasy in AFLP. The length distribution of AFLP fragments plays an important role. This distribution is highly skewed with more abundant short fragments. By simulation the expected similarity for unrelated genotypes is calculated. As much as 40% of the bands may be shared by chance in case of profiles with 120 bands. The collision problem is analogous to the birthday problem, which has a surprising solution. The collision problem is even more extreme, making it even more surprising. Profiles with only 19 bands contain collision(s) with probability 1/2. These findings have consequences for practice. In some cases it is better to prevent the occurrence of collisions by decreasing the number of bands, in other cases a correction for homoplasy and collision is preferred. Modified similarity coefficients are proposed, that estimate the fraction of homologous fragments, correcting for homoplasy and collision. Partially related to homoplasy and collision, we study the codominant scoring of AFLP in association panels. Examples of AFLP in lettuce and tomato serve as illustrations.
DNA helpt bij management : met een melkmonster op 21 dagen dracht controleren dankzij gensignalen? Interview met M. Smits
Booij, A. ; Smits, M.A. - \ 2009
Veeteelt 26 (2009)14. - ISSN 0168-7565 - p. 38 - 39.
melkveehouderij - melkkoeien - drachtigheidsperiode - melkmonsters - dierveredeling - genomen - selectie - signalen - functionele genomica - dairy farming - dairy cows - gestation period - milking portions - animal breeding - genomes - selection - signals - functional genomics
History and structure of the closed pedigreed population of Icelandic Sheepdogs
Oliehoek, P.A. ; Bijma, P. ; Meijden, A. van der - \ 2009
Genetics, Selection, Evolution 41 (2009). - ISSN 0999-193X - 12
hondenrassen - genetische diversiteit - genomen - bedrijfsvoering - dierziekten - honden - schaapherdershonden - dog breeds - genetic diversity - genomes - management - animal diseases - dogs - sheep dogs - genome - netherlands - disease
Background - Dog breeds lose genetic diversity because of high selection pressure. Breeding policies aim to minimize kinship and therefore maintain genetic diversity. However, policies like mean kinship and optimal contributions, might be impractical. Cluster analysis of kinship can elucidate the population structure, since this method divides the population in clusters of related individuals. Kinship-based analyses have been carried out on the entire Icelandic Sheepdog population, a sheep-herding breed. Results - Analyses showed that despite increasing population size and deliberately transferring dogs, considerable genetic diversity has been lost. When cluster analysis was based on kinships calculated seven generation backwards, as performed in previous studies, results differ markedly from those based on calculations going back to the founder-population, and thus invalidate recommendations based on previous research. When calculated back to the founder-population, kinship-based clustering reveals the distribution of genetic diversity, similarly to strategies using mean kinship. Conclusion - Although the base population consisted of 36 Icelandic Sheepdog founders, the current diversity is equivalent to that of only 2.2 equally contributing founders with no loss of founder alleles in descendants. The maximum attainable diversity is 4.7, unlikely achievable in a non-supervised breeding population like the Icelandic Sheepdog. Cluster analysis of kinship coefficients can provide a supporting tool to assess the distribution of available genetic diversity for captive population management
Genome-wide evaluation of populations
Daetwyler, H.D. - \ 2009
Wageningen University. Promotor(en): Johan van Arendonk; J.A. Woolliams, co-promotor(en): B. Villanueva. - [S.l. : S.n. - ISBN 9789085855286 - 187
dierveredeling - loci voor kwantitatief kenmerk - genomen - rundvee - inteelt - dierziekten - stamboom - genomica - diergenetica - genotyping - animal breeding - quantitative trait loci - genomes - cattle - inbreeding - animal diseases - pedigree - genomics - animal genetics - genotyping
Dit proefschrift onderzoekt het gebruik van moleculaire merkers voor genetische evaluatie van populaties. Moleculaire merkers kunnen worden gebruikt om de nauwkeurigheid van geschatte fokwaardes te verhogen. In het verleden was men gericht op het opsporen van een beperkt aantal zogenaamde QTL, delen van het genoom, die direct in verband staan met een kenmerk. Het doel was om deze QTL te benutten in fokprogramma’s met behulp van merker-ondersteunde selectie. Met het beschikbaar komen van grote hoeveelheden SNP-merkers kan gebruik worden gemaakt van een methode die gericht is op het gehele genoom, en bekend staat als “genome-wide evaluation” (GWE). Dit proefschrift presenteert resultaten van zowel QTL-detectie als GWE. Deterministische voorspellingen van nauwkeurigheid worden gepresenteerd en getest, en de invloed van de genetische structuur op nauwkeurigheid wordt onderzocht. Een methode wordt gepresenteerd voor het berekenen van missende genotypes, met als doel merkerdichtheid en nauwkeurigheid van GWE te verhogen. Daarnaast worden praktische toepassing van GWE en manieren om ontbrekende genetische variatie te kwantificeren bediscussieerd.
Genomic selection: een revolutie in fokkerij : selecteren op basis van DNA-merkers
Calus, M.P.L. ; Veerkamp, R.F. - \ 2009
V-focus 6 (2009)5. - ISSN 1574-1575 - p. 12 - 14.
rundveehouderij - dierveredeling - selectief fokken - genetische merkers - fokwaarde - dna - genomen - cattle husbandry - animal breeding - selective breeding - genetic markers - breeding value - dna - genomes
Genomic selection is in de fokkerijwereld het gesprek van de dag. Maar wat houdt het nu precies in? En wat voor gevolgen heeft het voor fokprogramma’s?
Selection at DNA level: Genomic selection brings about a revolution in animal breeding
Calus, M.P.L. ; Bastiaansen, J.W.M. ; Meuwissen, T.H.E. ; Veerkamp, R.F. - \ 2009
Cow Management 2009 (2009)jan/feb. - p. 6 - 9.
melkveehouderij - dierveredeling - selectief fokken - genomen - dna - genetische merkers - dairy farming - animal breeding - selective breeding - genomes - dna - genetic markers
10 years ago it was still a futuristic dream. Today, genomic selection is the hot topic in the world of animal breeding. But what precisely does it involve? Dutch researchers outline the background to this new technology.
A molecular cytogenetic study of intergenomic recombination and introgression of chromosomal segments in lilies (Lilium)
Nadeem Khan, M. - \ 2009
Wageningen University. Promotor(en): Richard Visser; Jaap van Tuyl. - [S.l. : S.n. - ISBN 9789085853800 - 121
lilium - cytogenetica - recombinatie - genomen - introgressie - hybridisatie - hybriden - genetische kartering - polyploïdie - koppelingskartering - lilium - cytogenetics - recombination - genomes - introgression - hybridization - hybrids - genetic mapping - polyploidy - linkage mapping
Lilies (Lilium L.) are one of the most important ornamental bulbous crops for cut flower industry
grown extensively in The Netherlands for last few decades. The genus Lilium consists of seven
different sections with about 80 species. The species within genus Lilium (2n = 2x = 24)
comprise a range of desirable and complementary characters. Besides being an important
horticultural crop, lily (Lilium) also serves as an interesting model plant for molecular
cytogenetic research and introgression breeding for several reasons like, i). Lily is a model crop
for interspecific hybridization and it includes plants of different taxonomic species each of which
possess valuable horticultural traits that need to be combined in the new cultivars. ii) Through
careful selection n and 2n gametes can be obtained in interspecific hybrids. iii) The genomes of
different species are so well differentiated genetically that the parental chromosomes can be
clearly identified in the F1 hybrids as well as in the progenies through DNA in situ hybridization
techniques. iv) The chromosomes are large enough and the number and position of
homoeologous recombination sites can be clearly detected. v) The large and easily identified
chromosomes in different lily species could be a potential source for the cytological mapping of
the Lilium genomes. Taking advantage of these favourable attributes of lily, a molecular
cytogenetic investigation was conducted to evaluate the amount of recombination and
introgression of characters between Longiflorum - Asiatic (LA) and Oriental - Asiatic (OA)
hybrids through the use of n and 2n gametes.
For this purpose different F1 Longiflorum × Asiatic (LA) and Oriental × Asiatic (OA)
hybrids were backcrossed with different Asiatic cultivars. Ovule and embryo rescue techniques
were employed to get backcross (BC) progenies. Most of the F1 LA appeared to be sterile but
some hybrids were able to produce only 2n gametes in considerable frequencies. However, in
rare occasions it was also found that normal meiosis took place in few of the LA hybrids which
resulted into the formation of normal n gametes. Ploidy level and intergenomic recombination
was studied in LA interspecific hybrids in order to assess the possibility of functional n gametes
and their potential use in introgression at diploid level in lily. A total of 104 BC1 LA
interspecific lily hybrids were obtained, 27 diploids (2n = 2x = 24), 73 triploids (2n = 2x = 36)
and 4 aneuploids (2x – 1, 2x + 2 or 2x + 3). Similarly, triploid BC1 (LAA) plants were
backcrossed to diploid Asiatic parents. As a result 14 diploid BC2 progenies were produced. The
intergenomic recombination and amount of introgression of respective genome (L and A) was
assessed in these diploid genotypes through GISH (Genomic in situ Hybridization). Extensive
intergenomic recombination was found among the chromosomes in LA hybrids. A large of
amount of L- genome was transmitted from F1 LA hybrids to their subsequent BC1 progenies.
However, very few segments of L- genome were introgressed from the BC1 diploid and triploid
(LAA) plants to the BC2 progenies (Chapter 2). GISH identifies a considerable amount of
recombination events amongst different interspecific lily hybrids (LA and OA) obtained from
functional 2n gametes. Based on recombination sites on different chromosomes identified by
GISH, cytological maps of three genomes of Lilium were constructed. For this purpose, BC
progenies of two diploid interspecific hybrids of lily, viz., Longiflorum × Asiatic (LA) and
Oriental × Asiatic (OA) were used. The BC progenies of LA hybrids consisted of both triploid
(2n = 3x = 36) and diploid (2n = 2x = 24) with some aneuploid genotypes and those of OA
hybrids mostly consisted of triploid (2n = 3x = 36) and some aneuploid genotypes. In LA
hybrids 248 recombination sites were cytologically localized on 12 different chromosomes of
each genomes (i.e., L and A). Similarly, 116 recombinant sites were marked on 12 chromosomes
each from the BC progenies of OA hybrids (O and A genomes). The distances of the
recombination sites from the centromeres are measured (in micrometres). Based on these
recombination sites four cytological maps were constructed. Since an Asiatic parent was
involved in both hybrids, viz., LA and OA, two maps were constructed for A genome which
were indicated as Asiatic (L) and Asiatic (O) and one each for Longiflorum (A) and Oriental (A)
genomes (Chapter 3).
With a view to generate genetic variation via homoeologous recombination in BC
progenies of LA and OA hybrids the most logical approach was the use of 2n gametes. 63 BC1
LA (LA × AA or AA × LA) and 53 OA (AA × OA) progeny plants were obtained after unilateral
sexual polyploidization. 16 genotypes from F2 LA populations were obtained after bilateral
sexual polyploidization through sib-mating of F1 LA hybrids. GISH was employed for the
identification of the parental genomes, mode of origin of these progenies and measurement of the
introgression in different interspecific lily hybrids. Most of the BC1 progeny plants (LA and OA)
had originated through 2n gametes by First Division Restitution (FDR) mechanism. However,
there were 12 genotypes in LA hybrids and four genotypes in OA hybrids that originated through
2n gametes formation as the result of Indeterminate Meiotic Restitution (IMR). A higher amount
of recombination was found in LA hybrids as compared to OA hybrids. Intergenomic
recombination was also determined in the sib-mated F2 LA population. In this case both parents
had contributed gametes with the somatic number of chromosomes (i.e., 2n-2n) thus confirming
the event of bilateral sexual polyploidization in interspecific LA hybrids. Based on these results,
the relevance of interspecific lily hybrids obtained from uni- and bilateral sexual
polyploidization leading to allotriploid and allotetraploid formation in interspecific lily hybrids is
discussed in the context of introgression and mapping (Chapter 4). Molecular markers are an important tool for the construction of genetic linkage maps, as the first step in the genetic
dissection of the required traits leading to crop improvement followed by the marker assisted
breeding in different plants. Lilium has one of the largest genome in plant kingdom and genetic
mapping in lilies is constrained by its large genome. DArT (Diversity Array Technology), a
molecular marker technique can detect and type DNA variation at several hundred genomic loci
in parallel without relying on genome sequence information. The DArT technique was developed
for Longiflorum × Asiatic (LA) lily hybrids to enable an efficient and effective genetic mapping
with the production of a large numbers of markers in microarrays-based assay. The restriction
enzyme PstI + TaqI combination generated the largest frequency of polymorphic genomic
representations for a genotyping array. Genomic representations from 88 F1 LA plants were used
to assemble a DArT genotyping microarray. A total of 687 DArT markers were developed and
382 polymorphic markers were mapped on 14 main linkage groups which is two more then the
haploid chromosome number (i.e. n = 12). The resulting linkage map with 382 DArT markers
spanned 1329 cM (3.5 cM/marker on average). The results highlighted the potential of DArT as
a genetic technique for genome profiling in the context of molecular breeding and genomics,
especially in crops with large genome sizes where other techniques proved to be less useful
The results of the present investigation are of practical implication. These results show
the advantages of the n gametes and their subsequent progenies which opened a new approach of
lily breeding ‘the analytic breeding’ in the allopolyploids. It also shows the possibility of using
certain triploid hybrids for further breeding. A comparison has been made between different
types of interspecific crosses, the amount of intergenomic recombination and introgressions of
chromosomal segments obtained after unilateral sexual polyploidization. Furthermore, bilateral
sexually polyploidization via sib-mated F1 hybrids producing 2n gametes has been proven. The
use of allotetraploids obtained from bilateral sexual polyploidization could be a novel approach
in the breeding of LA-hybrids. These allotetraploid with recombinant chromosomal segment
may be a potential source to generate genetic variation in subsequent progenies. The molecular
cytogenetic GISH and FISH techniques proved to be a powerful tool useful for the construction
of cytogenetic maps in interspecific crosses in crops with large genomes sizes like lily. These
techniques are also used for the identification and integration of genetic maps with chromosome
maps. FISH also helps to monitor the introgressed chromosome segment or marker of interest in
the subsequent progenies. Application of the DArT technique proved to be an effective method
to construct genetic linkage maps especially crops (like Lilium) with large genome sizes where
other techniques might be less useful.
The role of receptor-like proteins in Arabidopsis development
Wang, G. - \ 2009
Wageningen University. Promotor(en): Pierre de Wit; Gerco Angenent, co-promotor(en): Bart Thomma. - [S.l.] : S.n. - ISBN 9789085853848 - 188
arabidopsis - eiwitten - plantenontwikkeling - genen - genomen - arabidopsis - proteins - plant development - genes - genomes
An intriguing and long-standing question in developmental biology is how plant cells communicate with each other and sense signals from their surrounding environment. Through research over past decades, it became clear that plant cells use membrane-localized receptors to perceive signals from their environment, which subsequently results in the initiation of downstream signaling (Kobe and Kajava, 2001; Torii, 2005). The membrane-associated receptors often form in multimeric complexes that contain receptor-like proteins (RLP) (Song et al., 1997; Jeong et al., 1999; Fritz-Laylin et al., 2005) as well as receptor-like kinase (RLK) proteins (Shiu and Bleecker, 2001a; 2001b). This is the case for the development of the shoot apical meristem (SAM) involving the CLAVATA1 (CLV1) and CLAVATA2 (CLV2) receptor molecules, as well as a small secreted polypeptide CLAVATA3 (CLV3) (Clark et al., 1993; Clark et al., 1995; Clark et al., 1997; Kayes and Clark, 1998; Fletcher et al., 1999; Jeong et al., 1999; Brand et al., 2000; Schoof et al., 2000). The Arabidopsis gene CLV2 encodes an LRR RLP acting in a functional CLV receptor complex that is involved in restricting the size of shoot meristem (Jeong et al., 1999). A total of 57 AtRLPs, which share sequence similarity and domain composition, have been identified in the Arabidopsis genome (Wang et al., 2008). However, the function of most AtRLPs remains elusive despite a genome-wide functional study into the roles of AtRLPs has been carried out recently (Wang et al., 2008). Given that fact that many AtRLPs originated from duplication events (Fritz-Laylin et al., 2005), it is very likely that the lack of identification of biological functions for AtRLP genes may be explained by functional redundancy, a phenomenon that typically obscures studies employing a reverse genetics strategy, as has been described for many RLK gene family members (Cano-Delgado et al., 2004; Shpak et al., 2004; Albrecht et al., 2005; DeYoung et al., 2006; Hord et al., 2006). In the future, RNA interference (RNAi) or artificial microRNA (amiRNA) approaches to target the expression of multiple AtRLP genes simultaneously could be followed to circumvent the functional redundancy (Chuang and Meyerowitz, 2000; Miki and Shimamoto, 2005; Ellendorff et al., 2008; Ossowksi et al., 2008). Alternatively, these multiple mutants of closely related AtRLP genes or potential co-expressed AtRLP genes should be combined to reveal the function of these genes.
The clv2 mutant displays weaker, although similar phenotypes as clv1 and clv3 mutants (Kayes and Clark, 1998; Diévart et al., 2003; Chapters 2; 3.1), while loss-of-function mutants of clv3 are phenotypically stronger than clv1 or clv2 null mutants (Fletcher et al., 1999; Kayes and Clark, 1998; Dievart et al., 2003). In addition, CLV2 has a broader expression pattern, which may suggest a wider role for CLV2 in more developmental processes than only meristem development (Kayes and Clarks, 1998; Chapters 2; 3.1). Besides the broader role of CLV2, it is interesting to note that clv2 mutations, similar to clv1 mutations, are significantly affected by natural variation (Diévart et al., 2003; Chapters 3.1; 5), as has also been shown for the strubbelig (sub) and brassinosteroid insensitive1 (bri1) mutants (Chevalier et al., 2005; Cano-Delgado et al., 2004). The phenotype of clv2 in Col-0 (atrlp10) is also significantly enhanced by introduction into Ler background. Although our genetic interaction experiments indicated that the effect does not depend on the ER locus (Chapter 3.1), our observations imply that (a) CLV1/CLV2-modifying factor(s) exist(s) to co-regulate meristem development (Diévart et al., 2003; Chapters 3.1; 5). It would be interesting to identify these co-factor(s) in the future.
Several lines of evidence suggest a role for CLV2 in root development. Over-expression of CLV3, CLE19 and CLE40 leads to an arrest of root growth (Casamitjana-Martinez et al., 2003; Hobe et al., 2003; Fiers et al., 2004), while the clv2 mutant can suppress the short-root phenotype caused by ectopic expression of CLE19 (Fiers et al., 2005). In addition, clv2 failed to respond to exogenously supplied synthetic CLE peptide, which corresponds to the conserved CLE motif of the CLV3/ESR gene family (Fiers et al., 2005; Ito et al., 2006; Kondo et al., 2006), indicating that CLV2 is able to perceive the CLE ligands in the root. However, the clv2 mutant exhibits no visible root phenotype under normal growth conditions, suggesting that a redundant protein, most likely another AtRLP gene, compensates for the loss of CLV2 function in the root. We found that only a few AtRLP genes are expressed in the root, although their expression is quite low (Chapters 2; 3.1). Possibly, a root defect only will become apparent in combination of multiple mutants for these genes. This observation argues that a CLV-like pathway also operates in roots (Fiers et al., 2007), but no RLK involved in this process has been found, although some of the RLKs that are expressed in the root (Birnbaum et al., 2003; Nawy et al., 2005). As such, CORYNE/Suppressor of overexpression of LLP-2 (CRN/SOL2) and Barely Any Meristem1-3 (BAM1-3) might be the logical RLK candidates for the redundant role in root development, because of their pronounced expression in roots (DeYoung et al., 2006; Müller et al., 2008; Miwa et al., 2008). Specifically, like clv2 mutants, crn/sol2 mutants did not respond to CLE peptide treatments (Müller et al., 2008; Miwa et al., 2008), suggesting that CRN, like CLV2, is involved in transmitting CLE signals. Therefore, studies with different combinations of mutants of these genes will help to clarify their biological function in root development (Chapter 2; 3.1; 4).
Interestingly, WOX5, a homologue of WUS, marks the root Quiescent Centre (QC) identity and is expressed very early in the hypophysial cell (Sarkar et al., 2007), which is strikingly similar to the role of WUS in the shoot meristem (Haecker et al., 2004; Sarkar et al., 2007). Furthermore, it has been shown recently that POL and PLL, in addition to their role in Arabidopsis SAM maintenance (Song and Clark, 2005; Song et al., 2006), also act in the root meristem development through regulating the expression of the WUS homolog WOX5 (Song et al., 2008). These findings strengthen the hypothesis that a CLV-like pathway exists in the root meristem, which might include CLV2, CRN, WOX5, POL and PLL1. Similarly, our results as well as previous reports (Song and Clark, 2005; DeYoung et al., 2006; Müller et al., 2008) suggest that a CLV-related signaling pathway is involved in the regulation of leaf shape/size (Chapter 3.1) and pedicel length (Chapter 5). Interestingly, all these developmental pathways share some conserved factors such as POL and PLL, indicating common regulatory mechanisms exist in the developmental regulation of SAM, root meristem, leaf shape/size and pedicel growth.
We provide evidence that two CLV2-related AtRLPs, AtRLP2 and AtRLP12, were capable to rescue clv2 mutants when expressed under the control of the CLV2 promoter (Chapter 4), suggesting that functional specification of these two AtRLPs may reside, at least in part, in their cis-regulatory elements. The importance of variation in expression pattern for the specificity in function of closely-related genes from multi-gene families while the proteins are interchangeable, has been documented for many genes such as the CLV1-like genes (BAM1-3, DeYoung et al., 2006; Hord et al., 2006), ERECTA-family (ER and ERL1-2, Shapk et al., 2004) and BRL-family members (BRI1, BRL1and BRL3; Cano-Delgado et al., 2004). However, the double mutant combinations of atrlp2 and atrlp12 mutants with atrlp10/clv2 did not show additive effects on growth of meristems and other organs when compared to that of the atrlp10/clv2 mutant, suggesting that other AtRLPs, such as AtRLP3 and AtRLP11 that are duplication counterparts of AtRLP2 and AtRLP12 respectively, can also replace the function of CLV2 in the regulation of the meristem development (Chapter 4). Therefore, additional phenotypes and the role of other AtRLP family members will become apparent only in the absence of the entire CLV2 close-related members as has been shown for ER, ERL1 and ERL2
(Shpak et al., 2004).
Our studies in Chapter 4 revealed that several members of the AtRLP family can replace each other and are functionally equivalent. However, there are also family members with a similar protein domain organization, but that are clearly distinct from CLV2. This raised the question what determines the specificity in these proteins. We determined the function of the different domains by deletion analysis and generation of hybrid molecules (Chapter 4). CLV2 is still fully functional when the island domain is removed, while the C3-F region can be replaced by a close homologue (Chapter 4). Taken together, this study provided valuable information on the function of CLV2 domains that contribute to functional specificity and conservation. Despite these findings, little is known about the roles and specificity of other CLV2 domains, such as the transmembrane domain which is proposed to be the site for dimerization between CLV2 and CRN/SOL2 (Müller et al., 2008; Miwa et al., 2008). Of particular interest for future investigations are conserved residues flanking the LRR domain, which could be mutated to determine whether they are essential for CLV2 activity (Fritz-Laylin et al., 2005; van der Hoorn et al., 2005; Chapters 1; 3.1; 4).
Previous studies proposed that CLV2 dimerizes with CLV1 to form an active receptor complex that binds the CLV3 ligand and initiates the downstream signaling pathway required for the maintenance of the stem cell population in the shoot apical meristem (Jeong et al. 1999; Trotochaud et al., 1999; Rojo et al., 2002; Dievart and Clark, 2004). The CLV2 protein was regarded as a stabilizer for the CLV1 protein based on the observation that CLV1 protein levels were reported to be reduced by over 90% in clv2 loss-of-function mutants (Jeong et al., 1999). In addition to CLV1, the receptor kinase CRN/SOL2 acts closely together with CLV2 to transmit the CLV3 signal independently but in parallel with CLV1 (Müller et al., 2008; Miwa et al., 2008). CRN/SOL2 has a kinase domain which might create a fully functional transmembrane receptor kinase together with CLV2 through dimerization in the transmembrane domains (Müller et al., 2008). This raises the hypothesis that CLV3 could bind CLV2 directly. However, whether CLV3 peptide can directly bind to the extracelluar domain of CLV2 remains to be validated. Nevertheless, the CLV3 signal is probably transduced through two separate receptor complexes, comprising CLV1/CLV2 (or CLV1 alone) and CRN/CLV2 (Müller et al., 2008). Despite these observations, the role of CLV2 in these signaling pathways remains largely unresolved.
The CLV pathway is largely built on genetic data whereas direct biochemical evidence for the mode of action of the proteins involved is largely missing (Jeong et al. 1999; Trotochaud et al., 1999; Müller et al., 2008). Only very recently, it has been shown that the CLV3 peptide directly binds to the CLV1 ectodomain (Ogawa et al., 2008). In an effort to understand the physical interactions of the CLV proteins and their localization, we created fluorescently tagged versions of CLV1 and CLV2 (Chapter 5). These fusion proteins appeared to be targeted to the plasma membrane, displaying a common subcellular localization (Chapter 5). The functionality of the fusion proteins was confirmed by complementation of the respective mutants (Chapter 5). It has been postulated that the CLV1/CLV2 receptor complex resides in the plasma membrane to perceive the CLV3 ligand (Jeong et al. 1999; Trotochaud et al., 1999; Diévart et al., 2003). Our localization studies support this scenario. Unfortunately, it was not possible to determine a direct interaction between CLV1 and CLV2 in the framework of this Ph.D study. However, it is obvious that the interaction study can be the immediate next step using the available fluorescently-tagged CLV1 and CLV2 proteins and the stable transgenic lines generated in this study (Chapter 5). Furthermore, the study can be extended to visualize the components of the CLV signaling complex and follow their dynamics during plant growth. Furthermore, the labelled CLV-receptors can also be used for the isolation and identification of unknown components of the CLV receptor complex. For instance, it would be interesting to investigate whether CLV2 and CRN/SOL2 interact directly as proposed (Müller et al., 2008; Miwa et al., 2008). Indeed, a preliminary study using a similar approach supports the interaction of CLV2 and CRN/SOL2 (Y-F. Zhu and C-M. Liu, personal communication). It also intrigues to determine whether the CLV3 or other possible CLE(s) are directly interacting with the CLV2/CRN receptor complex. Undoubtedly, the tools generated in this study will be of great help for future experiments aiming to unravel the CLV signaling pathway.
Selecteren op DNA-niveau: genomic selection zorgt voor revolutie in fokkerij
Calus, M.P.L. ; Bastiaansen, J.W.M. ; Meuwissen, T.H.E. ; Veerkamp, R.F. - \ 2008
Veeteelt 25 (2008)18. - ISSN 0168-7565 - p. 12 - 15.
melkveehouderij - dierveredeling - selectief fokken - genomen - dna - merkers - dairy farming - animal breeding - selective breeding - genomes - dna - markers
10 jaar geleden waren het nog futuristische ideeën, nu is genomic selection in de fokkerijwereld het gesprek van de dag. Maar wat houdt het precies in? ASG-onderzoekers schetsen de achtergrond van de nieuwe techniek
Construction and use of a physical map of potato
Borm, T.J.A. - \ 2008
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Herman van Eck. - S.l. : S.n. - ISBN 9789085852377 - 139
solanum tuberosum - moleculaire kartering - genetische kartering - genomen - dna-fingerprinting - dna-bibliotheken - genetische merkers - maximale aannemelijkheid - moleculaire merkers - marker assisted breeding - aflp - solanum tuberosum - molecular mapping - genetic mapping - genomes - dna fingerprinting - dna libraries - genetic markers - maximum likelihood - molecular markers - marker assisted breeding - amplified fragment length polymorphism
Feeding the growing world population is one of the biggest challenges for the 21st century.
Potato, being the fourth crop in the human diet, after maize, wheat and rice, plays an
important role in this respect. Like other crops, potato is exposed to a range of potentially
yield-reducing factors: Pathogens, a (possibly changing) bad climate and averse soil
conditions. Research into the response of potato to these influences, often determined by
hereditary factors, is necessary to meet a growing demand for potatoes. A map of
genetically determined properties is crucial for this research. Several techniques are
available to produce maps – each with it's own merits and demerits, resulting in maps of
different qualities and with different resolutions. Two often used mapping techniques are
genetic mapping, where the inheritance of multiple traits (“markers”) is studied in
offspring using statistical analysis and the markers ordered accordingly, and physical
mapping on the basis of “Bacterial Artificial Chromosome” (BAC) libraries. BAC
libraries consist of a large number of individual bacterial strains (BAC clones), each
containing a randomly sampled section of DNA of the organism being studied. By
comparing individual BAC clones with each other, finding out where the donor organism's
(the organism being studied) DNA sections overlap, the BAC clones can be ordered into
groups or “contigs”. Comparison is often done on the basis of so called fingerprints – a
pattern consisting of DNA fragments of different lengths, resembling a bar-code pattern. A
similarity in fingerprint patterns between two BAC clones indicates that the BAC clones
contain similar (overlapping) sections of the donor organism's DNA. Recently an ultra
dense genetic map has been published, containing more than 10,000 markers produced
using “Amplified Fragment Length Polymorphism” (AFLPTM) marker technology. The
integrated physical and genetic map that is the subject of this thesis extends this genetic
map, and is in itself the starting point for determining the detailed DNA sequence of
potato, as is currently being undertaken by an international scientific collaboration within
the Potato Genome Sequencing Consortium (PGSC, http://www.potatogenome.net).
First step in creating this integrated physical and genetic map was creation, fingerprinting
and characterization of a BAC library, as described in chapter two. BACs were
individually fingerprinted using an AFLP based protocol, and (amongst others) these
AFLP BAC-fingerprints were compared to a theoretical model of the distribution of
fragment lengths in AFLP fingerprints to determine if fingerprinting was successful.
Correction and refinement of some of the mapping algorithms that were used to create the
genetic map are discussed in chapters three and four, resulting in refined genetic map
locations for the AFLP markers and the capability to process marker scores containing
arbitrary types of scoring ambiguities while conserving all available information. An
extension to the basic principle offers the possibility to also map AFLP markers derived
from different chromosomes that are indistinguishable on the basis of their AFLP
fragment length alone.
In chapter five, systematic differences in AFLP BAC fingerprints are discussed that are
caused by the use of different machines for capillary electrophoresis, by the use of
different fluorescent DNA labels and by different capillary position. These systematic
differences are (partially) corrected by using the (abundant) AFLP fingerprints of BAC
clones containing (part of) the potato chloroplast genome as a reference sample.
By ordering the AFLP fingerprints of individual BAC clones on the basis of fingerprint
similarity, a physical map is produced that is integrated with the genetic map using a
novel, ultra efficient, procedure described in chapter six. This procedure, “AFLP contig
matching” uses intricate experimental design and combinatorial analysis to obtain an
integrated physical and genetic map with the least amount of effort.
Cladosporium fulvum effector proteins and their role in pathogen virulence
Esse, H.P. van - \ 2008
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Bart Thomma. - [S.l. : S.n. - ISBN 9789085049272 - 214
solanum lycopersicum - tomaten - plantenziekteverwekkende schimmels - passalora fulva - genen - virulentie - genexpressie - genomen - gene silencing - gastheer-pathogeen interacties - plant-microbe interacties - solanum lycopersicum - tomatoes - plant pathogenic fungi - passalora fulva - genes - virulence - gene expression - genomes - gene silencing - host pathogen interactions - plant-microbe interactions
Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mould of tomato (Solanum esculentum). Chapter 1 is a “pathogen profile” describing the biology of the pathogen. During growth in the leaf apoplast, the intercellular space surrounding the mesophyll cells, the fungus secretes effector proteins that are thought to play a role in disease establishment. Eight of these effectors have been characterized in detail. For most of these effectors, cognate C. fulvum (Cf) resistance loci have been identified in tomato that mediate an immune response upon recognition of (the activity of) the cognate effector.
In chapter 2, a targeted proteomics approach to investigate the role of these effector proteins and to identify possible in planta targets is described. C. fulvum proteins were expressed as recombinant fusion proteins carrying various affinity–tags at either their C– or N–terminus. Although these fusion proteins were correctly expressed and secreted into the leaf apoplast, detection of affinity–tagged C. fulvum proteins failed and affinity–purification did not result in the recovery of these proteins. However, when using C. fulvum effector protein–specific antibodies, specific signals were obtained for the different proteins. It was therefore concluded that the stability of the in planta expressed recombinant fusion proteins is insufficient, which resulted in removal of the affinity–tag from the fusion proteins, irrespective of C– or N–terminal fusion or the nature of the affinity–tag. Similar observations were made when the fusion proteins were expressed in other Solanaceous species, but not when expressed in Arabidopsis thaliana.
Previous studies have demonstrated that Avr4 binds to chitin present in fungal cell walls, and that this binding by Avr4 can protect these cell walls against hydrolysis by plant chitinases. In chapter 3 it is described that Avr4–expression in Arabidopsis results in increased virulence of several fungal pathogens with exposed chitin in their cell walls, whereas the virulence of a bacterium and an oomycete remained unaltered. Heterologous expression of Avr4 in tomato increased the virulence of Fusarium oxysporum f. sp. lycopersici. Tomato GeneChip analysis was used to demonstrate that Avr4–expression in tomato results in the induced expression of only a handful of genes. Finally, silencing of the Avr4 gene in C. fulvum decreased fungal virulence on tomato. In conclusion, chapter 3 is the first report on the intrinsic function of a fungal avirulence protein that displays self-defense activity which is required for full pathogen virulence.
In chapter 4, a study on the intrinsic biological function of Avr2 is presented. The Avr2 effector interacts with the apoplastic tomato cysteine protease Rcr3, which is required for Cf–2–mediated immunity. In this chapter it is demonstrated that Avr2 is a genuine virulence factor of C. fulvum. Heterologous expression of Avr2 in Arabidopsis resulted in enhanced susceptibility towards a number of extracellular fungal pathogens that include Botrytis cinerea and Verticillium dahliae, and microarray analysis of unchallenged Arabidopsis plants showed that Avr2 expression triggered a global transcription profile that is reminiscent of pathogen challenge. Cysteine protease activity profiling revealed that Avr2 inhibits multiple extracellular Arabidopsis cysteine proteases. In tomato, Avr2 expression resulted in enhanced susceptibility not only towards natural Avr2–defective C. fulvum strains, but also towards Botrytis cinerea and Verticillium dahliae. Cysteine protease activity profiling in tomato revealed that Avr2 inhibits multiple extracellular cysteine proteases including Rcr3 and its close relative PIP1. Finally, silencing of the Avr2 gene in C. fulvum significantly compromised fungal virulence on tomato. This all shows that Avr2 is a genuine virulence factor of C. fulvum that inhibits several cysteine proteases required for plant basal defense in tomato.
Chapter 5 describes the discovery and characterization of a novel effector protein of C. fulvum, Ecp6. To discover novel C. fulvum effectors that might play a role in virulence, two–dimensional polyacrylamide gel electrophoresis (2D–PAGE) was used to visualize proteins secreted during C. fulvum–tomato interactions. Three novel C. fulvum proteins were identified; CfPhiA, Ecp6, and Ecp7. CfPhiA shows homology to proteins found on fungal sporogenous cells called phialides, while Ecp6 contains lysine motifs (LysM domains), which are recognized as carbohydrate–binding modules. Finally, Ecp7 encodes a small, cysteine–rich protein with no homology to known proteins. Heterologous expression of Ecp6 significantly increased the virulence of the vascular pathogen Fusarium oxysporum on tomato. Furthermore, by RNAi–mediated gene silencing it was demonstrated that Ecp6 is instrumental for C. fulvum virulence on tomato. Hardly any allelic variation was observed in the Ecp6 coding region of a worldwide collection of C. fulvum strains. Although none of the C. fulvum effectors identified so far have obvious orthologs in other organisms, conserved Ecp6 orthologs were identified in various fungal species. Homology based modelling suggests that the LysM domains of C. fulvum Ecp6 may be involved in chitin binding.
Chapter 6 presents global transcriptional profiling study to compare transcriptional changes in tomato during compatible and incompatible interactions with the foliar pathogenic fungus Cladosporium fulvum and the soil–borne vascular pathogenic fungus Verticillium dahliae. Although both pathogens colonize different host tissues, they display significant commonalities in their infection strategies as they both penetrate natural openings and grow strictly extracellular without the formation of haustoria. Furthermore, in incompatible interactions with both pathogens resistance is conveyed by extracellular transmembrane receptors that belong to the class of receptor–like proteins. For each of the two pathogens, the transcriptomes of the compatible and incompatible interaction largely overlapped. However, the C. fulvum–induced transcriptomes showed little overlap with the V. dahliae–induced transcriptomes, as most genes were uniquely regulated by one of the two pathogens. This also applied to both incompatible interactions, despite defense activation by the same type of resistance protein. Remarkably, of the relatively small subset of genes that was regulated by both pathogens a large portion showed an inverse regulation; induced by one pathogen and repressed by the other. With pathway reconstruction, interacting networks of tomato genes implicated in photorespiration, hypoxia and glycoxylate metabolism were identified that were repressed upon infection with C. fulvum and induced by V. dahliae. Similarly, auxin signaling was differentially affected by the two pathogens.
In chapter 7, the general discussion, the implications are of the data that are presented in this thesis are discussed for the use of C. fulvum as a model, and for fungal pathogens in general. Furthermore, the use of heterologous expression systems to study fungal effectors is briefly discussed. In several of the chapters presented in this thesis, the use of microarrays has been instrumental to investigate the biology of C. fulvum and the role of specific effectors secreted by the pathogen. Therefore, an overview of the currently available in silico tools for reconstruction of cellular pathways based on plant gene expression datasets is presented.
Ontwikkeling van een bodemgezondheids-chip
Bonants, P.J.M. ; Szemes, M. ; Speksnijder, A.G.C.L. ; Zijlstra, C. ; Overbeek, L.S. van; Doorn, R. van; Wubben, J.P. ; Doorn, J. van; Schoen, C.D. - \ 2008
detectie - dna - antagonisten - genen - genomen - pathogenen - nuttige organismen - bodemkwaliteit - moleculaire detectie - detection - dna - antagonists - genes - genomes - pathogens - beneficial organisms - soil quality - molecular detection
De ontwikkeling van een bodemgezondheids-chip kan een nuttige bijdrage leveren aan de detectie van een aantal targets: plantpathogenen, nuttige organismen (beneficials) en genen, die coderen voor antibiotica productie of antagonistische mechanismen
Genoom-analyse van ziektewerende bodems
Speksnijder, A.G.C.L. ; Overbeek, L.S. van - \ 2008
dna - detectie - antibiotica - genen - genomen - bodembiologie - biodiversiteit - genoomanalyse - bodemkwaliteit - agrobiodiversiteit - dna - detection - antibiotics - genes - genomes - soil biology - biodiversity - genome analysis - soil quality - agro-biodiversity
Onderzoek naar DNA-detectietechnieken, die zijn opgezet en uitgevoerd om antibiotica-genen en diversiteit van de microbiële gemeenschap in bodems te meten
Sonnenberg, A.S.M. - \ 2007
Paddestoelen : onafhankelijk vakblad voor Nederland en België 2007 (2007)6. - ISSN 1380-359X - p. 8 - 8.
genetica - genomen - dna - paddestoelen - agaricus bisporus - volgorden - genen - toepassing - onderzoek - genetics - genomes - dna - mushrooms - agaricus bisporus - sequences - genes - application - research
Eind 2007 zal een begin gemaakt worden met de ontrafeling van het hele genoom van de champignon (Agaricus bisporus). Het karwei zal waarschijnlijk in 2009 geklaard zijn. Dan zal de volgorde van alle 34 miljoen bouwstenen in het DNA (sequentie) van deze paddenstoel bekend zijn en tevens de locatie van alle genen
Identification of new biomarkers for early natural TSE detection using a genome-wide approach
Bossers, A. ; Harders, F.L. ; Keulen, L.J.M. van; Zijderveld, F.G. van - \ 2007
UK : DEFRA (Project final report to Medical Research Council )
schapenhouderij - schapen - dna - biologische technieken - dierziekten - schapenziekten - scrapie - genomen - microarrays - dna microarrays - biomarkers - sheep farming - sheep - biological techniques - animal diseases - sheep diseases - genomes
This research focuses on the genome-wide identification of new biomarkers (other than PrP) that might be useful to extend current TSE diagnostics especially in the preclinical phase. In addition this project might provide clues to better understand the TSE micro-pathogenesis. Specific aims: - generate a non-redundant sheep microarray from primary lymphoid tissues; − collect and preserve well documented material for RNA isolation from natural scrapie exposed or clean environments; − determine differential expressed genes between the collected samples and relate these to natural scrapie infection. These identified biomarkers might turn useful for the development of preclinical tests (at best on live animals) in the very early phase of natural scrapie infection. Such tests can support the control of scrapie in the field
Effectorvariatie in de Nederlandse Phytophthora infestans populatie
Versluis, H.P. ; Jiang, R.H.Y. ; Govers, F. - \ 2007
phytophthora infestans - genen - gewasbescherming - genomen - pathogenesis-gerelateerde eiwitten - phytophthora infestans - genes - plant protection - genomes - pathogenesis-related proteins
Uitgangspunten, resultaten en praktijk bij het onderzoek naar effectorvariatie in de Nederlandse Phytophthora infestans populatie
Voetsporen van evolutie: de dynamiek van effectorgenen in het Phytophthora-genoom
Jiang, R.H.Y. - \ 2007
Gewasbescherming 38 (2007)5. - ISSN 0166-6495 - p. 273 - 275.
aardappelen - solanum tuberosum - phytophthora infestans - genomen - phytophthora sojae - plantenziekteverwekkers - pathogenen - plantenziekteverwekkende schimmels - sojaproducten - wortelrot - gewasbescherming - potatoes - solanum tuberosum - phytophthora infestans - genomes - phytophthora sojae - plant pathogens - pathogens - plant pathogenic fungi - soyabean products - root rots - plant protection
Het geslacht Phytophthora omvat meer dan 65 verwoestende plantenpathogene soorten die ernstige schade toebrengen aan landbouwgewassen en aan planten, struiken en bomen in de natuur. Economisch belangrijke pathogenen zijn onder andere Phytophthora infestans, de veroorzaker van de aardappelziekte, Phytophthora sojae, die wortel- en stengelrot op sojaboon veroorzaakt. Een onlangs ondekte soort, Phytophthora ramorum, is verantwoordelijk voor het Sudden Oak Death-syndroom en verwoest eikenbomen langs de westkust van de Verenigde staten. Phytophthora behoort tot de oömyceten die, samen met plantenpathogene schimmels, de belangrijkste groep plantpathogenen vormen. Morfologische gezien lijken oömyceten en schimmels op elkaar maar ze behoren tot verschillende rijken, respectievelijk de Stramenopila en de Fungi. Convergente evolutie heeft ertoe geleid dat oömyceten en schimmels een vergelijkbaar wapenarsenaal hebben dat nodig is om planten aan te vallen
Phytophthora-genomics: nieuwe mogelijkheden en uitdagingen
Govers, F. ; Meijer, H.J.G. - \ 2007
Gewasbescherming 38 (2007)5. - ISSN 0166-6495 - p. 265 - 271.
aardappelen - solanum tuberosum - phytophthora infestans - plantenziekteverwekkende schimmels - dna - genomen - genetische analyse - pathogenen - plantenziekteverwekkers - organellen - onderzoek - gewasbescherming - genexpressieanalyse - wetenschappelijk onderzoek - potatoes - solanum tuberosum - phytophthora infestans - plant pathogenic fungi - dna - genomes - genetic analysis - pathogens - plant pathogens - organelles - research - plant protection - genomics - scientific research
Het onderzoek aan de ziekteverwekker Phytophthora infestans is in een stroomversnelling geraakt. Dit is grotendeels te danken aan de stormachtige ontwikkelingen in genomics en de nieuwe inzichten die uit het genoomonderzoek voortvloeien. De DNA-code van het genoom van Phytophthora infestans en van enkele verwante soorten is ontrafeld en via internet beschikbaar. We staan pas aan het begin van het ontginnen van een schat aan gegevens die in de genoomsequentie verborgen ligt maar er zijn nu al verrassende vindingen gedaan die kunnen leiden tot nieuwe strategiëen voor de bestrijding van de aardappelziekte. De moleculaire basis van de 'gen-om-gen'-interactie tussen aardappel en Phytophthora infestans wordt steeds duidelijker en we kunnen, op basis van geconserveerde motieven in effectoreiwitten, genen aanwijzen die een rol spelen in gastheerspecifiteit. Nu kunnen we bestaande theoriën gaan staven met experimentele bewijzen en voorspellingen doen over de duurzaamheid van resistentiegenen. We hebben genen gevonden die coderen voor eiwitten die niet voorkomen in andere organismen en daarom mogelijk interessante aangrijpingspunten zijn voor nieuwe Phytophthora-bestrijdingsmiddelen of een ingang bieden voor alternatieve vormen van bestrijding. Er is nu onomstotelijk bewijs dat Phytophthora, ondanks zijn schimmelachtige uiterlijk, niet tot schimmelrijk behoort maar groene voorouders heeft die over fotosynthetiserende organellen beschikten
Intergenomic recombination and introgression breeding in Longiflorum x Asiatic lilies
Shujun Zhou, - \ 2007
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Jaap van Tuyl. - [S.l.] : S.n. - ISBN 9789085046370 - 110
lilium - introgressie - plantenveredeling - recombinatie - sporogenie - soortkruising - genomen - kruisen - kruisingen - lilium - introgression - plant breeding - recombination - sporogony - interspecific hybridization - genomes - crossing - crosses
Lily, one of the economically most important ornamental crops, belongs to the genus Lilium of the family Liliaceae. There are about 80 species in Lilium which are categorized into seven sections, i.e., Lilium, Martagon, Pseudolirium, Archelirion, Sinomartagon, Leucolirion and Oxypetala. Usually, it is not so difficult to cross between the species within each section and the hybrids are fertile. However, it is very difficult to cross the species belonging to different sections. With cut style pollination followed by embryo rescue techniques, such distant interspecific crosses can be possible, but the hybrids are highly sterile. All modern lily cultivars have originated from hybridization among wild lily species. The three main lily cultivar groups, viz., Longiflorum, Asiatic and Oriental, originated from hybridization within one section, i.e., Leucolirion, Sinomartagon and Archelirion respectively. Up to now, about 150 Longiflorum, 4000 Asiatic cultivars, and 2000 Oriental cultivars have been registered. The genomes of Longiflorum (Leucolirion), Asiatic (Sinomartagon) and Oriental (Archelirion) are represented as L (Longiflorum), A (Asiatic) and O (Oriental) genome respectively. They possess quite different valuable traits, and one of the main goals of modern lily breedingareto combine the three distinctive groups in new cultivars. In this thesis, crosses among diploid Asiatic and Longiflorum cultivars, diploid F1 LA hybrids, triploid BC1 cultivars, allotetraploidand allopentaploid lilies were made. 11 diploid F1 LA hybrids and 19 triploids BC1 cultivars which were supplied by the Dutch lily breeding companies, and 23 new BC1, five BC2 and seven BC3 progenies were analyzed with conventional cytological methods, flow cytometry and genomic in situ hybridization.The configurations of metaphase I during meioses of the F1 LA hybrids are quantitatively variable, ranging from no bivalent to 12 bivalents in different pollen mother cells. This implies that LA hybrids have abnormal meiosis and normal meiosis, and indicates that LA hybrids have possibilities to produce aneuploid gametes, 2n gametes and n gametes (Chapter 2). Because the bivalents disjoin and the univalents divide simultaneously at anaphase I of the observed pollen mother cells, it is concluded that F1 LA hybrids have more potential to produce IMR 2n gametes than FDR 2n gametes I (Chapter 2). However, most of the BC1 progenies result from FDR 2n gametes and less from IMR 2n gametes. Probably, FDR 2n gametes have better viability than IMR 2n gametes because of chromosome and gene imbalance in the latter (Chapters 3 & 4).Besides the mode of 2n gamete formation, some crossover events, e.g., single, threestranddouble, four strand double, four strand triple crossover, etc, are clearly elucidated based on the GISH results from anaphase I of F1 LA hybrids (Chapter 2). The intergenomic recombinant chromosomes of triploid BC1 progenies mainly originate from single crossover (Chapters 3 & 4). The intergenomic recombinant chromosomes caused by other crossover events are confirmed in diploid BC1 progenies (Chapter 4).Based on GISH analysis of 19 BC1 cultivars from the Dutch lily breeding companies, 17 of the BC1 cultivars are eutriploid and two hypertriploid (Chapter 3). Nevertheless, among 45 new BC1 progenies, 10 of them are diploid, while the others are triploid (Chapter 4). This is the first reported finding that LA hybrid can produce functional haploid gametes. This finding might be valuable for lily introgression breeding. Only limited BC2 progenies, which originated from crosses between triploid BC1 cultivars and diploid Asiatic cultivars, were analyzed with GISH. They were predominantly diploid and they contained very few Longiflorum chromosomes or segments (Chapter 5).Allopentaploid lilies have relatively good male fertility as determined from their pollen germination. They were successfully crossed with Asiatic cultivars and Longiflorum cultivars. Most of the BC3 progenies were pseudoeuploids that possessed euploid chromosome numbers (2n=3x=36) but the parental genomes were aneuploid as a result of chromosome substitutions. Apart from this, one aneuploid (2n=3x+1=37) was also present. Both the pseudoeuploids and the aneuploids might contribute to genetic variation and are potentially useful for selection. Based on the results of lily interploid crosses (2x-3x, 2x-4x, 2x-5x and their reciprocals), diploid, triploid, tetraploid, and pentaploid lilies could be used as male parents when they had some degree of male fertility. Most diploid and triploid lilies could be used as female parents regardless of their male fertility. On the contrary, allotetraploid and allopentaploid lilies could hardly be used as female parents even though they had good fertility as estimated from pollen germination tests. The success and failure of interploid crosses in lilies depends on the viability of the gametes and the ploidy level of the secondary nucleus. From the process of tetrasporic eight-nucleate embryo sac formation in Lilium , it is derived that diploid, triploid, tetraploid, and pentaploid lilies produce tetraploid, hexaploid, octaploid and decaploid secondary nuclei in their embryo sacs respectively. Thus, It is suggested that tetraploid secondary nucleus might be ideal for lily endosperm development; hexaploid secondary nucleus are acceptable; but octaploid or higher secondary nuclei are not ideal (Chapter 5).The difference between 2x-4x and 4x-2x or 2x-5x and 5x-2x is obvious, because one was successful and the other not. A clear difference was also observed between 2x-3x and 3x-2x and between AA x LA and LA x AA. The difference is possibly caused by the frequency of functional gametes, ploidy level of endosperm and the interaction between embryo development and endosperm development (Chapter 5).2n gametes and n gametes might play different roles in lily breeding. The former is more useful for polyploid breeding; the latter may be valuable for introgression breeding. We could not over- or underestimate either of them, because the LA hybrids which could produce functional 2n gametes or n gametes are very limited. Especially, it is more difficult to find LA hybrids which produce functional n gametes. The advantages of 2n gametes over mitotic doubling in lily breeding have been well confirmed (Chapters 3 and 4). One of the further tasks is how to use the LA hybrids which produce viable n gametes in lily breeding.
Genetic mapping using the Diversity Arrays Technology (DArT) : application and validation using the whole-genome sequences of Arabidopsis thaliana and the fungal wheat pathogen Mycosphaerella graminicola
Wittenberg, A.H.J. - \ 2007
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Henk Schouten; Theo van der Lee. - [S.l.] : S.n. - ISBN 9789085046295 - 222
genetische kartering - technieken - nucleotidenvolgordes - genomen - arabidopsis thaliana - plantenziekteverwekkers - mycosphaerella - genetische merkers - plantenveredeling - genetic mapping - techniques - nucleotide sequences - genomes - arabidopsis thaliana - plant pathogens - mycosphaerella - genetic markers - plant breeding
Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds- to thousands of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. In contrast to many other gel-based marker technologies DArT markers can be sequenced readily. Chapter 1 gives an overview of the most widely used genetic marker methods and their limitations. In addition some background information is provided on the wheat infecting fungal pathogen Mycosphaerella graminicola , in which we applied DArT.
In Chapter 2 the DArT procedure is described in more detail. All steps in the DArT protocol are explained and discussed. In addition some adaptations to the DArT procedure with respect to the complexity reduction and use of adapter sequences are demonstrated on the basis of experiments performed in M. graminicola . Finally, the specifically developed software (DArTsoft) and the application of DArT markers in the mapping process are described.
Chapter 3demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana . Restriction fragments from a genomic representation of the ecotype Landsberg erecta (L er ) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypes
In Chapter 4DArT was used to construct two high-density genetic linkage maps of the haploid wheat pathogen M. graminicola . The isolate IPO323 that originates from a bread wheat field was crossed in planta with either another bread wheat isolate IPO94269 or the durum wheat isolate IPO95052. Host species specificity of the plant pathogenic fungus M. graminicola towards hexaploid bread wheat and tetraploid durum wheat is well-documented. Although M. graminicola has an important sexual cycle and in some parts of the world durum wheat and bread wheat are grown side by side, in nature most isolates are pathogenic to either bread wheat or durum wheat. The generation of progeny from the IPO323 x IPO95052 in planta cross was therefore unexpected as these parental isolates are avirulent to durum wheat and bread wheat, respectively. The segregation of markers was followed in the progenies of both crosses. In total 2078 markers (of which 1793 were DArT markers) could be mapped. The maps of the individual crosses showed absolute co-linearity to a constructed bridge map, with the exception of eight markers that appeared to reveal translocations between IPO95052 and IPO94269. Graphical genotyping enabled the identification of dispensable chromosomes in 15-20 % of the progenies, although these chromosomes were present in both parents. We demonstrate that this is due to aberrations (partly by non-disjunction) during meiosis. The loss of chromosomes did not seem to affect viability and pathogenicity. This is the first report on detailed meiotic events using high-density mapping in a haploid filamentous fungus. We show that DArT is a highly efficient and robust marker technology for genetic mapping. The DArT markers can be readily sequenced and are currently being deployed in the assembly of the 9X M. graminicola IPO323 genome sequence.
In Chapter 5 the genetic linkage map of the IPO323 x IPO95052 cross was used to map several quantitative trait loci (QTL) for host species specificity in M. graminicola .Virulence of the progeny (n=163) was tested on four bread wheat cultivars and three durum wheat cultivars. All progeny isolates grew normally in vitro,but in contrast to crosses performed with two bread wheat isolates, nearly 50 % were unable to cause disease on any of the generally susceptible bread wheat and durum wheat cultivars. Interestingly, other progeny caused disease on either bread wheat cultivars or durum wheat cultivars or both wheat species. This proves that the observed host species specificity was overcome in a single generation. In addition, some progeny were able to cause disease on the bread wheat cv. Shafir even though both parents are avirulent to this cultivar. Detailed analyses allowed the identification of QTLs that determine specificity using the high-density genetic linkage map containing 1144 molecular markers, described in the previous chapter. In total, we identified nine QTLs on seven linkage groups. Remarkably, no specific locus was identified that explains the host species specificity to either durum or bread wheat. One locus with a very high LOD value had a major effect on specificity towards all tested durum wheat cultivars, but was previously identified as a locus involved in cultivar specificity. To our knowledge, this is the first comprehensive QTL analysis on virulence performed on a plant pathogenic fungus. We conclude that specificity in M. graminicola is controlled by many genes and that the long-accepted notion of species specificity among isolates of M. graminicola may in fact be a mix of genetically inherited factors, whereby the distinction between host species specificity and cultivar specificity is not clear-cut.
Chapter 6shows the alignment of the DArT markers to the genome of M. graminicola that recently was sequenced by the US Department of Energy-Joint Genome Institute (DOE-JGI). Assembly (version 1.0) of the 9X shotgun sequence resulted in the identification of 129 scaffolds ranging in size from 99 kb to 6.36 Mbp and covering 41.9 Mbp. In this assembly a total of 36 telomeres were identified. Sequencing of DArT markers used for the construction of two high-density genetic linkage maps in M. graminicola provided us the opportunity to align the genetic maps with the physical (sequence) map. By performing BLAST and BLAT analysis of the sequences obtained, 1069 IPO323, 724 IPO95052 and 24 SSR markers could be positioned on the 22 largest scaffolds in the assembly allowing the most comprehensive alignment among sequenced fungal genomes. Comparison of the positions of these markers on the sequence map with the order of the markers on the genetic linkage maps resulted in a nearly complete co-linearity of the order of markers, covering >99 % of the current assembly. When a 2 cM confidence interval was applied, 99 % of the markers were co-linear with the genome sequence. The alignment provided evidence that scaffolds 7 and 17, 10 and 14 and also 12 and 22 belong to one chromosome and that scaffolds 4 and 9 were wrongly assembled in the first draft. Furthermore, we identified large differences in genetic versus physical distance in certain genomic regions demonstrating that recombination frequencies increase towards the telomeres. In addition, we identified significant differences in the recombination rates in the different crosses studied. The integration of the genetic maps with the physical map will: 1) assist in finishing the genome assembly; 2) facilitate map-based cloning strategies of genes involved in pathogenesis and 3) help to understand the processes that occur during meiosis and recombination.
Finally, Chapter 7 gives a general discussion. The advantages and disadvantages of the DArT marker method as well as alternative array-based technologies are discussed. We conclude that DArT is a cost effective marker technology which generates the extremely high quality data needed for high-density mapping. In addition the ease in which sequences of the markers are obtained, allow large-scale alignments between genetic and physical maps. This makes DArT the method of choice especially for species for which large genetic variation exists, limited financial resources are available or for complex polyploid genomes that may not be amenable to the whole-genome sequence approach. In addition the results obtained using the high-density genetic linkage maps from M. graminicola and the biological implications for the fungal population in respect to adaptation to the host are discussed.
Evolutionary study of plastid and mitochondrial DNA insertions into the nucleus of flowering plants
Noutsos, C. - \ 2007
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): D. Leister. - [S.l.] : S.n. - ISBN 9783832258818 - 108
planten - plastiden - mitochondria - mitochondriaal dna - dna - celkernen - genomen - bloeiende planten - plants - plastids - mitochondria - mitochondrial dna - dna - nuclei - genomes - flowering plants
The aim of this study was to characterize the structure of functional and non-functional nuclear inserts of organelle DNA as well the expression of photosynthetic genes which are transferred during endosymbiosis into the nucleus with the final scope to determine the impact of organelle DNA on gene and genome evolution.
In Chapter 1 a general description of the chloroplast organelle is given. Its functions emphasizing photosynthesis by which plants, algae, some bacteria, and protists convert the energy of sunlight to chemical energy. An overview on plastid membranes is provided as well. The origin of organelles is described. Mitochondria and chloroplast were once free living bacteria, a-proteobacteria or cyanobacteria, respectively. As a first endosymbiotic event, the mitochondrion was created by the endosymbiosis ofa proteobacteriumwith an ancestor cell containing no mitochondria. Later, a second
endosymbioticevent involving cyanobacteria and the primary ancestor cell, led to the creation of chloroplasts. Also, a description of the import machinery of chloroplasts and mitochondria is provided.
In Chapter 2 as a first approach the integration of relative large insertions of organelle DNA into the nucleus was studied using Arabidopsis and rice organelles and genomes. Thirteen integrants were identified in Arabidopsis and rice genomes. Nuclear genomes are exposed to a continuous influx of DNA from mitochondria and plastids. These insertions can occur during the illegitimate repair of double stranded breaks. After integration, nuclear organelle DNA is modified by point mutations and by deletions. Overall, the numbers of insertion and deletion events after integration of the thirteen
segmentsof organelle DNA into the nucleus are almost equal. Deletions are associated with the removal of DNA between perfect repeats, indicating that replication slippage has caused them. Two general types of nuclear insertions coexist; one is characterized by long sequence stretches that are colinear with organelle DNA, the other type consists of mosaics of organelle DNA, often derived from both plastids and mitochondria. The levels of sequence divergence of the two types exclude their common descent, implying that at least two independent modes of DNA transfer from organelle to nucleus operate.
In addition to the integration of large insertions of organelle DNA into the nucleus of organisms there are small pieces of organelle DNA integrated into functional genes. In Chapter 3 these insertions of organelle DNA into functional genes are described. This analysis was performed in human, yeast, rice and Arabidopsis. In general these insertions were small. In Arabidopsis there was only one insertion event per gene. On the contrary, in all other organisms more than one insertion in the genes, reported having insertions, could be detected. For all proteins, which had an organellar insertion, homologous proteins were analyzed and assigned lacking the organelle insertion, meaning that these organelle insertions might contribute to better functionality of the genes. Many of the genes needed for the proper function of organelles are nuclear encoded. It is believed that they were transferred during endosymbiosis from the organelles to the nucleus where they were exposed to several mutation events in order to adapt to the new environment. These genes acquire
atransit peptide in order to be targeted back to the organelle they were coming from.
In Chapter 4 the expression of nuclear encoded genes which are of chloroplast origin is studied. The expression of 3292 nuclear Arabidopsis genes, encoding mostly chloroplast proteins, were determined from 101 different environmental and genetic conditions. The 1590 most-regulated genes fell into 23 distinct groups of coregulated genes (regulons). With the exception of regulons 1 and 2, the rest are heterogeneous and consist of genes coding for proteins with different subcellular locations or contributing to several biochemical functions. The co-expression of nuclear genes coding for subunits of the photosystems or encoding proteins involved in the transcription/translation of plastome genes (particularly ribosome polypeptides) (regulons 1 and 2, respectively) implies the existence of a novel mechanism that coordinates plastid and nuclear gene expression and involves nuclear control of plastid ribosome abundance. The co-regulation of genes for photosystem and plastid ribosome proteins escapes a previously described general control of nuclear chloroplast proteins imposed by a transcriptional master switch, highlighting a mode of transcriptional regulation of photosynthesis which is different compared to other chloroplast functions. From an evolutionary standpoint, the results provided indicate that functional integration of the proto-chloroplast into the eukaryotic cell was associated with the establishment of different layers of nuclear transcriptional control.
The majority of the light harvesting complex genes are in regulon 1. In Chapter 5 a detailed expression analysis of the genes belonging to Lhc supergene family, which is of chloroplast origin, was performed by using poplar and Arabidopsis. The analysis was done in silico. Four rarely expressed Lhcgenes, Lhca5, Lhca6, Lhcb7, and Lhcb4.3 were studied in details. Those genes have high expression levels under different conditions and in different tissues than the abundantly expressed Lhca1 to 4 and Lhcb1 to 6 genes that code for the 10 major types of higher plant light-harvesting proteins. The pattern of the rarely expressed Lhc genes was always found to be more similar to that of PsbS, a subunit of photosystem II and the various light-harvesting-like genes, which might indicate distinct physiological functions for the rarely and abundantly expressed Lhc proteins. As the Lhcb4.3 gene seems to be present only in Eurosid species and as its regulation pattern varies significantly from that of Lhcb4.1 and Lhcb4.2, we conclude it to encode a distinct Lhc protein type, Lhcb8.
Chapter 6represents the general discussion of the thesis emphasizing on the roles that organelle insertions can have in the evolution of genes and genomes. Also open questions still existing on the continuous migration of DNA from the organelles to the nucleus are discussed.
In conclusion, DNA transfer from the organelles can have a neutral role by integrating into the nucleus and in areas where no functional elements exist. In addition, it gave rise to the creation of genes which are important for the proper function of the organelles. In this case the transfer of DNA took place in order to have better coordination of the regulation of the chloroplast and mitochondria. In addition there is transfer of small pieces of DNA into the open reading frame of functional genes which might contribute to better functionality of these genes where the insertions are taking place.
Scannen van de genenkaart : Johan van Arendonk: ''We moeten proefstieren blijven testen"
Arendonk, J.A.M. van - \ 2006
Veeteelt 23 (2006)6. - ISSN 0168-7565 - p. 50 - 51.
melkveehouderij - stieren (bulls) - ki stieren - melkveestieren - dierveredeling - selectie - genomen - fokwaarde - testen - interviews - dairy farming - bulls - ai bulls - dairy bulls - animal breeding - selection - genomes - breeding value - testing - interviews
Uitleg door de Wageningse hoogleraar Fokkerij en genetica op vragen als: Wat is genoomselectie? Zijn merkers overbodig geworden? Voor welke kenmerken biedt genoomselectie perspectief? Wat merken veehouders er in de praktijk van?
Bioinformatics for plant genome annotation
Fiers, M.W.E.J. - \ 2006
Wageningen University. Promotor(en): W. Stiekema, co-promotor(en): Jan-Peter (Jp) Nap. - [S.l.] : S.n. - ISBN 9789085045199 - 141
planten - bio-informatica - genoomanalyse - genomen - dna-sequencing - computeranalyse - genexpressieanalyse - plants - bioinformatics - genome analysis - genomes - dna sequencing - computer analysis - genomics
Large amounts of genome sequence data are available and much more will become available in the near future. A DNA sequence alone has, however, limited use. Genome annotation is required to assign biological interpretation to the DNA sequence. This thesis describes a variety of research topics for bioinformatics in the context of genome annotation. A review of the various topics and issues in the science of genome annotation is given in Chapter 2. The next chapter (3) describes the development of a system for automated, high throughput, genome annotation called Cyrille2. Chapter 4 focuses on interactive and intuitive visualization of different types of annotation data. Chapter 5 describes a new approach for the prediction of potential micro-RNAs (miRNAs) in a plant genome. Chapter 6 of this thesis focuses on proteins and the prediction of a particular protein feature, allergenicity.The overall scope of this thesis is broad and many of the challenges for bioinformatics in genome annotation are encountered. The primary challenges are reviewed in the final discussion of this thesis (Chapter 7) and concludes that these challenges fall into two main categories: communication and quality. Communication in bioinformatics for genome annotation is a major challenge on multiple levels; between computers, between humans and inbetween computers and humans. Each level brings its own challenges and requires different solutions. The quality of annotation data is the second major issue. Most annotations depend on previous annotations. The quality of these predictions obviously rely on the quality of the underlying data and are therefore susceptible to error propagation. A quality indication system that covers the data behind an annotation seems essential for the future of genome annotation.Current technological developments indicate dramatic increases in data production speed for genomics and related areas. Combined with the knowledge that only a small percentage of any genome is understood ensures that genome annotation will remain one of the more exciting and challenging fields of bioinformatics for decades to come.
Over verschillen en overeenkomsten
Groenen, M.A.M. - \ 2005
Wageningen : Wageningen Universiteit - 35
vee - genomen - dna - genen - genkartering - dna-sequencing - genetische variatie - moleculaire genetica - spraak - livestock - genomes - dna - genes - gene mapping - dna sequencing - genetic variation - molecular genetics - speech
Genoomonderzoek bij landbouwhuisdieren levert een belangrijke bijdrage aan een verhoogd fundamenteel wetenschappelijk inzicht in het biologisch functioneren van dier en mens en maakt het in toenemende mate mogelijk om binnen de fokkerij dieren te selecteren op basis van bekende variaties in het erfelijk materiaal (DNA)
|Plant species-level systematics : New perspectives on pattern & process
Bakker, F.T. ; Chatrou, L.W. ; Gravendeel, B. ; Pelser, P.B. - \ 2005
Liechtenstein : A.R.G. Gantner Verlag (Regnum vegetabile vol. 143) - ISBN 9783906166391 - 348
planten - taxonomie - biosystematiek - soorten - evolutie - fylogenetica - soortvorming - genomen - plants - taxonomy - biosystematics - species - evolution - phylogenetics - speciation - genomes
Genomic conflicts in Podospora anserina = Genomische conflicten in Podospora anserina
Gaag, M. van der - \ 2005
Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Fons Debets. - s.l. : S.n. - ISBN 9789085042556 - 152
pezizomycotina - schimmels - genomen - genetica - plasmiden - meiotic drive - meiose - uitkruisen - pezizomycotina - fungi - genomes - genetics - plasmids - meiotic drive - meiosis - outcrossing
This thesis deals with genomic conflicts raised by selfish elements in the ascomycete fungus Podospora anserina .Genomic conflicts arise when the effects of the selfish elements are opposite to the interests of the other parts of the genome. Two types of selfish elements are studied as well as certain characteristics of Podospora involved in the population dynamics of these elements, such as vegetative and sexual incompatibility, senescence and outcrossing.The natural habitat of Podospora anserina is dung of herbivores where it has an optimum growth temperature of 27 °C. The fungus can only reproduce sexually and the ascospores are the products of meiosis as well as the next generation of the fungus. Perithecia or fruiting bodies contain asci with four linearly arranged ascospores, which provide unique opportunities to analyse abnormal segregation and makes this fungus one of the genetic model organisms. Most ascospores are capable of completing the lifecycle of the fungus, as they contain two nuclei, each with one of the two mating types. This fungal trait is called pseudo or secondairy homothallism, and it allows sexual offspring to be produced by either selfing or outcrossing. Sometimes smaller single mating type ascospores are formed containing one nucleus and less cytoplasmic content and mitochondria. Colonies from these spores must outcross with another isolate to produce offspring. The fungal isolates used in this thesis were sampled from dung around Wageningen, the
Genomics and transcriptomics of White spot syndrome virus
Marks, H. - \ 2005
Wageningen University. Promotor(en): Just Vlak; R.W. Goldbach, co-promotor(en): M.C.W. van Hulten. - Wageningen : s.n. - ISBN 9789085043188 - 152
garnalen - penaeus monodon - dierenvirussen - transcriptie - genomen - genexpressieanalyse - shrimps - penaeus monodon - animal viruses - transcription - genomes - genomics
White Spot Syndrome Virus (WSSV) is a large enveloped DNA virus that infects shrimp and other crustaceans. The virions are approximately 275 x 120 nm in size and have an ovoid to bacilliform shape and a tail-like appendage at one end. Sequencing revealed that the circular, double stranded (ds) DNA genome of WSSV ranges between 293 and 307 kb in size depending on the WSSV isolate. For a sequenced isolate originating from
WSSV causes serious economic losses in shrimp culture, as 100% cumulative mortalities can be reached within 3-10 days under farming conditions. After its discovery in 1992 in Taiwan WSSV has quickly spread into Southeast-Asia and subsequently to shrimp farming areas all over the world. This thesis aims at obtaining fundamental insights in the genomic structure ("genomics") and transcription regulation ("transcriptomics") of WSSV. This in turn may provide better insight in the molecular basis of WSSV biology and epidemiology, which can be useful in the identification of targets for WSSV intervention strategies.
Alignment of the complete genome sequences of the isolates WSSV-TW, WSSV-CN and WSSV-TH, originating from Taiwan, China and Thailand, respectively, revealed that the sequences were very similar (over 99% sequence identity), suggesting that the isolates are variants of the same virus species ( Whispovirus ) and probably evolved recently from a common ancestor (Chapter 2). Two major polymorphic loci were identified, variable region (VR) ORF14/15 and VR ORF23/24, and both appeared to be genomic regions where large deletions occur. Further polymorphisms included loci with variable numbers of tandem repeats (VNTR loci). Next to VR ORF14/15 and VR ORF23/24, three of these loci, located in the regions coding for ORF75, ORF94 and ORF125, were identified as useful markers in epidemiological and ecological studies. The highly conserved genomic loci, e.g. the gene encoding the major structural virion protein VP26, are useful for reliable monitoring of WSSV infections in PCR based assays.
The observation that the isolate WSSV-TH contains a large deletion in VR ORF23/24 relative to the isolates WSSV-TW and WSSV-CN suggested the evolution and spread of WSSV from a common ancestor, provisionally located near the Southeast coast of
To study the possible effect of the genetic differences on the fitness and virulence of WSSV, two divergent WSSV isolates (TH-96-II and WSSV-TH) were compared (Chapter 4). TH-96-II was a newly characterized archival WSSV isolate from 1996, which has the largest genome size (~312 kb) of all WSSV isolates identified thus far. As TH-96-II does not contain deletions in either VR ORF14/15 or VR ORF23/24, it may be ancestral to all known WSSV isolates. WSSV-TH contains the smallest genome (~293 kb) identified at present, due to large deletions in VR ORF14/15 and VR ORF23/24. Comparison between TH-96-II and WSSV-TH, when administered to shrimp Penaeus monodon, showed a higher virulence and competitive fitness for the latter. This may suggest that the virus became more virulent over the years during the epidemic while moving south. This enhanced virulence is possibly caused by the continuous contact with susceptible animals, a behavior also seen with some other emerging viruses. Since the more virulent variant (WSSV-TH) has a smaller genome, it may replicate faster to reach a lethal dose. However, it is also possible that the observed differences in virulence are caused by other genetic polymorphisms between the two isolates.
As WSSV differs profoundly from other large ds DNA viruses and mainly contains unique genes, the mechanism of gene expression and transcription regulation of this new virus was investigated in the second part of this thesis. To study WSSV gene expression on a genome wide scale, a WSSV DNA microarray was constructed containing probes corresponding to nearly all putative WSSV ORFs (Chapter 5). Using a WSSV infection time course we could show expression of at least 79% of the WSSV ORFs included on the microarray in gill tissue of Penaeus monodon . Clustering of the transcription profiles of the individual genes showed the presence of two major classes of genes, a putative early and a putative late class, suggesting that the WSSV genes at large are expressed in a coordinated and cascaded fashion. Five genes encoding WSSV major virion proteins (VP28, VP26, VP24, VP19 en VP15), which clustered in the late class, were further confirmed to be late by RT-PCR (Chapter 6). Furthermore, the 5' and 3' ends of the mRNA of these late genes were determined for identification of common promoter motifs.
To search for common conserved WSSV promoter motifs associated with WSSV early or late gene expression, as determined by the microarrays, two in silico methods were employed (Chapter 7). The abundance of all 4 through 8 nucleotide motifs in the upstream sequences of WSSV genes relative to the complete genome was determined and the upstream sequences of early or late WSSV genes were analyzed for conserved sequences motifs using MEME. Both methods were complemented by alignments of empirically determined 5' ends of various WSSV mRNAs. The collective information shows that the upstream region of WSSV early genes, containing a TATA box and an initiator sequence, is reminiscent to Drosophila RNA polymerase II promoters, suggesting utilization of the cellular transcription machinery for generating early transcripts. The alignment of the 5' ends of known late genes, including the 5' ends determined in chapter 6, identified a degenerate consensus late transcription initiation motif (ATNAC). Of these genes, only one contained a functional TATA box. However, almost half of the WSSV late genes, as assigned by microarrays, did contain a TATA box in their upstream region. This may suggest the presence of two separate classes of late WSSV genes, one exploiting the cellular RNA polymerase II system for mRNA synthesis and the other generating messengers by a new virus-induced transcription mechanism.
Alignments of the 3' ends of various WSSV mRNAs suggest that there is no difference in polyadenylation between early and late mRNAs. The WSSV polyadenylation characteristics of both classes resemble regular polyadenylation in eukaryotic mRNAs, which is typically located 10 to 25 nt downstream of the sequence AATAAA.
In conclusion, the research performed for this thesis has led to a model on the mechanism of WSSV gene expression, and the promoter motifs involved (Chapter 8). The identification of genetic markers has led to more insight in the quick geographical spread of the virus, and the genetic characterization of WSSV isolates may add to the identification of virulence related factors on the WSSV genome. The fundamental insights obtained in the biology and epidemiology of WSSV in this thesis may help in the identification of WSSV genes which can be targets for WSSV intervention strategies.
Analysis of tomato spotted wilt virus genome transcription
Knippenberg, I.C. van - \ 2005
Wageningen University. Promotor(en): R.W. Goldbach, co-promotor(en): Richard Kormelink. - Wageningen : Ponsen & Looijen - ISBN 9789085040880 - 98
tomatenbronsvlekkenvirus - plantenvirussen - genomen - transcriptie - tomato spotted wilt virus - plant viruses - genomes - transcription
Tomato spotted wilt virus (TSWV) is the type species of the genus Tospovirus within the Bunyaviridae, a family of segmented negative strand RNA viruses. Although much ground has been covered in the past two decades, many questions concerning the mechanism of replication and transcription of this important group of viruses have thus far remained unanswered. Elucidation of the molecular mechanisms of viral transcription and replication requires manipulable systems in which these processes can be studied and unravelled. At the onset of the research described in this thesis, an in vitro assay was available in which RdRp (RNA-dependent RNA polymerase) activity ofpurified TSWV virions was observed, although it was unclear what kind of RNA synthetic activity was observed: transcription or replication.With the aim to unravel the molecular details of TSWV transcription, the in vitro assay was further developed into a well-defined transcription assay (Chapter 2). Transcription of TSWV, like that of all segmented negative strand RNA viruses, is initiated by cap snatching. In this process, a host mRNA (i.e. cap donor) is cleaved in its non-coding leader sequence by a virally encodedendonuclease,and the resulting capped leader is subsequently used to prime transcription of the viral genome. The existing in vitro system, in which only viral replication was observed, was modified by the addition ofrabbit reticulocyte lysate (RRL) which allowed viral transcription to take place. In this way a system was established in which the mechanism of cap snatching could be studied. Not only globin mRNAs present in the lysate, but also exogenously added capped RNA molecules were used as cap donors.The observed requirement for RRL seemed to imply a translational dependence of TSWV transcription. This possibility was further investigated in Chapter 3, using two inhibitors of protein synthesis. Surprisingly, in contrast to what has been observed for other Bunyaviruses, addition of these translation inhibitors had no effect on TSWV transcription in vitro. Moreover, no actual protein synthesis could be detected in the in vitro transcription assay, due to the assay conditions being incompatible with in vitro translation. These results indicated that TSWV transcription, unlike that of several other Bunyaviridae, is not coup led to translation.In the absence of RRL, viral replication was observed in the in vitro system (Chapter 2), although at a much lower rate than transcription. Not only RNPs (ribonucleoprotein complexes) as present in the lysed virus particles, but also purified cytoplasmic RNPs were active in RNA synthesis in the in vitro assay. However, in contrast to virion-RNPs, cytoplasmic RNPs were found to be highly active in replication (in the absence of RRL) but hardly active in transcription (in the presence of RRL). These findings imply the existence of RNPs in two different states: replication-mode and transcription-mode.As a first step towards understanding transcription termination, a rough mapping of the 3'-ends of the S RNA-encoded mRNAs was undertaken (Chapter 4). Viral mRNAs synthesised in the in vi/ra system were amplified by RT-PCR using primers for the leader sequence snatched from a-globin mRNAs in combination with a range of primers spanning the entire intergenic region (IR) of the S RNA segment. In addition, the size of the N mRNAs produced in infected plants was compared with the sizes of synthetic transcripts representing mRNAs terminated at defined positions in the intergenic region. The results indicated that transcription terminates near the 3'-end of the intergenic hairpin, yielding mRNAs that contain this hairpin structure at their 3' end, including a conserved sequence motif. These findings point toward a transcription termination mechanism reminiscent of that used by prokaryotes, where formation of a hairpin structure in the nascent transcript induces termination of transcription.Meanwhile, based on a concurrent PhD study, a model for TSWV cap snatching was proposed in which a cap donor (host mRNA leader) is required to have a single base complementarity to the viral temp late for transcription. The globin mRNAs in the RRL that are used as cap donor in the in vi/ra assay (Chapter 2) have 2 bases complementary to the viral temp late, which led to the question how far this complementarity could be extended. To investigate this, a capped transcript resembling a TSWV N mRNA, having a 15-nt stretch of complementarity, was tested and indeed found to be used as cap donor (Chapter 2), i.e. the 'viral mRNA' was re-snatched. The potential for re-snatching ofviral mRNAs occurring in vivo was investigated in Chapter 5. Competition assays using single-, double- and multiple-basepairing cap donors indicated a preference for donors with a long extent of base complementarity to the viral template. This implies that viral mRNAs would preferentially be used as cap donors, in other words re-snatching of viral mRNAs would prevail over snatching of host mRNAs. In addition, primer extension analyses on the products of a re-snatching reaction were used to examine the exact endonuclease cleavage site of re-snatching, indicating that endonuclease cleavage takes place after the first or second nucleotide complementary to the viral template. In chapter 6 the results presented in chapters 2-5 and their implications are discussed in relation to current knowledge on replication, transcription, and translation of negative and ambisense RNA viruses. The current model for the mechanism of cap snatching is fine-tuned further in view ofthe findings reported in Chapter 5.
Localization of candidate allergen genes on the apple (Malus domestica) genome and their putative allergenicity
Gao Zhongshan, - \ 2005
Wageningen University. Promotor(en): Evert Jacobsen; Richard Visser, co-promotor(en): Eric van de Weg; Luud Gilissen. - Wageningen : S.n. - ISBN 9789085042426 - 146
appels - malus pumila - allergenen - genomen - genen - allergieën - allergische reacties - voedselallergieën - plantenveredeling - rassen (planten) - dna-sequencing - genexpressieanalyse - apples - malus pumila - allergens - genomes - genes - allergies - allergic reactions - food allergies - plant breeding - varieties - dna sequencing - genomics
Apple is generally considered as a healthy food, but 2-3% European people can not eat this fruit because it provokes allergy reaction. Four classes of apple allergen genes have been identified, they are Mal d 1, Mal d 2, Mal d 3 and Mal d 4 . This thesis focuses on the genomic characterization of these candidate genes on the apple genome by linkage mapping through sequence-specific DNA markers. In total, 26 putative Mal d genes, 25 of them were mapped on 8 linkage groups:17 for Mal d 1 , two for Mal d 2 and Mal d 3 , and four for Mal d 4 .As for the patients in northern and central Europe, the major apple allergen is Mal d 1. Genetic analysis demonstrated the Mal d 1 genes on linkage group (Chromosomes) 16 are relevant. Furthermore, a molecular marker linked to low-Mal d 1 allergenic trait was identified.
Genomic variation in dairy cattle - Identification and use
Schrooten, C. - \ 2004
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Henk Bovenhuis. - [S.l.] : S.n. - ISBN 9789085040095
melkvee - genomen - genetische variatie - kwantitatieve kenmerken - loci - melkproductie - bouw (dier) - pleiotropie - genetische verbetering - genetische merkers - dierveredeling - moleculaire genetica - dairy cattle - genomes - genetic variation - quantitative traits - loci - milk production - conformation - pleiotropy - genetic improvement - genetic markers - animal breeding - molecular genetics
The development of molecular techniques has offered possibilities to identify quantitative trait loci (QTL). Studies in dairy cattle have mainly focused on milk production traits. This thesis first gives an overview of the main identified QTL for milk production traits. Subsequently, a study to detect QTL affecting 27 conformation traits and functional traits was performed. A granddaughter design consisting of 20 Holstein-Friesian grandsires and 833 sons was analyzed by multi-marker regression. This across-family analysis suggested the presence of 61 QTL. Ten of these QTL exceeded the genomewise significance threshold. These were mainly QTL affecting body size traits and udder conformation traits.
When QTL-information is used to select for a certain trait, genetic progress in other traits may be influenced as well, due to pleiotropic effects of QTL, or due to closely linked QTL. A method was developed to identify regions affecting multiple traits. The method is based on the covariance between marker contrasts from single-trait regression analysis for different traits. Application of this method to data on fifteen traits (milk production, udder conformation, udder health and fertility traits) in our granddaughter design resulted in 59 multiple trait quantitative trait regions (MQR). Most MQR were found on BTA 6, 13, 14, 19, 22, 23 and 25.
QTL-information can be used in breeding schemes (marker-assisted selection, MAS) to increase the rate of genetic improvement. A number of multi-stage dairy cattle breeding schemes was evaluated, studying the impact of increased preselection using QTL-information. Response in multi-stage MAS-schemes was 4.5% to 31.3% higher than response in corresponding schemes without QTL-information. In some of the MAS-schemes with a reduced number of progeny tested bulls, genetic progress was identical to or higher than genetic progress in the original schemes. The results indicate opportunities to improve current breeding schemes. The gains depend on the amount of genomic variation explained by QTL.
Currently available pedigrees and methods offer excellent opportunities to identify more QTL, thus increasing the fraction of the genomic variation explained by QTL. New initiatives, like sequencing the bovine genome, will further facilitate the identification of genomic variation, and its use.
Genetics of Erwinia resistance in Zantedeschia: impact of plastome-genome incompatibility
Snijder, R.C. - \ 2004
Wageningen University. Promotor(en): P. Stam, co-promotor(en): Pim Lindhout; Jaap van Tuyl. - [S.l.] : S.n. - ISBN 9789058089755 - 112
zantedeschia - ziekteresistentie - pectobacterium carotovorum subsp. carotovorum - plantenveredeling - soortkruising - incompatibiliteit - genetica - genomen - zantedeschia - disease resistance - pectobacterium carotovorum subsp. carotovorum - plant breeding - interspecific hybridization - incompatibility - genetics - genomes
Soft rot caused by Erwinia carotovora subsp. carotovora ( Pectobacterium carotovorum subsp. carotovorum ) is the most important disease of Zantedeschia spp. Cultivation measures can protect the crop partially, but a combination with resistant cultivars could result in better control. Resistant cultivars are not available, however. It is determined in this thesis that it is possible to breed for resistance caused by Erwinia. Resistance tests as well as a collection of plant material were developed to determine the genetic variation for resistance to Erwinia carotovora subsp. carotovora . The results of four resistance tests were in general concordance with experiences from practice. Althoughthe tubertests were most reproducible, the leaf disk test was used further research. The leaf disk test was not destructive, so seedlings and rare germplasm could be evaluated. Among the section Zantedeschia,Z..aethiopica appeared resistant to moderately resistant, while Z. odorata appeared susceptible. Only some cultivars of the section Aestivae ('Neroli', 'Coral Sunset' and 'Hazel Marie') appeared significantly less susceptible than the very susceptible reference 'Florex Gold'. Partial resistance was observed in Z. albomaculata subsp. macrocarpa and in Z. rehmannii . To determine the genetic basis of the partial resistance, hybrids were developed of species that have different resistance levels: Z. albomaculata (susceptible to partially resistant), Z. rehmannii (partially resistant), Z. elliotiana (susceptible) and Z. pentlandii (very susceptible). The families of these interspecific crosses (F 1 -hybrids) segregated for leaf colour (chlorophyll content) and vigour, dependent on the parents and the crossing direction. This segregation was not Mendelian, but depended on the crossing direction. Descendents of Z. rehmannii as mother were green (with a chlorophyll content that was similar as selfings of the parents) and vigorous. Descendents of Z. elliotiana as mother, however, were mainly pale green and these were less vigorous in some cases, depending on the father. The cause of these reciprocal differences was determined to evaluate the effect to the level of resistance. Plastome-specific CAPS-markers (Cleaved Amplified Polymorphic Sequence) were used to determine that there existed at leat three plastomes that differed in the level of compatibility to the genomes of the different species of section Aestivae . The segregation in leaf colour (chlorophyll content) within families of the same crossing direction originated from plastids that had inherited via pollen. This paternal influence in plastid inheritance was observed in 24 intespecific families of Z. rehmannii , Z. albomaculata , Z. elliotiana and Z. pentlandii. Thus was concludedthat biparentalininheritance of plastids is common common within interspecific crossings among section Aestivae . The plastome of Z. rehmannii was the most compatible to the other genomes. Plants with not fully compatible plastome-genome combinations had decreased vigour and level of resistance. Genetic analyses were therefore performed only on plants that did not suffer from plastome-genome incompatibility. The level of resistance of F 1 -hybrids was correlated to the level of resistance of the parents en transgression was observed in seedlings of Z. rehmannii and Z. albomaculata . This relation indicates a genetic basis for resistance with complementary or additive genes with great potential for resistance breeding.
Zooming in on the lettuce genome: species relationships in Lactuca s.l., inferred from chromosomal and molecular characters
Koopman, W.J.M. - \ 2002
Wageningen University. Promotor(en): L.J.G. van der Maesen; E. Jacobsen; R.G. van den Berg. - S.l. : S.n. - ISBN 9789058086761 - 196
lactuca - asteraceae - fylogenie - fylogenetica - klassering volgens erfelijke eigenschappen - herbaria - chromosome banding - dna - dna-fingerprinting - genomen - karyotypen - plantenveredeling - lactuca - asteraceae - phylogeny - phylogenetics - cladistics - herbaria - chromosome banding - dna - dna fingerprinting - genomes - karyotypes - plant breeding
Lactucasativa (cultivated lettuce) is the world's most important leafy salad vegetable. Apart from L. sativa , the genus Lactuca contains ca. 75 wild species, potentially useful to improve, for example, taste, texture, and disease resistance of cultivated lettuce. The wild species L. serriola (Prickly Lettuce), L. saligna (Least Lettuce), and L. virosa (Great Lettuce) are commonly used for lettuce improvement.
In preliminary experiments, we established that there is a close connection between evolutionary distances of wild species relative to cultivated lettuce, and their position in the lettuce gene pool (i.e., the possibility to hybridize them with cultivated lettuce). In the present thesis, we established evolutionary relationships among L. sativa and 22 wild species in order to predict this position.
We determined that L. sativa , L. serriola , L. dregeana , and L. altaica are closely related, and can be regarded as conspecific. L. aculeata is closely related to them, but is a distinct species. L. serriola , L. dregeana , L. altaica, and L. aculeata occupy the primary gene pool of cultivated lettuce. They can be easily hybridized with cultivated lettuce, and thus are readily accessible gene sources for lettuce improvement. L. saligna and L. virosa are less closely related to L. sativa , and occupy the secondary gene pool (i.e. hybridization with L. sativa is possible, but difficult). All primary and secondary gene-pool species can be classified in Lactuca sect. Lactuca subsect. Lactuca . We found that all tertiary gene-pool species (hybridization with L. sativa only possible with radical techniques) can be classified in the remaining sections of the genus Lactuca (sections Phaenixopus , Mulgedium, and Lactucopsis ). These sections are the most promising sources of wild species for future improvement of cultivated lettuce. In the experiments, the tertiary gene-pool species were represented by L. viminea , L. tatarica , L. sibirica , and L. quercina . Surprisingly, the species classified in Lactuca sect. Lactuca subsect. Cyanicae are not evolutionary close to cultivated lettuce. They are not part of the lettuce gene pool, and should be excluded from Lactuca .
To determine the evolutionary relationships among L. sativa and its wild relatives, we examined the genomes of the species at various levels, which provided additional information on genome evolution. We established, that in general the genome sizes in the group increased during evolution, while the ratio of AT/GC nucleotides decreased. Genome complexity for species with 2C DNA amounts below 8.5 pg was similar, but species with 2C DNA amounts exceeding 8.5 pg had more complex and less similar genomes. The species from the primary gene pool share a common ancestor, but the genomes of L. sativa / L. serriola , L. saligna , and L. virosa , evolved in different directions.
The present thesis demonstrates that with the proper combination of techniques, a plant systematic study can provide both practically applicable results and fundamental evolutionary insights, thus bridging the gap between fundamental and applied research.
The genome of Spodoptera exigua multicapsid nucleopolyhedrovirus : a study on unique features
IJkel, W.F.J. - \ 2001
Wageningen University. Promotor(en): R.W. Goldbach; J.M. Vlak; D. Zuidema. - S.l. : S.n. - ISBN 9789058084194 - 150
kernpolyedervirussen - baculovirus - genomen - moleculaire genetica - spodoptera exigua - nuclear polyhedrosis viruses - baculovirus - genomes - molecular genetics - spodoptera exigua
The Baculoviridae are a family of rod-shaped viruses with large circular double-stranded DNA genomes (Chapter 1). The family is subdivided into two genera, Granulovirus (GV) and Nucleopolyhedrovirus (NPV) on the basis of the type of body occluding the virions. NPVs are further subdivided in group I and II based on phylogenetic evidence of the DNA polymerase protein. Baculoviruses almost exclusively infect insects and are, therefore, attractive biological alternatives to chemical insecticides for insect pest control. The baculovirus Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) infects the beet army worm S. exigua (Lepidoptera: Noctuidae) and has been successfully used as a bioinsecticide to control this world-wide insect pest of agricultural importance. SeMNPV differs from many other baculoviruses in that it is mono-specific and highly virulent for S. exigua larvae. The research described in this thesis aimed at the molecular characterization of the baculovirus SeMNPV to gain insight in its gene content and organization in comparison to those of other baculoviruses. At the same time this study will support or reject its current taxonomic position using gene and genome phylogeny analyses and might reveal insight in the molecular mechanisms associated with the biological properties of SeMNPV.
As a start the complete nucleotide sequence of the DNA genome of SeMNPV, a putative group II NPV, was determined and analyzed (Chapter 2). The genome was composed of 135,612 bp containing 138 putative genes or open reading frames (ORFs). Major differences in SeMNPV gene content and arrangement were found compared with the group I NPVs Autographa californica (Ac), Bombyx mori (Bm ), Orgyia pseudotsugata (Op) and the group II NPV Lymantria dispar (Ld). Sixteen ORFs were unique to SeMNPV, while the remaining ORFs (122) all had a homolog in one or more of the nine baculoviruses sequenced to date (Chapter 7). Sixty-three ORFs were conserved among all nine baculoviruses and are likely to be essential for NPV multiplication and survival. Strikingly, two of these NPV 'core' genes, odv-e66 and p26 , were found duplicated in SeMNPV. Gene parity analysis of baculoviral genomes indicated that SeMNPV and LdMNPV are closely related and that they are only distantly related to group I NPVs. Therefore, SeMNPV can be considered as a group II NPV.
Two of the 16 unique SeMNPV genes, Se116 and Se117, share similarity on amino acid level, but are not related on nucleotide level. To investigate the function, if any, of the unique SeMNPV genes in general, Se116 and Se117 were analyzed and characterized (Chapter 3). Se116 and Se117 were expressed from early till late in infection both in cultured cells and in larvae of S. exigua. Their transcripts were polyadenylated and initiated from typical baculovirus early promoter motifs. Se116 and Se117 encoded proteins of 27 and 23 kDa, respectively, which were localized in the virogenic stroma of the nucleus. While the function of the Se116 protein remains enigmatic, the Se117 protein appeared to be a structural protein associated with nucleocapsids of occlusion-derived virus (ODV), but not of budded virus (BV). Further investigation will reveal if and how these proteins are involved in the SeMNPV virulence or host range determination.
The research on unique SeMNPV genes was extended (Chapter 4) by the characterization of another gene, Se17/18, unique among NPVs, but strikingly having a homolog (ORF129) in the granulovirus Xestia c-nigum (XcGV), which is only distantly related to SeMNPV. Se17/18 was transcribed in cultured S. exigua 301 cells from early till late in infection. However, in vivo transcripts could only be detected late in infection. These polyadenylated transcripts started in a region containing a baculovirus consensus early promoter motif. In contrast to the Se116 and Se117 proteins, the Se17/18 protein was primarily localized in the cytoplasm. A chicken polyclonal antiserum was raised that reacted specifically to Se17/18 protein expressed in E. coli . However, no immunoreactive protein was detected in SeMNPV-infected insect cells. The absence of immunoreactive Se17/18 protein implies that it is rapidly turned over in insect cell culture or that the gene is only active in larvae and possibly has a regulatory function.
A thorough analysis of the complete SeMNPV genome revealed that it lacked a homolog of the major budded virus glycoprotein gene gp64, that is found in AcMNPV and other group I NPVs. Upon infection, by representatives of this group, acidification of the endosome triggers fusion of the viral and endosomal membrane, which is mediated by the BV envelope glycoprotein GP64. Therefore, the entry mechanism of SeMNPV in cultured cells was examined. SeMNPV budded virus (BV) entered insect cells by endocytosis like BVs of group I NPVs. Furthermore, a functional homolog of the envelope fusion protein GP64 was identified in Se8 (76 kDa) and appeared to be the major envelope protein of SeMNPV BVs. Surprisingly, a 60 kDa cleavage product of this protein was present in the BV envelope. A furin-like proprotein convertase cleavage site was identified immediately upstream of the N-terminus of the mature Se8 protein and this site was also conserved in the LdMNPV homolog (Ld130) of Se8. Syncytium formation assays showed that Se8 and Ld130 alone were sufficient to mediate membrane fusion. Both proteins were primarily localized in the plasma membrane of insect cells, which was consistent with their fusogenic activity. If Se8 is cleaved by a cellular convertase the host could also play a role in the determination of virus host range and virulence.
The research on function of single SeMNPV genes and also the engineering of this virus for improved insecticidal activity or as expression vector have been hampered as defective viruses are quickly generated when using insect cell culture. These defective viruses lack 25 kb sequence information and are no longer active in vivo upon oral feeding. A novel procedure to isolate SeMNPV recombinants was adopted by alternate cloning between insect larvae and cultured cells. In this way a SeMNPV recombinant (SeXD1) was obtained infectious both in vivo and in cell culture and with an improved speed of kill. This recombinant lacked 10.6 kb of sequence information, including ecdysteroid UDP glucosyl transferase ( egt ), gp37 , chitinase and cathepsin genes, as well as several genes unique to SeMNPV. One of these unique genes was Se17/18. The result indicated, however, that these genes are dispensable for virus replication both in cell culture and in vivo . A mutant with a similar deletion was identified by PCR in the parental wild type SeMNPV isolate suggesting that genotypes with differential biological activities exist in field isolates of baculoviruses.
The research on SeMNPV described in this thesis, has provided a complete overview of its coding potential and insight in several features common to lepidopteran baculoviruses, such as 'core' genes, unique genes and clustering of conserved genes (Chapter 7). The initial characterization of several SeMNPV genes resulted in the identification of a novel ODV-specific nucleocapsid protein unique to SeMNPV and a novel major BV envelope fusion protein. The latter is the first baculovirus protein reported to be cleaved by a cellular furin-like proprotein convertase. The development of a novel procedure to generate recombinants in vivo is presumably applicable to many baculovirus species in order to obtain biologically active recombinants. Exploitation of this technique will enable the further characterization of (unique) SeMNPV genes by deletion, insertion and mutation by in vivo recombination. Understanding the function of SeMNPV genes will ultimately lead to the unravelling of the molecular basis underlying the mono-specificity and high virulence of SeMNPV for the beet army worm Spodoptera exigua .
Virion composition and genomics of white spot syndrome virus of shrimp
Hulten, M.C.W. van - \ 2001
Wageningen University. Promotor(en): J.M. Vlak; R.W. Goldbach. - S.l. : S.n. - ISBN 9789058085160 - 119
penaeus monodon - dierenvirussen - garnalen - genomen - eiwitten - taxonomie - envelopeiwitten - genexpressieanalyse - shrimps - penaeus monodon - animal viruses - genomes - proteins - envelope proteins - taxonomy - genomics
Since its first discovery in Taiwan in 1992, White spot syndrome virus (WSSV) has caused major economic damage to shrimp culture. The virus has spread rapidly through Asia and reached the Western Hemisphere in 1995 (Texas), where it continued its devastating effect further into Central- and South-America. In cultured shrimp WSSV infection can reach a cumulative mortality of up to 100% within 3 to 10 days.
One of the clinical signs of WSSV is the appearance of white spots in the exoskeleton of infected shrimp, hence its name.
WSSV has a remarkably broad host range, it not only infects all known shrimp species, but also many other marine and freshwater crustaceans, including crab and crayfish. Therefore, WSSV can be considered a major threat not only to shrimp, but also to other crustaceans around the world.
The WSSV virion is a large enveloped particle of about 275 nm in length and 120 nm in width with an ellipsoid to bacilliform shape and a tail-like extension on one end. The nucleocapsid is rod-shaped with a striated appearance and has a size of about 300 nm x 70 nm. Its virion morphology, nuclear localization and morphogenesis are reminiscent of baculoviruses in insects. Therefore, WSSV was originally thought to be a member of the Baculovirida e.
At the onset of the research presented in this thesis, only limited molecular information was available for WSSV, hampering its definitive classification as well as profound studies of the viral infection mechanism. As the first step towards unraveling the molecular biology of WSSV, terminal sequencing was performed on constructed genomic libraries of its genome.
This led to the identification of genes for the large (rr 1) and small (rr 2) subunit of ribonucleotide reductase, which were present on a 12.3 kb genomic fragment (Chapter 2). Phylogenetic analyses using the RR1 and RR2 proteins indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and further showed that WSSV and baculoviruses do not share a recent common ancestor.
Subsequently two protein kinase (p k) genes were located on the WSSV genome, showing low homology to other viral and eukaryotic pk genes (Chapter 3). The presence of conserved domains, suggested that these PKs are serine/threonine protein kinases. A considerable number of large DNA viruses contains one or more pk genes and these were used to construct an unrooted parsimonious phylogenetic tree. This tree indicated that the two WSSV pk genes originated most likely by gene duplication. Furthermore, the tree provided strong evidence that WSSV takes a unique position among large DNA virus families and was clearly separated from the Baculovirida e.
As a further step to analyze WSSV in more detail, its major virion proteins were analyzed. In general, structural proteins are well conserved within virus families and therefore represent good phylogenetic markers. Furthermore, knowledge on these proteins117 can lead to better insight in the viral infection mechanism. Five major proteins of 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), 19 kDa (VP19), and 15 kDa (VP15) in size were identified (Chapter 4, 5 and 6). VP26, VP24 and VP15 were found associated with the nucleocapsid, while VP28 and VP19 were found associated with the viral envelope. Partial amino acid sequencing was performed on these proteins to identify their respective genes in the WSSV genome.
The first structural genes to be identified on the WSSV genome were those coding for VP28 and VP26, which are most abundant in the virion (Chapter 4). The correct identification of these genes was confirmed by heterologous expression in the baculovirus insect cell expression system and detection by Western analysis using a polyclonal antiserum against total WSSV virions. Subsequently, VP24 was characterized (Chapter 5) and computer-assisted analysis revealed a striking amino acid and nucleotide similarity between VP24, VP26 and VP28 and their genes, respectively. This strongly suggests that these genes have evolved by gene duplication and subsequently diverged into proteins with different functions within the virion, i.e. envelope and nucleocapsid. All three proteins contained a putative transmembrane domain at their N-terminus and multiple putative N- and O-glycosylation sites. The putative transmembrane sequence in VP28 may anchor this protein in the viral envelope. The hydrophobic sequences may also be involved in the interaction of the structural proteins to form homo- or heteromultimers. In Chapter 6 the identification of the structural proteins VP19 and VP15 is described.
The VP19 polypeptide contained two putative transmembrane domains, which may anchor this protein in the WSSV envelope. Also this protein contained multiple putative glycosylation sites. N-terminal sequencing on VP15 showed that this protein was expressed from the second translational start codon within its gene and that the first methionine was cleaved off. As VP15 is a very basic protein and resembles histone proteins, it is tempting to assume that this protein functions as a DNA binding protein within the viral nucleocapsid.
None of the identified structural proteins showed homology to viral proteins in other viruses, which further supports the proposition that WSSV has a unique taxonomical position.
As the theoretical sizes determined of the various structural proteins, as derived from their genes, were smaller than the apparent sizes on SDS-PAGE, it was suspected that some of these proteins were glycosylated (Chapter 6). All five identified proteins were expressed in insect cells using baculovirus vectors, resulting in expression products of similar sizes as in the WSSV virion. The glycosylation status of the proteins was analyzed and this indicated that none of the five major structural proteins was glycosylated. This is a very unusual feature of WSSV, as enveloped viruses of vertebrates and invertebrates contain glycoproteins in their viral envelopes, which often play important roles in the interaction between virus and host, such as attachment to receptors and fusion with cell membranes.
To study the mode of entry and systemic infection of WSSV in the black tiger shrimp, Penaeus monodon, the role of the major envelope protein VP28 in the systemic infection in shrimp was studied (Chapter 7). An in vivo neutralization assay was performed in P. monodo n, using a specific polyclonal antibody generated against VP28. The VP28 antiserum was able to neutralize WSSV infection of P. monodon in a concentration-dependent manner upon intramuscular injection. This result suggests that VP28 is located on the surface of the virus particle and is likely to play a key role in the initial steps of the systemic infection of shrimp.
To analyze the genome structure and composition, the entire sequence of the double-stranded, circular DNA genome of WSSV was determined (Chapter 8). On the 292,967 nucleotide genome 184 open reading frames (ORFs) of 50 amino acids or larger were identified. Only 6% of the WSSV ORFs had putative homologues in databases, mainly representing genes encoding enzymes for nucleotide metabolism, DNA replication and protein modification. The remaining ORFs were mostly unassigned except for the five encoding the structural proteins. Unique features of the WSSV genome are the presence of an extremely long ORF of 18,234 nucleotides with unknown function, a collagen-like ORF, and nine regions, dispersed along the genome, each containing a variable number of 250-bp tandem repeats. When this WSSV genome sequence was compared to that of a second isolate from a different geographic location, the isolates were found to be remarkably similar (over 99% homology) (Chapter 9). The major difference was a 12 kbp deletion in the WSSV isolate, described here, which is apparently dispensable for virus infectivity.
To complete the taxonomic research on WSSV, its DNA polymerase gene was used in a phylogenetic study (Chapter 8), confirming the results of the phylogeny performed on PK.
To obtain a consensus tree, combined gene phylogeny analysis was performed using the rr 1, rr 2, pk and pol genes, which were also present in other large dsDNA virus families (Chapter 9). Based on this consensus tree no relationship was revealed for WSSV with any of the established families of large DNA viruses. The collective information on WSSV and the phylogenetic analysis suggest that WSSV differs profoundly from all presently known viruses and is a representative of a new virus family, with the proposed name 'Nimaviridae' (nima = thread).
The present knowledge on the WSSV genome and its major structural proteins, has created a good starting point for further studies on the replication strategy and infection mechanism of the virus, and last but not least, will open the way for the design of novel strategies to control this devastating pathogen.
Medicago truncatula, an intergenomic vehicle for the map-based cloning of pea (Pisum sativum) genes : comparative structural genomic studies of the pea Sym2-Nod3 region
Gualtieri González-Latorre, G.S. - \ 2001
Wageningen University. Promotor(en): A.H.J. Bisseling. - S.l. : S.n. - ISBN 9789058084392 - 146
pisum sativum - genen - genomen - genetische kartering - medicago truncatula - genetische modificatie - pisum sativum - genes - genomes - genetic mapping - medicago truncatula - genetic engineering
To determine the usefulness of M. truncatula as intergenomic vehicle for the positional cloning of pea genes it was studied whether these legumes are microsyntenic. These studies were focused on the pea Sym2 and Nod3 genomic regions. The M. truncatula orthologous genomic regions have been cloned and it was shown that these regions of the two legumes are microsyntenic. Both Sym2 and Nod3 play a key role in the pea- Rhizobium symbiosis, controlling Nod factor-structure dependent infection and autoregulation of nodule number, respectively.
A M. truncatula A17 BAC library was screened with a pea marker tightly linked to Sym2 and 11 clones were isolated. These clones formed three different contigs named C1, C2, and C3, which were extended to about twice their original size by chromosome walking resulting in contigs of 300 Kbp, 170 Kbp and 150 Kbp, respectively. Genetic and FISH mapping in M. truncatula revealed that the three contigs map on chromosome 5, and that C1 and C2 are tightly linked while C3 maps at a distance of 9 cM from C1/C2 on the same arm of this chromosome. By a combination of contig physical data and FISH it was estimated that C1 and C2 were separated by a gap of 30-40 Kbp. C1 and C2 were further linked to each other by screening an expanded version of the M. truncatula BAC library with the C1 and C2 contig-end subclones Mtg2511 and Mtg3556, respectively. Consequently, the small gap was closed by a 12 Kbp sequence linking C1/C2 which final size resulted in about 480 Kbp. Eight RFLP markers (including cDNAs and contig subclones) were isolated from C1/C2. Mapping of these markers using pea RILs and introgression lines demonstrated that C1/C2 represents the MedicagoSym2 -orthologous genomic region. Moreover, three markers showed recombinations between their pea homologous sequences and Sym2 , delimitating both the pea Sym2 region in the RILs and introgression lines, and the MedicagoSym2 -orthologous region . The MedicagoSym2 -orthologous region was delimitated to about 350 Kbp of C1/C2. In addition, by using a C2 subclone that encodes for a sequence highly homologous to the LRR-motif of the Cf4 and Cf9 tomato [ Licopersicon esculentum ] disease resistance proteins, a pea cDNA was isolated from a pea root hair library that also contains a LRR-domain highly homologous to that of Cf4 / Cf9 . The isolation of this pea RFLP marker demonstrates the use of M. truncatula as intergenomic positional-cloning vehicle of pea genes located within microsyntenic genomic regions. Furthermore, it was shown that the pea Sym2 -region is rich in Cf4 / Cf9 LRR-like sequences. The cloned C1/C2 Sym2 -orthologues candidates include receptor kinases and LRR-containing ( Cf4 / Cf9 -like, TMV -like) genes. Detailed analysis of 22 sequences from C1/C2 (including all RFLP markers) and the pea Sym2 -containing region showed that 4 of these sequences have homologues in c3, and that they are organized in clusters with a similar linear order in these contigs. This indicates that C1/C2 and C3 probably arose through duplication of a chromosomal segment.
By using the RNA differential display in combination with RILs and introgression lines, the tightly linked RFLP markers dd21.5 and Psc2.6 were isolated that are linked to the hypernodulating Nod3 locus and represent the closest markers mapping to the "south" of this locus on pea linkage group I in between Eil2 and Nod3 . These markers were used to identify the M. tuncatula A17 orthologous region with the aim to start the microsynteny-based positional cloning of Nod3 . The M. truncatula A17 BAC library was screened with these two markers. BAC clone 21F22 was isolated that contains the orthologue of dd21.5 . In addition 5 BAC clones were identified that co-hybridize with both dd21.5 and Psc2.6 , demonstrating the existence of M. truncatula genomic regions microsyntenic with the pea genomic region containing these two markers. BAC 21F22 was mapped by FISH on M. truncatula chromosome 4, revealing a local disruption of synteny at the dd21.5 locus between pea linkage group 1 and M. truncatula chromosome 5 that were syntenic for other markers (i.e. the markers isolated from the Sym2 -orthologous region, and the pea Eil2 marker). This finding reveals a chromosomal translocation that took place either in pea or in M. truncatula . However, it is unknown whether this translocation extends beyond the dd21.5 locus and includes the Nod3 locus. In addition to the strong FISH signal given by 21F22 on chromosome 4, weak signals were observed close to the telomeres of chromosome 5 and the position of one of these signals is comparable to the position of the C1/C2 Sym2 -orthologous region. Thus, it is possible that in spite of the translocation, chromosome 5 contains sequences with a low homology with those of the pea linkage group I segment containing dd21.5 . It remains to be determined whether the five microsyntenic BACs map in these chromosome 5 regions or whether they map in chromosome 4 and form a contig with BAC 21F22.
The data presented in this thesis set the basis for the microsynteny-based cloning of the Sym2 and Nod3 genes by using M.truncatula as intergenomic positional-cloning vehicle. In addition, the molecular resources generated in this thesis are useful to extend microsynteny studies at the Sym2 and Nod3 regions to other legume (e.g. Lotus japonicus ) and actinorhizal nodule-forming species, and also to non-nodulating related species from the Rosid I clade and non-nodulating less-related species (e.g. Arabidopsis and Brassica species that belong to the Rosid II clade). This will enable the identification of genes within these genomic regions that might be unique to nodule-forming plants engaged in symbiotic nitrogen fixation, representing a predisposition for nodulation at the ancestral root of the Rosid I clade. In other words, these studies will enable to answer one of the most interesting questions of plant biology and comparative genomics: whether the ability of legume and actinorhizal plants to establish a nodular symbioses, is given by unique properties that left their evolutionary "signatures" at the genome level.
|Stress-respons van voedselpathogenen bij milde conservering
Wouters, J.A. ; Bennik, M.H.J. ; Abee, T. - \ 2000
Voedingsmiddelentechnologie 33 (2000)26. - ISSN 0042-7934 - p. 11 - 14.
voedselbewaring thuis - voedselbewaring - houdbaarheid (kwaliteit) - kwaliteit - pathogenen - voedselmicrobiologie - voedingsmiddelen - genoomanalyse - genomen - listeria monocytogenes - bacillus cereus - voedselbederf - home food preservation - food preservation - keeping quality - quality - pathogens - food microbiology - foods - genome analysis - genomes - listeria monocytogenes - bacillus cereus - food spoilage
Milde proces- en conserveringsmethoden en het karakteriseren van bederfveroorzakende micro-organismen op bepaalde behandelingen. De genoomsequenties van genoemde pathogenen werden onderzocht in relatie tot hoge osmolariteit, lage temperatuur en ultra hoge druk (UHD)
A genetic and molecular analysis of two genes involved in flowering initiation of Arabidopsis = [Een genetische en moleculaire analyse van twee genen die betrokken zijn bij de bloei initiatie van Arabidopsis]
Soppe, W.J.J. - \ 2000
Agricultural University. Promotor(en): M. Koornneef. - S.l. : S.n. - ISBN 9789058082893 - 120
arabidopsis - arabidopsis thaliana - genetische analyse - genen - genexpressie - biotechnologie - bloei - fotoperiodiciteit - circadiaan ritme - moleculaire genetica - mutanten - genomen - arabidopsis - arabidopsis thaliana - genetic analysis - genes - gene expression - biotechnology - flowering - photoperiodism - circadian rhythm - molecular genetics - mutants - genomes
The transition from the vegetative to the reproductive phase (flowering initiation) in plants has a complex regulation which is affected by environmental and internal plant factors. The understanding of this process is not only of fundamental interest but could also lead to practical applications. The research into flowering initiation has a long history. The initial emphasis on physiological and biochemical studies led to the identification of different factors that influence flowering time. During the sixties, a genetic approach was initiated in different plant species. In Arabidopsis several late flowering mutants were isolated and genetically and physiologically characterised which revealed a complex regulation of flowering time by different pathways. These are the photoperiodic promotion pathway which promotes flowering under long day light conditions, the vernalisation promotion pathway which promotes flowering by low temperatures and the autonomous promotion pathway which promotes flowering independent of the environment. Due to its favourable genetic and molecular features, research on flowering initiation became focussed on Arabidopsis. Since the beginning of the nineties, several of the genes involved in the different pathways have been cloned, providing more information about the function of these genes in the cell and their relations with each other. Despite this increasing amount of information, the picture is still far from complete.
The aim of the work presented in this thesis is to increase our knowledge of flowering time regulation. It focussed on the genetic and molecular characterisation of the semi-dominant mutant fwa , which flowers late under long day light conditions and has been proposed to be part of the photoperiodic promotion pathway. One approach sought to identify additional genes that affect flowering by mutagenesis of the fwa mutant. In addition to three different intragenic revertants of fwa , this screen yielded a novel early flowering mutant.
This mutant was named early flowering in short days ( efs ). Its phenotypic characterisation has shown that the main role of the wild-type EFS gene is to delay flowering in plants that have entered the adult vegetative phase, which is considered to be the phase where plants are able to respond to environmental signals in order to flower. Consistent with this, efs mutant plants do not show an early flowering phenotype when grown under environmental conditions that lead to a shortened adult vegetative phase such as long days and vernalisation. To learn more about the role of EFS in relation to other genes involved in flowering initiation, double mutants were isolated. Their characterisation showed that efs is involved in the autonomous promotion pathway. This result, together with the lack of a vernalisation response, suggests that EFS is likely to represent a new element acting at a point close to the convergence of signals from the autonomous promotion pathway and the vernalisation promotion pathway.
The main topic of this thesis concerns the map based cloning of the FWA gene. By using plants which have a cross-over between FWA and surrounding markers, the FWA locus could be located in a region of about 60 Kb. Plant transformation experiments with cosmids spanning this region showed that the gene is located in the overlap of two cosmids. This overlap contained only one complete gene that encodes a homeodomain containing transcription factor. The altered expression of this gene in fwa mutants together with DNA mutations in the intragenic revertants of fwa-1 further proved that this gene is FWA .
Analysis of FWA revealed several interesting characteristics. Surprisingly, the mutant and wild-type alleles had an identical DNA sequence in the FWA region, excluding DNA mutations in the gene as a cause for the mutant phenotype. Furthermore, two direct repeated sequences were found in the 5' genomic region of FWA . In wild-type plants these repeats were heavily methylated, whereas they were completely un-methylated in the mutant alleles. In contrast to fwa mutant plants, which showed a high expression of FWA at all developmental stages, wild-type plants showed only a low expression of FWA in siliques and germinating seeds. Taken together, these findings suggest that loss of methylation of the FWA repeats in the fwa mutant causes a high level of expression of the gene, leading to a late flowering phenotype. A similar correlation of late flowering, FWA overexpression and hypomethylation of FWA repeats was found in late flowering plants which were derived from the ddm1 hypomethylation mutant. The late flowering phenotype of these plants had previously been mapped to the FWA region. Nevertheless, the correlation between hypomethylation of the FWA repeats and FWA expression was not found in germinating seeds of wild-type plants which showed expression of FWA but methylation of the repeats. Although this expression might come from residual mRNA produced earlier in developing seeds, it is possible that methylation of the repeats does not always prevent expression of FWA . Perhaps a different epigenetic mechanism early in development can induce expression of methylated genes.
The correlation of FWA expression with late flowering indicates that FWA is a repressor of flowering. Earlier studies had already shown that FWA does not only play a role in the initiation of flowering but also in flower meristems. However, the FWA transcript was not detected in flower buds or flowers and therefore, FWA might only affect this process when highly expressed in the fwa mutant. Possibly, FWA has no function in flowering initiation of wild-type. It might participate in a seed-specific process, as suggested by its expression in seeds. However, the lack of an obvious phenotype suggests that this role is minor or redundant with other genes.
The cloning of FWA revealed that the absence of methylation in the repeating sequences in the 5' region of the FWA gene leads to an enhanced expression in the fwa mutant. However, it did not become clear whether this correlation is direct or indirect. Also the importance of the methylation in wild-type plants is still unclear. It is possible that it has a role in the expression of the gene under specific environmental conditions.
The results discussed in this thesis have contributed to the existing knowledge of flowering initiation by the isolation of a mutant at a novel locus and the cloning of a previously known gene which are both involved in this process. In addition, the results suggest a possible role for DNA methylation in gene regulation of Arabidopsis.
The AVR9 elicitor peptide of the tomato pathogen Cladosporium fulvum : molecular aspects of recognition
Kooman - Gersmann, M. - \ 1998
Agricultural University. Promotor(en): P.J.G.M. de Wit; G.J.E.M. Honee. - S.l. : Kooman-Gersmann - ISBN 9789054857938 - 117
tomaten - passalora fulva - deuteromycotina - solanum lycopersicum - fysiologie - gastheer parasiet relaties - moleculaire biologie - plantenziektekunde - ziekteresistentie - genen - genomen - dematiaceae - tomatoes - passalora fulva - deuteromycotina - solanum lycopersicum - physiology - host parasite relationships - molecular biology - plant pathology - disease resistance - genes - genomes - dematiaceae
The interaction between the fungal pathogen Cladosporium fulvum and tomato has been used as a model system to study the molecular basis of gene-for-gene relationships. C. fulvum is a specialized, biotrophic pathogen, which causes leaf mold on tomato. Under humid conditions conidia of C. fulvum germinate and form runner hyphae on the lower side of the leaf. If no resistance genes of the plant match any of the avirulence genes of the fungus, the interaction is compatible and infection will proceed. However, when both a resistance gene and its matching avirulence gene are present, the plant recognizes the fungus and the interaction is incompatible. In an incompatible interaction active defense responses, including the hypersensitive response (HR) are initiated, which inhibit fungal growth effectively. Avirulence genes encode lace-specific elicitors, which are present in intercellular washing fluids obtained from compatible interactions of C. fulvum and tomato (De Wit and Spikman, 1982). Injection of these intercellular washing fluids in tomato plants resistant to the C. fulvum strain from which the washing fluids were obtained, results in specific necrosis at the site of injection. The race-specific elicitor AVR9 was isolated and purified (Scholtens-Toma and de Wit, 1988). AVR9 specifically induces necrosis on tomato genotypes carrying the Cf-9 resistance gene. The encoding AVR9 gene was isolated, and it was shown that this gene specifically determines avirulence of C. fulvum on tomato plants carrying the Cf-9 resistance gene (Van den Ackerveken et al., 1992; Marmeisse et al., 1993). The Avr9 gene encodes a 63-amino acid pre-proprotein containing one potential glycosylation site (Van den Ackerveken et al., 1993). Different forms of the AVR9 elicitor were found, of which the mature AVR9 elicitor of 28 amino acids is predominantly present in C. fulvum-infected tomato plants (Van den Ackerveken et al., 1993). The global structure of the AVR9 peptide shows 3 antiparallel β-sheets and 3 disulfide bonds that are arranged in a cystine knot (Vervoort et al., 1997).
In the research project described in this thesis, we studied AVR9 elicitor perception in tomato plants that carry the Cf-9 resistance gene and compared the results to those obtained with tomato plants lacking this gene. Previously, several research groups had shown that elicitors are recognized through plants receptors, which are localized on the plasma membrane (summarized in chapter 1). To find and characterize the receptor for AVR9, the peptide was labeled with iodine-125 and binding to tomato membranes was studied, as presented in chapter 2. 125 I-AVR9 showed specific, saturable, and reversible
As described in chapters 3, 4, and 5, experiments were performed to prove or reject one of these two hypotheses. To determine whether the high-affinity binding site for AVR9 is indeed a functional receptor, we studied the correlation between binding affinity and necrosis-inducing activity of mutant AVR9 peptides. We determined structure-activity relationships of the AVR9 peptide by independently substituting each amino acid of AVR9 by alanine, using a site-directed mutagenesis approach. In addition, surfaceexposed amino acid residues of AVR9 were substituted by other amino acids. Activity of mutant Avr9 constructs was studied by expressing the constructs in MM-Cf9 tomato plants using the potato virus X (PVX) expression system, and assessing the severity of necrosis induced by each PVX::Avr9 construct. This allowed direct identification of amino acid residues of AVR9 that are essential for elicitor activity. We identified amino acid substitutions resulting in AVR9 mutants with higher, similar or lower elicitor activity compared to the wild-type AVR9 peptide. Mutants of the amino acid residues Phe21 and Leu24 had completely lost elicitor activity. Necrosis-inducing activity of isolated AVR9 peptides correlated well with the necrosis induced by the corresponding PVX::Avr9 constructs. It was concluded the PVX expression system is ideally suited to analyze necrosis-inducing activity of AVR9 peptides. We analyzed whether there is a correlation between elicitor activity of the mutant AVR9 peptides and their affinity to the binding site in membranes of tomato. Therefore, Nicotiana clevelandii plants were inoculated with a selection of PVX::Avr9 constructs and mutant AVR9 peptides were purified from these plants. In addition, some AVR9 peptides were chemically synthesized. Characterization by Electrospray Mass Spectrometry, Circular Dichroism-, and 'H-NMR- spectroscopy revealed that both the in planta produced and the synthetic mutant peptides were correctly folded. AVR9 peptides purified from PVX::Avr9-infected N. clevelandii contained one N-acetylglucosamine, although small amounts of non- glycosylated AVR9 peptides were also detected. The glycosylated AVR9 peptides showed lower affinity to the binding site than the non-glycosylated AVR9 peptides, whereas they did not differ significantly in necrosis-inducing activity. For both the non- glycosylated and glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membranelocalized binding site and their necrosis-inducing activity in MM-Cf9 tomato was found, i.e. peptides with higher affinity to the binding site showed higher necrosis-inducing activity. This correlation suggested that the characterized high-affinity binding site for AVR9 is indeed a functional receptor that initiates the AVR9- CF-9-dependent HR in MM-Cf9 plants.
In chapter 5, we studied whether the Cf-9 resistance gene or (one of) its homologues code for an AVR9 binding site. We tested binding of AVR9 to microsomal membranes of a variety of solanaceous and non-solanaceous plant species and analyzed these species for the presence of Cf-9-homologues. All solanaceous species tested contain homologues of the Cf-9 resistance gene and membranes of these plants contain a highaffinity binding site for AVR9. However, a high affinity binding site for AVR9 is also present on membranes of the non-solanaceous plant species cucumber, barley and oat, which do not contain homologues of the Cf-9 resistance gene. Membranes of tobacco, transgenic for the Cf-9 resistance gene, showed no change in the number of AVR9 binding sites. Arabidopsis does not have a binding site for AVR9 and membranes of Arabidopsis, transgenic: for the Cf-9 resistance gene, also showed no AVR9 binding. From this we concluded that not only the Cf-9 resistance gene, but also its homologues are not required for high-affinity binding of AVR9. Based on the presented data, we have developed a model, explaining recognition of AVR9 in MM-Cf-9 tomato (chapter 6). This model predicts that the high-affinity binding protein either 'presents' the AVR9 elicitor to the Cf-9-encoded protein or that binding of AVR9 induces a conformational change of the high-affinity binding protein. The latter results in recruitment of Cf-9 into the AVR9- receptor complex. Subsequently, signal cascade(s) resulting in HR will be initiated.
Analysis of the chicken genome : mapping of monogenic traits
Ruyter-Spira, C.P. - \ 1998
Agricultural University. Promotor(en): E.W. Brascamp; M.A.M. Groenen; M. Georges. - S.l. : S.n. - ISBN 9789054859598 - 134
kippen - pluimvee - genomen - genkartering - genetische merkers - microsatellieten - koppeling - verenkleed - kleur - dwerggroei - nucleotidenvolgordes - genexpressie - insulineachtige groeifactor - biotechnologie - fowls - poultry - genomes - gene mapping - genetic markers - microsatellites - linkage - plumage - colour - dwarfism - nucleotide sequences - gene expression - insulin-like growth factor - biotechnology
The development of genetic linkage maps in farm animals is progressing rapidly. Linkage maps can be used to identify genetic loci responsible for genetic variation in traits of economic importance. The ultimate goal is to find the underlying genes involved in these traits. To achieve this, the so called positional candidate gene approach is gaining in importance. This approach is based on the genetic localization of a trait using genetic linkage analysis in livestock species. Subsequent comparative mapping of the trait locus with the gene-rich maps of the human and the mouse may reveal candidate genes for the trait in question.
For the construction of comparative maps the genetic localization of many genes needs to be determined. In this thesis, the development of highly informative microsatellite markers from expressed sequences, derived from a brain and embryonic cDNA library of the chicken, is described. In addition to this, a preliminary comparative map of the chicken is presented.
The second objective of this thesis was to develop a quick and reliable method in order to localize monogenic traits. To achieve this, an already existing technique, called bulked segregant analysis, which has originally been used for the localization of monogenic traits in plants using random amplified polymorphic DNA markers (RAPDs), and restriction fragment length polymorhisms (RFLPs), was combined with the use of fluorescently labelled microsatellite markers. This method prooved to be very sensitive in detecting linked markers at greater genetic distances and the genetic map locations of the monogenic traits Dominant White ( I ) and Autosomal dwarfism ( adw ) were succesfully determined.
Comparative mapping revealed that adw is located in a chromosomal region that is conserved between chicken, human and mouse. Interestingly, in the mouse the phenotype "Pygmy" , which shows a striking similarity to the Autosomal Dwarf phenotype in chickens, is also located in this region. The Pygmy phenotype arises from the inactivation of the High Mobility Group I-C ( HMGI-C ) gene. In the human, the HMGI-C gene is also located in the same conserved chromosomal segment. Fluorescent in situ hybridization of chicken metaphase chromosomes using the chicken HMGI-C gene as a probe, showed that the chicken HMGI-C gene is indeed located in the region of the adw locus. However, northern blot analysis showed no difference in the expression of the HMGI-C gene between adw and wild type chicken embryos. Also no mutations in the HMGI-C mRNA were detected. Finally, other candidate genes for both adw and I are proposed.
Isolation and characterisation of starch biosynthesis genes from cassava (Manihot esculenta Crantz)
Munyikwa, T.R.I. - \ 1997
Agricultural University. Promotor(en): E. Jacobsen; R.G.F. Visser. - S.l. : Munyikwa - ISBN 9789054858416 - 128
koolhydraten - polysacchariden - biosynthese - genen - genomen - manihot esculenta - cassave - carbohydrates - polysaccharides - biosynthesis - genes - genomes - manihot esculenta - cassava
Cassava (Manihot esculenta Crantz) is a tropical crop grown for its starchy thickened roots, mainly by peasant farmers, in the tropics, for whom it is a staple food. There is an increasing demand for the use of cassava in processed food and feed products, and in the paper and textile industries amongst others. This thesis describes research on the cloning of the genes encoding ADP-glucose pyrophosphorylase small and large subunits (AGPase B and S, respectively) and granule bound starch synthase II (GBSSII). These genes and their products were extensively characterised to determine their role in starch biosynthesis in cassava. Functional verification of the genes was carried out by transforming potato and cassava followed by analysis of the starch produced by the transgenic plants.
In Chapter 1 cassava production in the world in general and in Zimbabwe in particular is examined against the backdrop of new cloning and transformation strategies to improve starch quality and quantity. The development of cassava cultivars whose starches have novel physico-chemical properties by genetic modification of the process of starch biosynthesis is examined therein. The main criteria for these new cultivars to emerge are set forth as being: the availability of cloned and characterised starch biosynthesis genes, a universally applicable transformation and regeneration procedure for cassava, transfer to appropriate cassava cultivars, and biosafety analysis of transgenic cassava plants before disbursement to farmers.
The cloning of the cassava starch biosynthesis genes encoding granule bound starch synthase II (GBSSII) and the large and small subunits of ADP-glucose pyrophosphorylase (AGPase) is described in Chapters 2 and 3. The cloning of GBSSII reveals that there is indeed a second isoform of this enzyme in cassava as in other plants species. While sharing very little amino acid sequence homology with cassava GBSSI the GBSSII isophorm shares high amino acid sequence homology to other GBSSII genes from pea and potato. Cassava GBSSII seems to be more important in leaf tissue where it is more highly expressed than in tuber tissue where GBSSI predominates. Mapping of GBSSII revealed that this is a single copy gene located on the male derived linkage group T of the cassava mapping population.
Cloning of the cassava genes coding for the small (B) and large subunit (S) of AGPase revealed interesting aspects about the cassava enzyme. The cassava AGPase is likely to be heterotetrameric in constitution as had been found in other plant species. Comparison of the cassava AGPase sequences with those of already cloned AGPases revealed that AGPase B is more similar to small subunit genes from other plants than to cassava AGPase S coding for the large subunit (Chapter 3). Segregation analysis of a cassava mapping population revealed that AGPase S is a single copy gene that is localised on the female derived linkage group E of the cassava genetic map. Both genes are expressed in all cassava tissues but AGPase B was shown to have a higher steady state mRNA level than AGPase S especially in leaf and tuber tissue. Post-transcriptional control of small subunit polypeptide levels could be inferred from the discrepancy between AGPase B mRNA and polypeptide levels. The AGPase enzyme activity was much higher in young cassava leaves than older leaves and tubers. Cassava leaf AGPase activity was increased 3 fold by the addition of 3-PGA (3-phospho-glycerate) and inhibited by up to 90% in the presence of inorganic phosphate (Pi). The tuber enzyme was relatively unaffected by 3PGA, but was highly inhibited by Pi.
In order to verify the biological role of the AGPase B gene antisense constructs were made of the cassava AGPase B behind a CaMV35S promoter (chapter 3). This was transferred into potato plants by Agrobacterium tumefaciens. While the 224 transgenic antisense AGPase B potato plants did not differ in appearance from normal potato plants, 45 transgenic plants, however, had more numerous and smaller tubers than control plants. Antisense plants with reduced AGPase B mRNA levels had 1.5 to 3 times less starch than tubers from the control plants. The levels of the soluble sugars in the antisense plants increased significantly (up to 10 times more glucose, 6 times the amount of fructose, and 5 times the amount of sucrose) when compared to those found in control plants. These results show that a heterologous gene from cassava can have an antisense effect in potato, but that the number of plants required to find plants exhibiting maximum antisense effect has to be very large. This is probably due to sequence homology differences between the cassava AGPase B and potato AGPase B genes which share only 68% amino acid sequence homology.
Chapter 5 describes the further development of an efficient, time and labour saving protocol for transforming cassava based on stringent selection of the luciferase (firefly) marker gene. In addition the first reported transformation of cassava with a gene (AGPase B) other than a marker gene is described. An antisense construct was made for transforming cassava. This consisted of the cassava AGPase B gene which was placed in antisense orientation behind the CaMV35S promoter. This was then coupled to the luciferase gene driven by another CaMV35S promoter. After particle bombardment of cassava FEC transgenic tissue was selected using three different selection regimes: non stringent luciferase selection, stringent luciferase selection and combined chemical (phosphinothrycin) and luciferase selection. Stringent luciferase selection whereby luciferase positive FEC units were precisely pinpointed, isolated and cultured was found to be the most effective and time saving method. It was possible to generate cultures having more than 90% luciferase positive FEC tissue after 12 weeks of stringent LUC selection, compared to 45% and <1 % for combined selection and non stringent selection respectively. The number of luciferase positive mature embryos generated was directly proportional to the percentage of luciferase positive tissue in the original FEC culture. Stringent luciferase selection enabled the time taken for production of transgenic cassava plants to be reduced to 28-36 weeks as compared to 8 months to a year with no stringent selection or LUC/PPT selection.
Cassava plants carrying the AGPase B antisense gene had extremely low levels of starch, compared to control plants, as shown by iodine staining of in vitro induced thick stems. In plants exhibiting the highest AGPase B antisense effect, starch formation was limited only to the epidermal layer. These results functionally confirm the identity of cassava AGPase B as well as emphasising the critical role of AGPase in starch formation in cassava.
A discussion about the significance and implications of cloning cassava genes and producing transgenic cassava for culture in developing countries is carried out in Chapter 6. While there are clearly many economic and nutritional benefits to producing transgenic cassava, for resource poor farmers, many people in the South are not aware of the biosafety implications of growing transgenic crops. It is further emphasised that discussions and debate should be initiated to make local communities aware of the issues surrounding transgenic crops and their products. In addition it is recommended that some form of international legal framework be set up to ensure that resource poor farmers are not disadvantaged by the patenting of material originating from their communities by individuals and companies in the North. This thesis clearly demonstrates how it will be possible in the near future to produce new cassava cultivars carrying the appropriate genes to affect pronounced changes on tuber productivity and starch quality.
Marker assisted elucidation of the origin of 2n-gametes in diploid potato
Bastiaanssen, H.J.M. - \ 1997
Agricultural University. Promotor(en): E. Jacobsen; M.S. Ramanna. - S.l. : Bastiaanssen - ISBN 9789054857594 - 150
haploïdie - polyploïdie - mutaties - plantenveredeling - genomen - solanum tuberosum - aardappelen - haploidy - polyploidy - mutations - plant breeding - genomes - solanum tuberosum - potatoes
This thesis describes the selection and evaluation of diploid potato clones (2n=2x=24) that produce unreduced or 2n-gametes with 24 chromosomes instead of the normal reduced n-gametes with 12 chromosomes. To elucidate the modes of origin of the 2n-gametes, the progenies derived from such gametes were analysed for different marker loci of one chromosome. Besides the already known mechanisms of First and Second Division Restitution (FDR and SDR) in potato, the multilocus analysis showed the additional mechanism of Post-Meiotic Restitution (PMR) resulting in completely homozygous 2n-gametes. The FDR and SDR 2n-gametes were used for gene-centromere mapping. Multilocus analysis of SDR 2n-gametes showed the map position of the centromere of chromosome 8 in relation to the RFLP-loci. These genetic analyses also demonstrated the occurrence of a single crossover per chromosome arm (high level of chiasma interference). In the light of these findings, it is possible to construct more critical genetic maps of potato.
Genetic analysis of seed development in Arabidopsis thaliana = [Genetische analyse van de zaadontwikkeling in Arabidopsis thaliana]
Leon - Kloosterziel, K. - \ 1997
Agricultural University. Promotor(en): M. Koornneef. - S.l. : Leon-Kloosterziel - ISBN 9789054857709 - 119
genen - genomen - mutaties - mutagenese - mutagenen - plantenfysiologie - plantenontwikkeling - vruchten - rijp worden - brassicaceae - zaadzetting - zaden - formatie - kieming - zaadkieming - kiemrust - genes - genomes - mutations - mutagenesis - mutagens - plant physiology - plant development - fruits - ripening - brassicaceae - seed set - seeds - formation - germination - seed germination - seed dormancy
This thesis deals with the genetic aspects of seed development in Arabidopsisthaliana. Mutants affected in several aspects of seed development and, more specifically, in seed maturation have been isolated by various selection procedures. The mutants have been analyzed genetically, physiologically, and morphologically. Some of the mutants are impaired in the biosynthesis or sensitivity to the plant hormone, abscisic acid (ABA). All ABA-related mutants show reduced seed dormancy, indicating the important role of this hormone in the establishment of dormancy. In a direct screen for reduced dormancy, two mutants (rdo) with reduced dormancy were found. These were not ABA-deficient and showed the same sensitivity to ABA, ethylene, auxin, and cytokinin as the wild-type. In contrast to this embryo-determined reduced dormancy, reduced dormancy can also originate in an altered seed coat (testa), like in the altered testa shape ( ats ) mutant. Here, the altered testa shape is caused by a defect in the development of the integuments. Extreme ABA-insensitive mutants ( abi3 ) have green seeds that fail to complete many other aspects of seed maturation, including the induction of dormancy and desiccation tolerance, and the accumulation of seed storage proteins and lipids. In addition to abi3 mutants, lec and fus mutants exhibit such a severely disturbed seed maturation as well, with dark purple seeds due to anthocyanin accumulation. The fus3 mutant shows normal ABA-sensitivity. These various seed maturation mutants indicate that specific genes, some acting dependently and some acting independently from ABA, are responsible for seed maturation programs. The seed maturation mutants were subjected to a physiological and biochemical analysis. A GA-deficient mutant was combined with these mutants. Analysis of these double mutants indicated that seeds of the abi3 and lec mutants did not require GA for germination, in contrast to fus3 seeds. This correlates with ABA-sensitivity for germination. The composition of storage proteins and carbohydrates in abi3 , lec, and fus3 mutant seeds has been compared. The abi3 , lec, and fus3 mutants all showed severely reduced storage proteins. The desiccation intolerance of these seed maturation mutants was not correlated with the lack of specific carbohydrates. Furthermore, the mutants had a higher total content of carbohydrates. This is probably a consequence of the lower levels of storage lipids and proteins.
The transmission of cytoplasmic genes in Aspergillus nidulans
Coenen, A. - \ 1997
Agricultural University. Promotor(en): R.F. Hoekstra. - S.l. : Coenen - ISBN 9789054856542 - 106
aspergillus - cytoplasmatische overerving - plasmiden - genen - genomen - eiwitsynthese - cytoplasma - aspergillus - cytoplasmic inheritance - plasmids - genes - genomes - protein synthesis - cytoplasm
This manuscript concerns the spread of selfish cytoplasmic genes in the fungus Aspergillus nidulans. A.nidulans is a common soil fungus that grows vegetatively by forming a network (mycelium) of hyphae and reproduces via sexual ascospores and asexual conidiospores. Cytoplasmic genes are genes that are located in the cell cytoplasm and not in the cell nucleus where most genes are situated. The cytoplasmic genes investigated in this research are the genomes of mitochondria and viruses. Selfish genes are genes that are maintained in a population despite a negative effect on the fitness of their host.
A possibility for the spread of selfish genes is created when they can compensate their negative effect on host fitness with an enhanced transmission rate. Because cytoplasmic genes can be transmitted independently from nuclear genes they can enhance their transmission rate in ways that nuclear genes cannot. Therefore there is evolutionary selection on mechanisms that regulate the transmission of cytoplasmic genes, thus preventing the spread of selfish cytoplasmic genes. Heterokaryon incompatibility may be such a mechanism. I have investigated the genetics of heterokaryon incompatibility and the transmission of mitochondria and viruses in A. nidulans. The implications of the results for the spread of selfish cytoplasmic genes are discussed.
Genetics of heterokaryon incompatibility
Contact between two fungal mycelia can result in their fusion and the formation of a heterokaryon (a mycelium containing nuclei from both strains). Such mycelial fusion creates possibilities for the horizontal transmission of cytoplasmic genes (transmission between individuals of the same generation). In most fungal species heterokaryon formation is restricted by a heterokaryon incompatibility mechanism. In A.nidulans heterokaryon incompatibility is regulated by heterokaryon incompatibility genes (het genes). Strains with different alleles for one or more het genes cannot form a heterokaryon, they belong to different heterokaryon compatibility groups (hcg's).
A sample of 24 isolates collected in England and Wales in 1992 was found to contain 20 hcg's. Only 2 of these hcg's were present in the 20 previously described hcg's (Chapter 6). Due to this large amount of variation most pairwise strain combinations will be heterokaryon incompatible.
Genetic analysis revealed the existence of partial heterokaryons, heterokaryons that grow less vigorously than 'normal' heterokaryons (Chapter 2). At first it was thought that partial heterokaryon incompatibility was caused by partial-het genes. Later it was discovered that partial heterokaryon incompatibility between parent and progeny could be induced by sexual reproduction (Chapter 3).
I attempted to isolate strains in which heterokaryon incompatibility as a result of an allelic difference for het gene A or het gene B was suppressed (Chapter 3). 1 was successful for het gene B but not for het gene A. This may be because het gene A is an essential gene. All suppressor mutations were intragenic. Suppression of het gene B results in heterokaryon compatibility with both alleles of het gene B. Strains that had switched from compatibility with allele B' of het gene B to compatibility with allele B of het gene B were also isolated. This indicates that the two alleles are highly similar.
Biparental inheritance creates possibilities for the spread of selfish mitochondrial genes. If mitochondria are inherited biparentally the progeny contain the mitochondria from both parents. Selfish mitochondria can enhance their transmission rate by winning the ensuing competition between the maternal and the paternal mitochondria. In A. nidulans mitochondrial inheritance is strictly uniparental (Chapter 5). All of the +-* 10000 ascospores in a fruitbody are inherited from the maternal parent. An investigation of more than a hundred fruitbodies did not reveal the presence of a single paternal mitochondrion. Selfish mitochondria cannot spread through A.nidulans populations by biparental inheritance.
The horizontal transmission of mitochondria is greatly restricted. Horizontal transmission within a kg was observed in a very low frequency and horizontal transmission between hcg's was never observed (Chapter 6). Selfish mitochondria cannot spread through A. nidulans populations by horizontal transmission.
The combination of uniparental inheritance and horizontal transmission within a kg creates a possibility for the spread of selfish mitochondria. The recombination of het genes during sexual reproduction results in the presence of the maternal mitochondria in all the hcg's included in the progeny. Consequently a selfish mitochondrion can spread by transmission within a hcg. However due to the low transmission rate within a hcg and the presumed rarity of sexual reproduction under natural conditions the spread of selfish mitochondria will be severely restricted. It is doubtful whether such a small enhancement of the mitochondrial transmission rate will compensate for the negative effects of selfish mitochondria, on host fitness.
There are no reports of viruses being found in sexual aspergilli despite their ubiquity in asexual aspergilli. For this research we transferred viruses from the asexual A..niger to the sexual A..nidulans by protoplast fusion (Chapter 4). Virus infection was not observed to have any effect on the fitness of Infected fungi. This shows that the absence of viruses from A..nidulans isolates is not the result of resistance to virus infection.
The horizontal transmission of viruses within a hcg is highly efficient. The transmission between hcg's is restricted but not prevented by heterokaryon incompatibility. Heterokaryon incompatibility in itself will not prevent the spread of viruses through A. nidulans populations.
Viruses are excluded from ascospores (Chapter 4). This will not prevent the spread of viruses through A. nidulans populations because viruses are included in the conidiospores. However the exclusion of viruses from ascospores and the recombination of het genes during sexual reproduction does allow the formation of new virus-free hcg's. If virus-free hcg's are created faster than they are infected by horizontal transmission this will result in the exclusion of viruses from A..nidulans populations. This could explain the exclusion of viruses from populations of sexual aspergilli.
Plant genes involved in the establishment of an actinorhizal symbiosis
Ribeiro, A.I.F. - \ 1997
Agricultural University. Promotor(en): A. van Kammen; T. Bisseling. - S.l. : Ribeiro - ISBN 9789054856665 - 108
genen - genomen - frankia - planten - symbiose - genes - genomes - frankia - plants - symbiosis
Actinorhizal multilobe nodules are induced by actinomycetes of the genus Frankia on the roots of several dicotyledenous species belonging to eight different plant families. Each nodule lobe is a modified lateral root, without a root cap, with a central vascular cylinder, and with infected and uninfected cortical cells.
To isolate plant genes involved in the establishment of an actinorhizal symbioses, we have differentially screened an A. glutinosa nodule cDNA library with nodule- and root cDNA, respectively. Several cDNAs, representing genes expressed at elevated levels in nodules compared to roots, as determined by RNA gel blot analysis, were isolated and sequenced. The localization of the corresponding mRNAs in the nodule was examined by in situ hybridization. Whenever necessary, model systems such as yeast and Arabidopsis were used to analyse the functions of the encoded proteins.
Two of the isolated cDNA clones corresponded to sucrose synthase and enolase, enzymes involved in carbon metabolism. The corresponding genes were expressed in all plant tissues but at markedly elevated levels in nodules. In situ hybridization showed that in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the vascular bundle.
Another cDNA clone, pAgthil, was shown to encode a homolog of yeast Thi4, which is involved in thiazole biosynthesis. The corresponding gene, agthil, was found to be expressed at high levels in nodules and shoot tips of A. glutinosa, while being expressed at low levels in roots, flowers, and developing fruits. In nodules, agthil mRNA was localized in the infected cortical cells and in the pericycle of the nodule vascular system. A homolog of this gene, ara6/tz was identified in Arabidopsis thaliana. ara6/tz maps in a region of chromosome 5 of Arabidopsis containing the tz locus. This is consistent with the observations that ara6/tz transcription was impaired in two out of five Arabidopsis tz mutant lines. ara6/tz is expressed at high levels in chloroplast-containing parenchymatic cells of leaves, inflorescence shoots and flowers of Arabidopsis, and at lower levels in the vascular system. The function of AgThi1 was demonstrated by yeast complementation studies, in which AgThi1 was able to rescue a yeast thi4 mutant.
cDNAs encoding glutamine synthetase (GS) and acetylomithine transaminase (AOTA), both involved in nitrogen metabolism, were isolated. GS is the enzyme responsible for ammonium assimilation, while AOTA is involved in the biosynthesis of citrulline, the nitrogen transport form in Alnus. GS mRNA was found in all tissues tested with the highest levels in nodules, where it was present in the infected cells as well as in the cells of the pericycle of the vascular system. AOTA transcripts were detected at very low levels in roots and at high levels in nodules, where it was confined to the infected cells. These data suggested that in A. glutinosa nodules, ammonium assimilation takes place in both the infected cells and in the pericycle of the vascular system, and citrulline biosynthesis occurs mainly in the infected cells. Ammonium assimilation in the pericycle is likely to be related to nitrogen transport.
One of the few nodule-specific genes, i.e., genes that are not expressed in roots, ag12, was shown to be expressed in nodules at the highest levels in infected cells before the onset of nitrogen fixation. Sequencing showed that ag12 encodes a serine protease of the subtilisin (EC 184.108.40.206) family. A homolog of ag12, ara12, was identified in Arabidopsis. ara12 was expressed in all organs, with the highest expression levels in the beginning of silique development. To assess the importance of this protease in other actinorhizal symbioses, the expression pattern of its homolog, cg12, was examined in nodules of Casuarina glauca and found to be similar to that of ag12. These results are discussed in view of the phylogenetical relationship of Alnus and Casuarina.
|Marktintroductie van genetisch gemodificeerde voedingsmiddelen. Doelstellingen demonstratieproject
Kok, E.J. ; Noordam, M.Y. ; Noteborn, H.P.J.M. - \ 1996
Voeding 57 (1996)7/8. - ISSN 0042-7926 - p. 20 - 23.
voedsel - voedselindustrie - voedselinspectie - voedselvoorziening - voedseltechnologie - voedingsmiddelen - genetische modificatie - genomen - overheidsbeleid - haploïdie - gezondheidszorg - bedrijfsvoering - marketing - maaltijden - mutaties - voeding - plantenveredeling - polyploïdie - volksgezondheid - recombinant dna - food - food industry - food inspection - food supply - food technology - foods - genetic engineering - genomes - government policy - haploidy - health care - management - meals - mutations - nutrition - plant breeding - polyploidy - public health
Aandacht voor het demonstratieproject "Risicoanalyse van genetisch gemodificeerde voedingsmiddelen als basis voor marktintroductie". Verslag van een workshop
An En/Spm based transposable element system for gene isolation in Arabidopsis thaliana
Aarts, M.G.M. - \ 1996
Agricultural University. Promotor(en): M. Koornneef; A. Pereira. - S.l. : Aarts - ISBN 9789054856221 - 138
genen - genomen - brassicaceae - genes - genomes - brassicaceae - cum laude
At the start of the research described in this thesis, the main aim was to develop, study and apply an efficient En/Spm-I/dSpm based transposon tagging system in Arabidopsis thaliana to generate tagged mutants and to provide insights in the possibilities for future applications of such a transposon tagging system in studying plant gene functions. The first step was the introduction of an active En/Spm-I/dSpm system into Arabidopsis. Initially a very simple T-DNA construct was transformed, containing a nearly full En-1 element, without left and right border sequences, and with its promoter replaced by the stronger CaMV 35S promoter. As the same construct harboured a non-autonomous I/dSpm element, only one T-DNA transformation was needed. Transformation of this 'in cis two- element En/Spm-I/dSpm system' yielded one transformant with two T-DNA insertion loci, TEn2 and TEn5, each having one, respectively, five 35S -En/Spm t ransposase gene copies (Chapter 1). The transposition activity of the I/dSpm elements turned out to be surprisingly high. Instead of a germinal excision frequency, which was generally used to express the activity of heterologous transposable element systems, the term independent transposition frequency or itf was coined, as a measure accounting for the entire transposition process. Although not always easy to compare for different transposable element systems, an average itf of over 10%, as was found for this En/Spm-I/dSpm system (Chapter 1), has not been reported for any of the Ac-Ds based heterologous transposon tagging systems developed for Arabidopsis (Bancroft et al., 1992; Bhatt et al., 1996; Fedoroff and Smith, 1993; Honma et al., 1993; Long et al., 1993b; Swinburne et al., 1992). Obtaining a high transposition frequency in Ac-Ds systems is hampered by the fact that the transposase acts as an inhibitor of Ac or Ds transposition when its expression exceeds a certain level. Apparently such autoregulatory mechanism is not present in the 'in cis two-element En/Spm-I/dSpm system'.
The need for a more sophisticated system diminished with the availability of this simple, but efficient En/Spm-I/dSpm transposon system and it was therefore studied in more detail to determine: 1) the ability to transpose continuously even after many plant generations; 2) the distribution of elements after transposition; 3) the ability to transpose to transcribed regions and 4) the ability to cause mutations. To start with the first issue, transposition has been studied in up to 12 generations, starting from the primary transformant. In all these generations, there was no apparent reduction in itf, demonstrating a continuous transposition of I/dSpm elements, irrespective of the generation number. The second issue, the distribution of elements after transposition, is another important aspect of a transposable element system. For maize transposons it is reported that the insertion site is preferentially physically and often genetically linked to the excision site (Dooner and Belachew, 1989; Peterson, 1970). This is not so remarkable considering that the proteins that perform the transposition steps will have a higher chance of encountering a nearby site on the genome instead of a distant DNA sequence. Like in maize (Peterson, 1970), the I/dSpm elements show a preference for insertion in genetically linked sites (Chapter 1), but the preference is not very explicit. Based on the observations of transpositions from tagged genes to sites within a few cM or only several kb away, and on the analogy to the En/Spm-I/dSpm elements in maize, the overall estimate is that about 30% of the elements transpose to sites genetically linked to the excision site. The mapped elements (Chapter 5) show a fairly even distribution over the genome, although there seems to be some clustering of elements (from different origin) to certain genomic regions, like the top of chromosome 4, the bottom of chromosome 1 and the lower half of chromosome 5.
In accordance with the idea that DNA must be in an open confirmation to allow the access of transposase proteins before transposition (Zhang and Spradling, 1993), there are many indications that I/dSpm elements insert in regions of the Arabidopsis genome containing genes: a) I/dSpm flanking DNAs rarely contain repetitive sequences, but are mostly single copy sequences, as are genes (Chapters 1 and 5); b) about half of the I/dSpm elements are inserted in relatively conserved genomic regions, with no RFLPs for five restriction enzymes (Chapters 1 and 5); c) at least one third of the examined I/dSpm elements is inserted in close vicinity of transcriptionally active genomic sequences (Chapter 7). Insertion in unique, conserved and often transcribed DNA may not seem surprising for a plant species with little repetitive DNA and a small genome with a high gene density (Meyerowitz, 1989). However, a high frequency of insertion into genic regions of the genome offers the best chances for gene tagging.
The most important aspect of a transposable element system is the possibility to generate tagged mutants. The En/Spm-I/dSpm system is mutagenic, with as much as 12 tagged mutants found so far. Most of these were obtained after screening for random mutant phenotypes. When screening for specific mutants, such as reduced seed dormancy, tagged alleles of the ABI3 and LEC1 genes were found (M. Koornneef et al., unpublished results). Mutants at the CER6 locus were obtained by targeted tagging, using the nearby ap1::I/dSpm allele as the I/dSpm element donor (A. Pereira, unpublished results). These selected and targeted transposon tagging experiments are illustrative examples of the feasibility to efficiently isolate tagged mutants of a special phenotypic or genotypic class.
Ideally, a population saturated with different I/dSpm insertions can be made, allowing the isolation of mutants for virtually every gene. Such a 'mutation machine' can be further used for PCR based targeted gene inactivation. This novel technique, which was originally developed for Drosophila melanogaster (see O'Hare, 1990), exploits the abundance of transposons for the identification of insertions in genes with known DNA sequence, but no known mutant phenotype. In general, DNAs from multidimensional pools of individuals from a large population are used for a PCR using two primers. One primer is specific for the transposon terminus (directed outwards), the other is specific for the target gene. A fragment can only be amplified when a transposon insert is close enough to the target primer. This technique has been shown to work in Drosophila (Ballinger and Benzer, 1989; Kaiser and Goodwin, 1990), Caenorhabditis elegans (Zwaal et al., 1993), Petunia hybrida (Koes. et al., 1995) and maize (Das and Martienssen, 1995), using 'mutation machine' transposable element systems.
Two I/dSpm tagged genes that have been studied in great detail are the MS2 gene (Chapters 2 and 3) and the CER1 gene (Chapter 4). Mutants for both genes display a conditional male sterile phenotype, which is the only mutant phenotype for the ms2 mutant, but for the cer1 mutant it is a pleiotropic effect of a deficiency in epicuticular wax biosynthesis. The ms2 mutants are occasionally able to self-fertilize, especially in high relative humidity and late in plant development, but seed set rarely reaches more than a few percentages of wild-type seed set. This in contrast to the cer1 mutants, which are male sterile in low relative humidity (≤50% RH) and fertile in high relative humidity (≥95% RH). Fertility cannot be completely restored by environment in the ms2 mutants due to the drastic effect of the mutation on pollen development. The MS2 gene is expressed in the tapetum around the time of microspore release from the microspore mother cells. The gene is needed for the development of a proper exine layer, protecting the microspore from harmful environmental influences. Consequently the few ms2 microspores that are produced have very feeble pollen walls, which leaves only very few pollen grains intact for fertilization.
The CER1 gene acts much later in pollen development. Phenotypically cer1 and wild-type pollen cannot be distinguished, apart from a difference in gerniination ability (Chapter 4). As cer1 pollen germination is like wild type when applying CER1 pollen or by pollinating in high relative humidity, there appears to be a substance missing from the pollen coat that is required for the necessary rehydration of a pollen grain. Although essential under low relative humidity conditions, this is only a minor defect, which can be easily overcome.
Besides the similarity in mutant phenotypes, the MS2 gene and the CER1 gene share the characteristic of encoding proteins with homology to enzymes in the fatty acid biosynthesis pathway. The MS2 protein has most resemblance with the wax fatty acid reductase protein from the desert shrub jojoba, which is involved in the conversion of wax fatty acids to wax alcohols (Chapter 3). The CER1 protein shares structural features of fatty acid desaturases, and it has a proposed function as a decarbonylase, converting long carbon chain aldehydes to alkanes (Chapter 4). There are more examples of a correlation between male fertility and wax biosynthesis, as also other cer mutants such as cer3, 6, 8 and 10 are known to be disturbed in male fertility. It demonstrates the general importance of fatty acid biosynthesis for male gametogenesis.
The last part of this thesis has been devoted to further applications of the En/Spm-I/dSpm tagging system in Arabidopsis for the analysis of plant gene functions. The first description of transposable elements as controlling elements (McClintock, 1948), was based on the effect transposons had on the expression of maize genes. Especially (d)Spm insertions were known to cause Spm dependent or suppressible gene expression (Fedoroff, 1989). This effect is now also described for the En/Spm- I/dSpm system in Arabidopsis (Chapter 6). As in maize, an En/Spm suppressible allele contains an anti-parallel I/dSpm element insertion, which can be spliced from the mRNA. This knowledge can be further used to design an artificial gene expression system, in which an introduced gene containing an anti-parallel I/dSpm element, can be negatively controlled in the presence of an En/Spm transposase source. The reverse effect, En/Spm dependent gene expression, is now also described for Arabidopsis (Chapter 6), but the mechanism for dependence is not yet understood. The availability of the En/Spm dependent lad::I/dSpm mutant will be useful for further research.
A more general way to study gene expression is the use of gene traps as detectors of gene activity. The pilot experiments described in chapter 7, have shown the possibility to adapt I/dSpm elements as gene traps. Especially their advantage in detecting genes without the need for mutation, and the possibility of studying the activity of genes which are lethal as homozygous mutants, are important additional properties of gene trap systems over "traditional" transposon tagging. In combination with its efficient transposition behaviour, an En/Spm-I/dSpm based gene trap tagging system seems an attractive alternative for the existing Ac-Ds or T-DNA based gene trap systems.
Summarizing, an En/Spm-I/dSpm transposon tagging system has been well developed for Arabidopsis and many of its basic characteristics are studied and described. I/dSpm tagged mutants can be found with reasonable frequencies, either by random, selected or targeted tagging strategies. The cloning and characterization of two genes affecting male fertility has been described. Further ways to improve tagging frequencies, based on phenotypic or on genotypic selection have been discussed. In addition, the system can be exploited to study plant gene expression and gene function either by En/Spm controlled activity of I/dSpm tagged genes, or by using I/dSpm gene detector elements.
Basic aspects of potato breeding via the diploid level
Hutten, R.C.B. - \ 1994
Agricultural University. Promotor(en): J.G.T. Hermsen. - S.l. : Hutten - ISBN 9789054852926 - 93
haploïdie - polyploïdie - mutaties - plantenveredeling - genomen - solanum tuberosum - aardappelen - haploidy - polyploidy - mutations - plant breeding - genomes - solanum tuberosum - potatoes
In this thesis research is presented on all steps in a potato breeding program via the diploid level: dihaploid induction, selection at the diploid level and sexual polyploidization. In spite of the significant seed parent x pollinator interaction estimated, IVP 101 was found to have a significant higher dihaploid induction ability than the widely used pollinators IVP 35 and IVP 48. Significant differences in dihaploid production ability between seed parents were also found. Dihaploid populations from 31 varieties or breeding lines were evaluated for occurrence and frequency of mutant phenotypes, for tuberization, flowering, pollen stainability, 2n-pollen production and resistance to Ro-1. The expression of yield, yield components, vine maturity, under water weight and chip colour at the diploid and tetraploid level and the parental effects on these characters of 4x . 2x progenies were investigated to find an answer to the question whether at the diploid level other selection criteria should be employed than at the tetraploid level when selecting tetraploid breeding lines. For yield direct selection at the diploid level had no effect on the performance of tetraploid progeny. For under water weight more stringent and for vine maturity less stringent selection criteria were required at the diploid level than at the tetraploid level. The frequently reported superiority of FDR 2n-gametes versus SDR 2n-gametes could be confirmed for yield only. For vine maturity, under water weight and chip colour no considerable differences were found between means of FDR and SDR progenies from reciprocal 4x-2x crosses.
Vrielink, R. ; Heusden, S. van - \ 1993
Wageningen : CPRO-DLO - 23
gegevens verzamelen - schatting - genen - genomen - handboeken - solanum lycopersicum - meting - moleculaire biologie - registreren - tomaten - data collection - estimation - genes - genomes - handbooks - solanum lycopersicum - measurement - molecular biology - recording - tomatoes
Genes and sequences involved in the replication of cowpea mosaic virus RNAs
Eggen, R. - \ 1989
Agricultural University. Promotor(en): A. van Kammen; R.W. Goldbach. - S.l. : Eggen - 119
koebonenmozaïekvirus - replicatie - dna-replicatie - genen - genomen - cowpea mosaic virus - replication - dna replication - genes - genomes
The aim of the studies described in this thesis was to gain more insight in the complex molecular mechanisms underlying the RNA replication of the cowpea mosaic virus genome. Previously the replication of CPMV RNA has been examined extensively with crude membrane fractions prepared from CPMV infected cowpea leaves (Zabel, 1978; Dorssers, 1983). These studies resulted in the identification of a host-encoded RNA-dependent RNA polymerase (Mr 130K), with unknown biological function, and the virus-encoded replicase (Mr 110K). As integral constituent of a membrane bound replication complex (RCX) the viral replicase was only capable of elongating viral plus-sense RNA chains that have already been Initiated invivo . Furthermore, the invitro activity was not representative for the invivo in situation in that no single-stranded (as) progeny RNA production was observed. To resolve the mechanism of initiation of CP14V RNA synthesis and to examine the role of different virus-encoded proteins and specific genomic RNA sequences in this process a replication system that also accepts exogenously added RNA templates is required. Despite exhaustive attempts it has not yet been possible to reconstitute polymerase activity after the removal of the tightly bound template RNA from crude replication complexes isolated from CPMV infected cowpea leaves. In chapter 2 we have explained that CPMV RNA replication probably requires a primer and a template for the Initiation of RNA synthesis. Based on this Idea we have tried to reconstitute polymerase activity with a variety of mixtures of primers and templates. Wildtype CPMV-RNA and plus or minus-stranded transcript RNAs were tested as exogenously added templates. The primers used in the assays were oligo(U) or a short RNA stretch corresponding with the first 94 nucleotides of the viral B-RNA. In addition a mixture of poly(A) and oligo(U) as used in the research on RNA replication of poliovirus, has been tested in our system. Since the viral replicase activities, isolated from CPMV infected cowpea tissue, were low, incomplete and overshadowed by the strong host-encoded RNA-dependent RNA polymerase activity, the invitro RNA replication experiments were difficult to perform and it was troublesome to interprete the results. In an attempt to overcome these problems the invitro RNA replication assays and reconstitution experiments were extended to crude membrane fractions, prepared from another systemic host of CPMV, Chenopodiumamaranticolor . Indeed the host-encoded RNA-dependent RNA polymerase activity was less prominent In this plant and it was shown that due to the greater stability of the CPMV-RCX in this host,
ss progeny RNA was produced. However, as a result of the more stable RCX too, It was very difficult to remove the endogenous RNA template and hence reconstitution of polymerase activity with exogenously added templates was not possible. As an alternative approach to analyse the function of viral proteins in theCPMV RNA replication, we have started to examine the possibility of reconstituting a system for viral RNA synthesis fromCPMV encoded proteins produced in Escherichiacoli . For that purpose fragments of cDNA clones containing the coding regions of the B-RNA encoded 87K putative core polymerase and the 110K viral replicase were used to produce sizable levels of these proteins in E.coli . Such an expression system may generate a template-dependent activity due toCPMVproteins which are not associated with membranes and hence could be more amenable to study the RNA replication process. This RNA synthetic activity has been demonstrated successfully for polioviral polymerase expressed in E.coli . Although the expectedCPMV encoded proteins were synthesized, they did not show any polymerase activity, contrasting with the highly active polio polymerase, simultaneously obtained in the same way. Since poliovirus andCPMV have analogous features of the genomic RNA and their expression strategy and show a comparable functional organization In their polyproteins, it has been assumed that both viruses probably would have a similar RNA replication mechanism. The results however contradict this Idea and indicate that the analogy betweenCPMVand pollovirus can not be extended to homologous mechanistical aspects of the virus multiplication. This hypothesis Is strengthened by the differences In the expression mechanisms (Wellink, 1988). Probably as a result of the split genome ofCPMV, whereas pollovirus has a genome consisting of a single RNA molecule, gene expression will be differently regulated. This makes sense since for the production of progeny virus a single RNA molecule provided with a single VPg is required versus 60 copies of each of the capsid proteins. The divided genome ofCPMV enables a regulated production of these proteins, whereas for poliovirus such regulation is not possible. Since the expression of virus-encoded proteins Is needed for viral RNA replication, it is plausible to assume that differences in the regulation of gene expression may result In different RNA replication mechanisms for poliovirus andCPMV. Although the 87K and 110K B-RNA-encoded proteins produced in E.coli appear to be not capable or not sufficient for polymerase activity, the expression system has shown to be very useful for the production of separate virusencoded proteins, among which the active viral protease. Based on both the expression experiments and the inability to prepare a template-dependent replication system, it seems a plausible hypothesis that the processing products87Kand110K,already folded in a certain way, are not able to (re)initiatie RNA replication on an added template, but that a larger precursor protein from which the polymerase is simultaneously cleaved and incorporated into an active RCX is needed. If this hypothesis Is true it would be impossible to observe complementation between two mutant B-RNA molecules, each encoding only a part of the open reading frame such that the two mutant RNA molecules together produce the complete set of B-RNA encoded proteins. These experiments still have to be performed. As polymerase activity is only expected upon binding of the proteins to a template and upon subsequent initiation of complementary RNA strand synthesis, it would be worthwhile to study these processes Independently. Firstly, one can examine the binding between specific polymerase proteins, which may be Isolated from the E.coli expression system, and templates, followed by reconstitution of a functional replication complex by the addition of suitable primers like specific oligonucleotides or VPg in various forms and membranes which can be isolated from plant or synthesized invitro . For the examination of specific nueleotide sequences involved in the viral RNA replication Infectious transcripts produced from full-length DNA copies of B- and M-RNA have been exploited(Vos, 1987).Both the infection procedure and the specific infectivity of the transcripts were improved, resulting in a system suitable to introduce site specific mutations in the viral RNAs and to analyse the effects of such mutations on the RNA replication invivo . The potential of the Infectious transcripts has been demonstrated by the analysis of genomic RNA sequences with putative RNA replication signals. By subtile modifications, a function In the RNA replication could be described to an homologous nucleotide-stretch which is present in the3'region of both B- and M-RNA. The Importance of these nucleotides in the viral RNA multiplication was strengthened by the reversion of the mutated stretches to the wildtype sequence in this area during replication cycles invivo . As the reversion was a stepwise process, the reversion probably occurs by the preferential multiplication of those RNA molecules which have advantageous point mutations in this nucleotide stretch. Alternatively, recombination with the homologous area in the wildtype M-RNA transcript could be the underlying mechanism for reversion. When Band M-RNA transcripts with identical mutations in this region will be used as inoculum, one can discriminate between these two possibilities. Whether the nucleotide stretch Indeed forms the supposed hairpin, which may protect the RNA against degradation or alternatively have a specific signal function In RNA replication or encapsidation, remains an interesting question to be solved. A major advantage for further studies on the RNA replication of CPMV Is that this virus has a genome consisting of two RNA molecules. B-RNA can replicate Independently and contains the genetic information needed for the RNA multiplication. M-RNA contains the information for the cell to cell transport of the virus and needs B-RNA encoded proteins for its multiplication. This dependency must be its multiplication. This dependency must be exploited In future experiments by modifying the M-RNA sequence at specific places while keeping the B-RNA encoded replication machinery intact. Such studies may considerably contribute to the fully understanding of the complex RNA replication mechanism of CPMV and related viruses.
|Manipulatie van het gehalte 2n-gameten in pollen van diploide solanums
Eijlander, R. - \ 1988
Wageningen : SVP (SVP rapport nr. 291) - 15
genomen - haploïdie - mutaties - plantenveredeling - stuifmeel - polyploïdie - aardappelen - solanum tuberosum - technieken - genomes - haploidy - mutations - plant breeding - pollen - polyploidy - potatoes - solanum tuberosum - techniques
|Onderzoek aan produktie van haploiden bij Brassica : verslag van bezoek aan Canada, 1 t/m 22 juni 1987
Duijs, J.G. - \ 1987
Wageningen : IVT (Rapport / Instituut voor de Veredeling van Tuinbouwgewassen nr. 237) - 15
koolsoorten - canada - embryokweek - genomen - haploïdie - mutaties - plantenveredeling - polyploïdie - onderzoek - weefselkweek - cabbages - canada - embryo culture - genomes - haploidy - mutations - plant breeding - polyploidy - research - tissue culture
The production and evaluation of monohaploid potatoes (2n=x=12) for breeding research on cell and plant level
Uijtewaal, B.A. - \ 1987
Agricultural University. Promotor(en): J.G.T. Hermsen; E. Jacobsen. - S.l. : Uijtewaal - 112
genomen - haploïdie - hybriden - soortkruising - mutaties - plantenveredeling - polyploïdie - aardappelen - solanum tuberosum - somatische hybridisatie - genomes - haploidy - hybrids - interspecific hybridization - mutations - plant breeding - polyploidy - potatoes - solanum tuberosum - somatic hybridization
The use of monohaploid potato clones in research in theory has many potential advantages in comparison with the use of heterozygous di- and tetraploids. Because in monohaploids each chromosome is single, recessive as well as dominant alleles are detectable and can be selected for. In addition, monohaploids offer a unique opportunity to produce homozygous clones. The aim of the study presented in this thesis was to determine which are the pros and cons of using monohaploids in practical breeding and fundamental research, and which are the consequences of monoploidy and homozygosity in a normally highly heterozygous crop such as potato.
Large-scale production of monohaploids appeared to be possible by prickle pollination, but turned out to be genotype-specific. It was shown that the ability for monohaploid production can be transferred to the progeny by crossing. During the investigation reported herein more than 500 monohaploids have been produced that could be used for further research.
In order to combine different selected monohaploid genotypes that will warrant high-level heterozygosity, methods for protoplast isolation, culture and fusion have been adjusted to the material used. Several monohaploid genotypes could be regenerated from protoplasts to plants but always the ploidy level raised to 2x or even 4x. After protoplast fusion, hybrid fusion products could readily be selected because of a differential regeneration capacity. By using isozyme markers for detection of hybridity the first regenerants after protoplast fusion were shown to be hybrids. A difference of at least four weeks in regeneration time was found between the first heterozygous (hybrids) and the first homozygous (parental) genotypes. Some regenerants were triploid. This indicates that at least one monohaploid protoplast was involved in fusion. The majority however was tetraploid and this was probably the result of somatically doubling x+x fusion products. Only in some genotype combinations also increased callus growth rates were found.
The plant vigour in homo- and heterozygotes suggests that dominance effects are stronger than additive gene effects. Owing to sterility problems and a relatively bad plant performance, homozygous and there fore also indirectly monohaploid potato clones are of little importance for practical breeding. However, for fundamental research, monohaploids are very useful: (i) they can be produced on a large scale from specific diploid lines, (ii) they can be maintained in vitro on the monohaploid level for at least 2-3 years via shoot tip propagation, and (iii) protoplast isolation and fusion, and plant regeneration from protoplast derived calli has proven to be possible.
|Wellicht meer melk met tetraploid Engels raaigras.
Lantinga, E.A. - \ 1987
Boerderij/Veehouderij 72 (1987). - ISSN 0169-0213 - p. 22VE - 23VE.
diervoedering - genomen - haploïdie - lolium - melkproductie - mutaties - plantenveredeling - plantengemeenschappen - polyploïdie - onderzoek - vegetatie - animal feeding - genomes - haploidy - lolium - milk production - mutations - plant breeding - plant communities - polyploidy - research - vegetation
Om meer informatie te verkrijgen over de geschiktheid van tetraploide weidetypen van Engels raaigras voor blijvend grasland werd door de vakgroep Landbouwplantenteelt en Graslandkunde van de Landbouwuniversiteit Wageningen een onderzoek opgezet. Hieruit kwam naar voren dat bij een te verwachten hogere opname van tetraploid gras door melkkoeien theoretisch de melkproduktie met een kg per koe per dag zal toenemen. Proefopzet en berekeningswijze zijn uiteengezet
Cytogenetic approaches to breeding and propagation of male sterile parent lines for hybrid varieties of rye
Vries, J.N. de - \ 1984
Landbouwhogeschool Wageningen. Promotor(en): J. Sybenga. - Wageningen : De Vries - 128
combining ability - cytogenetica - genomen - mutaties - rogge - secale cereale - selectie - combining ability - cytogenetics - genomes - mutations - rye - secale cereale - selection
Van rogge is bekend, dat belangrijke opbrengst- en kwaliteitsverbeteringen zijn te bereiken, wanneer op een effectievere manier dan tot nu toe gebruik zou kunnen worden gemaakt van de aanwezige 'bastaard groeikracht, of 'heterosis'. Om de mogelijkheden van heterosis volledig uit te buiten, moet men hybride rassen kweken. Een bijkomend voordeel van hybride rassen is hun uniformiteit.
Het zaaizaad van een hybride ras ontwikkelt zich op planten die niet in staat zijn functioneel stuifmeel te vormen. De bestuiving van deze 'mannelijk steriele' (m.s.)-lijn, of 'moederlijn', gebeurt door een vaderlijn waarvan de combinatiegeschiktheid met de m.s.- lijn na uitvoerige proefnemingen is komen vast te staan.
Vooral bij windbestuivers als rogge is volstrekte mannelijke steriliteit van de moederlijn een vereiste: 66n enkele mannelift fertiele - overigens niet tot de vaderlijn behorende - plant tussen de planten van de moederlijn kan tientallen m.s.-buurplanten bestuiven. Het zaad dat uit zoln bestuiving voortkomt, is geen hybride zaaizaad, maar wordt wel meegeoogst en heeft uiteindelijk een negatieve invloed op prestatie en uniformiteit van het hybride ras.
Gedurende de afgelopen tien jaar is in rogge een systeem ontwikkeld, dat het mogelijk maakt, m.s.-lijnen op een betrouwbare en betrekkelijk eenvoudige - en dus commercieel aantrekkelijke - manier in stand te houden en te vermeerderen. In dit systeem wordt gebruik gemaakt van cytoplasmatische mannelijke steriliteit. Omdat mnmenteel slechts 66n steriliserend cytoplasma operationeel is, is de genetische basis van hybride rassen, die zijn veredeld met dit systeem, bijzonder smal en dus kwetsbaar.
Voor de hybride veredeling van een aantal andere gewassen zijn alternatieve methoden ontwikkeld of voorgesteld, die in principe ook in rogge toepasbaar zijn. In dit proefschrift zijn een aantal aspecten van de problemen, die zich bij de toepassing van deze alternatieven kunnen voordoen, nader belicht.
Alle voorgestelde alternatieven hebben met elkaar gemeen, dat gebruik wordt gemaakt van een monofactorieel recessief overervend, chromosofflaal gelocaliseerd m.s.-gen. Het dominante allel van dit gen ligt op extra chromosoommateriaal met een van normaal afwijkende structuur, terwijl het normale chromosoomcomplement de drager is van het recessieve allel. Het doel van deze constructie is, te voorkomen dat het dominante allel via het stuifmeel wordt overgedragen op de nakomelingschap. Daarvoor is nodig, dat stuifmeel met het extra materiaal niet aan de bevruchting kan deelnemen ('certatie'). Bovendien moeten de structurele afwijkingen in het extra materiaal verhinderen, dat door recombinatie het dominante allel op de normale chromosomen terecht komt. Wordt aan beide voorwaarden voldaan, dan is de vermeerdering van een volledig mannelijk steriele ouderlijn mogelijk, door normale, homozygoot recessieve m.s-planten te bestuiven met pollen van fertiele planten met een chromosomale en genotypische constitutie als hierboven beschreven. Planten met dergelijke 'gebalanceerde chromosoomsystemen' kunnen door zelfbestuiving worden vermeerderd, omdat eicellen met extra chromosoommateriaal wél reproductief zijn. Om bij de vermeerdering zoveel mogelijk arbeid te besparen, zou gebruik gemaakt kunnen worden van een recessief over-ervend gen dat letaliteit veroorzaakt. Net als bij een m.s.-gen, moet het dominante allel op het extra chromosoommateriaal en het recessieve allel op de normale chromosomen liggen. Deze constructie leidt ertoe, dat na zelfbestuiving uitsluitend planten met extra chromosoommateriaal in leven blijven.
Recombinatie in gebalanceerde chromosoomsystemen kan worden bestudeerd door splitsende nakomelingschappen van kruisingen tussen planten met deze systemen en dragers van monofactorieel overervende morfologische kenmerken te onderzoeken. Daartoe is allereerst geprobeerd 17 van dergelijke genen - waaronder een m.s.-gen - te localiseren op één van de zeven roggechromosomen (hoofdstuk 1). In 13 gevallen lukte dat, en bij negen genen kon tevens worden vastgesteld op welke chromosoomarm ze liggen. Vier genen konden niet worden gelocaliseerd. Dit onderzoek resulteerde in de eerste genenkaarten voor de chromosomen 2R en 5R van rogge.
Bij de in hoofdstuk 1 beschreven genlocalisatie werd gebruik gemaakt van reciproke translocaties: permanente uitwisselingen tussen niet-homologe chromosomen. Reeds lang is bekend, dat in het chromosoomsegment tussen het punt van uitwisseling ('translocatiebreukpunt') en het centromeer recombinatie doorgaans gereduceerd is. Dergelijke 'interstitible' segmenten zouden derhalve geschikt zijn als plaats voor een m.s.-gen. Een duidelijke uitzondering op de regel van lage interstitiële recombinatie wordt behandeld in hoofdstuk 2, waar het splitsingsgedrag in de nakomelinschap van een kruising tussen een translocatie en een interstitieel gelocaliseerd gen wordt beschreven. Zowel op grond van de waargenomen splitsing, als op basis van de frequenties van karakteristieke chromosoomconfiguraties gevormd tijdens de metafase van de reductiedeling ('MI-configuraties') in de translocatieheterozygoot kon worden geconcludeerd, dat bij deze translocatie een extreem hoge chiasmafrequentie in het interstitAle segment optreedt. Een vanuit theoretisch oogpunt belangwekkende gevolgtrekking uit dit onderzoek was, dat de centromeerorAntatie van translocatiemultivalenten in planten met (sub-)metacentrische chromosomen door interstitigle chiasmavorming kan veranderen van 'bij voorkeur zig-zag' in 'geen voorkeur'.
Een aantal voorwaarden waaraan voldaan moet worden om twee typen gebalanceerde chromosoomsystemen te construeren, is beschreven in de hoofdstukken 3 en 5. Hoofdstuk 3 behandelt de isolatie van vier verschillende tertiaire trisomen: planten die naast het normale chromosoomcomplement een translocatiechromosoom bevatten. Tertiaire trisomen komen in een lage frequentie voor in de nakomelingschap van translocatieheterozygoten. Uit de experimenten beschreven in hoofdstuk 3 bleek, dat de aanwezigheid van een extra chromosoom in een translocatieheterozygoot deze frequentie aanzienlijk kan verhogen. Voorwaarde is,dat dit extra chromosoom compleet is: de aanwezigheid van een extra Itelocentrisch' chromosoom (bestaande uit slechts één van de twee armen van een normaal chromosoom) bleek de ontstaansfrequentie van tertiaire trisomen niet wezenlijk te beïnvloeden. Tevens kwam naar voren, dat de kans op tertiaire trisomen in de nakomelingschap groter is naarmate bij een MI-configuratie in de ouderplanten meer chromosomen betrokken zijn (tot zeven stuks in dit onderzoek). De vier gelsoleerde tertiaire trisomen bevatten alle een extra translocatiechromosoom dat korter is dan de gemiddelde lengte van de normale chromosomen; tertiaire trisomen met een 'lang' translocatiechromosoom extra konden niet worden verkregen.
De isolatie van vier verschillende telotertiaire compenserende trisomen is beschreven in hoofdstuk 5. Het ontbreken van een normaal chromosoom wordt in dit type trisoom gecompenseerd door de aanwezigheid van een translocatie- en een telocentrisch chromosoom. De vier telotertiaire compenserende trisomen kwamen voor in de nakomelingschap van een aantal translocatieheterozygoten, waarin een telocentrisch chromosoom extra aanwezig was, of waarin twee telocentrische chromosomen de plaats in hadden genomen van een normaal chromosoom. Geheel in overeenstemming met wat op grond van theoretische overwegingen verwacht mocht worden, werd gevonden dat de frequentie van telotertiaire compenserende trisomen hoger is, naarmate het uitgewisseld segment van het translocatiechromosoom met de compenserende rol korter is. Een belangrijke constatering was, dat telotertiaire compenserende trisomen niet alleen korte, maar ook lange translocatiechromosomen kunnen bevatten. In vergelifting met tertiaire trisomen (hoofdstuk 3) betekent dit, dat uitgaande van een gegeven aantal translocaties, tweemaal zoveel verschillende telotertiaire compenserende trisomen geconstrueerd kunnen worden, waardoor de mogelijkheid om een geschikte plaats voor een m.s.-gen te vinden, wordt vergroot.
Het gedrag van de vier tertiaire trisomen uit hoofdstuk 3 wordt beschreven in hoofdstuk 4. Het extra translocatiechromosoom van elk van deze trisomen bevat een stuk van chromosoom 5R met daarop het dominante allel van het 'tigrina' gen. De normale chromosomen dragen de recessieve allelen, zodat er sprake is van vier lgebalanceerdel tertiaire trisomen. Het tigrina gen veroorzaakt een gele dwarsstreping van het blad ('tijgering'). In zelfbevruchtingsnakomelingschappen van de vier gebalanceerde tertiaire trisomen werd geconstateerd dat recombinatie tussen dit gen en het translocatiebreukpunt erg laag is, zelfs in de twee trisomen waarbij gen en breukpunt aan weerszijden van het centromeer liggen, en ondanks het feit dat het tigrina gen betrekkelijk ver van het centromeer afligt. Aangezien onderzoek aan MI-configuraties uitwees dat overkruising in de chromosoomarm met het tigrina gen zeer frequent optreedt, moest worden geconstateerd dat een groot deel van de overkruisingen plaats heeft bij het uiteinde van deze chromosoomarm. Dit is in overeenstemming met conclusies uit ander onderzoek aan rogge, waarbij werd gevonden dat deze 'terminale chiasmalocalisatie' ook in andere chromosoomarmen voorkomt. Wordt hierdoor het gebied waarin een m.s.-gen zou kunnen liggen op zich al vergroot, ook de interstitiele segmenten van de vier bestudeerde extra translocatiechromosomen komen voor de locatie van het M.s gen in aanmerking: slechts in één tertiair trisoom kon worden aangetoond, dat interstitiele chiasmavorming optrad, in een frequentie van 1%. Overigens vond enige recombinatie ook plaats in een tertiair trisoom waarbij gen en breukpunt vlak bij elkaar lagen.
Bij drie van de vier tertiaire trisomen kon worden aangetoond, dat het extra chromosoom via het stuifmeel op de nakomelingschap wordt overgebracht, echter in lage frequenties. Mannelijke transmissie kon niet worden vastgesteld bij het vierde tertiaire trisoom, maar door de proefopzet kon een geringe mannelijke transmissie onopgemerkt blijven. Aanwijzingen werden verkregen dat het transmissieniveau door selectie is te beinvloeden. Bovendien bleek dat in inteeltlijnen trisomen minder groeikrachtig zijn dan normale planten zonder extra chromosoommateriaal, waardoor bij voldoende dicht zaaien van de moederlijn een gering aantal trisomen zou kunnen worden weggeconcurreerd doornormaal groeikrachtige m.s-planten. Hiervan wordt in de hybride zaaizaadproductie van gerst gebruik gemaakt. Sporadisch traden tijdens de reductiedeling van tertiaire trisomen onregelmatigheden op, resulterend in nakomelingen met een gewijzigde chromosoomsamenstelling. Meestal waren deze planten niet in staat nakomelingen voort te brengen, en gezien hun geringe groeikracht zouden ook zij waarschijnlijk geblimineerd worden in concurrentie met normale buurplanten.
Onderzoek aan de vier telotertiaire compenserende trisomen uit hoofdstuk 5 leidde in hoofdzaak tot dezelfde conclusies ten aanzien van recombinatie, chiasmalocalisatie, transmissie en meiotische stabiliteit als voor de onderzochte tertiaire trisomen.
Hybride rogge is alleen economisch aantrekkelijk, wanneer de extra zaaizaadkosten van een hybride ras worden gecompenseerd door de extra baten verkregen door het verbouwen ervan. De zaaizaadproductie moet daarom tegen zo laag mogelijke kosten worden gerealiseerd. Hiervoor zijn arbeidsbesparende letaliteitsgenen beschikbaar of kunnen met betrekkelijk gemak worden g&induceerd door mutagene behandelingen. Ook het aantal m.s.-genen zou langs deze weg kunnen worden verhoogd. Vooralsnog lijkt het aantal beschikbare translocaties en andere chromosoomafwijkingen voldoende groot om enkele voor hybrideveredeling geschikte gebalanceerde chromosoomsystemen op te zetten. Wel dient het dominante m.s.-allel absoluut gekoppeld te zijn met de translocatiebreukpunten van het extra chromosoommateriaal om mannelijke fertiliteit in de moederlijn te voorkomen. Indien, om wat voor reden dan ook, een dergelijke koppeling niet te verwezenlijken is, maar een iets zwakkere wel, dient nagegaan te worden of een beperkte afname van de prestatie en de uniformiteit van de hybride te toleren is.
Pseudogamic production of dihaploids and monoploids in Solanum tuberosum and some related species
Breukelen, E.W.M. van - \ 1981
Landbouwhogeschool Wageningen. Promotor(en): J.G.T. Hermsen. - Wageningen : Centre for Agricultural Publishing and Documentation - ISBN 9789022007624 - 121
haploïdie - polyploïdie - mutaties - plantenveredeling - genomen - solanum tuberosum - aardappelen - haploidy - polyploidy - mutations - plant breeding - genomes - solanum tuberosum - potatoes
Attempts were made to maximize frequencies of dihaploids from Solanum tuberosum, obtained through pseudogamy after pollination with S.phureja. Factors influencing dihaploid frequencies were studied: genetics of the pollinator effect, genetics of the seed parent effect and interaction between the two effects on dihaploid frequencies. Temperature influences were determined in a growth chamber experiment. The mechanism of dihaploid formation was studied with the aid of cytological techniques. The pollinator effect was confirmed. Five or more loci were involved and the within-locus interaction was intermediate. High numbers of hybrids had a negative but small effect on numbers of dihaploids. The seed parent effect was also confirmed. The frequency of dihaploids was determined by the sporophyte rather than by the gametophyte of the seed parent. No interaction was found between the pollinator and seed parent effect on the dihaploid frequency. Low temperature had a positive effect on the dihaploid frequency via the pollinator, but no effect was found via the seed parent. Not the 2n-pollen but the n-pollen proved to be instrumental in dihaploid induction. Monoploids were produced from diploid S. tuberosum and S. verrucosum using several S. phureja genotypes as pollinator. The n-pollen induced the haploids in this case as well. Doubled monoploids were obtained with good female fertility.
|Vegetatieve vermeerdering en chromosoomverdubbeling in vitro bij kruisbloemigen
Zuidgeest, J.H.M. - \ 1977
Wageningen : SVP - 9
brassicaceae - embryokweek - genomen - haploïdie - mutaties - plantenveredeling - polyploïdie - weefselkweek - brassica - brassicaceae - embryo culture - genomes - haploidy - mutations - plant breeding - polyploidy - tissue culture - brassica
|Verslag van bezoeken aan genenbanken in de DDR en de BRD
Hogenboom, N.G. ; Jong, P.C. de; Pet, G. - \ 1975
Wageningen : [s.n.] (Rapport / Instituut voor de veredeling van tuinbouwgewassen no. 119) - 13
databanken - genen - genomen - duitsland - databases - genes - genomes - germany