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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Identification of metabolites involved in heat stress response in different tomato genotypes
Paupière, Marine J. - \ 2017
University. Promotor(en): Richard Visser, co-promotor(en): Arnaud Bovy; Yury Tikunov. - Wageningen : Wageningen University - ISBN 9789463431842 - 168
solanum lycopersicum - tomatoes - genotypes - heat stress - heat tolerance - pollen - metabolomes - metabolites - metabolomics - tomaten - genotypen - warmtestress - hittetolerantie - stuifmeel - metabolomen - metabolieten - metabolomica

Tomato production is threatened by climate change. High temperatures lead to a decrease of fruit set which correlates with a decrease of pollen fertility. The low viability of tomato pollen under heat stress was previously shown to be associated with alterations in specific metabolites. In this thesis, we used untargeted metabolomics approaches to broaden the identification of metabolites affected by heat stress. We assessed the suitability of pollen isolation methods for metabolomics analysis and considered the pitfalls for our further analysis. We explored the developmental metabolomes of pollen and anthers of different tomato genotypes under control and high temperature conditions and identified that microsporogenesis is a critical developmental stage for the production of mature and fertile pollen grain under heat stress. Several metabolites were putatively associated with tolerance to high temperature such as specific flavonoids, polyamines and alkaloids. These metabolites can be further used as markers in breeding programs to develop new genotypes tolerant to high temperatures.

Invloed van gewassen op bodemkwaliteit: Variatie tussen genotypen : een verkennende literatuurstudie voor ruwvoedergewassen
Wiel, Clemens C.M. van de; Linden, Gerard van der; Sukkel, Wijnand - \ 2017
Wageningen : Wageningen Plant Research (Rapport / Wageningen Plant Research 668) - 17
voederplanten - ruwvoer (forage) - bodemkwaliteit - genotypen - fodder plants - forage - soil quality - genotypes
Gewassen beïnvloeden de bodem waarop ze geteeld worden. In deze verkennende literatuurstudie is onderzocht wat er bekend is over variatie tussen verschillende genotypen/plantenrassen met betrekking tot hun effect op bodemkwaliteit voor een aantal ruwvoedergewassen, in het bijzonder Maïs (Zea mays), Engels raaigras (Lolium perenne) en Witte klaver (Trifolium repens). Dergelijke variatie zou mogelijkheden kunnen bieden om via veredeling en het inzetten van specifieke rassen de bodemkwaliteit te verbeteren. Er is betrekkelijk weinig onderzoek vanuit deze invalshoek gedaan. De focus van de studie was gericht op variatie in wortelarchitectuur, en wortelexudatie en wortelafsterving/nieuwvorming in relatie tot het organische stof gehalte en beschikbaarheid van nutriënten, en de bodem biota, met name mycorrhiza.
Development and application of a 20K SNP array in potato
Vos, Peter - \ 2016
University. Promotor(en): Richard Visser; Fred van Eeuwijk, co-promotor(en): Herman van Eck. - Wageningen : Wageningen University - ISBN 9789462579569 - 166
solanum tuberosum - potatoes - genotypes - single nucleotide polymorphism - data analysis - plant breeding - linkage disequilibrium - genome analysis - tetraploidy - aardappelen - genotypen - gegevensanalyse - plantenveredeling - verstoord koppelingsevenwicht - genoomanalyse - tetraploïdie

In this thesis the results are described of investigations of various application of genome wide SNP (single nucleotide polymorphism) markers. The set of SNP markers was identified by GBS (genotyping by sequencing) strategy. The resulting dataset of 129,156 SNPs across 83 tetraploid varieties was used directly to map traits, but also as a basis for the development of a 20K SNP array in Potato (Solanum tuberosum L.). Subsequently this array, named SolSTW, was used to collect genotypic data from 569 potato genotypes. This dataset offered insight in the breeding history of potato, population structure, linkage disequilibrium (LD) and the potential of GWAS (genome wide association studies) in potato.

In Chapter 2 we describe to development of the SolSTW 20K Infinium SNP array. One third of the SNPs on this array originate from the well-known SolCAP 8303 SNP array. The other SNPs are a subset from a targeted re-sequencing project of 83 tetraploid potato varieties. Because of the high SNP density in potato only a limited number of SNPs is suitable for assay development on a SNP array. An obvious outcome is that flanking SNPs contribute to assay failure, particularly for assays with SNPs located in introns. We used fitTetra software to cluster the distribution of captured signals of each marker into the expected five genotypic classes (nulliplex, simplex, duplex, triplex, quadruplex), resulting in a dataset with 14,530 SNP markers. Subsequently the genotypic data obtained with the SolSTW array was used to characterize a set of 569 potato varieties, advanced breeding clones and progenitors. This resulted in the identification of several footprints of potato breeding. Firstly SNPs were dated i.e. the year of market release of the first variety showing polymorphism for a SNP locus is an indication of the ancestry of a SNP. In such a way we identified SNPs with an ancestry tracing back to heirloom varieties, and SNPs (post-1945 SNPs) tracing back to wild species used in modern introgression breeding. Secondly, the changes in allele frequency were calculated over time. Most SNPs show a relative stable allele frequency over time, and very limited genetic variation is removed from the gene-pool of potato i.e genetic erosion is almost absent. Therefore we conclude that 100 years of breeding has not been able to get rid of non-beneficial genetic variation. Only a limited number of SNPs show a rapid increased in allele frequency, which can be explained by positive selection for disease resistance by breeders, or the more frequent use of several founders.

Better understanding of the genome wide decay of Linkage Disequilibrium (LD) and population structure offers relevant knowledge to perform and interpret the results of a genome wide association study (GWAS) (Chapter 3). Linkage disequilibrium (LD) is a complex phenomenon, and the influence of the factors shaping LD in tetraploids is hardly studied. Therefore we used simulated data to disentangle and therewith understand often-confounded factors underlying LD-decay. We simulated datasets differing in number of haplotypes in a population, and differing in percentage of haplotype specific SNPs. In these simulations we observed that the choice of an estimator of LD-decay has a major effect on the outcome of an LD-decay estimate, while the true LD-decay remains the same. Based on the simulation we conclude that a 90% percentile and a so-called D1/2 (the distance where 50% of the initial LD is decayed) performed best to estimate and compare LD-decay in potato. To understand the various aspects of LD-decay in the variety panel of 537 varieties, the panel was subdivided in several groups based on the age of a variety and the population structure groups. This resulted in the identification of LD-decay over time, i.e in relatively young varieties the average size of the LD-blocks is smaller. The differences between subpopulations were smaller and are most likely the effect of the population structure. We also observed that there are very long LD-blocks caused by introgression breeding and that different a priori MAF-thresholds also can influence the outcome of LD-decay estimation.

Having both LD-decay and population structure defined a genome wide association study (GWAS) was conducted (Chapter 4). For this purpose α-solanine and α-chaconine were measured in potato tubers. Subsequently the sum of both (total SGA) and the ratio between the two were used to discover QTLs for these traits in a GWAS. Additionally we used three bi-parental populations to validate the GWAS results. Total SGA content was confounded with population structure and therefore it was difficult to explain all phenotypic variation with SNP markers. Two QTLs (Sgt1.1 and Sgt11.1) were identified which could be validated in one of the segregating populations. The ratio between α-solanine and α-chaconine was not confounded with population structure, resulted in the identification of two major-effect QTLs (Sgr7.1 & Sgr8.1) located near the candidate genes SGT1 and SGT2, which are known for being responsible in the final steps towards either α-solanine or α-chaconine. The QTL Sgr8.1 could be validated, however similar phenotypes were explained by different haplotypes in two populations. We show that population structure, low frequent alleles and genetic heterogeneity may explain to some degree the missing heritability in GWAS in potato.

In Chapter 5 we describe how the method of graphical genotyping, which is widely used in diploid bi-parental populations, can be applied in a variety panel of tetraploid varieties. We show that a few discrete filtering steps in Excel can be used to display patterns that are visual representations of introgression segments and the locations of historical recombination events. Using this method we identified introgression segments from Solanum vernei including the Gpa5 locus on chromosome 5 and Solanum stoloniferum introgression segment including a gene involved in resistance to Potato Virus Y on chromosome 11. This method requires that the haplotypes that cause the phenotypic effect have to be identical by descent (IBD).

In the final chapter 6 the results of chapter 2 to 5 are discussed. We look forward on how our results can be used in future research and applied in marker-assisted breeding. Additionally some new GWAS results are presented for tuber flesh colour, foliage maturity and resistance to Globodera pallida pathotype 3.

Feather pecking and monoamines - a behavioral and neurobiological approach
Kops, M.S. - \ 2014
University; Utrecht University. Promotor(en): B. Olivier; O. Güntürkün, co-promotor(en): S.M. Korte; Liesbeth Bolhuis. - Utrecht, The Netherlands : Utrecht University - ISBN 9789039361283 - 172
pluimveehouderij - hennen - verenpikken - diergedrag - dierenwelzijn - pluimvee - diergezondheid - dierlijke productie - serotonine - dopamine - fenotypen - genotypen - neurotransmitters - invloeden - poultry farming - hens - feather pecking - animal behaviour - animal welfare - poultry - animal health - animal production - serotonin - phenotypes - genotypes - influences
Severe feather pecking (SFP) remains one of the major welfare issues in laying hens. SFP is the pecking at and pulling out of feathers, inflicting damage to the plumage and skin of the recipient. The neurobiological profile determining the vulnerability of individual hens to develop into a severe feather pecker is unknown, although brain monoamines such as serotonin (5-HT) and dopamine (DA) seem to play a role. Previous studies related lower 5-HT and DA turnover ratios to an increased risk to develop SFP.In this thesis, monoamine levels in brain areas involved in emotional regulation and motor control were compared between phenotypically and genetically selected high and low feather peckers at different ages. It was found that adult high feather peckers had higher monoaminergic activity (lower metabolite levels and/or turnover ratios) in comparison to low feather peckers, which is in contrast with results on young hens. Differences were seen in several brain areas, namely the dorsal thalamus, medial striatum, amygdala, caudocentral nidopallium, and the somatomotor arcopallium, but to a lesser extend or not in the caudolateral nidopallium and the hippocampus. To investigate the exact neurobiological mechanism behind severe feather pecking further extracellular levels of 5-HT and DA and their metabolites were measured by in vivo microdialysis. Up till now, microdialysis has only been executed in young chickens, but this thesis describes the first microdialysis study performed in adult laying hens. It was found that adult severe feather peckers had a higher baseline release of 5-HT in the caudal nidopallium, a large associative area in the chicken’s forebrain. This result could not be explained by the amount of 5-HT presynaptically stored, as both high and low SFP lines displayed a similar 5-HT release after d-fenfluramine administration. This confirms that genetic selection on SFP has altered the serotonergic system in feather pecking-phenotypes. With clear phenotypic and genotypic differences in brain areas related to emotional regulation and motor control, it can be assumed that brain deficits at a young age increase an individual’s vulnerability to stressful environmental changes, which is associated with the prevalence of SFP later in life. The cause of the inversion of neurochemical patterns in young and adult high and low feather pecking hens remains to be elucidated. Perhaps this inversion is caused by development itself. On the other hand, higher behavioral patterns (SFP and other types of allopecking) observed in the high feather pecking chickens might have influenced the monoaminergic activity since the brain influences behavior and vice versa. Altogether, this thesis demonstrates the importance of considering the impact of genetic selection and also environmental conditions on brain neurotransmission and with that, on the vulnerability of individual chickens to develop SFP. Both the serotonergic and dopaminergic systems are involved in the development of SFP. With SFP being a multifactorial problem both genotype and phenotype have to be taken into account. Furthermore, in vivo microdialysis is a valuable approach to investigate why individual laying hens start SFP. This will lead to further understanding and ultimately in the reduction of SFP.
Linkages between plant traits and soil ecology in the rhizosphere and through litter decomposition
Brolsma, K.M. - \ 2014
University. Promotor(en): Ellis Hoffland, co-promotor(en): Ron de Goede. - Wageningen : Wageningen University - ISBN 9789462571068 - 112
bodemecologie - rizosfeer - ligstro - decompositie - wortels - bodembiologie - nematoda - solanum tuberosum - globodera pallida - biofumigatie - genotypen - soil ecology - rhizosphere - litter - decomposition - roots - soil biology - biofumigation - genotypes
Environmental regulation of seed performance
He, H. - \ 2014
University. Promotor(en): Harro Bouwmeester; J.C.M. Smeekens, co-promotor(en): Henk Hilhorst; Leonie Bentsink. - Wageningen University : Wageningen University - ISBN 9789462570337 - 185
arabidopsis - zaden - kiemrust - zaadkieming - milieueffect - genetica - genotypen - temperatuur - seeds - seed dormancy - seed germination - environmental impact - genetics - genotypes - temperature

The seed stage is an essential episode in the life cycle of higher plants. The environmental cues that seeds experience during their development are important components of their life history. The parental environment, from pre-fertilization until seed dispersal affects performance of the dry mature seed and, therefore, affects the life cycle of the next generation. The evolutionary response to environmental perturbations has resulted in genetic changes in order to increase the fitness of the population, which is called ‘adaptation’. The aims of this study were to increase our understanding of how environments regulate seed performance, both on the long term, i.e. through adaptation of seed performance traits to local conditions and on the short term, i.e. by acclimation of plants to different seed maturation environments.

The ideal hen for organic and free range systems
Maurer, V. ; Amsler, Z. ; Zeltner, E. ; Leenstra, F.R. ; Niekerk, T.G.C.M. van; Reuvekamp, B.F.J. ; Bestman, M.W.P. ; Verwer, C.M. ; Sambeek, F. ; Galea, F. - \ 2014
pluimveehouderij - biologische landbouw - huisvesting van kippen - hennen - scharrelhouderij - genotypen - diergezondheid - poultry farming - organic farming - chicken housing - hens - free range husbandry - genotypes - animal health
The aim of LowInputBreeds is to develop a system to evaluate layer genotypes in free range and organic systems and to optimise the management of those systems.
Toprunderen door genomische kennis
Veerkamp, R.F. ; Calus, M.P.L. ; Roos, S. de; Geus, B. de - \ 2014
V-focus 2014 (2014)1. - ISSN 1574-1575 - p. 30 - 31.
rundveehouderij - dierveredeling - dierenwelzijn - duurzame veehouderij - duurzame ontwikkeling - bedrijfsresultaten in de landbouw - dna - genotypen - dierlijke productie - tracking en tracing - cattle husbandry - animal breeding - animal welfare - sustainable animal husbandry - sustainable development - farm results - genotypes - animal production - tracking and tracing
Met de introductie van genomics is het mogelijk om op basis van DNA-testen te voorspellen wat de genetische waarde van een dier in de populatie is. Breed4Food, een samenwerking van vier private partijen en Wageningen UR, wil de kennis over genomische informatie versnellen en samen delen. Dit artikel beschrijft de activiteiten, die worden opgepakt voor de rundveehouderij.
Grain filling, starch degradation and feeding value of maize for ruminants
Ali, M. - \ 2013
University. Promotor(en): Paul Struik; Wouter Hendriks, co-promotor(en): John Cone. - Wageningen : Wageningen UR - ISBN 9789461738196 - 177
maïs - zea mays - groeifasen, rijp - genotypen - zetmeelvertering - pensvertering - voedingswaarde - herkauwersvoeding - korrels (granen) - maïskuilvoer - kuilvoerbereiding - gewasfysiologie - voedingsfysiologie - maize - maturity stage - genotypes - starch digestion - rumen digestion - nutritive value - ruminant feeding - kernels - maize silage - silage making - crop physiology - nutrition physiology

Keywords; Maize (Zea mays L), Genotypes, Grain filling, Growth temperature, Kernels, Gas production, Starch degradation, Oven-drying, Silage, Ensiling temperature, Ensiling duration, Feeding value, Lactating cows

Maize (Zea mays L.) is a major component in the ration of dairy cows in many parts of the world. The currently increasing economic importance of maize has highlighted the need to determine its nutritional value, and to assess the factors influencing its nutritive value. Genotypic make-up (especially differences in starch and endosperm), growing conditions, maturity stage at harvest, and post handling processes, like oven-drying, can influence nutritive value of maize kernels. Similarly, ensiling temperature and duration can affect feeding value of maize silage. This thesis is divided into three parts; the first aim was to characterize the dry matter (starch) accumulation of maize different genotypes in different environments under controlled (glasshouse) and on different locations (sand and clay) in field conditions. Maize genotypes used were different in starch structure and composition, and in type of endosperm. Starch structure refers to amylose and amylopectin; and composition refers to their proportions, whereas type of endosperm defines levels of vitreousness. The vitreousness is the ratio of vitreous (hard) to floury (soft) endosperm. Six maize genotypes, differing in amylose content and vitreousness, were grown under three contrasting day/night temperature regimes during grain filling and harvested at different maturity stages from two greenhouse experiments. Similar investigations were carried on another set of genotypes grown on sandy and clay soils and with different sowing times under field conditions. Water contents and dry matter (starch) accumulation were significantly influenced by growth temperature, genotype, soil type and sowing time (P<0.0001). The second aim of thesis was to establish a relationship between rumen in vitro starch degradation (feeding value) of maize kernels and different factors, like genotype, growth temperature during grain filling, and maturity stage. Oven-dried kernels of six maize genotypes, from the two greenhouse experiments mentioned before were investigated. Starch content was measured using an enzymatic method and the gas production technique was used to assess starch degradation in rumen fluid of dairy cows. The extent of starch degradation at different incubation times was calculated from measured gas production data (6, 12 and 20 h, respectively) and a published equation. At each maturity stage, whole kernel and starch degradation in rumen fluid depended on the genotype (P<0.0001), growing conditions (P<0.0001), starch content (P<0.0001) and starch amount (P<0.0001) in the kernels. The same but fresh (not oven-dried) maize kernel samples were investigated using gas production technique to determine the impact of oven-drying on rumen in vitro starch degradation of maize kernels. Oven-drying significantly (P<0.0001) influenced the rumen in vitro starch degradation in maize kernels various incubation times, with more starch being degraded in the fresh than in the oven-dried maize kernels, although the differences were small. There was a consistent and highly significant (P<0.009 to 0.0002) interaction between oven-drying and genotype, with the high-amylose genotype showing larger effects of oven-drying than the other genotypes. The third aim of thesis was to investigate effect of ensiling temperature and duration on feeding value of maize silage. Samples of maize whole plants (dry matter 33%) were collected from the medium vitreous endosperm cultivar, grown in different seasons on sandy soils. Maize plants were chopped and ensiled in mini silos at three different temperatures. Samples from the silos were taken after 0 (not ensiled, i.e. control), 4, 8 and 16 weeks of ensiling. The gas production technique was used to evaluate the influence of the ensiling temperature and duration of ensiling on the degradation of the fresh ground silage samples in rumen fluid. The final pH of the silages and the gas production was significantly influenced by ensiling temperature in both seasons (P<0.0001). Gas production and pH decreased with an increase in ensiling duration (P<0.0001). The relationship between pH and gas production was quadratic and depended on the ensiling temperature (P<0.002). It was found that ensiling temperature and ensiling duration determine the rate of change and final pH, and play a significant role in feeding value of maize silage. The finding of thesis can be used to determine the exact feeding value of maize kernels and silage, and also can be used as a tool to revise the current feeding evaluation systems i.e. shift from oven-dried to fresh samples.

Optimizing genomic selection for scarcely recorded traits
Pszczola, M.J. - \ 2013
University. Promotor(en): Johan van Arendonk, co-promotor(en): Mario Calus; T. Strabel. - Wageningen : Wageningen UR - ISBN 9789461737663 - 158
melkvee - genomen - selectief fokken - genetische verbetering - fokwaarde - fenotypen - genotypen - kenmerken - voeropname - dierveredeling - dairy cattle - genomes - selective breeding - genetic improvement - breeding value - phenotypes - genotypes - traits - feed intake - animal breeding

Animal breeding aims to genetically improve animal populations by selecting the best individuals as parents of the next generation. New traits are being introduced to breeding goals to satisfy new demands faced by livestock production. Selecting for novel traits is especially challenging when recording is laborious and expensive and large scale recording is not possible. Genetic improvement of novel traits may be thus limited due to the small number of observations. New breeding tools, such as genomic selection, are therefore needed to enable the genetic improvement of novel traits. Using the limited available data optimally may, however, require alternative approaches and methodologies than currently used for conventional breeding goal traits. The overall objective of this thesis was to investigate different options for optimizing genomic selection for scarcely recorded novel traits. The investigated options were: (1) genotype imputation for ungenotyped but phenotyped animals to be used to enlarge the reference population; (2) optimization of the design of the reference population with respect to the relationships among the animals included in it; (3) prioritizing genotyping of the reference population or the selection candidates; and (4) using easily recordable predictor traits to improve the accuracy of breeding values for scarcely recorded traits. Results showed that: (1) including ungenotyped animals to the reference population can lead to a limited increase in the breeding values accuracy; (2) the reference population is designed optimally when the relationship within the reference are minimized and between reference population and potential selection candidates maximized; (3) the main gain in accuracy when moving from traditional to genomic selection is due to genotyping the selection candidates, but preferably both reference population and selection candidates should be genotyped; and (4) including the predictor traits in the analysis when it is recorded on both reference population and selection candidates can lead to a significant increase in the selection accuracy. The key factors for successful implementation of selection for a novel trait in a breeding scheme are: (1) maximizing accuracy of genotype prediction for ungenotyped animals to be used for updating the reference population; (2) optimizing the design of the reference population; (3) determining easy to record indicator traits that are also available on the selection candidates (4) developing large scale phenotyping techniques; and (5) establishing strategies and policies for increasing the engagement of farmers in the recording of novel traits.

A taste of pepper: genetics, biochemistry and prediction of sweet pepper flavor
Eggink, P.M. - \ 2013
University. Promotor(en): Richard Visser, co-promotor(en): Arnaud Bovy; Chris Maliepaard; J.P.W. Haanstra. - S.l. : s.n. - ISBN 9789461737243 - 159
capsicum annuum - paprika's - wilde verwanten - genotypen - chemische samenstelling - smaak - geur en smaak - capsicum baccatum - introgressie - plantenveredeling - sweet peppers - wild relatives - genotypes - chemical composition - taste - flavour - introgression - plant breeding

This PhD project started with the composition of a diverse panel of genotypes that represented, (i) roughly the flavor variation in the commercial Capsicum annuum breeding program of Rijk Zwaan, (ii) parents of available mapping populations and (iii) some genotypes that were expected to have extraordinary flavors. The complete set consisted of 35 genotypes of which 24 genotypes were non-pungent. Volatile and non-volatile compounds as well as some breeding parameters were measured in mature fruits of all genotypes throughout the growing season. In addition, from three harvests the non-pungent genotypes were evaluated for taste by a trained descriptive sensory panel.

The biochemical profiling with use of SPME-GC-MS allowed visualization of between- and within-species volatile compound variation. Principal components analysis (PCA) on the intensity patterns of 391 putative volatile compounds revealed individual grouping of C. chinense, C. baccatum var. pendulum and C. annuum, indicating potentially interesting volatile variation present in the former two groups. A large group of saturated and unsaturated esters were mainly responsible for the individual grouping of the C. chinense accessions. Due to the elevated acid concentrations and aberrant volatile profiles of the C. baccatum var. pendulum accessions PEN45 and PEN79, the two BIL populations derived from these accessions were identified as interesting candidates for further study. Compared to e.g. Mazurka the citrate concentration of the C. baccatum accessions was 2.5-3 times higher and the malate concentrations were even up to 12 times higher (Chapter 2).

Based on the non-pungent genotypes, we found highly correlated clusters of volatiles and non-volatiles, which could be related to metabolic pathways and common biochemical precursors (Chapter 3). Contrasts between genotypes were caused by both qualitative and quantitative differences in these metabolic clusters, with the phenolic derivatives, higher alkanes, sesquiterpenes and lipid derived volatiles forming the major determinants. For the description of the non-pungent genotypes the panelists used fourteen attributes to describe the flavor sensation in the mouth/throat, which were the texture attributes crunchiness, stickiness of the skin, toughness and juiciness, the basic taste attributes sweetness and sourness and the retronasal flavor attributes aroma intensity, grassiness, green bean, carrot, fruity/apple, perfume, petrochemical and musty. The variation in flavor could be reduced into two major sensory contrasts, which were a texture related contrast and the basic sweet-sour contrast. The structure of the PCA plots resulting from the analysis with one harvest (Chapter 3) and the analysis with the combined three harvests (Chapter 4) remained almost identical, indicating the stability of these contrasts. To relate the sensory attributes to the metabolite data and to determine the importance of the individual compounds we used Random Forest regression on the individual harvests and on the three harvests together. Several predictors for the attributes aroma, fruity/apple, sourness and sweetness were found in common between harvests, which we proposed as key-metabolites involved in flavor determination of sweet pepper (Chapter 4). This list contains compounds with known relations to attributes, like sweetness and sugars, but also several compounds with new relations. In this respect we have demonstrated for the first time, that the metabolites p-menth-1-en-9-al, (E)-β-ocimene, (Z)-2-penten-1-ol, and 1-methyl-1,4-cyclohexadiene are related to fruity/apple taste and/or sweetness of pepper. For sourness the only compound with a consistent significant contribution was an unknown C6H8O2 compound. We postulated therefore the hypothesis that in pepper the effect of sourness related metabolites is masked by other volatile and non-volatile compounds or texture differences (Chapter 3). Subsequently in Chapter 4 we described a clear sweetness-sourness interaction and demonstrated that the masking effect of fructose and other sugars explained why we did not find organic acids contributing to the prediction of sourness. The major sensory attributes were also predicted between harvests. The Random Forest predictions of the texture related attributes (juiciness, toughness, crunchiness and stickiness of the skin) and sweetness were very good. The predictions of the attributes aroma intensity, sourness and fruity/apple were somewhat lower and more variable between harvests, especially in the second harvest. In general, we concluded that prediction of attributes with higher heritabilities works better and is more consistent over harvests (Chapter 4).

Based on the results of the initial experiments (Chapter 2) the species C. baccatum was chosen for further study. To exploit the potential flavor wealth of C. baccatum PEN45 we combined interspecific crossing with embryo rescue, resulting in a multi-parent BC2S1 population, that was characterized for sensory and biochemical variation (Chapter 5). We developed a population specific genetic linkage map for QTL mapping of characterized traits. Because of the complex structure of our BC2S1 mapping population we encountered several limitations, such as accidental co-segregation, underrepresentation of color linked markers and pre-selection leading to skewness, which might have resulted in false positive or missed QTLs. Despite these limitations, we were still fairly well able to map several biochemical, physical and sensory traits, as demonstrated at first for the (monogenic) control traits red color and pungency in the BC2S1 mapping population and in second instance by validation of genetic effects via an experiment with near-isogenic lines (NILs).This two-step approach turned out to be very powerful, since it led to the identification of the main results from this thesis: (i) Asmall C. baccatum LG3 introgression causing an extraordinary effect on flavor, which resulted in significantly higher scores for the attributes aroma, flowers, spices, celery and chives. In an attempt to identify the responsible biochemical compounds few consistently up- and down-regulated metabolites were detected, including the well-known pepper compound 2-isobutyl-3-methoxypyrazine (down) and 6-methyl-4-oxo-5-heptenal (up); (ii) Two introgressions (LG10.1 and LG1) had major effects on terpenoid content of mature fruits, affecting at least fifteen different monoterpenes; (iii) A second LG3 fragment resulted in a strong increase in Brix (total soluble solids) without negative effects on fruit size (Chapter 5).

In Chapter 6 some extra sensory results of the pungent genotypes are given and a comparison between the two C. baccatum pendulum BILs (PEN45 and PEN79 derived) is made in light of the overall results. Finally the perspectives for breeding are discussed and presented in the form of a flowchart for flavor improvement.

Genetic analysis of drought stress response in Arabidopsis thaliana and Brassica rapa
El-Soda, M. - \ 2013
University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - S.l. : s.n. - ISBN 9789461737441 - 146
brassica campestris - arabidopsis thaliana - droogte - droogteresistentie - stressreactie - genetische analyse - genotypen - genotype-milieu interactie - drought - drought resistance - stress response - genetic analysis - genotypes - genotype environment interaction

Drought is the major abiotic stress affecting plant growth and limiting crop productivity worldwide. Plants have evolved three adaptive strategies, drought escape, drought avoidance and drought tolerance, to cope with drought. Knowledge on how Quantitative Trait Loci (QTL), or genes underlying these strategies interact with their environments will significantly increase our understanding and the success of breeding for drought tolerance. This thesis focused on phenotyping shoot and root traits ofA. thaliana and B. rapa grown on sand and in greenhouses,to further understand how plants can adapt to natural drought stress. In chapter 2, an already existing ArabidopsisRIL population was selected based on the differential root drought response of the two parental lines, Sha and Col, to be evaluated under different water regimes. Chapter 3 illustrated the use of GWAS in identifying candidate genes that are associated with pant response to drought.. In order to apply the same methodology in crop breeding, chapter 4 introduces a contribution to the genetic mapping of a new B. rapa RIL population, consisting of 160 lines and genotyped with 270 different markers was achieved. The morphological and physiological responses of this population to drought was evaluated in chapter 5. The results presented in the present thesis demonstrate that QxE is an important component of the genetic variance and can play a great role in improving drought tolerance in future breeding programs. In general, several QTL and SNPs were mapped either with main effect or with interaction with environments QxE. Many of the mapped QTL showed conditional neutrality and antagonestic pleiotropy.

Identification of traits and QTLs contributing to salt tolerance in barley (Hordeum vulgare L.)
Nguyen Viet Long, L. - \ 2012
University. Promotor(en): Richard Visser, co-promotor(en): Gerard van der Linden; Oene Dolstra. - S.l. : s.n. - ISBN 9789461734099 - 137
hordeum vulgare - gerst - zouttolerantie - loci voor kwantitatief kenmerk - groei - plantensamenstelling - genotypen - chromosomen - homeostase - plantenveredeling - barley - salt tolerance - quantitative trait loci - growth - plant composition - genotypes - chromosomes - homeostasis - plant breeding

Salinity is the most severe abiotic stress perceived by plants and affects about 800 million hectares of land worldwide, including 20% of the world’s highly productive irrigated land. Significant crop yield losses are observed due to salinity. Salinization is increasing because of poor irrigation management and climate change. Improving salt tolerance in crops is for these reasons an important target for plant breeding in the near future. However, salinity tolerance in plants is not easy to breed for due to its interaction with many physiological processes controlled by many genes and their interaction with the environment. Barley is a good model crop to study different mechanisms conferring salt tolerance in cereals. A traditional QTL mapping approach in combination with a new association mapping method allowed us to efficiently explore the genetics and genetic diversity of salt tolerance in barley. Improvements of the association mapping technology highly increased detection power and mapping accuracy. The traits and QTLs identified in this thesis point out both osmotic and ionic stress tolerant genes as important targets for salt tolerance breeding. This thesis provides tools to plant breeders for the application of marker-assisted introgression breeding of salt tolerance genes in their breeding programs. Some QTLs were found to be syntenic with the important QTLs/genes for salt tolerance found in wheat and rice such as Na+ and K+ transporter gene families. Other QTLs were new and suggest the presence of novel genes that play an important role in plant ion homeostasis, transportation of Cl- and Ca2+ and osmotic tolerance. We demonstrated that association mapping can be a powerful approach to dissect the complexity of salt tolerance in barley. The newly available high-density SNP map of barley and the barley genome sequence in the near future further increases the accuracy of mapping studieswith the association panel and will greatly facilitate the cloning of the genes underlying salt tolerance in barley. This thesis thus contributes to better a understanding of the physiological and genetic basis of salt tolerance and improved breeding strategies for the development of salt tolerant varieties.

Discovery and genotyping of existing and induced DNA sequence variation in potato
Uitdewilligen, J.G.A.M.L. - \ 2012
University. Promotor(en): Richard Visser, co-promotor(en): Herman van Eck; Anne-Marie Wolters. - S.l. : s.n. - ISBN 9789461732330 - 165
solanum tuberosum - aardappelen - dna-sequencing - dna - nucleotidenvolgordes - genotyping - plantenveredeling - genotypen - fenotypen - tetraploïdie - potatoes - dna sequencing - nucleotide sequences - plant breeding - genotypes - phenotypes - tetraploidy

In this thesis natural and induced DNA sequence diversity in potato (Solanum tuberosum) for use in marker-trait analysis and potato breeding is assessed. The study addresses the challenges of reliable, high-throughput identification and genotyping of sequence variants in existing tetraploid potato cultivar panels using traditional Sanger sequencing and next-generation massively parallel sequencing (MPS), and the application of this knowledge in the form of genetic markers. Furthermore, it explores the efficiency of ethyl methanesulphonate (EMS) mutagenesis combined with high resolution melting (HRM) DNA screening to induce and discover novel sequence variants in potato genotypes.
Discovery and genotyping of sequence diversity in outcrossing autotetraploid species like potato is complex. In autotetraploid species, genotyping implies the quantitative identification of five alternative allele copy number states. In Chapter 1, several methodologies to identify and genotype DNA sequence variants, and the application of these sequence variants is discussed. This chapter provides an introduction to genotyping-by-sequencing (GBS) and the determination of allele copy number.
In Chapter 2 the sequence diversity in three genes of the carotenoid pathway is assessed in diploid and tetraploid potato genotypes using direct Sanger sequencing. To investigate the genetics and molecular biology of orange and yellow flesh colour in potato, association analysis between SNP haplotypes and flesh colour phenotypes was performed, and the inheritance and gene expression of associated alleles was studied. We observed among eleven beta-carotene hydroxylase 2 (CHY2) alleles one dominant allele with a major effect, changing white into yellow flesh colour. In contrast, none of the lycopene epsilon cyclase (LCYe) alleles seemed to have a large effect on flesh colour. Analysis of zeaxanthin epoxidase (ZEP) alleles showed that a recessive allele with a non-LTR retrotransposon sequence in intron 1 reduced the expression level of the ZEP gene and caused accumulation of zeaxanthin. Genotypes combining presence of the dominant CHY2 allele with homozygosity for the recessive ZEP allele produced orange-fleshed tubers that accumulate large amounts of zeaxanthin.
Sanger amplicon sequencing was applied in Chapter 3 to evaluate the sequence diversity in α-Glucan Water Dikinase (StGWD), a candidate gene underlying a QTL involved in potato starch phosphate content. Sanger sequences of two StGWD amplicons from a global collection of 398 commercial cultivars and progenitor lines were used to identify 16 unique haplotypes. By assigning tag SNPs to these haplotypes and by determining the allele copy number of identified sequence variants, we inferred the four-allele genetic composition for almost all cultivars assayed at this locus. This allowed genetic diversity parameters like the average number of different alleles present in a single cultivar (Ai=3.1) and the average intra-individual heterozygosity (Ho=0.765) to be estimated for this locus. Pedigree analysis confirmed that the identified haplotypes are identical by descent (IBD) and offered insight in the breeding history of elite potato germplasm. Haplotype association analysis led to the identification of two StGWD alleles causing altered starch phosphate content, which was further verified in diploid and tetraploid mapping populations containing the relevant alleles. One of these alleles (Allel H) increases the fraction of starch that is phosphorylated, while the other one (Allele A) decreases it.
To scale up the discovery and genotyping of sequence variants, and to make it more whole-genome oriented, Chapter 4 reports on massively parallel sequencing (MPS) of approximately 800 genes scattered over the potato genome and resequenced in 83 tetraploid potato cultivars and a monoploid reference accession. We show that by combining MPS with genome complexity reduction and indexed sequencing, sufficient read depth for GBS can be achieved for reliable discovery and genotyping of sequence variants in individual tetraploid potato genotypes. With a custom designed, SureSelect enrichment library, 1.44 Mb of DNA sequence was targeted. The genes targeted were mainly single-copy genes, selected based on putative gene functions in both primary and secondary metabolic pathways, potato quality traits and biotic and abiotic stresses, and included a large set of conserved orthologous sequence genes (COSII) useful for genetic anchoring and phylogenetic studies. The indexed and enriched DNA libraries were sequenced on a Illumina HiSeq. After filtering and processing the raw sequence data, 12.4 Gb of high-quality sequence data was mapped to the potato genome, covering 2.1 Mb of the genome sequence with a median average read depth of 63× per cultivar. We detected over 129,000 sequence variants in these data and determined allele copy number of the variants in individual potato samples. The accuracy of the sequence-based allele copy number estimates was verified by a low-density SNP genotyping assay. This showed that for reliable genotyping a read count-based genotype quality score is best applied and a read depth of 80× is recommended for determining allele copy number in autotetraploid potato. Average nucleotide diversity (π=10.7×10-3 genome-wide, ≈1 variant/93 bp between two random alleles) varied along the twelve potato chromosomes, and individual genes under selection were identified. As an example for application of GBS for genome-wide association analysis (GWAS), the identified sequence variants and genotype data were tested in a marker-trait association analysis with plant maturity and tuber flesh colour. This led to the identification of alleles accounting for significant phenotypic variation in these traits.
In Chapter 5 we applied the chemical mutagen EMS to diploid potato by two different treatments, a pollen and a seed treatment. We screened the resulting populations for novel mutations using HRM analysis. A pollen treatment with EMS dissolved in a sucrose solution was found to induce mutations only at a low frequency (only one mutation discovered after screening >2.7 Mb of sequence). In planta selection of the most vital mutagenized pollen seems to have lowered the mutation density to a frequency that is not suitable for reverse genetics studies. Treatment of potato seeds with EMS on the other hand provided a high density of novel mutations (1 mutation/65 kb), discovered in the M1 generation. In contrast to most EMS mutagenesis studies, we directly screened the M1 generation of the seed-treated population. In six candidate genes involved in potato starch and frying quality traits, 65 novel sequence variants were discovered. In all six genes, missense mutations that are predicted to damage protein function were discovered, and for four genes five premature stop codon mutations were identified. We attempted to stabilize and transfer 27 putatively interesting mutations to the M2 and M3 generation for further evaluation. Genetically stable M2 and M3 plants have been generated for 10 (37%) of these mutations. The estimated density of M1 mutations that are transferable to the M2 generation (one “accessible” mutation/118-176 kb) is higher than the mutation densities obtained in most other plant species, for which the M2 generation has been screened. The results of this chapter thus demonstrate that screening the M1 generation offers a good alternative to the commonly applied M2 screening for the rapid generation of novel genetic variation at a high density, without too much complication in recovering mutations in the M2 generation.
In the concluding Chapter 6, results of preceding chapters are evaluated, and the prospects of the findings for potato research and breeding are discussed.

Hoge resolutie typering van Coxiella burnetii
Janse, I. ; Bossers, A. ; Roest, H.I.J. ; Rotterdam, B. van - \ 2011
Bilthoven : RIVM (RIVM briefrapport / Rijksinstituut voor Volksgezondheid en Milieu 330302001/2011) - 38
q-koorts - coxiella burnetii - epidemiologie - genotypen - virulentie - geiten - schapen - q fever - epidemiology - genotypes - virulence - goats - sheep
Dit rapport beschrijft het onderzoek wat uitgevoerd is in het kader van het project ‘Hoge Resolutie Typering Coxiella burnetii’’. Het doel van dit project was om de genoomsequenties van een aantal Nederlandse isolaten van de bacterie Coxiella burnetii, de veroorzaker van Q-koorts, in kaart te brengen om hiermee de ontwikkeling van verbeterde typeringsmethodes mogelijk te maken. Op basis van genoomsequenties kan onderzocht worden of de Nederlandse uitbraken bijvoorbeeld samenhangen met veranderde virulentiekenmerken. Genoom informatie is ook essentieel om een verbeterde, op de Nederlandse situatie toegespitste typering mogelijk te maken.
Een ontmoeting met de Nederlanders: populatiegenetica van Phytophthora infestans in Nederland gedurende het laatste decennium
Lee, T.A.J. van der; Ying, L. ; Bosch, G.B.M. van den; Gent-Pelzer, M.P.E. van; Evenhuis, A. ; Jacobsen, E. ; Huang, S. ; Flier, W.G. ; Kessel, G.J.T. - \ 2011
Gewasbescherming 42 (2011)4. - ISSN 0166-6495 - p. 180 - 181.
phytophthora infestans - nederland - genetische diversiteit - genotypen - plantenziekteverwekkende schimmels - netherlands - genetic diversity - genotypes - plant pathogenic fungi
Een set van meer dan duizend P. infestans-isolaten afkomstig van aardappelproductievelden en onderzoeksvelden werd verzameld in de periode 2000-2009. De genetische diversiteit van de Nederlandse populatie werd bepaald. De resultaten laten zien dat elk jaar als gevolg van de seksuele cyclus een groot aantal nieuwe genotypen wordt gevormd, waarvan er slechts enkele zo succesvol zijn dat ze lokaal of regionaal een epidemie veroorzaken.
Moleculaire risk assessment Escherichia coli O157 in Nederland
Franz, E. ; Wal, F.J. van der; Hoek, A. ; Heuvelink, A.E. - \ 2011
Bilthoven : RIVM (RIVM briefrapport / Rijksinstituut voor Volksgezondheid en Milieu 330411001/2011) - 15
escherichia coli - risicoschatting - virulentie - voedselveiligheid - genotypen - risk assessment - virulence - food safety - genotypes
Infecties door Shiga toxine-producerende Escherichia coli O157 (STEC O157) zijn een bedreiging voor de volksgezondheid gezien de ernstige klinische gevolgen bij voornamelijk jonge kinderen en de potentie voor voedselgerelateerde uitbraken. Verschillende STEC O157 isolaten kunnen erg verschillen in genetische samenstelling. In deze studie is de mate van genotypische gelijkenis tussen STEC O157 isolaten uit (gezonde) runderen en humane ziektegevallen onderzocht aan de hand van vier moleculaire typeringsessays.
The genetic architecture of gene expression in Caenorhabditis elegans
Viñuela Rodriguez, A. - \ 2011
University. Promotor(en): Jaap Bakker, co-promotor(en): Jan Kammenga. - [S.l.] : S.n. - ISBN 9789085858386 - 119
caenorhabditis elegans - genexpressie - stressfactoren - genetische analyse - transcriptie - loci voor kwantitatief kenmerk - genotypen - fenotypen - genexpressieanalyse - polygene overerving - gene expression - stress factors - genetic analysis - transcription - quantitative trait loci - genotypes - phenotypes - genomics - polygenic inheritance

Most organisms are exposed to a continuously changing environment throughout their life. For instance the ambient temperature is usually not constant and many species are exposed to a diverse range of anthropogenic stressors like toxic compounds. Moreover, individuals are prone to genetic changes due to mutation and allelic recombinations. All these factors might affect particular phenotypes, while others remain unchanged. This thesis provides insight into how phenotypic traits are affected by external stress factors and allelic recombinations in the nematode Caenorhabditis elegans (Nematoda; Rhabditidae). Because phenotypes and their variation may be explained by variation in gene expression, this thesis explored the architecture of gene expression and some of the elements that contribute to gene expression.

Chapters 2 and 3 focus on environmental stressors with i) a specific target (two organophosphorus pesticides) and ii) a non-target mode of action (temperature) to study their influence on gene expression. A single genotype, the canonical wild type strain Bristol (N2) was used to study the effect of interacting pesticides by exposing nematodes to a toxicant mixture at two different temperatures Analysis revealed common transcriptional responses related to detoxification, stress, innate immunity, and transport of lipids to all treatments. It was found that for both pesticides these similar processes were regulated by different gene transcripts in single and combined treatments. These results also showed that the effect of a mix of low doses of pesticides is not a summed effect of the single components. Moreover, increased temperature elevates the toxic consequences to the pesticides exposures. This toxicity gain is attributed to an elevated uptake and accumulation of the toxicants in the organisms. These results support the idea that the observed higher toxicity of pesticides with temperature might be a consequence of gene-environment interactions affecting detoxification genes. Together, thefirst part of this thesis illustrates the intense crosstalk between gene pathways in response to interacting environmental stressors in C. elegans.

The second part of the thesis elaborates on the influence of different genotypes as multiple perturbations on gene expression. How the genotype-phenotype relationship progresses with age was investigated using a quantitative genetics approach (genetical genomics). We performed a genetic mapping strategy of gene transcription variation (expression-QTL, eQTL) to explore the dynamics of regulatory loci affecting genome-wide gene expression at three different ages. We used a recombinant inbred line (RIL) population generated from a cross between the C. elegans strain N2 and the wild type CB4856 in Chapter 4. Also, we investigated the influence of age to reveal a genotype-by-age effect (gxaeQTL) on gene expression. The total number of detected eQTL decreased with age whereas the variation in expression increased. In developing worms, the number of genes with increased expression variation (1282) was similar to the ones with decreased expression variation (1328). In aging worms the number of genes with increased variation (1772) was nearly 5 times higher than the number of genes with a decreased expression variation (373). Furthermore, the number of cis-acting eQTL in juveniles decreased by almost 50% in old worms whereas the number of trans-acting loci decreased by ~27%, indicating that cis-regulation becomes relatively less frequent than trans-regulation in aging worms. Our findings demonstrate that eQTL patterns are strongly affected by age and suggest that gene network integrity declines with age. To better understand the changes in the gene network with age, gene expression profiles of N2 and CB4856 were generated for Chapter 5. We explored gene expression heritability and transgression as genetic parameters for the analysis of gene expression divergence in different genotypes. The average broad sense heritability was similar in developing and aging worms; but the gene expression variance that can be attributed to genetic variance in each gene changes with age. It can be proposed that regulation became more polygenic in aging worms. These changes explain the decrease in detected eQTLs. Likewise, it explains the imbalance between highly heritable genes and eQTLs in aging worms.

Chapter 6 discusses the main conclusion of this thesis in the context of the robustness theory. Robustness in biological systems provides the potential to survive severe environmental and genetic perturbations in the form of cryptic genetic variation. The variation we observed in gene transcripts due to external and internal perturbations not always translated to physiological phenotypic variation. In some cases however, the mechanisms underlying phenotypic robustness failed and phenotypic variation was observed. Such genetic cryptic variation was revealed as new molecular and physiological phenotypes.

Agrobacterium-mediated transformation of Mycosphaerella fijiensis, the devastating Black Sigatoka pathogen of bananas
Díaz-Trujillo, C. ; Adibon, H. ; Kobayashi, K. ; Zwiers, L.H. ; Souza, M.T. ; Kema, G.H.J. - \ 2010
Gewasbescherming 41 (2010)3. - ISSN 0166-6495 - p. 151 - 151.
mycosphaerella fijiensis - fungiciden - bananen - genotypen - fenotypen - rhizobium - genetische transformatie - genoomanalyse - fungicides - bananas - genotypes - phenotypes - genetic transformation - genome analysis
Mycosphaerella fijiensis, M. musicola en M. eumusae veroorzaken de Sigatoka-ziekte in banaan. Op dit moment is de toepassing van fungiciden de enige optie om deze ziekte te bestrijden. Het PRPB (Pesticide Reduction Program for Banana) investeert in de ontwikkeling van technieken voor de genotype- en fenotypebepaling van M. fijiensis. Hierbij wordt gebruikt gemaakt van ATMT (Agrobacterium tumefaciens-mediated transformation).
Pepino mosaic virus: an endemic pathogen of tomato crops
Hanssen, I.M. - \ 2010
University. Promotor(en): Pierre de Wit; B. Lievens, co-promotor(en): Bart Thomma. - [S.l. : S.n. - ISBN 9789085855576 - 170
solanum lycopersicum - tomaten - Pepinomozaïekvirus - genotypen - genetische variatie - symptomen - ziekteoverdracht - in zaden overlevende virussen - transcriptomics - tomatoes - pepino mosaic virus - genotypes - genetic variation - symptoms - disease transmission - seedborne viruses
Owing to their large population size and short generation time, viruses generally have a huge potential to evolve and adapt under natural selection pressure. Despite tremendous efforts in human, animal and plant health management, viral diseases remain difficult to control and eradicate. Moreover, existing control strategies are compromised by the continuous emergence of new viruses. In Chapter 1 emerging viruses of tomato crops are reviewed. This includes Pepino mosaic virus (PepMV), a Potexvirus with a single stranded RNA genome, a rapidly emerging virus which has become one of the most important viral diseases in tomato production worldwide over the recent years.
Infection by PepMV can cause a broad range of symptoms on tomato plants, of which especially the typical fruit marbling can lead to significant economical losses. Presently, five PepMV genotypes (EU, LP, CH2, US1 and US2) have been described worldwide, three of which (EU, LP and US2) have previously been reported to occur in Europe. As nature and severity of PepMV symptoms are highly variable, economical damage caused by PepMV is difficult to assess and the identification of factors contributing to symptom severity is warranted. In Chapter 2 the genetic diversity of the PepMV population in Belgian greenhouses is studied and related to the symptom development in tomato crops. Previously, no correlation has been found between different PepMV genotypes and the symptomatology of infected plants. A novel assay based on restriction fragment length polymorphism (RFLP) was developed to discriminate the different PepMV genotypes. Both RFLP and sequence analysis revealed the occurrence of two genotypes, the EU genotype as well as the CH2 genotype, within the tomato production in Belgium. Surprisingly, a clear dominance of the CH2 genotype in the Belgian PepMV population was found, although this genotype has previously not been found in commercial tomato production. Whereas no differences were observed in symptom expression between plants infected by one of the two genotypes, co-infection with both genotypes resulted in more severe PepMV symptoms. Furthermore, our study revealed that PepMV recombinants frequently occur in such mixed infections.
So far, it remained unclear whether different PepMV isolates can cause differential symptom severity. Therefore, PepMV symptomatology of different isolates was studied in Chapter 3. Based on the survey described in Chapter 2, four isolates that differed in symptom expression in the crop of origin were selected for greenhouse trials. The selected isolates were inoculated onto tomato plants grown in separate plastic tunnels. PepMV symptom development was assessed regularly and extensive sampling followed by ELISA analyses, genotyping and nucleotide sequencing was performed to study viral presence and variation in PepMV sequences throughout the trial period. Two isolates (EU mild and CH2 mild) that were selected based on mild symptom expression in the crop of origin caused only mild symptoms in the trial, while two other isolates (CH2 aggressive and EU+CH2) that were selected for severe symptom display, caused considerably more severe symptoms. Sequence identity between the mild and the aggressive CH2 isolates was as high as 99.4%. Results of this study show that differential symptom expression can, at least partially, be attributed to the PepMV isolate, which may be related to minor differences at the nucleotide level between isolates.
In Chapter 4, seed transmission of PepMV in tomato is demonstrated. Fruit was harvested from the greenhouse trials described in Chapter 3 and more than 100,000 seeds were extracted and cleaned using an enzymatic treatment without disinfection. Infection assays using indicator plants confirmed the presence of infectious virus particles on the seeds. In the framework of a European project, seeds were distributed to 10 different laboratories in three separate batches, and germinated for seedling analyses by ELISA. In total over 87,000 plants were tested, and 23 PepMV-infected plants were detected, indicating an overall transmission rate of 0.026 %. Our results clearly show that PepMV can be transmitted from contaminated seeds to seedlings, highlighting the risk of using seeds from PepMV-infected plants, and revealed the potential for seed transmission to contribute to spread of PepMV.
In Chapter 5, the potential of three mild PepMV isolates, belonging to the CH2, EU and LP genotypes, to protect a tomato crop against the aggressive CH2 isolate (Chapter 3) as the challenge isolate, was assessed in greenhouse trials. After challenge infection, enhanced symptom display was recorded in plants that were pre-inoculated with a protector isolate that belonged to a different genotype (EU, LP) than the challenge isolate. A quantitative genotype-specific TaqMan assay revealed that in these plants, the accumulation of the challenge isolate only temporarily slowed down. By contrast, efficient cross-protection was obtained using the mild isolate of the CH2 genotype, and in this case the challenge isolate was barely detectable in the pre-inoculated plants. These results suggest that the interaction between PepMV isolates largely depends on RNA sequence homology and that post-transcriptional gene silencing plays an important role in cross-protection.
As plant viruses are obligate intracellular parasites that hijack host cellular functions and resources for their replication and movement, they generally induce a wide variety of alterations in host gene expression and cell physiology. In Chapter 6, we used a custom-designed Affymetrix tomato GeneChip array that contains probe sets to interrogate over 22,000 tomato transcripts to study transcriptional changes in response to inoculation with the highly similar (99.4% nucleotide sequence identity) mild and aggressive CH2 isolates that are characterized in Chapter 3. Interestingly, our results show that both isolates induce differential transcriptomic responses in the tomato host despite accumulation to similar viral titers. PepMV inoculation resulted in an extensive transient repression of host genes which clearly affected primary metabolism. Especially the defense response intensity was higher upon inoculation with the aggressive isolate, and defense was mediated by salicylic acid signaling rather than by jasmonate signaling. Our results furthermore show that PepMV differentially regulates the RNA silencing pathway, suggesting a role for PepMV-encoded silencing suppressors, and the ubiquitination pathway. In addition, perturbation of pigment biosynthesis, as monitored by differential regulation of the flavonoid/anthocyanin and lycopene biosynthesis pathways, was monitored, which can be associated with the typical PepMV-induced marbling of tomato fruit.
Finally, Chapter 7, the general discussion, is a PepMV pathogen profile in which the results obtained in this work are discussed and integrated into a review on the current knowledge on this highly successful pathogen of tomato crops.

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