Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Unraveling the genetics of Botrytis cinerea resistance in Gerbera hybrida
    Fu, Yiqian - \ 2017
    Wageningen University. Promotor(en): R.G.F. Visser, co-promotor(en): P.F.P. Arens; J.M. van Tuyl. - Wageningen : Wageningen University - ISBN 9789463431811 - 159
    gerbera - plant pathogenic fungi - botrytis cinerea - disease resistance - genetic mapping - transcriptomics - quantitative trait loci - plant breeding - gerbera - plantenziekteverwekkende schimmels - botrytis cinerea - ziekteresistentie - genetische kartering - transcriptomica - loci voor kwantitatief kenmerk - plantenveredeling

    Gerbera hybrida is one of the top five cut flowers. It is well-known to people for its variation in flower color and patterning. Gerbera breeding at the moment is done using conventional methods which are based on a phenotypic selection. This has drawbacks in breeding speed and efficiency, especially for complex traits like disease resistance. Gerbera gray mold, promoted by high humidity during the production in greenhouses or by an accumulation of condensate during transportation, is a considerable threat to the gerbera production. Gerbera gray mold is caused by Botrytis cinerea and plant resistance to B. cinerea is considered to be a polygenic trait that needs the contribution of multiple loci, and on top of that is highly affected by the environment. Conventional breeding might be inefficient for improving Botrytis resistance in gerbera.

    In this study, the transcriptomes of four parents of two gerbera populations were sequenced using Illumina paired-end sequencing. Transcriptome data provides a resource for genetic dissection and an insight to explore gene functions for this ornamental crop. To identify the QTL regions leading to the phenotypic variation in Botrytis resistance, and establishing a relationship between marker genotype and phenotypic variation for marker assisted selection (MAS), genetic linkage maps were constructed with SNP markers in the two F1 segregating populations. A total of 20 QTLs were identified in the parental maps of the two populations. The number of QTLs found and the explained variance of most QTLs detected reflects the complex mechanism of Botrytis disease response. Narrowing down the QTL region and identifying the causal gene(s) underlying a QTL could maximize the effective use of MAS in breeding. Homologs of known functional genes involved in Botrytis resistance from other species were obtained in gerbera and SNP markers identified and mapped. Twenty-nine candidate genes were mapped and seven candidate genes could be mapped on both populations. Seven candidate genes were located in the vicinity of the QTLs detected. The co-localization of QTLs with CGs gives an indication that these candidate genes could probably be involved in resistance to Botrytis and provide a more precise possibility to use MAS in gerbera breeding in the future. A tobacco rattle virus (TRV) based gene silencing system which was used to inspect the function of two candidate genes. The two CGs are the homologs of the genes responsible for Botrytis resistance in tomato and both mapped in QTL regions related to Botrytis resistance in gerbera ray floret test. Silencing the two genes by VIGS, showed smaller lesion sizes upon Botrytis infection on gerbera ray florets compared with the controls.

    The entire research went from the generation of four parental transcriptome data sets to development of SNP markers (Chapter 2), construction of genetic maps and to mapping QTLs for Botrytis resistance (Chapter 3). This was further on combined with candidate gene searching in other crops, querying and mapping homologous genes (Chapter 4) and characterizing the candidate genes which co-localized with QTLs (Chapter 5). The whole process not only helped us to unravel the genetics of Botrytis resistance in gerbera and develop genetic tools for gerbera improvement, but also could serve as guidance for developing marker-assisted selection for other ornamental plants from the beginning.

    The allo-octoploid strawberry: simply complex
    Dijk, Thijs van - \ 2016
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Eric van de Weg. - Wageningen : Wageningen University - ISBN 9789462579637 - 185
    fragaria ananassa - strawberries - polyploidy - microsatellites - linkage mapping - genome analysis - quantitative trait loci - genetic mapping - flowering - plant breeding - fragaria ananassa - aardbeien - polyploïdie - microsatellieten - koppelingskartering - genoomanalyse - loci voor kwantitatief kenmerk - genetische kartering - bloei - plantenveredeling

    The garden strawberry (Fragaria x ananassa) is a fruit species that was developed through human intervention less than 300 years ago. Currently, it is the most important soft fruit in both production as well as value and renowned for its deliciousness. There are many challenges in growing such a delicate fruit, many of which have been overcome through improved cultivation techniques and breeding. The perishability of the product is, however, not the only challenge faced by strawberry breeders. In terms of genome composition, strawberry appears to have accumulated a wonderful array of obstacles to genetic studies. It is a vegetatively propagated allo-octoploid outbreeder, and only few crop species are worse of in this respect. Many of the molecular genetic ground work is therefore performed in its diploid ancestor, the woodland strawberry Fragaria vesca, which was sequenced in 2011. However, since nearly all strawberries that are eaten are octoploid, genetic research can’t linger at the wild diploids forever. In this thesis we developed new tools and analysis methods for genetic studies in the allo-octoploid strawberry and subsequently applied these methods in the detection of marker-trait associations.

    The purpose of Chapter 2 was to develop a method to interpret the complex peak patterns generated by microsatellites in octoploid strawberry in such a way that we ended up with as much information as one would expect to retrieve from a microsatellite in a diploid system. This information could then be used to generate high quality linkage maps for the different sub-genomes and allow for easy alignment and comparison. We named the method MADCE, which stands for Microsatellite Allele Dose & Configuration Establishment. In the MADCE methodology, we first need to determine the dose of each allele present in an individual. For this we used the signal of fluorescent microsatellite peaks in relation to the total fluorescent signal generated by all peaks for that microsatellite. We then used the disomic inheritance of strawberry to establish the allelic configuration of each different homoeologue (subgenome). The repulsion of alleles from the same subgenome in offspring allowed us to form subgenomic pairs of alleles. We found that in single cross mapping populations, the deployment of our method was fairly easy due to the high number of offspring that can be used to establish repulsion between alleles. However, for pedigreed breeding germplasm this was another matter, as generally only few offspring were available. For this we added some additional tricks to the MADCE method, although some uncertainty about the configuration would remain for problematic lines and alleles.

    In Chapter 3 we used the MADCE method from Chapter 2 to generate a genome wide linkage map for the Holiday x Korona (HxK) mapping population. This linkage map was to be used in subsequent experiments for QTL discovery as well as provide the strawberry community with a highly detailed map consisting not only of marker distances, but allele and haplotype configuration of the parents Holiday and Korona as well. The haplotype information revealed that inbreeding (homozygosity) levels in Holiday were similar to the levels expected from its pedigree, but that inbreeding levels of Korona were more than three times higher than expected, which could be resultant from selection pressure enacted by breeders. Selection pressure could also be causal to our discovery that the kinship between the two cultivars was twice as high as expected from their shared ancestry. Another discovery was a large inversion on one of the subgenomes of linkage group 2 (D). Up until the publication of our linkage map this inversion had not been reported in other linkage maps. Another innovation was our attempt at giving a biological or evolutionary meaning to the denomination of the linkage groups by arranging them according to similarity to the diploid ancestor F. vesca, based on F. vesca derived primer amplification efficiencies. The HxK map has been used in several (ongoing) research projects outside of our research group and has contributed to the development of the 90k Axiom SNP array for cultivated strawberry.

    In Chapter 4 we performed a QTL mapping study for disease resistance against the problematic pathogen Phytophthora cactorum, which causes crown rot in strawberry plants. In this study we used two different mapping populations: the Holiday x Korona (HxK) population from the previous chapter as well as E1998-142 x Elsanta (ExEls), developed more specifically for the purpose of finding resistance against P. cactorum. The HxK and ExEls populations were phenotyped over three years (2008, 2010 and 2011) under different seasons and conditions. The correlation between years for was quite low for both populations (ranging from 0.18 to 0.47), indicating a large environmental effect on disease pressure. Results from the QTL analysis showed that most QTLs were small in effect and only just above the statistical significance threshold. Only for ExEls we uncovered two QTLs with relatively high significance levels, but none were significant in all three years. Because of the high environmental influence, and the desire to have QTLs that are robust over environments, we used the average of all three years (AOTY) as an additional phenotype. When we used the AOTY trait, the QTL on LG7D became stronger than for any of the individual years. Whereas for the LG7C QTL the significance dropped to just below threshold levels. These results indicated that removing environmental noise through averaging over experiments is a good way to uncover the most reliable and therefore more valuable to a breeding program.

    In Chapter 5 we investigated the genetics behind two different flowering habits that are grown commercially worldwide: seasonal flowering habit (SF) and perpetual flowering (PF) These varieties initiate flowering under long days, and can therefore produce fruit for a much longer period: throughout the summer and early fall. Evidence from literature and practical breeding suggested that PF is under dominant control. We decided to treat PF as a qualitative trait and divided two small mapping populations into PF and SF individuals. After screening several microsatellites, we found one locus that completely cosegregated with the PF trait at the bottom of LG4D. At the moment of mapping, a paper was published which mapped the same trait to the same location. We found that there were two very clear candidate genes within our QTL interval, FaCDF2 and FaFT2, which were homologous to genes that are major factors in the flowering pathway of Arabidopsis and many other plant species. We then sequenced the FaCDF2 gene from a number of distinct PF and SF cultivars. This resulted in the discovery of two quite distinct allelic variants, one of which was present in all PF cultivars. However this variant was also present in some of the SF cultivars, indicating that either FaCDF2 is not the causal gene, or that other loci can have a qualitative effect on the switch from SF to PF. We then performed microsatellite haplotyping on hundreds of cultivars and this revealed that all PF varieties of all origins carry the same haplotype in the PF QTL region, and that there weren’t any recombinations between the candidate genes FaCDF2 and FaFT2, which are 250kb apart on the physical genome. This makes it still undecided which of these two candidate genes are causal to the PF trait. Another interesting result from the haplotyping was that the complete PF haplotype was present with moderate frequency in SF varieties as well. Not only does this suggest a common origin, it also complicates the establishment of a theory for the mechanisms behind perpetual flowering in cultivated strawberry. So far we have not been able to establish whether the PF haplotype that is present in SF cultivars is functionally distinct from the PF haplotype in PF cultivars. All we know is that it does not confer perpetual flowering in these SF cultivars, and further experiments would be needed to find out the exact mechanism behind perpetual flowering.

    In the general discussion (Chapter 6), the results of this thesis were placed in the broader context of science in general and plant breeding in particular.

    The origin, versatility and distribution of azole fungicide resistance in the banana black Sigatoka pathogen Pseudocercospora fijiensis
    Chong Aguirre, Pablo A. - \ 2016
    Wageningen University. Promotor(en): Gert Kema; Pedro Crous. - Wageningen : Wageningen University - ISBN 9789462578791 - 289
    pseudocercospora - plant pathogenic fungi - fungicides - pesticide resistance - defence mechanisms - genetic diversity - genetic mapping - sensitivity - musa - bananas - fungal diseases - disease control - pseudocercospora - plantenziekteverwekkende schimmels - fungiciden - resistentie tegen pesticiden - verdedigingsmechanismen - genetische diversiteit - genetische kartering - gevoeligheid - musa - bananen - schimmelziekten - ziektebestrijding

    Pseudocercospora fijiensis causes black Sigatoka disease of banana. It is one of the most damaging threats of the crop requiring excessive fungicide applications for disease control as the major export “Cavendish” clones are highly susceptible. The consequence of this practice is the reduced efficacy of disease management strategies due to increasing levels of fungicide resistance. In this thesis the history and current practices of black Sigatoka disease management as well as the underlying mechanisms of fungicide resistance to a major group of fungicides are described. We discovered that both target site mutations and promotor insertions are crucial for modulating sensitivity. The more insertions, the higher the expression of the gene and the more resistant the strain. Using this information, we advocate modern monitoring techniques and improved disease control strategies as well as the urgent need for innovative banana breeding to develop resistant varieties for a sustainable global banana production.

    Development of a breeding strategy for nitrogen use efficiency in spinach (Spinacia oleracea L.)
    Chan Navarrete, J.R. - \ 2016
    Wageningen University. Promotor(en): Edith Lammerts van Bueren, co-promotor(en): Oene Dolstra; Gerard van der Linden. - Wageningen : Wageningen University - ISBN 9789462577961 - 161
    spinacia oleracea - spinach - plant breeding - nitrogen - nutrient use efficiency - genetic mapping - nitrogen fertilizers - fertilizer application - genetic diversity - nitrogen response - spinacia oleracea - spinazie - plantenveredeling - stikstof - nutriëntengebruiksefficiëntie - genetische kartering - stikstofmeststoffen - bemesting - genetische diversiteit - stikstofrespons

    Spinach (Spinacia oleracea L.) is one of the most consumed leafy vegetables worldwide and it is considered to be highly nutritious. Spinach is a short-cycle leafy crop that has a high demand for nitrogen in order to rapidly come to a harvestable product that has the required dark green colour within a reasonable harvest window. In commercial production of spinach the recovery of N is poor, which may result in environmental pollution. To increase sustainability of both organic and conventional spinach cultivation there is a need to reduce the dependency on high levels of nitrogen. Growers therefore urgently need cultivars with a satisfactory yield under reduced N input conditions. Nitrogen use efficiency (NUE), defined as the ability to produce high biomass per unit N applied, is low in spinach. The present study aims to evaluate spinach genotypes for selectable traits under varying N supply and provide tools and knowledge to facilitate the development of varieties with good yield, quality and stability under low N input. To minimise environmental variation affecting the identification of traits related to NUE a screening method was developed using a hydroponics system. The genetic diversity for NUE related traits was first studied with 24 commercial cultivars under contrasting levels of N supply based on the Ingestad model with a steady-state N application. This demonstrated that the hydroponics screening strategy as a pre-screening tool enabled reliable detection of heritable variation among cultivars for NUE-related traits under optimal as well as suboptimal N input. Shoot dry weight and leaf area were preferred selectable traits for the detection of heritable differences contributing to NUE in spinach. The effect of N application strategy was examined in seven cultivars grown under hydroponics conditions with low and high N levels supplied either as a single bulk N application resembling N fertilization in field cultivation, or a steady-state N application according to Ingestad. The latter application strategy provided more stable and reproducible conditions for determination of genetic differences in NUE under low N conditions for a short-cycle leafy vegetable crop. Several tools for molecular genetic evaluation of NUE in spinach were provided as well, including a SNP marker set for marker-assisted breeding, a genetic mapping population with a corresponding genetic map, and the identification of two major QTL regions contributing to growth under low N conditions. With these tools, an efficient strategy for breeding for NUE efficiency in spinach would include screening under controlled conditions at high and low N using leaf area, biomass and root to shoot ratio as selectable traits, and QTL identification of genetic factors that can be targeted and combined using marker-assisted selection. An in depth genotype by environment interaction analysis using six field trials showed that environmental factors like temperature, soil, and management strongly influence nitrogen availability in the soil in a short cycle crop like spinach. This severely complicates selection and breeding for NUE of spinach under field conditions, and emphasizes the importance of performing trials under better controllable conditions for genetic dissection of NUE and discovery of genetic factors contributing to NUE. It also underscores the importance of validating these findings in various field trials. Multi-environment field trials with different levels of N fertilization will then allow selection of cultivars that combine stable performance under various low input growing conditions with high yields under more favorable conditions.

    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.

    Ecogenomics of plant resistance to biotic and abiotic stresses
    Davila Olivas, N.H. - \ 2016
    Wageningen University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462576575 - 259
    016-3932 - arabidopsis thaliana - defence mechanisms - drought resistance - insect pests - plant pathogenic fungi - stress - stress response - transcriptomics - genomics - genetic mapping - arabidopsis thaliana - verdedigingsmechanismen - droogteresistentie - insectenplagen - plantenziekteverwekkende schimmels - stress - stressreactie - transcriptomica - genomica - genetische kartering
    Summary

    In natural and agricultural ecosystems, plants are exposed to a wide diversity of abiotic and biotic stresses such as drought, salinity, pathogens and insect herbivores. Under natural conditions, these stresses do not occur in isolation but commonly occur simultaneously. However, plants have developed sophisticated mechanisms to survive and reproduce under suboptimal conditions. Genetic screenings and molecular genetic assays have shed light on the molecular players that provide resistance to single biotic and abiotic stresses. Induced defenses are attacker specific and phytohormones play an essential role in tailoring these defense responses. Because phytohormones display antagonistic and synergistic interactions, the question emerges how plants elicit an effective defense response when exposed to conflicting signals under multiple attack. Recent studies have shed light on this issue by studying the effects of combinations of stresses at the phenotypic, transcriptomic and genetic level. These studies have concluded that the responses to combined stresses can often not be predicted based on information about responses to the single stress situations or the phytohormones involved. Thus, combined stresses are starting to be regarded as a different state of stress in the plant. Studying the effects of combinations of stresses is relevant since they are more representative of the type of stresses experienced by plants in natural conditions.

    In a coordinated effort, responses of Arabidopsis thaliana to a range of abiotic and biotic stresses and stress combinations have been explored at the genetic, phenotypic, and transcriptional level. For this purpose we used an ecogenomic approach in which we integrated the assessment of phenotypic variation and Genome-Wide Association (GWA) analysis for a large number of A. thaliana accessions with an in-depth transcriptional analysis. The focus of this thesis is especially on (but not limited to) three stresses, i.e. drought, herbivory by Pieris rapae caterpillars, and infection by the necrotrophic fungal pathogen Botrytis cinerea. These stresses were chosen because the responses of A. thaliana to these three stresses are highly divergent but at the same time regulated by the plant hormones JA and/or ABA. Consequently, analysis of responses to combinatorial stresses is likely to yield information on signaling nodes that are involved in tailoring the plant’s adaptive response to combinations of these stresses. Responses of A. thaliana to other biotic and abiotic stresses are included in an integrative study (Chapter 6).

    We first investigated (Chapter 2) the extent of natural variation in the response to one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips, Myzus persicae aphids) and two combined stresses (drought plus P. rapae, and B. cinerea plus P. rapae). Using 308 A. thaliana accessions originating from Europe, the native range of the species, we focused on the eco-evolutionary context of stress responses. We analyzed how the response to stress is influenced by geographical origin, genetic relatedness and life-cycle strategy, i.e. summer versus winter annual. We identified heritable genetic variation for responses to the different stresses. We found that winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars and to the combined stresses of drought followed by P. rapae and infection by the fungus B. cinerea followed by herbivory by P. rapae. Furthermore, we found differential responses to drought along a longitudinal gradient.

    We further investigated, using A. thaliana accession Col-0, how phenotypic and whole-genome transcriptional responses to one stress are altered by a preceding or co-occurring stress (Chapters 3 and 4). The whole-transcriptomic profile of A. thaliana triggered by single and combined abiotic (drought) and biotic (herbivory by caterpillars of P. rapae, infection by B. cinerea) stresses was analyzed by RNA sequencing (RNA-seq). Comparative analysis of plant gene expression triggered by single and double stresses revealed a complex transcriptional reprogramming. Mathematical modelling of transcriptomic data, in combination with Gene Ontology analysis highlighted biological processes specifically affected by single and double stresses (Chapters 3). For example, ethylene (ET) biosynthetic genes were induced at 12 h by B. cinerea alone or drought followed by B. cinerea inoculation. This induction was delayed when plants were pretreated with P. rapae by inducing ET biosynthetic genes only 18 hours post inoculation. Other processes affected by combined stresses include wound response, systemic acquired resistance (SAR), water deprivation and ABA response, and camalexin biosynthesis.

    In Chapter 4, we focused on the stress imposed by P. rapae herbivory alone or in combination with prior exposure to drought or infection with B. cinerea. We found that pre-exposure to drought stress or B. cinerea infection resulted in a significantly different timing of the caterpillar-induced transcriptional changes. Additionally, the combination of drought and P. rapae induced an extensive downregulation of A. thaliana genes involved in defence against pathogens. Despite the larger reduction in plant biomass observed for plants exposed to drought plus P. rapae feeding compared to P. rapae feeding alone, this did not affect weight gain of this specialist caterpillar.

    In Chapter 5, we used univariate GWA to (1) understand the genetic architecture of resistance to the different stresses and (2) identify regions of the genome and possible candidate genes associated with variation in resistance to those stresses. In Chapter 5 a subset of the stresses addressed in Chapter 1 (i.e. drought, herbivory by P. rapae and P. xylostella, and the combined stresses drought plus P. rapae and B. cinerea plus P. rapae) were investigated. Results from GWA were integrated with expression data generated in Chapters 3 and 4 or available from the literature. We identified differences in genetic architecture and QTLs underlying variation in resistance to (1) P. rapae andP. xylostella and (2) resistance to P. rapae and combined stresses drought plus P. rapae and B. cinerea plus P. rapae. Furthermore, several of the QTLs identified contained genes that were differentially expressed in response to the relevant stress. For example, for P. xylostella one of the QTLs contained only two genes encoding cysteine proteases (CP1 and CP2). The expression data indicated that these genes were induced by P. rapae and P. xylostella herbivory.

    In Chapter 6, the genetic architecture underlying plant resistance to 11 single stresses and some of their combinations was investigated. First, the genetic commonality underlying responses to different stresses was investigated by means of genetic correlations,, revealing that stresses that share phytohormonal signaling pathways also share part of their genetic architecture. For instance, a strong negative genetic correlation was observed between SA and JA inducers. Furthermore, multi-trait GWA identified candidate genes influencing the response to more than one stress. For example, a functional RMG1 gene seems to be associated with susceptibility to herbivory by P. rapae and osmotic stress since loss of function mutants in RMG1 displayed higher resistance to both stresses. Finally, multi-trait GWA was used to identify QTLs with contrasting and with similar effects on the response to (a) biotic or abiotic stresses and (b) belowground or aboveground stresses.

    Finally, In Chapter 7, I discuss the feasibility of obtaining plants that are resistant to multiple stresses from the point of view of genetic trade-offs and experimental limitations. The ecogenomic approach for gene discovery taken in this thesis is discussed, and recommendations are especially given on the use of herbivorous insects in quantitative genetic studies of stress resistance. Furthermore, alternatives to the use of insects in quantitative genetic studies of stress resistance are discussed and proposed. Finally, I discuss the feasibility of using an ecogenomic approach to study stress responses in other plant species than the model plant of molecular genetics, A. thaliana.

    A wealth of candidate genes was generated by taking an ecogenomic approach, in particular transcriptome analysis and GWA analysis. Functional characterization of these genes is a next challenge, especially in the context of multiple stress situations. These genes constitute a rich source of potential factors important for resistance to abiotic, biotic and combined stresses that in the future may be applied for crop improvement.

    Mapping moves on Arabidopsis : from natural variation to single genes affecting aphid behaviour
    Kloth, K.J. - \ 2016
    Wageningen University. Promotor(en): Marcel Dicke; Harro Bouwmeester, co-promotor(en): Maarten Jongsma. - Wageningen : Wageningen University - ISBN 9789462576483 - 269
    016-3933 - arabidopsis thaliana - insect pests - aphidoidea - pest resistance - genetic mapping - gene expression - quantitative traits - functional genomics - feeding behaviour - insect plant relations - arabidopsis thaliana - insectenplagen - aphidoidea - plaagresistentie - genetische kartering - genexpressie - kwantitatieve kenmerken - functionele genomica - voedingsgedrag - insect-plant relaties
    Exploiting wild relatives of S. lycopersicum for quality traits
    Víquez Zamora, A.M. - \ 2015
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Sjaak van Heusden; Arnaud Bovy. - Wageningen : Wageningen University - ISBN 9789462574410 - 167
    solanum lycopersicum - tomaten - solanum pimpinellifolium - wilde verwanten - germplasm - kenmerken - gewaskwaliteit - genomica - genetische kartering - plantenveredeling - solanum lycopersicum - tomatoes - solanum pimpinellifolium - wild relatives - germplasm - traits - crop quality - genomics - genetic mapping - plant breeding

    Exploiting wild relatives of S. lycopersicum for quality traits

    Ana Marcela Víquez Zamora

    Tomatoes are consumed worldwide and became a model for crop plant research. A part of the research aims at expanding genetic diversity in tomato; this can be done by incorporating useful genes found in wild germplasm. In this thesis we focused on exploring the variation between commercial tomatoes and accessions of wild relatives. Especially, we focused on the species Solanum pimpinellifolium. We explored the genome and the metabolome of accessions of S. pimpinellifolium and several varieties of S. lycopersicum. Finally a crossing population between the species was genetically analyzed. Clear differences were identified. We found genome regions related to differences between round and cherry tomatoes, TYLCV resistance, flavour and the phenylpropanoid pathway. S. pimpinellifolium certainly harbours useful genetic variability that can be (re-) introduced in tomato. Our results give an insight in the physical positions of metabolite related QTLs that can be used by breeders to exploit S. pimpinellifolium to improve tomato quality.

    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

    Summary

    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.

    Regulation and natural functions of lipopeptide biosynthesis in Pseudomonas
    Song, C. - \ 2015
    Wageningen University. Promotor(en): Francine Govers, co-promotor(en): Jos Raaijmakers. - Wageningen : Wageningen University - ISBN 9789462572690 - 173
    pseudomonas fluorescens - lipoproteïnen - biosynthese - genetische kartering - genregulatie - genomica - transcriptomica - verdedigingsmechanismen - protozoa - mutanten - pseudomonas fluorescens - lipoproteins - biosynthesis - genetic mapping - gene regulation - genomics - transcriptomics - defence mechanisms - protozoa - mutants

    Summary

    Lipopeptides (LPs) are surface-active, antimicrobial compounds composed of a lipid moiety linked to a short linear or cyclic oligopeptide. In bacteria, LPs are synthesized by large nonribosomal peptide synthetases (NRPSs) via a thiotemplate process. Compared to the understanding of LP biosynthesis, little is known about the genetic regulation.

    The aims of this PhD thesis were to identify new regulatory genes of LP biosynthesis and to unravel the natural functions of LPs in plant-associated Pseudomonas species. Using a combination of various ‘omics’-based technologies, we identified two small RNAs, designated RsmY and RsmZ, that, together with the repressor proteins RsmA and RsmE, regulate the biosynthesis of the LP massetolide in the rhizosphere bacterium Pseudomonas fluorescens SS101. Four other regulatory genes (phgdh, dnaK, prtR and clpA) of massetolide biosynthesis were identified via random mutagenesis. Mutations in each of these four genes caused a deficiency in massetolide production, swarming motility and biofilm formation, two natural functions associated with the production of LPs in Pseudomonas. Results further indicated that the ClpAP protease complex regulates massetolide biosynthesis via the pathway-specific, LuxR-type regulator MassAR, the heat shock proteins DnaK and DnaJ, and proteins of the TCA cycle.

    LPs exhibit broad-spectrum antimicrobial activities and have diverse natural functions for the producing bacteria. LPs of P. fluorescens were shown to play an important role in defense against protozoan predation. Genome-wide transcriptome analysis revealed that 55 and 73 genes were up- and down-regulated respectively in P. fluorescens strain SS101 upon grazing by the protozoan predator Naeglaria americana. The up-regulated genes included the LP biosynthesis genes massABC, but also genes involved in alkane degradation and in putrescine catalysis. Putrescine induced encystment of the protozoa, possibly providing a second line of defense against predation. MALDI imaging mass spectrometry (IMS) and live colony NanoDesi mass spectrometry further revealed, in real time, site-specific LP production at the interface of Pseudomonas-protozoa interactions. When the closely related strain P. fluorescens SBW25 was exposed to N. americana, similar overall transcriptional and metabolic responses were observed as found for strain SS101, but also strain-specific responses were apparent. These results indicate that closely related bacterial strains exhibit common and unique transcriptomic and metabolic responses to protozoan predation. Next to defense against competitors and predators, LPs are well-known for their role in swarming motility, a flagella-driven multicellular behavior of bacteria. Orfamide-deficient mutants of P. protegens Pf-5, either with deletions in the biosynthesis gene ofaA or in the regulatory gene gacA, cannot swarm on their own but ‘hitch-hike’ with parental strain Pf-5. However, distinctly different spatial distributions in co-swarming colonies were observed for these two mutants, with the ofaA mutant moving behind the wild type and the gacA mutant predominating on the edge of the swarming colony. Subsequent experimental evolution assays showed that repeated swarming cycles of strain Pf-5 drives parallel evolution toward fixation of spontaneous gacS/gacA mutants on the edge, ultimately causing colony collapse. Transcriptome analyses revealed that genes associated with resource acquisition, motility, chemotaxis and efflux were significantly upregulated in these regulatory mutants. Moreover, microscopic analysis showed that gacA mutant cells were longer and more flagellated than wild type and ofaA mutant cells, which may explain their predominance on the edge of co-swarming colonies. Collectively, these results indicated that adaptive convergent evolution through point mutations is a common feature of range-expanding microbial populations and that the putative fitness benefits of these spontaneous mutations during dispersal of bacteria into new territories are frequency-dependent.

    Genetic investigation of the nonhost resistance of wild lettuce, Lactuca saligna, to lettuce downy mildew, Bremia lactucae
    Boer, E. den - \ 2014
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Marieke Jeuken; Rients Niks. - Wageningen : Wageningen University - ISBN 9789462572072 - 186
    lactuca saligna - schimmelziekten - bremia lactucae - ziekteresistentie - terugkruisen - inteeltlijnen - genetische kartering - lactuca sativa - lactuca saligna - fungal diseases - bremia lactucae - disease resistance - backcrossing - inbred lines - genetic mapping - lactuca sativa

    Abstract

    Downy mildew (Bremia lactucae) in lettuce (Lactuca sativa) is a devastating foliar disease causing high losses in lettuce cultivation. The wild lettuce and nonhost species, Lactuca saligna, is absolute resistant to downy mildew and cross-fertile with L. sativa, albeit with a low success rate and occasional reduced fertility and/or vitality in later inbred generations. This exceptional availability of hybrid plant offsprings creates a unique opportunity to study nonhost resistance by a genetic approach.

    The L. saligna nonhost resistance genes might be more durable than the classical monogenic race-specific R genes that are mainly used in lettuce breeding. The identification of genes conferring nonhost resistance is a crucial step in its understanding and usage in breeding.

    In this thesis the quantitative resistances of three backcross introgression lines (BILs), carrying an individual 30 to 50 cM long introgression segment from L. saligna in a L. sativa background, were fine mapped. Disease evaluation of sub-BILs with smaller introgression segments revealed that the resistance of all three BILs was explained by 17 sub-QTLs with a smaller and plant stage dependent effect, some segments reducing, others even promoting downy mildew infection.

    Further the potential of stacking quantitative resistances of eight BILs per combinations of two was tested under field conditions. Only three out of ten double-combinations resulted in an increased resistance level compared to their parental individual lines, from which one had additive and two had epistatic interactions between the introgressions.

    As the studies on individual QTL effects of BILs did not reveal potential genetic interactions that could explain the complete resistance of L. saligna, a novel approach was set out to search for indications of epistatic interactions. ‘Selective genotyping’ was applied on the phenotypic disease extremes of large F2 offsprings, in which multi-locus interactions between L. saligna alleles are still prevalent. In a kind of bulked segregant analysis approach four major resistance regions were identified. Preliminary results showed epistatic interactions between the regions on Chromosome 6 and 1 and between Chromosome 6 and 7.

    During the development of sub-BILs, a digenic hybrid incompatibility was observed: plants carrying a L. saligna segment on Chromosome 6 always required a L. saligna segment on Chromosome 4. Segregation analysis suggested a prezygotic reproductive barrier by non-transmission of one specific hybrid gametophyte (male and female).

    In cooperation with the research group of Guido van den Ackerveken of University Utrecht a lettuce germplasm screening was conducted for candidate downy mildew effector proteins, which interact with resistance genes in lettuce and trigger a defence response. One of two responsive effector proteins, ‘BLG01’, triggered a hypersensitive cell death response in most tested L. saligna accessions and its response was mapped on Chromosome 9.

    Despite the complex interactions between resistance QTLs, this thesis research has delivered many insights that are important steps forward towards understanding the incompatible interaction between B. lactucae and L. saligna and its future application in resistance breeding.

    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.

    Laying the foundations for dough-based oat bread
    Londoño, D.M. - \ 2014
    Wageningen University. Promotor(en): Richard Visser; Rob Hamer, co-promotor(en): Rene Smulders; Luud Gilissen. - Wageningen : Wageningen University - ISBN 9789462570085 - 139
    avena sativa - haver - deeg - bakkwaliteit - coeliakie - avenine - genen - genetische kartering - bèta-glucaan - wei-eiwit - plantenveredeling - avena sativa - oats - doughs - baking quality - coeliac syndrome - avenin - genes - genetic mapping - beta-glucan - whey protein - plant breeding

    The motivation to perform this study was to generate the fundamentals to use oats for bread-making applications. This will offer consumers a healthier alternative product to wheat bread in their daily diet, because oat foods, especially through their high amount of soluble fibre (notably beta-glucans) contribute to the reduction of blood cholesterol levels and of blood glucose rise after the meal. Oats also have a high content of (poly-) unsaturated fatty acids that contribute to maintaining normal blood cholesterol levels. One specific target group that would benefit from the development of good quality oat bread are people with celiac disease (CD). Oats is widely consumed by them, even though its safety has been subject of some debate for a long time. Two peptides from oat avenins can be recognized as T cell epitopes by few CD patients, and differential signals of gluten-specific monoclonal antibodies and in-vitro T cells to oat varieties have suggested the existence of differences in immunogenicity. These health and food safety issues have been addressed in the General Introduction.

    Bread is consumed all over the world. So far, production of large-volume bread is only possible with wheat. The quality of existing oat bread is below to what consumers are used to with wheat bread. This is partly due to the lack of knowledge regarding the functionality of oats for other purposes than porridge and breakfast cereals, which are the most common applications. These applications do not represent a big technological challenge as bread does, because bread-making requires a system able to hold gas during proving and baking. In wheat, this is conferred by gluten proteins that form a viscoelastic network with the capacity to expand and to maintain itself after expansion. Oats lack gluten proteins with network-forming capacity. Current oat bread applications rely on batter systems and on the use of additives to increase viscosity for stabilization of gas cells.

    This thesis consists of two parts. The first part concerns the safety of oats for people with celiac disease (Chapter 2). This was studied by cloning and sequencing avenin genes from 13 Avena species with combinations of the three genomes (A, C, D) that are also present in the hexaploid cultivated A. sativa. We identified up to 10 avenin genes in a single hexaploid oat plant. Avenin proteins clustered in four groups of which two contained the two avenin CD epitopes. All Avena species examined harbored avenins of these two groups, so it is unlikely to find oat cultivars that are devoid of the avenin CD epitopes. None of the internationally agreed gluten CD epitopes from wheat, rye and barley were found to be present in oat avenins. Some epitope variants with two and three amino acid substitutions occurred, but they were predicted to not resist proteolysis in the gastro-intestinal tract and will therefore not be of clinical relevance. Perfect recognition sites of antibodies R5 and G12 (which are used in commercial gluten detection kits) were also not present in avenins. Thus, monoclonal antibody signals to oat are probably due to cross-reactivity or promiscuous recognition of avenin peptides, and such signals should not be interpreted as differences in immunogenicity of oat varieties for CD patients.

    The second part of this thesis focussed on the study of the technological properties of oats. Oats have been used as an addition to wheat-based dough or in an oat-based batter system. However, while for wheat the dough-making parameters necessary to obtain good quality bread have been defined through a long history of research, this is not the case for oats. To fill this gap, this thesis studied the technological properties of oats using a systematic approach. First, we developed a dough testing system that allowed us to assess the dough-making properties of oat flour in a standardized way (Chapter 3). For this we used wheat as a model. We reproduced various quality profiles of wheat flour using combinations of oat flour and vital gluten. Then, we selected a dough system made of 87.2% oat flour and 12.8% gluten as our standard dough test system. This dough system was sensitive to differences among oat cultivars. Thus, having developed a tool that could detect differences regarding dough-making properties among oat cultivars, the next step was to try to explain those differences in terms of compositional factors. We decided to start our exploration with beta-glucans, because these fibres are one of the oat components that attract interest because of their health benefits. We studied the impact of beta-glucans on dough rheology (Chapter 4) following two strategies: (i) using the developed standard dough system containing gluten; and (ii) by removing the gluten from the system and replacing these proteins by alternative network-forming compounds. In both systems, beta-glucans affected dough rheology. Increasing their concentration resulted in an increase of dough stiffness and in a reduction of dough extensibility. Beta-glucans negatively influenced the elastic properties that additional wheat gluten conferred to oat dough. Low beta-glucan (<2%) oat flour had better extensibility properties than oat meal dough or oat flour dough enriched with beta-glucans. The effect was governed by its concentration and its molecular weight (which determines viscosity). Medium-viscosity beta-glucans had a less negative impact than high-viscosity (high molecular weight) beta-glucans. Overall, our findings indicate that beta-glucans are a key component determining rheology of oat-based dough systems.

    Chapter 5 addressed the effect of particle size distribution on dough-making properties. We found that oat meal is not the best material for bread-applications because it produces a very stiff and short dough. Re-milling did not change this pattern. In contrast, complete removal of the bran from the oat meal did improve dough-making properties, which indicated that dough rheology was negatively impacted by the bran. Large and medium size bran particles were more harmful than fine bran particles. Large and medium bran contained 8% beta-glucans, while fine bran contained 1.6% only. We concluded that oat meal is not appropriate for bread applications. Fractionation of the milled product is an interesting alternative to produce low-beta-glucan flour for bread-making purposes, and the bran can be used to enrich other food products with beta-glucans. This chapter also addressed whether kilning and milling methods applied to oat grains could affect bread-making purposes. Infrared (IR) and steam kilning both affected dough-making properties of oat grains in the standard dough system. The effect of steam kilning was on water absorption only. Non-kilned and steam-kilned grains showed similar extensibility behavior. In contrast, IR kilning affected water absorption and harmed completely the dough extensibility properties of oat grains. Flour from IR kilned grains made a very stiff and short dough. Thus, IR kilning is definitely not suitable for bread applications.

    Finally, in Chapter 6, we addressed the need for good quality gluten-free oat bread. As further research is required for better understanding of the oat dough system, we studied the rheological properties of oat flour relevant for leavening with gluten alternatives. Whey protein particles (WPP) had appeared to be successful in enhancing viscoelastic properties of wheat starch dough, allowing loaves with specific volumes of ca 3.7 mL/g. We studied whether WPP could have a similar positive effect on oat flour dough. WPP increased the resistance to extension and the gas retention capacity of oat flour dough. However, in our small scale baking experiments, WPP did not increase loaf specific volume and had a negative effect on gas production. On the other hand, WPP improved crumb texture. WPP are promising as a structuring agent in oat dough, but the process should be further optimized.

    In the General Discussion we pay attention to the food safety issue of oats for people with coeliac disease. Our analysis across the genus Avena of avenin genes and proteins produced an important new and supporting argument to the safety of oats, as they appeared to contain none of the generally agreed celiac disease-related gluten epitopes from wheat, barley and rye. With this analysis we also could explain the positive signals for the presence of gluten (as described in the literature for several oat varieties on the basis of the R5 and the G12 antibody assay and on T cell tests) as being the result of cross-reactivity or promiscuity, without having clinical relevance. The data in this thesis therefore support the advice to gradually introduce the consumption of oats into the daily diet of people with coeliac disease. Further, we discuss the results and the consequences of our technological research on oat flour dough. It appeared that beta-glucans have a serious negative effect on the rheology of the oat dough, which indicates the need for further research on improvement of the balance between optimum application of beta-glucans for health (high amounts and of high molecular weight is better) and for baking quality (low amounts and of low molecular weight is better). Also the pre-treatment of oat flour (notably kilning and milling) and the application of whey protein particles to replace gluten require further optimization. Here the developed standard oat flour dough model system will be a useful tool.

    Breeding for pepper fruit quality: a genitical metabolomics approach
    Wahyuni, Y. - \ 2014
    Wageningen University. Promotor(en): Raoul Bino, co-promotor(en): Arnaud Bovy; E. Sudarmonowati; A.R. Ballester. - Wageningen : Wageningen University - ISBN 9789461739582 - 193
    capsicum annuum - capsicum frutescens - capsicum chinense - capsicum baccatum - plantenveredeling - metabolomica - gewaskwaliteit - secundaire metabolieten - gezondheid - genetische kartering - rijp worden - capsicum annuum - capsicum frutescens - capsicum chinense - capsicum baccatum - plant breeding - metabolomics - crop quality - secondary metabolites - health - genetic mapping - ripening

    A diverse collection of 32 pepper accessions was analysed for variation in health-related metabolites, such as carotenoids, capsaicinoids, flavonoids and vitamins C and E. For each of the metabolites analysed, there was a lot of variation among the accessions and it was possible to identify accessions with high amounts of specific metabolites. While all accessions contained high levels of vitamin C, accession C. chinenseAC2212 was found to be an excellent source of vitamin E, whereas C. annuumLong Sweet accumulated high levels of the flavonoid quercetin. A genetical metabolomics approach was used to study the genetic basis of metabolic traits in a segregating F2 population based on a cross between two contrasting pepper genotypes. This led to the identification of several metabolites QTL hotspots. The genetic basis for the accumulation of several flavonoids in pepper fruit was further investigated, by combining metabolic, gene expression and candidate gene-based marker data. This provided valuable insight into the key genes important for flavonoid accumulation in pepper fruit. The results of this study will help breeders to assist future breeding programs aimed at optimizing the levels of nutritional compounds in pepper fruit.

    Towards generating broad-spectrum resistance to pathogens in plants: studies on a down-stream signalling NB-LRR of tomato
    Sueldo, D.J. - \ 2014
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Matthieu Joosten; Wladimir Tameling. - Wageningen : Wageningen University - ISBN 9789461738974 - 209
    solanum lycopersicum - tomaten - ziekteresistentie - verdedigingsmechanismen - receptoren - pathogenesis-gerelateerde eiwitten - bindende eiwitten - virulentie - mutanten - genetische kartering - solanum lycopersicum - tomatoes - disease resistance - defence mechanisms - receptors - pathogenesis-related proteins - binding proteins - virulence - mutants - genetic mapping
    Bioinformatics assisted breeding, from QTL to candidate genes
    Chibon, P.Y. - \ 2013
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Richard Finkers. - S.l. : s.n. - ISBN 9789461737366 - 149
    plantenveredeling - bio-informatica - moleculaire veredeling - marker assisted breeding - loci voor kwantitatief kenmerk - genetische kartering - gegevensverwerking - ontologieën - plant breeding - bioinformatics - molecular breeding - marker assisted breeding - quantitative trait loci - genetic mapping - data processing - ontologies

    Over the last decade, the amount of data generated by a single run of a NGS sequencer outperforms days of work done with Sanger sequencing. Metabolomics, proteomics and transcriptomics technologies have also involved producing more and more information at an ever faster rate. In addition, the number of databases available to biologists and breeders is increasing every year. The challenge for them becomes two-fold, namely: to cope with the increased amount of data produced by these new technologies and to cope with the distribution of the information across the Web. An example of a study with a lot of ~omics data is described in Chapter 2, where more than 600 peaks have been measured using liquid chromatography mass-spectrometry (LCMS) in peel and flesh of a segregating F1apple population. In total, 669 mQTL were identified in this study. The amount of mQTL identified is vast and almost overwhelming. Extracting meaningful information from such an experiment requires appropriate data filtering and data visualization techniques. The visualization of the distribution of the mQTL on the genetic map led to the discovery of QTL hotspots on linkage group: 1, 8, 13 and 16. The mQTL hotspot on linkage group 16 was further investigated and mainly contained compounds involved in the phenylpropanoid pathway. The apple genome sequence and its annotation were used to gain insight in genes potentially regulating this QTL hotspot. This led to the identification of the structural gene leucoanthocyanidin reductase (LAR1) as well as seven genes encoding transcription factors as putative candidates regulating the phenylpropanoid pathway, and thus candidates for the biosynthesis of health beneficial compounds. However, this study also indicated bottlenecks in the availability of biologist-friendly tools to visualize large-scale QTL mapping results and smart ways to mine genes underlying QTL intervals.

    In this thesis, we provide bioinformatics solutions to allow exploration of regions of interest on the genome more efficiently. In Chapter 3, we describe MQ2, a tool to visualize results of large-scale QTL mapping experiments. It allows biologists and breeders to use their favorite QTL mapping tool such as MapQTL or R/qtl and visualize the distribution of these QTL among the genetic map used in the analysis with MQ2. MQ2provides the distribution of the QTL over the markers of the genetic map for a few hundreds traits. MQ2is accessible online via its web interface but can also be used locally via its command line interface. In Chapter 4, we describe Marker2sequence (M2S), a tool to filter out genes of interest from all the genes underlying a QTL. M2S returns the list of genes for a specific genome interval and provides a search function to filter out genes related to the provided keyword(s) by their annotation. Genome annotations often contain cross-references to resources such as the Gene Ontology (GO), or proteins of the UniProt database. Via these annotations, additional information can be gathered about each gene. By integrating information from different resources and offering a way to mine the list of genes present in a QTL interval, M2S provides a way to reduce a list of hundreds of genes to possibly tens or less of genes potentially related to the trait of interest. Using semantic web technologies M2S integrates multiple resources and has the flexibility to extend this integration to more resources as they become available to these technologies.

    Besides the importance of efficient bioinformatics tools to analyze and visualize data, the work in Chapter 2also revealed the importance of regulatory elements controlling key genes of pathways. The limitation of M2S is that it only considers genes within the interval. In genome annotations, transcription factors are not linked to the trait (keyword) and to the gene it controls, and these relationships will therefore not be considered. By integrating information about the gene regulatory network of the organism into Marker2sequence, it should be able to integrate in its list of genes, genes outside of the QTL interval but regulated by elements present within the QTL interval. In tomato, the genome annotation already lists a number of transcription factors, however, it does not provide any information about their target. In Chapter 5, we describe how we combined transcriptomics information with six genotypes from an Introgression Line (IL) population to find genes differentially expressed while being in a similar genomic background (i.e.: outside of any introgression segments) as the reference genotype (with no introgression). These genes may be differentially expressed as a result of a regulatory element present in an introgression. The promoter regions of these genes have been analyzed for DNA motifs, and putative transcription factor binding sites have been found.

    The approaches taken in M2S (Chaper 4) are focused on a specific region of the genome, namely the QTL interval. In Chapter 6, we generalized this approach to develop Annotex. Annotex provides a simple way to browse the cross-references existing between biological databases (ChEBI, Rhea, UniProt, GO) and genome annotations. The main concept of Annotex being, that from any type of data present in the databases, one can navigate the cross-references to retrieve the desired type of information.

    This thesis has resulted in the production of three tools that biologists and breeders can use to speed up their research and build new hypothesis on. This thesis also revealed the state of bioinformatics with regards to data integration. It also reveals the need for integration into annotations (for example, genome annotations, protein annotations, and pathway annotations) of more ontologies than just the Gene Ontology (GO) currently used. Multiple platforms are arising to build these new ontologies but the process of integrating them into existing resources remains to be done. It also confirms the state of the data in plants where multiples resources may contain overlapping. Finally, this thesis also shows what can be achieved when the data is made inter-operable which should be an incentive to the community to work together and build inter-operable, non-overlapping resources, creating a bioinformatics Web for plant research.

    Cytological, genetic and agronomic characterization of a barley reciprocal translocation
    Farré Martinez, A. - \ 2012
    Wageningen University. Promotor(en): Fred van Eeuwijk, co-promotor(en): I. Romagosa; Hans Jansen. - S.l. : s.n. - ISBN 9789461734112 - 131
    gerst - cytogenetica - reciproke translocatie - agronomische kenmerken - genetische kartering - toegepaste statistiek - biometrie - biostatistiek - barley - cytogenetics - reciprocal translocation - agronomic characteristics - genetic mapping - applied statistics - biometry - biostatistics

    Reciprocal translocations (RT) are one of the most common structural chromosomal rearrangements occurring in plant species. Spontaneous RT are extremely uncommon in cultivated barley. In fact, ‘Albacete’ is the only extensively cultivated barley variety known to carry a RT without any major reduction in fitness. Previous results have shown that 1H and 3H chromosomes are involved in this translocation which may have a potential interest for plant breeding. A depth understanding of this RT could allowed us to know whether this translocation is involved or not in their drought tolerance and find some interesting phenotypic attributes that could be transfer to other barley genotypes to develop specific germoplasm adapted to stress.

    In a first step, a set of 230 doubled haploid lines derived from the cross between ‘Albacete’ and the non-translocation barley variety ‘Barberousse’ was used to develop a statistical-genetic approach for the construction of a linkage maps in populations obtained from RT heterozygotes. A preliminary linkage study revealed six main linkage groups, compared to the seven linkage groups on the barley consensus map. One of the linkage groups contained markers of chromosomes 1H and 3H. This union was due to the presence of a RT between chromosomes 1H and 3H in the variety ‘Albacete’. The results of this study revealed that the position of the translocation breakpoints was placed around the centromeric regions for both chromosomes.

    In a second step, a combination of cytological and molecular genetic approaches was used in order to know how large the segments are that are involved in the interchange between the two chromosomes. The results obtained in this study indicate that the translocation is quite large with breakpoints located on the long arms of chromosomes 1H and 3H resulting in the RT 1HS.1HL-3HL and 3HS.3HL-1HL. The gene content around the translocation breakpoints was estimated at approximately 1,100 and 710 gene models for 1H and 3H, respectively.

    A large number of doubled haploid lines derived from four crosses involving ‘Albacete’ as one of the parents were used to phenotypically characterize the effects of the RT on some interesting agronomic traits.The results suggested that the RT alone is not causing the drought tolerance of ‘Albacete’; thousand kernel weight is the only trait which is clearly enhanced in the lines carrying the RT. Further work combining the results from this study with QTL analysis was carried out to phenotypically characterize the effects of the reciprocal translocation and QTL simultaneously. For that, an integrated map was constructed making used of a modified version of an algorithm developed by Jansen (2005). Finally, specific QTL methodology for the simultaneous analysis of these interconnected populations was developed; two traits: days to heading (independent of the RT) and thousand kernel weight (affected by the RT) were used as an example.

    Genetic control and variation in turkey: molecular insights in selection
    Aslam, M.L. - \ 2012
    Wageningen University. Promotor(en): Martien Groenen, co-promotor(en): John Bastiaansen; Richard Crooijmans. - S.l. : s.n. - ISBN 9789461733092 - 184
    meleagris - kalkoenen - genetische variatie - moleculaire veredeling - selectie - genetische kartering - loci voor kwantitatief kenmerk - moleculaire technieken - dierveredeling - meleagris - turkeys - genetic variation - molecular breeding - selection - genetic mapping - quantitative trait loci - molecular techniques - animal breeding


    The turkey (Meleagris gallopavo) is an important agricultural species that is largely used as a meat type bird as egg production of this species is very low. Turkey is the second largest contributor to the world’s poultry meat production after chicken. Understanding the etiology and biology underlying production and health traits is very important for the genetic improvement of these traits in the desired direction and to avoid undesired side-effects. The aim of the research described in this thesis was to interrogate the genetics of turkey traits related to meat production and to investigate the genetic diversity of commercial and heritage turkey populations.
    Different analyses were performed that included the estimation of genetic and (common) environmental variances for growth (body weight as well as growth curve traits), breast meat yield and meat quality traits in turkeys. I describe the construction of a single nucleotide polymorphism (SNP) based linkage map of turkey and its comparison with the physical map of chicken to investigate genome structural differences between these highly important poultry production species. Two inter-, and 57 intra-chromosomal rearrangements between these two species were confirmed or discovered which is a low number in comparison to mammals and lead to the conclusion that turkey and chicken have highly conserved genomic structure. I used the linkage map of turkey together with individual phenotypes to map quantitative trait loci (QTL) in the same population for the traits described above. Results showed quantitative trait loci on 21 of the 27 turkey chromosomes covered by the linkage map. Forty-five quantitative trait loci were detected across all traits and these were found in 29 different regions on the 21 chromosomes. The next step, after the analyses on the reference population was to investigate the genomic variation in turkeys Next generation sequencing was used to investigate genome variation and the discovery of genome-wide signatures of selection in the turkey respectively. Sequencing was performed on 32 individuals from eleven different turkey populations (seven commercial, three heritage and a South Mexican wild population). Analysis of next generation sequencing data resulted in the detection of 5.49 million putative SNPs compared to the reference genome. The average frequency of heterozygous nucleotide positions in individual turkeys was 1.07 Kb-1 which is substantially lower than in chicken and pigs. The SNPs were subsequently used for the analysis of genetic diversity between the different populations. Genetic diversity analysis using pairwise Nei’s genetic distance among all the individuals from the 11 turkey populations showed that all of the commercial lines branched from a single node relative to the heritage varieties and the ancestral turkey population, indicating that commercial lines appear to share a common origin.
    After assessing genome wide variation and diversity between breeds, the SNP data from ten of the turkey populations (29 individuals) was used to detect selective sweep regions. Across the turkey populations, 54 genomic regions with significant evidence for a selective sweep were detected. These sweeps were distributed over 14 different chromosomes. This study has investigated the genetics i.e. analysis of variances and QTL mapping related to economically important traits in turkey production and the genomic variation of turkey. Furthermore, this study has also created resources e.g. millions of discovered SNPs for subsequent genomic work in the turkey such as to discover variant (s) for both minor and major effects on traits of economic importance, and a high-resolution linkage map can be developed.


    Characterising the cellulose synthase complexes of cell walls
    Mansoori Zangir, N. - \ 2012
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade. - S.l. : s.n. - ISBN 9789461732958 - 162
    planten - celwanden - cellulose - biosynthese - enzymen - enzymactiviteit - eiwitten - katalyse - genetische kartering - genomica - plants - cell walls - cellulose - biosynthesis - enzymes - enzyme activity - proteins - catalysis - genetic mapping - genomics

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as the catalytic subunits of the complex. The objective of the research presented in this thesis was to generate more in-depth knowledge in cellulose biosynthesis and to this aim better characterize and understand the cellulose synthase complex and its components by notably investigating the similarities and differences between the CESAs in the primary and secondary cellulose complex and identifying the various interacting proteins forming the complex in the plant cell wall. KORRIGAN and specific isoforms of sucrose synthase were shown to be co-localized and physically interact with the CESAs in the Cellulose Synthase Complex at the plasma membrane supporting their participation in cellulose biosynthesis in Arabidopsis.

    Development of genomic resources for ornamental lilies (Lilium L.)
    Shahin, A. - \ 2012
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Jaap van Tuyl; Paul Arens. - S.l. : s.n. - ISBN 9789461733009 - 169
    lilium - sierplanten - plantenveredeling - transcriptomica - genetische kartering - genotyping - genetische merkers - nucleotidenvolgordes - genomica - lilium - ornamental plants - plant breeding - transcriptomics - genetic mapping - genotyping - genetic markers - nucleotide sequences - genomics

    Lily (Lilium L.) is a perennial bulbous ornamental, belonging to subclass Monocotyledonae and family Liliaceae. Lily, according to statistics of Dutch auctions, is the fifth most important cut flower and the second in flower bulbs based on acreage. This species has been extensively used for cytogenetic studies, but molecular genetic studies are limited. The heterogenic nature and the very complex and huge genome (36 Gb) of lily might be the reason for this. To improve the efficiency of breeding and selection in this species, and set up the basis for genetic studies in Lilium, genomic resources are needed.

    Next generation sequencing (NGS) technology (454 pyro-sequencing) was used to sequence the transcriptomes (RNA-seq) of four lily cultivars: ‘Connecticut King’, ‘White Fox’, ‘Star Gazer’, and Trumpet that belong to the four most important hybrid groups: Asiatic, Longiflorum, Oriental, and Trumpet respectively. Successfully, 52,172 unigenes with an average length of 555 bp were developed and used for a wide range of genetic and genomic studies: SNP marker identification for genetic mapping, gene annotation, and comparative genomic studies.

    Combining NGS with SNP genotyping techniques to accelerate genetic studies is of considerable interest in different species. In this study, thousands of SNPs out of the 52,172 lily unigenes were identified. Genotyping technique KASPar (KBiosciences competitive Allele Specific PCR) was used to genotype two lily mapping populations: ‘LA (L. longiflorum ‘White Fox’ x Asiatic hybrid ‘Connecticut King’) and AA (‘Connecticut King’ x ‘Orlito’) using 225 SNP markers selected from ‘Connecticut King’ unigenes. Genotyping success rate was 75.5% (170 SNP markers worked), polymorphic SNP rate was 45% (102 SNP markers), and mapped SNP marker rate was 42% (94 SNP mapped) in LA population and 38% (85 SNP mapped) in AA population. Thus, we validated a subset of the putative SNP makers and showed the usability of this type of markers to improve genetic maps for complex genomes like that of lily.

    The SNP markers together with the available AFLP (amplified fragment length polymorphisms), DArT (diversity arrays technology), and NBS (nucleotide binding site) markers were used to build reference genetic maps for these two lily populations. These maps represent the first reasonably saturated maps that cover 89% of the lily genome with an average marker density of one marker per 4 cM. The availability of more SNP markers for genotyping, opens the door for further enriching these genetic maps and thus improve the marker density.

    The genetic maps were used to map and understand the genetic of several horticultural traits in Lilium. Fusarium oxysporum and lily mottle virus (LMoV) are considered as very serious diseases in Lilium and as such present important targets for breeding. Six putative QTLs (quantitive trait loci) were identified for Fusarium resistance in AA population, from which QTL1 was the strongest (explains ~25 % of phenotypic variation). In LA population, QTL1 was also confirmed. Thus, QTL1 is a strong and reliable QTL in both populations and it can be used to develop markers for most of the Fusarium resistance for molecular assisted breeding (MAB) applications. The LMoV was mapped as a marker on the AA genetic maps, however, no close markers to this trait (i.e. distance of the closest marker to LMoV was 9 cM) were identified yet. Several ornamental traits: lily flower color ‘carotene’ (LFCc), flower spots (lfs), stem color (LSC), antherless phenotype (lal), and flower direction (up-side facing, lfd) were phenotyped and mapped. Some of these traits showed to be recessive traits (spots, antherless, and flower direction) and controlled by a single gene. Developing markers for recessive traits is valuable since such markers allow the identification of suitable breeding parents so the presence of the recessive trait can be either enhanced or repressed. A more complex trait is flower longevity because it is a function of: the number of buds per inflorescence, the expansion and opening of the buds, the life span of the individual flowers, and also the life-span of the leaves. Moreover, senescence in Lilium is ethylene-insensitive and the regulator(s) of its vase life is not known yet. Our study showed that vase life of individual lily flowers increased significantly by the exogenous application of sugars. Abscisic acid (ABA) level, furthermore, increased dramatically in lily flowers at senescence compared with anthesis. This indicates that ABA might be the main regulator of vase life in lily. However, more experiments should be conducted to prove this conclusion.

    The genomic resources developed for lily together with genomic resources developed for Tulipa L., in the same way, offered a valuable source of information to conduct comparative genomic studies within and between these two genera. We initiated the first step towards linking molecular genetic maps of Lilium and Tulipa using transcriptome sequences generated by 454 pyro-sequencing. Orthologous genes between lily and tulip were identified (10,913 unigenes) based on sequence data of four lily cultivars and five tulip cultivars. Next, common SNP and EST-SSR markers between the parents of lily mapping populations (AA and LA population) and the parents of tulip mapping population (‘Kees Nelis’ (T. gesneriana) x ‘Cantata’ (T. fosteriana)) based on these orthologous sequences were generated. A total of 229 common SNP and 140 common EST-SSR markers were identified. Genotyping and mapping these markers in the populations of both genera will link the genetic maps of Lilium and Tulipa and thus allow insight into the preservation of gene order, structure, and ‘putative’ functional homology in addition to evolutionary processes.

    Also, these genomic resources can be used to increase the resolution of, and support for, phylogenetic trees. We selected a set of orthologous genes of Lilium (19 genes, 11,766 bp containing 433 polymorphic sites), of Tulipa (20 genes, 10,347 bp containing 216 polymorphic sites), and of the orthologous genes between the two genera (7 genes, 5,790 bp containing 587 polymorphic sites). These sets are uniquely present in the sequences and informative in estimating the genetic divergence of the two genera, thus they can be used to genotypes more species per genera to build genera and maybe family trees later on. The nucleotide polymorphism rate of Lilium was twice as high as that of Tulipa, on average one substitution per 26 bp for Lilium compared with one substitution per 48 bp for Tulipa. NGS provide a valuable source for large numbers of phylogenetic informative substitutions that might revolutionize the phylogenetic, population genetic, and biodiversity studies. However, the use of bi-allelic information from multiple loci in phylogenetic studies is still challenging and it needs to be studied further.

    Moreover, having such high numbers of sequence data, allows us to test some evolutionary hypotheses such as positive selection: selection during domestication/breeding processes might be imprinted in the species genome, which can be examined based on omega (dn/ds) values. The higher the omega value the stronger the indication of positive selection. Positive selection was recorded in Lilium and Tulipa genomes when this small subset of gene contigs (46) of the two genera was tested. Our hypothesis could not be confirmed, however to draw final conclusions on this matter, omega values for many more genes of the two genera have to be measured.

    Finally, a wealth of putative molecular markers (SNPs and SSRs) has become available that can have direct applications for breeding in these genera. SNP markers are important since they are user friendly, efficient, transferable, and co-dominant markers. Applying high throughput genotyping technology to genotype the two lily populations improved the coverage of the two genetic maps. Also, genotyping the same SNP markers in the two populations facilitated the comparisons between the linkage groups of the two populations and will allow the construction of a consensus map. Consequently, exchange of genetic knowledge (mainly QTLs) between the two populations will be easier. The thousands of SNPs identified in the genome of the four lily cultivars opens the door for combining the current linkage mapping studies with association studies which will have a direct impact on improving the resolution of mapping and on MAB applications in Lilium.


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