Prospects of whole-genome sequence data in animal and plant breeding
Binsbergen, Rianne van - \ 2017
Wageningen University. Promotor(en): R.F. Veerkamp; F.A. Eeuwijk, co-promotor(en): M.P.L. Calus. - Wageningen : Wageningen University - ISBN 9789463431903 - 220
next generation sequencing - dna sequencing - quantitative trait loci - cattle - genomics - solanum lycopersicum - animal breeding - plant breeding - next generation sequencing - dna-sequencing - loci voor kwantitatief kenmerk - rundvee - genomica - solanum lycopersicum - dierveredeling - plantenveredeling
The rapid decrease in costs of DNA sequencing implies that whole-genome sequence data will be widely available in the coming few years. Whole-genome sequence data includes all base-pairs on the genome that show variation in the sequenced population. Consequently, it is assumed that the causal mutations (e.g. quantitative trait loci; QTL) are included, which allows testing a given trait directly for association with a QTL, and might lead to discovery of new QTL or higher accuracies in genomic predictions compared to currently available marker panels. The main aim of this thesis was to investigate the benefits of using whole-genome sequence data in breeding of animals and plants compared to currently available marker panels. First the potential and benefits of using whole-genome sequence data were studied in (dairy) cattle. Accuracy of genotype imputation to whole-genome sequence data was generally high, depending on the used marker panel. In contrast to the expectations, genomic prediction showed no advantage of using whole-genome sequence data compared to a high density marker panel. Thereafter, the use of whole-genome sequence data for QTL detection in tomato (S. Lycopersicum) was studied. In a recombinant inbred line (RIL) population, more QTL were found when using sequence data compared to a marker panel, while increasing marker density was not expected to provide additional power to detect QTL. Next to the RIL population, also in an association panel it was shown that, even with limited imputation accuracy, the power of a genome-wide association study can be improved by using whole-genome sequence data. For successful application of whole-genome sequence data in animals or plants, genotype imputation will remain important to obtain accurate sequence data for all individuals in a cost effective way. Sequence data will increase the power of QTL detection in RIL populations, association panels or outbred populations. Added value of whole-genome sequence data in genomic prediction will be limited, unless more information is known about the biological background of traits and functional annotations of DNA. Also statistical models that incorporate this information and that can efficiently handle large datasets have to be developed.
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.
Anaerobic microbial processes for energy conservation and biotransformation of pollutants
Luz Ferreira Martins Paulo, Lara da - \ 2017
Wageningen University. Promotor(en): A.J.M. Stams, co-promotor(en): D.Z. Sousa. - Wageningen : Wageningen University - ISBN 9789463431125 - 234
anaerobic microbiology - anaerobes - energy conservation - biotransformation - pollutants - heavy metals - sulfates (inorganic salts) - nickel - cobalt - methanosarcina barkeri - genomics - polymerase chain reaction - anaërobe microbiologie - anaërobe micro-organismen - energiebehoud - biotransformatie - verontreinigende stoffen - zware metalen - sulfaten (anorganische zouten) - nikkel - kobalt - methanosarcina barkeri - genomica - polymerase-kettingreactie
Anaerobic microbial processes are commonly applied in the treatment of domestic and industrial wastewaters. Anaerobic digestion (AD) of wastewater has received a great deal of attention, but many aspects related to the complex interactions between microorganism, and how that is affected by the presence of certain toxic, are not yet fully understood. A particular case of this is the effect of heavy metals or chlorinated compounds. These compounds are known to have a strong impact in methanogens, a phylogenetic diverse group responsible for the last step of the AD process. The negative effect of sulphate towards methanogenesis is mainly related to outcompetition of methanogens by sulphate-reducing bacteria (SRB), or to toxicity caused by the sulphide generated from sulphate reduction. Heavy metals are part of many enzymes and cofactors and, in low concentrations, may beneficiate microbial activity. However, high concentrations of metals may disrupt enzyme function and structure. In cases where metal concentration is high, the presence of sulphate or sulphide might be favourable because sulphide precipitate with metals and detoxify the environment. In Chapter 2 we provide a review on the current knowledge on the effects of heavy metals and sulphate on AD, with special focus on methanogenesis. From this literature study, it came out that the influence of some metals, such as Co, is not extensively studied and that the potential of biologically produced sulphide as metal detoxification method in AD is still quite unexplored. In Chapter 3 we explored different strategies to improve methane production. Low concentrations of Ni and Co were supplemented to anaerobic sludge and the impact on methane production was evaluated. Although in contrast with other studies, no beneficial effect of metal supplementation was observed. Further on, the impact of high concentrations of Ni and Co added to anaerobic sludge was evaluated, as well as the use of sulphide as a detoxification strategy. This was evaluated in terms of impact on methane production and in changes in the microbial communities. The results showed that sulphide can be used as a method for metal detoxification, but in the case of biological produced sulphide, the competition between SRB and methanogens needs to be considered.
Chlorinated compounds are widely used and commonly found in wastewaters. Several methanogenic metal-containing cofactors are reported to be involved in reductive dechlorination. Therefore, in Chapters 4 and 5 the potential of metal supplementation to enhance the dechlorination process was studied. In Chapter 4, the enrichment of methanogenic cultures able to perform reductive dechlorination of 1,2-dichloroethene (DCE) and tetrachlorethene (TCE) using different inoculum sources and substrates is described. Differences in physiological performance and in the microbial communities were evaluated. The results showed that the microbial community can be influenced by inoculum and substrate as well as by the chlorinated compound used. The enriched cultures presenting the best dechlorination performance were selected and used for metal supplementation studies with Ni, Co, and Fe. The results showed a clear positive impact of metal addition, both on methane production and reductive dechlorination. Further research on metal supplementation to enhance dechlorination was performed in pure cultures of Methanosarcina barkeri, a methanogen known to be able to reduce DCE (Chapter 5). In this case, it was observed that metal supplementation could improve methane production and reductive dechlorination, but the effect is dependent on the metal and concentration used. It was found that methanogenesis and reductive dechlorination can be affected in a different way by the same metal.
Finally, in Chapter 6 the impact of sulphate on a methane-producing bioelectrochemical system (BES), an emerging technology that can be applied to wastewater treatment, was studied. The results showed an unexpected fast sulphate removal in the system and a limited impact caused by sulphate addition on methane production. The sulphate removal could only partially be explained by microbial activity, but the results demonstrated the ability of microbial communities to evolve and adapt to new operational conditions.
In conclusion, the work presented in this thesis gave insights on the impact of heavy metals and sulphate in methanogenic systems. Furthermore, different approaches to maximise methane production were evaluated. In particular, it was shown that metal supplementation can be a promising strategy to improve anaerobic microbial processes, such as methanogenesis and reductive dechlorination.
Milk progesterone measures to improve genomic selection for fertility in dairy cows
Tenghe, Amabel Manyu Mefru - \ 2017
Wageningen University. Promotor(en): R.F. Veerkamp; B. Berglund, co-promotor(en): D. J. de Koning; A.C. Bouwman. - Wageningen : Wageningen University - ISBN 9789463431330 - 179
dairy cows - fertility - progesterone - milk - genomics - genetic improvement - heritability - genetic parameters - dairy performance - reproductive traits - animal genetics - animal breeding - dairy farming - melkkoeien - vruchtbaarheid - progesteron - melk - genomica - genetische verbetering - heritability - genetische parameters - melkresultaten - voortplantingskenmerken - diergenetica - dierveredeling - melkveehouderij
Improved reproductive performance has a substantial benefit for the overall profitability of dairy cattle farming by decreasing insemination and veterinary treatment costs, shortening calving intervals, and lowering the rate of involuntary culling. Unfortunately, the low heritability of classical fertility traits derived from calving and insemination data makes genetic improvement by traditional animal breeding slow. Therefore, there is an interest in finding novel measures of fertility that have a higher heritability or using genomic information to aid genetic selection for fertility. The overall objective of this thesis was to explore the use of milk progesterone (P4) records and genomic information to improve selection for fertility in dairy cows. In a first step, the use of in-line milk progesterone records to define endocrine fertility traits was investigated, and genetic parameters estimated. Several defined endocrine fertility traits were heritable, and showed a reasonable repeatability. Also, the genetic correlation of milk production traits with endocrine fertility traits were considerably lower than the correlations of milk production with classical fertility traits. In the next step 17 quantitative trait loci (QTL) associated with endocrine fertility traits, were identified on Bos taurus autosomes (BTA) 2, 3, 8, 12, 15, 17, 23, and 25 in a genome-wide association study with single nucleotide polymorphisms. Further, fine-mapping of target regions on BTA 2 and 3, identified several associated variants and potential candidate genes underlying endocrine fertility traits. Subsequently, the optimal use of endocrine fertility traits in genomic evaluations was investigated; using empirical and theoretical predictions for single-trait models, I showed that endocrine fertility traits have more predictive ability than classical fertility traits. The accuracy of genomic prediction was also substantially improved when endocrine and classical fertility traits were combined in multi-trait genomic prediction. Finally, using deterministic predictions, the potential accuracy of multi-trait genomic selection when combining a cow training population measured for the endocrine trait commencement of luteal activity (C-LA), with a training population of bulls with daughter observations for a classical fertility trait was investigated. Results showed that for prediction of fertility, there is no benefit of investing in a cow training population when the breeding goal is based on classical fertility traits. However, when considering a more biological breeding goal for fertility like C-LA, accuracy is substantially improved when endocrine traits are available from a limited number of farms.
Susceptibility pays off: insights into the mlo-based powdery mildew resistance
Appiano, Michela - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Yuling Bai; Anne-Marie Wolters. - Wageningen : Wageningen University - ISBN 9789462579484 - 265
solanum lycopersicum - tomatoes - disease resistance - susceptibility - oidium neolycopersici - genes - gene expression - genomics - molecular breeding - plant breeding - solanum lycopersicum - tomaten - ziekteresistentie - vatbaarheid - oidium neolycopersici - genen - genexpressie - genomica - moleculaire veredeling - plantenveredeling
Powdery mildew (PM) is a worldwide-occurring plant disease caused by ascomycete fungi of the order Erysiphales. A conspicuous number of plant species are susceptible to this disease, the occurrence of which is increasing due to the influence of climate change. Symptoms are easy to recognize by the powdery whitish fungal structures growing on the surface of plant organs. Severe infections cause significant losses in crops, such as tomato, cucumber and wheat, as well as in ornamentals, like rose and petunia. Accordingly, breeding crops with a robust immunity to this disease is of great economic importance.
A significant step in this direction was the discovery of mlo (mildew locus o) mutant alleles of the barley HvMlo gene, which are responsible for the non-race specific resistance to the barley PM pathogen, Blumeria graminis f.sp. hordei (Bgh). During the years, this recessively inherited resistance was observed to be durable, contrary to the short life-span of resistances conferred by dominant resistance (R-) genes used in barley breeding programs. Studies on the histological mechanisms of the mlo-based resistance showed that the PM pathogen was stopped during penetration of the cell wall by the formation of a papilla. This structure prevents the formation of the feeding structure of the pathogen, called a haustorium.
After sequencing many plant genomes, we are discovering that MLO genes are not only typical of this cereal, but are ubiquitously present in higher plant species in multiple copies per species, forming a gene family. The impairment of some members of a number of ever increasing plant species lead to broad-spectrum resistance towards their adapted PM pathogens. For example, in tomato the ol-2 gene, naturally harbored by the cherry tomato Solanum lycopersicum var. cerasiforme, represents the loss-of-function allele of the SlMLO1 gene, conferring resistance to the PM pathogen Oidium neolycopersici (On). Consequently, the use of mlo mutants represents a suitable alternative to the classical use of R-genes in breeding programs.
In Chapter 2, we describe the in silico identification of the complete tomato SlMLO gene family using the available information in the SOL genomic network database. In total, 16 tomato SlMLO members were cloned from leaf, root, flower and fruit of the susceptible tomato cv. Moneymaker to confirm the sequences retrieved from the database and to verify their actual expression in these tissues. We observed the presence of various types of splicing variants, although their possible functional meaning has not been investigated. Motif analyses of each of the translated protein sequences and phylogenetic studies highlighted, on one hand, amino acid stretches that characterize the whole MLO family, and, on the other hand, stretches conserved in MLO homologs that are phylogenetically related. Following a gene expression study upon On inoculation, we identified members of the SlMLO family that are upregulated few hours after pathogen challenge. Except SlMLO1, none of the three newly identified homologs in clade V, thus phylogenetically close to SlMLO1, are induced. Interestingly, two homologs, each found in different clades, are upregulated similarly to SlMLO1. Using an RNAi approach, we silenced the additional clade V-SlMLO homologs, namely SlMLO3, SlMLO5 and SlMLO8, to investigate their possible role in PM resistance. We observed that none of these homologs if individually silenced, leads to PM resistance. However, if SlMLO5 and SlMLO8 are silenced together with SlMLO1, a significantly higher level of resistance is achieved compared to plants carrying the ol-2 allele. The role of SlMLO3 could not be verified. We, therefore, concluded that there are three SlMLO genes in tomato unevenly contributing to the PM disease, of which SlMLO1 has a major role.
Chapter 3 focuses on the components of the tomato mlo-based resistance. In Arabidopsis, it is known that four members of the SNARE protein family, involved in membrane fusion, are involved in mlo-based resistance. In this chapter, we focused on the identification of tomato homologs of the Arabidopsis syntaxin PEN1 (AtSYP121). Among the group of syntaxins identified in tomato, two were closely related to each other and also to AtPEN1, denominated SlPEN1a and SlPEN1b. Another Arabidopsis syntaxin that shows a high level of homology with PEN1, called SYP122, was also found to group together with the newly identified SlPEN1 genes. However, the role of SYP122 in plant immunity was not shown in literature. After obtaining individual silencing RNAi constructs, we transformed the resistant ol-2 line, and we challenged the obtained transformants with the adapted PM On, and the non-adapted Bgh. Interestingly, we observed a significant On growth and an enhanced Bgh cell entry only in SlPEN1a silenced plants but not in SlPEN1b silenced ones. We performed a protein alignment of tomato and Arabidopsis functional and non-functional PEN sequences. The presence of three differently conserved non-synonymous amino-acid substitutions is hypothesised to be responsible for the specialization in plant immune function.
In Chapter 4 and Chapter 5, we build up a body of evidence pointing to the fact that the function of the MLO susceptibility genes is highly conserved between monocot and dicot plant species.
In Chapter 4 we started by identifying and functionally characterizing two new MLO genes of Solanaceous crops affected by the PM disease, tobacco (Nicotiana tabacum) and eggplant (Solanum melongena). We named them NtMLO1 and SmMLO1 in the respective species, as they are the closest homologs to tomato SlMLO1. By overexpressing these genes in the resistant ol-2 line, we obtained transgenic plants that were susceptible to the PM pathogen On. This finding demonstrates that both heterologous MLO proteins can rescue the function of the impaired ol-2 allele in tomato. In addition, we found in tobacco NtMLO1 an amino acid (Q198) of critical importance for the susceptibility function of this protein.
In Chapter 5, we used the same approach adopted in Chapter 4 to show that other MLO proteins of more distant dicot species, like pea PsMLO1, can rescue the loss-of-function of the tomato ol-2 allele. And finally, we stretched this concept also to monocot MLO proteins, using barley HvMlo. While performing these experiments, we could verify that the function of the monocot and dicot susceptibility MLO proteins does not rely on the presence of class-specific conservation. The latter can be the reason for the phylogenetic divergence, placing monocot MLO proteins in clade IV and dicot MLO proteins in clade V of the phylogenetic MLO tree. However, functional conservation might depend on crucial shared amino acids of clade IV and V MLO proteins. Therefore, we also conducted a codon-based evolutionary analysis that resulted in the identification of 130 codons under negative selection, thus strongly maintained during evolution.
In Chapter 6 we introduce the PM disease in cucumber caused by Podosphaera xanthii (Px). We cloned the candidate susceptibility gene for PM in cucumber, CsaMLO8, from susceptible and resistant genotypes. The latter was described as an advanced cucumber breeding line characterized by hypocotyl resistance. In this line, we found the presence of aberrant splicing variants of the CsaMLO8 mRNA due to the insertion in its corresponding genomic region of a Class LTR retrotransposon. Heterologous expression of the wild-type cucumber allele in the tomato ol-2 line restored its PM susceptibility, while the heterologous expression of the aberrant protein variant failed to do so. This finding confirms that the resistance of the advanced cucumber breeding line is due to the disruption of the coding region of this gene. We also showed that the expression of CsaMLO8 in the susceptible genotype is induced by Px in hypocotyl tissue, but not in cotyledon or leaf. Finally, by examination of the resequencing data of a collection of 115 cucumber accessions, we found the presence of the TE-containing allele in 31 of them among which a wild cucumber accession that might have been used in breeding programs to obtain resistance to the PM disease in cucumber.
In Chapter 7 a novel loss-of-function allele of the SlMLO1 gene is described, designated m200. This allele was found in a resistant plant (M200) from a mutagenized tomato Micro-Tom (MT) population obtained with the chemical mutagen ethyl methanesulfonate (EMS). The m200 mutation corresponds to a nucleotide transversion (T à A) which results in a premature stop codon. The length of the predicted SlMLO1 protein in the M200 plant is only 21 amino acids, thus much shorter than the predicted protein of the previously described ol-2 allele, consisting of 200 amino acids. Thanks to the development of a High-Resolution Melting (HRM) marker designed to detect the m200 mutation, we observed that this allele confers recessively inherited resistance in backcross populations of the resistant M200 plant with MT and Moneymaker. Histological study showed that the resistance of the m200 mutant is associated with papilla formation. Finally, we compared the rate of On penetration in epidermal cells of m200 plants with the one of plants carrying the ol-2 allele and the transgenic plants in which multiple SlMLO homologs were silenced, generated in Chapter 2.
Ultimately, in Chapter 8 the results of the previous chapters are discussed in the context of 1) practical applications in breeding programs aimed at introducing the mlo-based resistance in new crops, 2) possible research aimed at unraveling the function of the MLO protein and 3) the role of other SNARE proteins.
Volatile-mediated interactions in the rhizosphere
Cordovez da Cunha, Viviane - \ 2016
Wageningen University. Promotor(en): Francine Govers; Jos Raaijmakers, co-promotor(en): V.J. Carrion. - Wageningen : Wageningen University - ISBN 9789462579019 - 219
rhizosphere bacteria - rhizosphere fungi - microbial interactions - volatile compounds - suppressive soils - actinobacteria - streptomyces - microbacterium - thanatephorus cucumeris - growth stimulators - biological control - defence mechanisms - genomics - transcriptomics - rizosfeerbacteriën - rizosfeerschimmels - microbiële interacties - vluchtige verbindingen - ziektewerende gronden - actinobacteria - streptomyces - microbacterium - thanatephorus cucumeris - groeistimulatoren - biologische bestrijding - verdedigingsmechanismen - genomica - transcriptomica
Plants and microorganisms are constantly engaged in highly dynamic interactions both above- and belowground. Several of these interactions are mediated by volatile organic compounds (VOCs), small carbon-based compounds with high vapor pressure at ambient temperature. In the rhizosphere, VOCs have an advantage in intra- and interorganismal signaling since they can diffuse through soil pores over longer distances than other metabolites and are not dependent on water availability. The research described in this PhD thesis explored how beneficial and pathogenic microorganisms that live in the rhizosphere and endosphere modulate plant growth, development and resistance via the production of VOCs. In vitro and in vivo bioassays as well as different ‘omic’ approaches, such as volatomics, transcriptomics and genomics, were employed to investigate underlying mechanisms of VOC-mediated microbe-microbe and microbe-plant interactions.
To investigate the diversity and functions of microbial VOCs, a disease-suppressive soil was used as the source of the VOC-producing microorganisms. Previous metagenomics studies reported Actinobacteria, in particular Streptomyces and Microbacterium species, as the most abundant bacterial genera found in a soil naturally suppressive to the fungal root pathogen Rhizoctonia solani. VOCs of several Streptomyces isolates inhibited hyphal growth of R. solani and in addition, promoted plant growth. Coupling the Streptomyces VOC profiles with their effects on fungal growth pinpointed methyl 2-methylpentanoate and 1,3,5-trichloro-2-methoxy benzene as antifungal VOCs. Also Microbacterium isolates showed VOC-mediated antifungal activity and plant growth promotion. VOC profiling of Microbacterium sp. EC8 revealed several sulfur-containing compounds and ketones such as dimethyl disulfide, trimethyl trisulfide and 3,3,6-trimethylhepta-1,5-dien-4-one (also known as Artemisia ketone). Genome analysis of strain EC8 revealed genes involved in sulfur metabolism. Resolving the role of the identified compounds and genes in VOC-mediated plant growth promotion and induced resistance will be subject of future studies. VOC-mediated chemical warfare underground has been proposed as a key mechanism of natural disease-suppressive soils. The results presented in this thesis indeed point in that direction. However, to more conclusively determine the role of the identified Actinobacterial VOCs in soil suppressiveness to R. solani, it will be important to demonstrate that the fungicidal VOCs are actually produced in situ at the right place and at sufficient concentrations to suppress plant infection by the pathogenic fungus.
In agriculture, VOCs and VOC-producing microorganisms provide a potential alternative to the use of pesticides to protect plants and to improve crop production. In the past decades, several in vitro studies have described the effects of microbial VOCs on other (micro)organisms. However, little is still known on the potential of VOCs in large-scale agriculture and horticulture. The results described in this thesis show that VOCs from Microbacterium sp. EC8 stimulate the growth of Arabidopsis, lettuce and tomato, but do not control damping-off disease of lettuce caused by R. solani. Significant biomass increases were also observed for plants exposed only shortly to the bacterial VOCs prior to transplantation of the seedlings to soil. These results indicate that VOCs from strain EC8 can prime plants for growth promotion without direct contact and prolonged colonization. Furthermore, the induction of the plant growth-promoting effects appeared to be plant tissue specific. Root exposure to the bacterial VOCs led to a significant increase in plant biomass whereas shoot exposure did not result in significant biomass increase of lettuce and tomato seedlings. Genome-wide transcriptome analysis of Arabidopsis seedlings exposed to VOCs from this bacterium showed an up-regulation of genes involved in sulfur and nitrogen metabolism and in ethylene and jasmonic acid signaling. These results suggest that the blend of VOCs of strain EC8 favors, in part, the plant’s assimilation of sulfate and nitrogen, essential nutrients for plant growth, development and also resistance.
Similar to beneficial microorganisms, plant pathogenic microorganisms have also evolved strategies to modulate growth and defense of their hosts. For instance, compounds secreted by pathogens may suppress or interfere with plant defense. In this thesis I show that R. solani produces an array of VOCs that promote growth, accelerate development, change VOC emission and reduce insect resistance of plants. Plant growth-promoting effects induced by the fungal VOCs were not transgenerational. Genome-wide transcriptome analysis of Arabidopsis seedlings revealed that exposure to fungal VOCs caused up-regulation of genes involved in auxin signaling, but down-regulation of genes involved in ethylene and jasmonic acid signaling. These findings suggest that this soil-borne pathogen uses VOCs to predispose plants for infection by stimulating lateral root formation and enhancing root biomass while suppressing defense mechanisms. Alternatively, upon perception of VOCs from soil-borne pathogens, plants may invest in root biomass while minimizing investments in defense, a trade-off that helps them to speed up growth and reproduction and to survive pathogen attack.
In conclusion, the research presented in this thesis shows that both plants and microorganisms engage via VOCs in long-distance interactions and that beneficial and pathogenic soil microorganisms can alter plant physiology in different ways. Here, I provided a first step in identifying microbial genes involved in the regulation of biologically active VOCs as well as candidate plant genes involved in VOC perception and signal transduction. How plants sense and differentiate among VOCs from beneficial and pathogenic soil microorganisms will be an intriguing subject for future studies.
Statistical methods for QTL mapping and genomic prediction of multiple traits and environments: case studies in pepper
Alimi, Nurudeen Adeniyi - \ 2016
Wageningen University. Promotor(en): Fred van Eeuwijk, co-promotor(en): Marco Bink. - Wageningen : Wageningen University - ISBN 9789462579361 - 153
capsicum - statistical analysis - statistics - genomics - quantitative trait loci - quantitative traits - quantitative methods - genetics - crop yield - capsicum - statistische analyse - statistiek - genomica - loci voor kwantitatief kenmerk - kwantitatieve kenmerken - kwantitatieve methoden - genetica - gewasopbrengst
In this thesis we describe the results of a number of quantitative techniques that were used to understand the genetics of yield in pepper as an example of complex trait measured in a number of environments. Main objectives were; i) to propose a number of mixed models to detect QTLs for multiple traits and multiple environments, ii) to extend the multi-trait QTL models to a multi-trait genomic prediction model, iii) to study how well the complex trait yield can be indirectly predicted from its component traits, and iv) to understand the ‘causal’ relationships between the target trait yield and its component traits.
The thesis is part of an EU-FP7 project “Smart tools for Prediction and Improvements of Crop Yield” (SPICY- http://www.spicyweb.eu/). This project generated phenotypic data from four environments using 149 individuals from the sixth generation of recombinant inbred lines obtained from intraspecific cross between large – fruited inbred pepper cultivar ‘Yolo Wonder’ (YW) and the hot pepper cultivar ‘Criollo de Morelos 334’ (CM 334). A total of 16 physiological traits were evaluated across the four trials and various types of genetic parameters were estimated. In a first analysis, the traits were univariately analyzed using linear mixed model. Trait heritabilities were generally large (ranging between 0.43 – 0.96 with an average of 0.86) and mostly comparable across trials while many of the traits displayed heterosis and transgression. The same QTLs were detected across the four trials, though QTL magnitude differed for many of the traits. We also found that some QTLs affected more than one trait, suggesting QTL pleiotropy (a QTL region affecting more than one trait). We discussed our results in the light of previously reported QTLs for these and similar traits in pepper.
We addressed the presence of genotype-by-environment interaction (GEI) in yield and the other traits through a multi-environment (ME) mixed model methodology with terms for QTL-by-environment interaction (QEI). We opined that yield would benefit from joint analysis with other traits and so deployed two other mixed model based multi-response QTL approaches: a multi-trait approach (MT) and a multi-trait multi-environment approach (MTME). For yield as well as the other traits, MTME was superior to ME and MT in the number of QTLs, the explained variance and accuracy of predictions. Many of the detected QTLs were pleiotropic and showed quantitative QEI. The results confirmed the feasibility and strengths of novel mixed model QTL methodology to study the architecture of complex traits.
The QTL methods considered thus far are not well suited for prediction purposes as only a limited set of QTL-related markers are used. Since the main interest of this research includes improvement of yield prediction, we explored both single-trait and multi-trait versions of genomic prediction (GP) models as alternatives to the QTL-based prediction (QP) models. This was termed direct prediction. The methods differed in their predictive accuracies with GP methods outperforming QP methods in both single and multi-traits situations. We borrowed ideas from crop growth model (CGM) to dissect complex trait yield into a number of its component traits. Here, we integrated QTL/genomic prediction and CGM approaches and showed that the target trait yield can be predicted via its component traits together with environmental covariables. This was termed indirect prediction. The CGM approach seemed to work well at first sight, but this is especially due to the fact that yield appeared to be strongly driven by just one of its components, the partitioning to fruit.
An alternative representation of the biological knowledge of a complex target trait such as yield is provided by network type models. We constructed both conditional and unconditional networks across the four environments to understand the ‘causal’ relationships between target trait yield and its component traits. The final networks for each environment from both conditional and unconditional methods were used in a structural equation model to assess the causal relationships. Conditioning QTL mapping on network structure improved detection of refined genetic architecture by distinguishing between QTL with direct and indirect effects, thereby removing non-significant effects found in the unconditional network and resolving QTL pleiotropy. Similar to the CGM topology, yield was established to be downstream to its component traits, indicating that yield can be studied and predicted from its component traits. Thus, the genetic improvements of yield would benefit from improvements on the component traits.
Finally, complex trait prediction can be enhanced by a full integration of the methods described in the different chapters. Recent research efforts have been channelled to incorporating both multivariate whole genome prediction models and crop growth models. Further research is required, but we hope that the present thesis presents useful steps towards better prediction models for complex traits exhibiting genotype by environment interaction.
Ecophysiology and environmental distribution of organohalide-respiring bacteria
Lu, Y. - \ 2016
Wageningen University. Promotor(en): Hauke Smidt, co-promotor(en): Siavash Atashgahi. - Wageningen : Wageningen University - ISBN 9789462578418 - 239
bacteria - halides - ecophysiology - phylogenetics - genomics - lakes - halogens - pollutants - bacteriën - haliden - ecofysiologie - fylogenetica - genomica - meren - halogenen - verontreinigende stoffen
Organohalide-respiring bacteria (OHRB) are able to breathe natural and anthropogenically produced organohalides persistent in a broad range of oxygen-depleted environments. Therefore, these microorganisms are of high interest for organohalide-contaminated site bioremediation and natural halogen and carbon cycle. Nevertheless, to assess and adjust in situ bioremediation strategies and to enhance current understanding about the role of OHRB in natural habitats, thorough understanding of their ecophysiology and interaction with surrounding biotic and abiotic forces is necessary. To this end, this thesis focused on exploring ecophysiology and environmental distribution of OHRB in pristine and contaminated sites and unraveling their interactions with the co-existing microbial guilds in the community and geochemical parameters by application of a suite of physiological, molecular and geochemical analyses.
Based on a comprehensive overview of currently known organohalide-respiring isolates and their environmental distribution, the presence of yet unknown OHRB in extreme environments was proposed as the known organohalide-respiring isolates survive/thrive at a moderate range of pH and salinity in laboratory culture. Therefore, the OHRB were surveyed in alkaline and hypersaline sediments collected from Lake Strawbridge, Western Australia, that was known to emit organohalides. As a result, for the first time, the dechlorination of chloroform and perchloroethene (PCE) to dichloromethane and trichloroethene, respectively， was documented from an alkaline hypersaline pristine environment.
Corrinoids are essential cofactors for the activity of reductive dehalogenase enzymes. Ironically, some OHRB are reported to be corrinoid auxotrophs. Using transcriptional analysis and shotgun proteomics, here we show corrinoid auxotrophy in Dehalobacter restrictus PER-K23T. This detrimental deficiency seems to be compensated by up-regulation of relevant cobalamin salvaging and transport pathways to ensure sufficient corrinoid supply under partial corrinoid starvation. Hence, such OHRB incapable of de novo corrinoid synthesis will be dependent on non-dechlorinating community members to fulfill their nutritional needs indicating paramount importance of syntrophic interactions in supporting robust growth and activity of OHRB.
Bacterial community analysis of chlorinated benzene dechlorinating consortia derived from contaminated harbour sludge suggested members of the Bacteroidetes phylum and Clostridiales order as well as sulfate-reducing Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to OHRB i.e. D. mccartyi and Dehalobacter. However, despite well-controlled lab condition, syntrophic interactions could be influenced by geochemical parameters under field settings. Accordingly, analysis of geochemical and microbial determinants of OHR at a site biostimulated by glycerol injection further verified supportive role of fermenters and sulfate reducers under highly reduced condition following biostimulation. However, towards the end of field experiment, reducing condition faded and sulfate increased concurrent with the appearance of Epsilonproteobacteria and Deferribacteres as putative oxidizers of reduced sulfur compounds. The latter guilds might serve as detoxifiers of sulfide and thereby stimulate D. mccartyi, but could also be inhibitory as successors of the more important syntrophic fermenting and sulfate reducing bacteria.
In conclusion, this thesis expands our understanding of ecophysiology and environmental distribution of OHRB, addressing their presence in pristine environments as well as providing further evidence for their dependencies on other microbial community members in order to meet their nutritional requirements. Hence, research described here strengthens the scientific foundation for evaluating and optimizing strategies for the bioremediation of organohalide-contaminated sites and expands the natural niche of OHRB to extreme pristine environments.
An evolutionary and functional genomics study of Noccaea caerulescens, a heavy metal hyperaccumulating plant species
Wang, Y. - \ 2016
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - Wageningen : Wageningen University - ISBN 9789462578562 - 190
brassicaceae - genomics - hyperaccumulator plants - heavy metals - genes - genetic variation - brassicaceae - genomica - hyperaccumulerende planten - zware metalen - genen - genetische variatie
Noccaea caerulescens is the only known Zn/Cd/Ni hyperaccumulator. The Ganges accession (2n = 14) has an, yet unpublished, genome size of ~319 Mb, with 29,712 predicted genes representing 15,874 gene families. This species is distributed mainly in Europe. Three ecotypes can be distinguished: two metallicolous ecotypes, resident to serpentine soil (Ni enriched) and calamine soil (Zn/Cd enriched), and a non-metallicolous ecotype, growing on regular, non-metalliferous soils. The physiological differences that underlie variation in heavy metal accumulation and tolerance are well-understood, and the molecular basis of hyperaccumulation and tolerance has been explored by transcript profiling in the presence of metals and by comparative transcriptome analysis using N. caerulescens and non-hyperaccumulators such as Arabidopsis thaliana. The genetic variation which emerged during the evolution of metal hyperaccumulation has not yet been investigated. The work described in this thesis considers the identification of genetic variation under selection for Zn/Cd hyperaccumulation and tolerance by next generation resequencing of the wild metallicolous (calamine) and non-metallicolous populations and the generation of a mutant N. caerulescens library for functional analysis. The regulation of flowering time was also investigated, using early flowering mutants selected from the mutant library.
Host-plant resistance to western flower thrips in Arabidopsis
Thoen, Manus P.M. - \ 2016
Wageningen University. Promotor(en): Marcel Dicke; Harro Bouwmeester, co-promotor(en): Maarten Jongsma. - Wageningen : Wageningen University - ISBN 9789462578807 - 191
arabidopsis thaliana - host plants - insect pests - frankliniella occidentalis - defence mechanisms - pest resistance - genomics - genome analysis - host-seeking behaviour - optical tracking - data analysis - insect plant relations - arabidopsis thaliana - waardplanten - insectenplagen - frankliniella occidentalis - verdedigingsmechanismen - plaagresistentie - genomica - genoomanalyse - gedrag bij zoeken van een gastheer - optisch sporen - gegevensanalyse - insect-plant relaties
Western flower thrips is a pest on a large variety of vegetable, fruit and ornamental crops. The damage these minute slender insects cause in agriculture through feeding and the transmission of tospoviruses requires a sustainable solution. Host-plant resistance is a cornerstone of Integrated Pest Management (IPM). Plants have many natural defense compounds and morphological features that aid in the protection against herbivorous insects. However, the molecular and physiological aspects that control host-plant resistance to thrips are largely unknown.
A novel and powerful tool to study host-plant resistance to insects in natural populations is genome-wide association (GWA) mapping. GWA mapping provides a comprehensive untargeted approach to explore the whole array of plant defense mechanisms. The development of high-throughput phenotyping (HTP) systems is a necessity when large plant panels need to be screened for host-plant resistance to insects. An automated video-tracking platform that could screen large plant panels for host-plant resistance to thrips, and dissect host-plant resistance to thrips in component traits related to thrips behavior, was developed. This phenotyping platform allows the screening for host-plant resistance against thrips in a parallel two-choice setup using EthoVision tracking software. The platform was used to establish host-plant preference of thrips with a large plant population of 345 wild Arabidopsis accessions (the Arabidopsis HapMap population) and the method was optimized with two extreme accessions from this population that differed in resistance to thrips. This method can be a reliable and effective high throughput phenotyping tool to assess host-plant resistance to thrips in large plant populations. EthoAnalysis, a novel software package was developed to improve the analyses of insect behavior. There were several benefits from using EthoAnalysis to analyze EthoVision data. The detailed event statistics that could be extracted from EthoAnalysis allows researchers to distinguish detailed differences in moving and feeding behavior of thrips. The potential of this additional information is discussed in the light of quantitative genetic studies.
Stress resistance was studied in the HapMap population on a total of 15 different biotic and abiotic stresses ranging from biotic stresses like insects and nematodes, to abiotic stresses like drought and salt. A multi-trait GWA study to unravel the genetic architecture underlying plant responses to the different stresses was performed. A genetic network in this study revealed little correlation between the plant responses to the different insect herbivores studied (aphids, whiteflies, thrips and caterpillars). For thrips resistance a weak positive correlation with resistance to drought stress and Botrytis, and a negative correlation with resistance to parasitic plants were observed. One of the surprising outcomes of this study was the absence of shared major QTLs for host-plant resistance and abiotic stress tolerance mechanisms. RESISTANCE METHYLATED GENE 1 (RMG1) was one of the candidate genes in this multi-trait GWA study that could be controlling shared resistance mechanisms against many different stresses in Arabidopsis. RMG1 is a nucleotide-binding site Leucine-rich repeat (NB-LRR) disease resistance protein and its potential relation to several resistance/tolerance traits was successfully demonstrated with T-DNA insertion lines.
The 15 stresses were used in a comparison with a metabolomics dataset on this Arabidopsis HapMap population. It was discovered that levels of certain aliphatic glucosinolates correlated positively with the levels of resistance to thrips. This correlation was further investigated with the screening of a RIL (Recombinant Inbred Line) population for resistance to thrips, several knockout mutants and the analysis of co-localization of GWA mapping results between glucosinolates genes and thrips resistance. In a GWA analysis, the C4 alkenyl glucosinolates that correlated the strongest with thrips resistance mapped to the genomic regions containing genes known to regulate the biosynthesis of these compounds. However, thrips resistance did not co-localize with any of the GSL genes, unless a correction for population stratification was omitted. Additional screening of a Cvi x Ler RIL population showed a QTL for thrips resistance on chromosome 2, but no co-localization with any known glucosinolate genes, nor with thrips resistance loci identified by GWA mapping. Knock-out mutants and overexpressors of glucosinolate synthesis genes could also not confirm a causal link between glucosinolates and resistance to thrips. It is possible that the crucial factors that control resistance to thrips may not have been present in sufficient quantities or in the right combinations in the mutants, RILs and NIL screened in this study. Alternatively, the correlation between thrips feeding damage and glucosinolate profiles could be based on independent geographical clines. More research should be conducted to assess which of these explanations is correct.
In the general discussion, the results from this thesis are discussed in a broader perspective. Some prototypes of new phenotyping platforms that could further aid screening for resistance to thrips in the future are presented. Natural variation in host-plant resistance to thrips is compared to the variation in host-plant resistance to aphids and caterpillars. The geographic distribution of host-plant resistance to thrips is not evident in the other insects, in line with the distribution of glucosinolate profiles and other climate factors. The chapter concludes with some suggestions for future research in the field of host-plant resistance to thrips.
Mechanistic dissection of plant embryo initiation
Radoeva, T.M. - \ 2016
Wageningen University. Promotor(en): Dolf Weijers, co-promotor(en): Sacco de Vries. - Wageningen : Wageningen University - ISBN 9789462578135 - 183
embryogenesis - embryos - plants - auxins - genes - genomics - arabidopsis - cell suspensions - in vivo experimentation - zygotes - monozygotic twins - embryogenese - embryo's - planten - auxinen - genen - genomica - arabidopsis - celsuspensies - in vivo experimenten - zygoten - monozygote tweelingen
Land plants can reproduce sexually by developing an embryo from a fertilized egg cell, the zygote. After fertilization, the zygote undergoes several rounds of controlled cell divisions to generate a mature embryo. However, embryo formation can also be induced in a variety of other cell types in many plant species. These non-zygotic embryos go through analogous developmental phases and are morphologically similar to the zygotic embryo. Despite its fundamental importance and enormous application potential, the mechanisms that alter cell fate from non-embryonic to embryonic are elusive. In the past decades, a variety of different model systems have been used to identify regulators of embryo induction, but it is unclear if these act in a common network. We recently found that inhibition of auxin response in the extra-embryonic suspensor cells cell-autonomously and predictably triggers a switch towards embryo identity. In my thesis I have used the suspensor-derived embryogenesis as a uniform model system to study the crucial first reprogramming step of embryo initiation process.
Through genome-wide transcriptional profiling upon local (suspensor-specific) auxin response inhibition (Chapter 2) and through testing the ability of fifteen known embryogenesis inducers to promote embryo formation in suspensor cells (Chapter 3), we suggest that suspensor to embryo transformation requires a defined set of genetic regulators. The results obtained in my thesis provide essential tools and basis for further research and are a step forward to understanding the first step of embryo initiation process and to unravel the mystery of totipotency in plants.
Akkermansia species : phylogeny, physiology and comparative genomics
Ouwerkerk, J.P. - \ 2016
Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): Clara Belzer. - Wageningen : Wageningen University - ISBN 9789462577411 - 178
akkermansia - akkermansia muciniphila - gastrointestinal microbiota - phylogeny - physiology - genomics - dna sequencing - nucleotide sequences - transcriptomes - antibiotic resistance - genome annotation - akkermansia - akkermansia muciniphila - microbiota van het spijsverteringskanaal - fylogenie - fysiologie - genomica - dna-sequencing - nucleotidenvolgordes - transcriptomen - antibioticaresistentie - genoomannotatie
The gastrointestinal tract is lined with a mucus layer, which is colonized by a distinct mucosal microbial population. The anaerobic gut bacterium Akkermansia muciniphila is a well-described member of the mucosal microbiota and has been shown to be a human gut symbiont. In the mucus layer this gut symbiont is likely exposed to the oxygen that diffuses from mucosal epithelial cells. We showed that A. muciniphila has an active detoxification system to cope with reactive oxygen species and can use oxygen for respiration at nanomolar oxygen concentrations, with cytochrome bd as terminal oxidase.
Until now, the type strain A. muciniphila MucT was the only cultured representative of this species. We isolated and characterized six new A. muciniphila strains from faecal samples of four different human subjects. These A. muciniphila strains showed minimal genomic and physiologic divergence while retaining their mucin degrading and utilisation capacities. Apart from the human gastrointestinal tract, we detected Akkermansia species in intestinal samples of numerous mammals. An additional ten new A. muciniphila strains were isolated from seven different mammalian species and showed high genomic and physiologic similarity to type strain A. muciniphila MucT. Apart from Akkermansia species, other Verrucomicrobia were identified within the gastrointestinal tract of non-human mammals. Furthermore, we obtained an Akkermansia isolate from the reticulated python, which had a similar mucin degrading capacity as the human strain A. muciniphila MucT but showed more efficient galactose utilization. On the basis of further phylogenetic, physiological, and genomic characterisations, strain PytT was found to represent a novel species within the genus Akkermansia, for which the name Akkermansia glycaniphilus sp. nov. is proposed.
Overall, A. muciniphila strains isolated from intestinal samples of human and other mammals show very limited genomic and physiologic divergence. This together with the widely-spread global presence of A. muciniphila and the dependence on mucin for optimal growth, points towards a conserved symbiosis. This conserved symbiosis might be indicative for the beneficial role of this organism in respect to the host metabolic health. This is in line with the observation that A. muciniphila has been negatively associated with obesity and its associated metabolic disorders.
In mice, treatment with viable A. muciniphila cells reversed high-fat diet-induced obesity. We described a scalable workflow for the preparation and preservation of high numbers of viable cells of A. muciniphila under strict anaerobic conditions for therapeutic interventions. Moreover, we developed various quality assessment and control procedures aimed to ensure the use of viable cells of A. muciniphila at any location in the world. These viable cells were used in a pilot study in humans in which no adverse events were observed. This is promising for future applications of A. muciniphila as a new therapeutic, leading towards the potential treatment of unhealthy states of the microbiota.
The fish egg microbiome : diversity and activity against the oomycete pathogen Saprolegnia
Liu, Y. - \ 2016
Wageningen University. Promotor(en): Francine Govers; Jos Raaijmakers, co-promotor(en): Irene de Bruijn. - Wageningen : Wageningen University - ISBN 9789462577671 - 169
salmon - fish eggs - marine microorganisms - microbial diversity - bioinformatics - genomics - saprolegnia - oomycota - fish diseases - suppression - fungal antagonists - zalm - visseneieren - mariene micro-organismen - microbiële diversiteit - bio-informatica - genomica - saprolegnia - oömycota - visziekten - onderdrukking - schimmelantagonisten
Prof. dr. F. Govers (promotor); Prof. dr. J.M. Raaijmakers (promotor); Dr. I. de Bruijn (co-promotor); Wageningen University, 13 June 2016, 170 pp.
The fish egg microbiome: diversity and activity against the oomycete pathogen Saprolegnia
Emerging oomycete pathogens increasingly threaten biodiversity and food security. This thesis describes the study of the microbiome of Atlantic salmon (Salmo salar L.) eggs and analyses of the effects of infections by the oomycete pathogen Saprolegnia on the microbial architecture. A low incidence of Saprolegniosis was correlated with a relatively high abundance and richness of specific commensal Actinobacteria. Among the bacterial community, the isolates Frondihabitans sp. 762G35 (Microbacteriaceae) and Pseudomonas sp. H6 significantly inhibited hyphal attachment of Saprolegnia diclina to live salmon eggs. Chemical profiling showed that these two isolates produce furancarboxylic acid-derived metabolites and a lipopeptide viscosin-like biosurfactant, respectively, which inhibited hyphal growth of S. diclina in vitro. Among the fungal community, the fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride. Both a quantitative and qualitative difference in the Trichoderma population between Saprolegnia-infected and healthy salmon eggs was observed, which suggested that mycoparasitic Trichoderma species could play a role in Saprolegnia suppression in aquaculture. This research provides a scientific framework for studying the diversity and dynamics of microbial communities to mitigate emerging diseases. The Frondihabitans, Pseudomonas and Trichoderma isolates, and/or their bioactive metabolites, are proposed as effective candidates to control Saprolegniosis.
Mapping and fine-mapping of genetic factors affecting bovine milk composition
Duchemin, S.I. - \ 2016
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Henk Bovenhuis; Marleen Visker; Willem F. Fikse. - Wageningen : Wageningen University - ISBN 9789462577305 - 190
dairy cows - dairy cattle - milk composition - milk fat - genetic factors - quantitative trait loci - genomics - genetic mapping - animal genetics - melkkoeien - melkvee - melksamenstelling - melkvet - genetische factoren - loci voor kwantitatief kenmerk - genomica - genetische kartering - diergenetica
Duchemin, S.I. (2016). Mapping and fine-mapping of genetic factors affecting bovine milk composition. Joint PhD thesis, between Swedish University of Agricultural Sciences, Sweden and Wageningen University, the Netherlands
Bovine milk is an important source of nutrients in Western diets. Unraveling the genetic background of bovine milk composition by finding genes associated with milk-fat composition and non-coagulation of milk were the main goals of this thesis. In Chapter 1, a brief description of phenotypes and genotypes used throughout the thesis is given. In Chapter 2, I calculated the genetic parameters for winter and summer milk-fat composition from ~2,000 Holstein-Friesian cows, and concluded that most of the fatty acids (FA) can be treated as genetically the same trait. The main differences between milk-fat composition between winter and summer milk samples are most likely due to differences in diets. In Chapter 3, I performed genome-wide association studies (GWAS) with imputed 777,000 single nucleotide polymorphism (SNP) genotypes. I targeted a quantitative trait locus (QTL) region on Bos taurus autosome (BTA) 17 previously identified with 50,000 SNP genotypes, and identified a region covering 5 mega-base pairs on BTA17 that explained a large proportion of the genetic variation in de novo synthesized milk FA. In Chapter 4, the availability of whole-genome sequences of keys ancestors of our population of cows allowed to fine-mapped BTA17 with imputed sequences. The resolution of the 5 mega base-pairs region substantially improved, which allowed the identification of the LA ribonucleoprotein domain family, member 1B (LARP1B) gene as the most likely candidate gene associated with de novo synthesized milk FA on BTA17. The LARP1B gene has not been associated with milk-fat composition before. In Chapter 5, I explored the genetic background of non-coagulation of bovine milk. I performed a GWAS with 777,000 SNP genotypes in 382 Swedish Red cows, and identified a region covering 7 mega base-pairs on BTA18 strongly associated with non-coagulation of milk. This region was further characterized by means of fine-mapping with imputed sequences. In addition, haplotypes were built, genetically differentiated by means of a phylogenetic tree, and tested in phenotype-genotype association studies. As a result, I identified the vacuolar protein sorting 35 homolog, mRNA (VPS35) gene, as candidate. The VPS35 gene has not been associated to milk composition before. In Chapter 6, the general discussion is presented. I start discussing the challenges with respect to high-density genotypes for gene discovery, and I continue discussing future possibilities to expand gene discovery studies, with which I propose some alternatives to identify causal variants underlying complex traits in cattle.
Regulation of cucumber (Cucumis sativus) induced defence against the two-spotted spider mite (Tetranychus urticae
He, J. - \ 2016
Wageningen University. Promotor(en): Harro Bouwmeester; Marcel Dicke, co-promotor(en): Iris Kappers. - Wageningen : Wageningen University - ISBN 9789462576810 - 211
cucumis sativus - cucumbers - induced resistance - plant pests - tetranychus urticae - mites - defence mechanisms - herbivore induced plant volatiles - herbivory - metabolomics - terpenoids - genomics - cucumis sativus - komkommers - geïnduceerde resistentie - plantenplagen - tetranychus urticae - mijten - verdedigingsmechanismen - herbivoor-geinduceerde plantengeuren - herbivorie - metabolomica - terpenen - genomica
Plants have evolved mechanisms to combat herbivory. These mechanisms can be classified as direct defences which have a negative influence on the herbivores and indirect defence that attracts natural enemies of the attacking herbivores. Both direct and indirect defences can be constantly present or induced upon attack. This study, using cucumber (Cucumis sativus) and the two-spotted spider mite (Tetranychus urticae) as model, aimed to reveal the molecular mechanisms underlying the induced defence during herbivory, with emphasis on transcriptional changes and the involved TFs, the enzymatic function of the genes associated with volatile biosynthesis, and their promoters which regulate their expression.
Ecophysiology of novel intestinal butyrate-producing bacteria
Bui, Thi Phuong Nam - \ 2016
Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): Caroline Plugge. - Wageningen : Wageningen University - ISBN 9789462577015 - 202
butyrates - butyric acid bacteria - intestines - microbial interactions - faecal examination - mice - man - infants - genomics - intestinal physiology - microbial physiology - biochemical pathways - lysine - sugar - butyraten - boterzuurbacteriën - darmen - microbiële interacties - fecesonderzoek - muizen - mens - zuigelingen - genomica - darmfysiologie - microbiële fysiologie - biochemische omzettingen - lysine - suiker
The human intestinal tract harbours a trillion on microbial cells, predominantly anaerobes. The activity and physiology of these anaerobes is strongly associated with health and disease. This association has been investigated for a long time.However, this has not been fully understood. One of the reasons is the limited availability of cultured representatives. It is estimated that there may be more than 3000 species colonised in the gut of healthy individuals, however, only a bit over 1000 species have been isolated and characterised. Among the intestinal microbes, butyrate-producing bacteria are of special interest as the butyrate produced, is crucial to maintain a healthy gut. In addition, butyrate-producing bacteria have shown a reverse correlation with several intestinal diseases. In Chapter 2 we described a novel species Anaerostipes rhamnosivorans 1y2T isolated from an infant stool. This strain belonged to genus Anaerostipes within Clostridium cluster XIVa. A. rhamnosivorans had a capability of converting rhamnose into butyrate that is unique within intestinal butyrate-producing bacteria. The genomic analysis also revealed the entire rhamnose fermentation pathway as well as the acetyl-CoA pathway for butyrate production. This bacterium is able to produce butyrate from a wide range of sugars as well as lactate plus acetate. In Chapter 3, we described the microbial interactions between A. rhamnosivorans and Bacteriodes thetaiotaomicron in dietary pectins; Blautia hydrogenotrophica in lactate and small amount of acetate; Methanobrevibacter smithii in glucose. We observed that A. rhamnosivorans was able to benefit from its partners in all cocultures for butyrate production. This is likely due to its high metabolic flexibility. While the interaction between A. rhamnosivorans and B. thetaiotaomicron appeared as syntrophy, the interaction between A. rhamnosivorans and hydrogenotrohic microbes were cross-feeding type where hydrogen was transferred between two species. The latter resulted in an increase in butyrate level. In Chapter 4 we described a novel species Intestinimonas butyriciproducens SRB521T representing a novel genus Intestinimonas from a mouse caecum within Clostridium cluster IV. This bacterium produced butyrate and acetate as end products from Wilkins-Chalgren-Anaerobe broth.
Butyrate production is assumed to derive from carbohydrate employing acetyl-CoA pathway. No gut bacterium is known to convert proteins or amino acids to butyrate although butyrogenic pathways from amino acid degradation have been detected in the human gut using metagenomic approach. In Chapter 5 we discovered a novel butyrate synthesis pathway from the amino acid lysine and the Amadori product fructoselysine in Intestinimonas butyriciproducens AF211 that was isolated from human stool. This strain appeared to grow much better in lysine as compared to sugars although lysine and acetyl-CoA pathways were both detected in its complete genome. Moreover, the strain AF211 was able to metabolise efficiently fructoselysine into butyrate, and acetate was found to affect the fructoselysine fermentation, representing the impact of the environmental conditions where acetate is abundant in the gut. While the lysine pathway was found in the gut of many individuals, the fructoselysine pathway was present in only half of those samples. The finding that strain I. butyriciproducens AF211 is capable of the butyrogenic conversion of amino acid lysine and fructoselysine, an Amadori product formed in heated foods via the Maillard reaction, indicated a missing link that coupling protein metabolism and butyrate formation. As this Amadori product has been implicated to play a role in aging process, the use of strain AF211 as fructoselysine clearance in the gut needs further investigation. In Chapter 6 we performed genomic and physiological comparison between the I. butyriciproducens strain AF211 (human isolate) and SRB521T (mouse isolate). I. butyriciproducens was the most abundant species within the Intestinimonas genus and highly prevalent in humans based on metadata analysis on 16S amplicons. We confirmed that the butyrogenesis from lysine was a shared characteristic between the two I. butyriciproducens strains. We also observed the host specific features including tolerance to bile, cellular fatty acid composition, more efficient capability of converting sugars into butyrate, especially galactose and arabinose, in the human strain AF211. In addition, genomic rearrangements as well as variations in bacteriophages differed among strains.
Genomic selection in egg-laying chickens
Heidaritabar, M. - \ 2016
Wageningen University. Promotor(en): Martien Groenen, co-promotor(en): John Bastiaansen. - Wageningen : Wageningen University - ISBN 9789462576704 - 220
hens - genomics - genetic variation - selective breeding - quantitative traits - breeding value - animal genetics - animal breeding - hennen - genomica - genetische variatie - selectief fokken - kwantitatieve kenmerken - fokwaarde - diergenetica - dierveredeling
Heidaritabar, M. (2016). Genomic selection in egg-laying chickens. PhD thesis, Wageningen University, the Netherlands
In recent years, prediction of genetic values with DNA markers, or genomic selection (GS), has become a very intense field of research. Many initial studies on GS have focused on the accuracy of predicting the genetic values with different genomic prediction methods. In this thesis, I assessed several aspects of GS. I started with evaluating results of GS against results of traditional pedigree-based selection (BLUP) in data from a selection experiment that applied both methods side by side. The impact of traditional selection and GS on the overall genome variation as well as the overlap between regions selected by GS and the genomic regions predicted to affect the traits were assessed. The impact of selection on genome variation was assessed by measuring changes in allele frequencies that allowed the identification of regions in the genome where changes must be due to selection. These frequency changes were shown to be larger than what could be expected from random fluctuations, indicating that selection is really affecting the allele frequencies and that this effect is stronger in GS compared with BLUP. Next, concordance was tested between the selected regions and regions that affect the traits, as detected by a genome-wide association study. Results showed a low concordance overall between the associated regions and the selected regions. However, markers in associated regions did show larger changes in allele frequencies compared with the average changes across the genome. The selection experiment was performed using a medium density of DNA markers (60K). I subsequently explored the potential benefits of whole-genome sequence data for GS by comparing prediction accuracy from imputed sequence data with the accuracy obtained from the 60K genotypes. Before sequencing, the selection of key animals that should be sequenced to maximize imputation accuracy was assessed with the original 60K genotypes. The accuracy of genotype imputation from lower density panels using a small number of selected key animals as reference was compared with a scenario where random animals were used as the reference population. Even with a very small number of animals as reference, reasonable imputation accuracy could be obtained. Moreover, selecting key animals as reference considerably improved imputation accuracy of rare alleles compared with a set of random reference animals. While imputation from a small reference set was successful, imputation to whole-genome sequence data hardly improved genomic prediction accuracy compared with the predictions based on 60K genotypes. Using only those markers from the whole-genome sequence that are more likely to affect the phenotype was expected to remove noise from the data, but resulted in slightly lower prediction accuracy compared with the complete genome sequence. Finally, I evaluated the inclusion of dominance effects besides additive effects in GS models. The proportion of variance due to additive and dominance effects were estimated for egg production and egg quality traits of a purebred line of layers. The proportion of dominance variance to the total phenotypic variance ranged from 0 to 0.05 across traits. Also, the impact of fitting dominance besides additive effects on prediction accuracy was investigated, but was not found to improve accuracy of genomic prediction of breeding values.
Breeding against infectious diseases in animals
Rashidi, H. - \ 2016
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Herman Mulder; P.K. Mathur. - Wageningen University - ISBN 9789462576452 - 179
livestock - infectious diseases - animal breeding - selective breeding - disease resistance - tolerance - genetic variation - breeding value - genetic correlation - traits - genomics - animal genetics - vee - infectieziekten - dierveredeling - selectief fokken - ziekteresistentie - tolerantie - genetische variatie - fokwaarde - genetische correlatie - kenmerken - genomica - diergenetica
Infectious diseases in farm animals are of major concern because of animal welfare, production costs, and public health. Farms undergo huge economic losses due to infectious disease. The costs of infections in farm animals are mainly due to production losses, treatment of infected animals, and disease control strategies. Control strategies, however, are not always successful. Selective breeding for the animals that can mount a defence against infection could therefore be a promising approach. Defensive ability of an animal has two main mechanisms: resistance (ability to control the pathogen burden) and tolerance (ability to maintain performance when pathogen burden increases). When it is difficult to distinguish between resistance and tolerance, defensive ability is measured as resilience that is the ability to maintain performance during a disease outbreak regardless of pathogen burden. Studies have focused on the genetics of resistance and resilience with little known about the genetics of tolerance and its relationship with resistance and resilience. The objectives of this thesis were to: 1) estimate the genetic variation in resistance, tolerance, and resilience to infection in order to assess the amenability of these traits for selective breeding in farm animals, 2) estimate the genetic correlation between resistance, tolerance and resilience and 3) detect genomic regions associated with resistance, tolerance, and resilience.
In chapter 2, we studied the variation among sows in response to porcine reproductive and respiratory syndrome (PRRS). First a statistical method was developed to detect PRRS outbreaks based on reproduction records of sows. The method showed a high sensitivity (78%) for disease phases. Then the variation of sows in response to PRRS was quantified using 2 models on the traits number of piglets born alive (NBA) and number of piglets born dead (LOSS): 1) bivariate model considering the trait in healthy and disease phases as different traits, and 2) reaction norm model modelling the response of sows as a linear regression of the trait on herd-year-week estimates of NBA. Trait correlations between healthy and disease phases deviated from unity (0.57±0.13 – 0.87±0.18). The repeatabilities ranged from 0.07±0.027 to 0.16±0.005. The reaction norm model had higher predictive ability in disease phase compared to the bivariate model.
In chapter 3 we studied 1) the genetic variation in resistance and tolerance of sheep to gastrointestinal nematode infection and 2) the genetic correlation between resistance and tolerance. Sire models on faecal nematode egg count (FEC), IgA, and pepsinogen were used to study the genetic variation in resistance. Heritability for resistance traits ranged from 0.19±0.10 to 0.59±0.20. A random regression model was used to study the reaction norm of sheep body weight on FEC as an estimate of tolerance to nematode infection. We observed a significant genetic variance in tolerance (P<0.05). Finally a bivariate model was used to study the genetic correlation between resistance and tolerance. We observed a negative genetic correlation (-0.63±0.25) between resistance and tolerance.
In chapter 4, we studied the response to selection in resistance and tolerance when using estimated breeding values for resilience. We used Monte Carlo simulation to generate 100 half-sib families with known breeding values for resistance (pathogen burden) and tolerance. We used selection index theory to predict response to selection for resistance and tolerance: 1) when pathogen burden is known and selection is based on true breeding values for resistance and tolerance and 2) when pathogen burden is unknown and selection is based on estimated breeding values for resilience. Using EBV for resilience in absence of records for pathogen burden resulted in favourable responses in resistance and tolerance to infections, with more emphasis on tolerance than on resistance. However, more genetic gain in resistance and tolerance could be achieved when pathogen burden was known.
In chapter 5 we studied genomics regions associated with resistance, resilience, and tolerance to PRRS. Resistance was modelled as sire effect on area under the PRRS viremia curve up to 14 days post infection (AUC14). Resilience was modelled as sire effects on daily growth of pigs up to 28 days post infection (ADG28). Tolerance was modelled as the sire effect on the regression of ADG28 on AUC14. We identified a major genomics region on chromosome 4 associated with resistance and resilience to PRRS. We also identified genomics regions on chromosome 1 associated with tolerance to PRRS.
In the general discussion (chapter 6) I discussed: 1) response to infection as a special case of genotype by environment interaction, 2) random regression model as a statistical tool for studying response to disease, 3) advantages and requirements of random regression models, and 4) selective breeding of farm animals for resistance, tolerance, and resilience to infections. I concluded that random regression is a powerful approach to estimate response to infection in animals. If the adequate amount of data is available random regression model could estimate breeding values of animals more accurately compared to other models. I also concluded that before including resistance and tolerance into breeding programs, breeders should make sure about the added values of including these traits on genetic progress. Selective breeding for resilience could be a pragmatic approach to simultaneously improve resistance and tolerance.
Phenotypic and genetic diversity of the species Lactobacillus rhamnosus
Ceapa, C.D. - \ 2016
Wageningen University. Promotor(en): Michiel Kleerebezem; Jan Knol; J. Lambert. - Wageningen : Wageningen University - ISBN 9789462576285 - 195
fermentation products - probiotics - intestinal microorganisms - lactic acid bacteria - strains - medicinal properties - genomics - nutrition and health - fermentatieproducten - probiotica - darmmicro-organismen - melkzuurbacteriën - stammen (biologisch) - medicinale eigenschappen - genomica - voeding en gezondheid
The thesis explores the diversity of Lactobacillus rhamnosus, a species from which strains are studied for their anti-inflammatory, anti-allergic, and diarrhea preventing effects. The work combines observations on the behavior of the bacteria in a simplified laboratory setting (use of carbohydrates, immune modulation effects, anti-pathogenic effects) with genomic information obtained by sequencing, with the aim to pinpoint genes that could be relevant for bacterial survival and metabolic capacities. Phenotypic and genotypic profiling analyses congruently revealed that carbohydrate metabolism and transport is essential for this species’ adaptation to the environment. Genotype–phenotype correlation analysis enabled us to predict and then experimentally verify genes responsible for the utilization of L-Sorbose, L-Fucose α-D-Methyl Glycoside.
Multi-population genomic prediction
Wientjes, Y.C.J. - \ 2016
Wageningen University. Promotor(en): Roel Veerkamp; Mario Calus. - Wageningen : Wageningen University - ISBN 9789462576193 - 267
cum laude - dairy cattle - genomics - prediction - quantitative trait loci - genetic improvement - breeding value - selective breeding - animal breeding - animal genetics - melkvee - genomica - voorspelling - loci voor kwantitatief kenmerk - genetische verbetering - fokwaarde - selectief fokken - dierveredeling - diergenetica
Cum laude graduation