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.

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    We will mail you new results for this query: keywords==genetische analyse
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Genetic studies towards elucidation of drought tolerance of potato
Tessema, Biructa Bekele - \ 2017
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Gerard van der Linden. - Wageningen : Wageningen University - ISBN 9789463431958 - 195
solanum tuberosum - potatoes - drought resistance - plant breeding - genetic analysis - quantitative trait loci - aardappelen - droogteresistentie - plantenveredeling - genetische analyse - loci voor kwantitatief kenmerk

Drought is a major threat to agricultural production, which makes drought tolerance a prime target for breeding approaches towards crop improvement. Drought is a complex polygenic trait and poses a challenge for drought tolerance breeding. Improving crops for drought tolerance at least requires the knowledge of the physiological mechanisms of the contributing traits and their genetic control. Thus, identification of genetic variation for drought tolerance is the first step towards drought tolerance breeding. The effect of drought stress on potato tuber yield and quality is very significant as potato is considered sensitive to water shortage. To understand the genetic factors underlying drought tolerance in potato, we performed drought stress experiments under green house and field conditions with moderate drought and severe drought stress conditions, respectively. In the field, potato genotypes were exposed to severe drought stress for two consecutive years starting from tuber initiation, which progressed to severe drought stress. In addition, we examined potato cultivars for moderate drought tolerance under greenhouse conditions where water application was reduced 50-60% from optimum amount starting from stolon formation. Morphological and physiological trait data were collected that allowed precise monitoring of the drought response of potato. Phenotypic data collected under severe drought stress conditions which includes traits like shoot and root biomass (fresh and dry), yield and chlorophyll content were used for QTL mapping while data collected under moderate drought stress conditions was used for genome wide association mapping. With QTL mapping, 60 QTLs were identified controlling those traits both under well-watered and drought stress conditions. In the drought tolerance evaluation of the potato cultivars under greenhouse conditions we identified significant marker trait associations for both above- and belowground traits. Many of the QTLs detected for drought tolerance traits were specific to either moderate or severe drought tolerance conditions. However, a few QTLs showed an overlap between these drought stress environments. This demonstrates the presence of common genomic regions controlling drought tolerance traits under moderate and severe drought stress conditions. In addition, from the two years of field drought stress experiments we selected a subset of genotypes that showed contrasting responses to drought stress. We used these genotypes to further examine the relationship between canopy development and tuber yield under severe drought stress conditions. Canopy development was measured for several time points and the data were used for curve fitting. From the curve-fit, parameters related to the different developmental phase of canopy were extracted. We observed that there is positive correlation between canopy parameters and tuber yield under drought stress conditions. The evaluation of potato for drought tolerance under field and greenhouse conditions has resulted in the identification of several QTLs that can be interesting to be used for enhancing drought tolerance in potato. Furthermore, the use of model derived parameters gave a better insight into the relationship between canopy development and tuber yield under water stress conditions.

Genetic improvement of longevity in dairy cows
Pelt, Mathijs van - \ 2017
Wageningen University. Promotor(en): Roel Veerkamp, co-promotor(en): T.H.E. Meuwissen. - Wageningen : Wageningen University - ISBN 9789463430821 - 188
dairy cows - longevity - genetic improvement - breeding value - genetic analysis - survival - animal models - animal genetics - melkkoeien - gebruiksduur - genetische verbetering - fokwaarde - genetische analyse - overleving - diermodellen - diergenetica

Improving longevity helps to increase the profit of the farmer, and it is seen as an important measure of improved animal welfare and sustainability of the sector. Breeding values for longevity have been published since in 1999 in the Netherlands. For AI-companies and farmers it is necessary that breeding values are accurately estimated and will remain stable for the rest of life. However, current breeding values for longevity of bulls seem to fluctuate more than expected. The main aim of this thesis was to revisit the genetics of longevity and develop a genetic evaluation model for longevity, where breeding values reflect the true breeding value quicker during early life and therefore breeding values become more stable. Genetic parameters were estimated for survival up to 72 months after first calving with a random regression model (RRM). Survival rates were higher in early life than later in life (99 vs. 95%). Survival was genetically not the same trait across the entire lifespan, because genetic correlations differ from unity between different time intervals, especially when intervals were further apart. Survival in the first year after first calving was investigated more in depth. Survival of heifers has improved considerably in the past 25 years, initially due to the focus on a high milk production. More recently, the importance of a high milk production for survival has been reduced. Therefore functional survival was defined as survival adjusted for within-herd production level. For survival the optimum age at first calving was around 24 months, whereas for functional survival calving before 24 months resulted in a higher survival. Over years, genetic correlations between survival in different 5-yr intervals were below unity, whereas for functional survival genetic correlations did not indicate that survival changed over years. This suggested that a genetic evaluation using historical data should analyze functional survival rather than survival. A new genetic evaluation system for longevity was developed based on a RRM analyzing functional survival. Based on the correlation between the first breeding value of a bull and his later breeding values, the ranking of bulls was shown to be more stable for RRM than the current genetic evaluation. Bias in breeding value was observed, mainly for bulls with a large proportion of living daughters. Adjusting for within-herd production level reduced this bias in the breeding values greatly. Before implementing this new model for genetic evaluation, the cause of this bias needs to be further investigated.

Genetische analyse van het Veluws Heideschaap
Verweij, Marjolein ; Maurice - Van Eijndhoven, M.H.T. ; Oldenbroek, J.K. ; Windig, J.J. ; Zon, Saskia van - \ 2016
Zeldzaam huisdier 41 (2016)3. - ISSN 0929-905X - p. 20 - 21.
schapenhouderij - schapen - inteelt - schapenrassen - rammen - genetische analyse - veluws heideschaap - sheep farming - sheep - inbreeding - sheep breeds - rams - genetic analysis - veluwe heath sheep
De SZH heeft dertig jaar geleden geholpen om de rammencirkel voor het Veluws heideschaap op te zetten met als doel de inteelttoename laag te houden. Afgelopen jaar heeft de SZH in samenwerking met het Centrum voor Genetische Bronnen Nederland (CGN) onderzocht wat de rammencirkel heeft opgeleverd bij het Veluws heideschaap.
Genetische analyse van de Stabij
Doekes, H.P. ; Oldenbroek, J.K. ; Windig, J.J. - \ 2016
Zeldzaam huisdier 41 (2016)1. - ISSN 0929-905X - p. 18 - 19.
zeldzame rassen - genetische analyse - hondenrassen - friese stabij - dierveredeling - rare breeds - genetic analysis - dog breeds - frisian stabyhoun - animal breeding
De Stabij is een van de negen oorspronkelijke Nederlandse hondenrassen die een belangrijke rol vervullen in het Fairfok-programma van de Raad van Beheer voor de Kynologie. De SZH ondersteunt dat programma door de rasverenigingen van kennis en advies te voorzien en heeft met het CGN een genetische analyse gemaakt van het Stabijras.
Genetische analyse van het Markiesje
Doekes, H.P. ; Oldenbroek, J.K. ; Windig, J.J. - \ 2016
Zeldzaam huisdier 41 (2016)2. - ISSN 0929-905X - p. 20 - 21.
hondenrassen - zeldzame rassen - genetische analyse - dierveredeling - stamboeken - dog breeds - rare breeds - genetic analysis - animal breeding - herdbooks
Het Markiesje is een van de negen oorspronkelijke Nederlandse hondenrassen. Het vervult een belangrijke rol in het Fairfok-programma. De SZH ondersteunt dat programma door de rasverenigingen van kennis en advies te voorzien.
Maintenance of Borrelia burgdorferi s.l. diversity in enzootic cycles
Swart-Coipan, Elena - \ 2016
Wageningen University. Promotor(en): Willem Takken; Herbert Prins, co-promotor(en): H. Sprong. - Wageningen : Wageningen University - ISBN 9789462579576 - 180
borrelia burgdorferi - life cycle - tickborne diseases - metastigmata - ixodes ricinus - disease vectors - rodents - mammals - genetic analysis - multilocus sequence typing - lyme disease - levenscyclus - ziekten overgebracht door teken - vectoren, ziekten - knaagdieren - zoogdieren - genetische analyse - multi-locus sequentie typering - lyme-ziekte

Lyme borreliosis is the most prevalent vector-borne disease in the temperate regions of the northern hemisphere. The bacteria that cause it are members of the Borrelia burgdorferi sensu lato complex, a group of spirochaetes which are transmitted by hard ticks of the Ixodes ricinus complex. In several European countries, including The Netherlands, the incidence of Lyme borreliosis has been on the rise for the last decades. The acarological risk of human infection with Borrelia burgdorferi s.l. has been defined as the density of infected questing nymphs. This definition assumes that the distribution of the various genospecies of Borrelia in Lyme borreliosis is reflected in their distribution in questing ticks; furthermore, it assumes that all Borrelia genospecies are considered equally hazardous for humans. In order to define effective intervention strategies for controlling the disease, more insight in the transmission dynamics of tick-borne pathogens, both between animals and ticks, but also from ticks to humans is needed. As part of a Dutch research programme – “Shooting the messenger” – this PhD thesis focussed on linking the transmission cycles of Lyme spirochaetes to the different clinical manifestations of Lyme borreliosis. To that end, I explored aspects of the ecology and molecular adaptations of B. burgdorferi s.l. at various scales, from complex to genospecies level.

The ecological adaptations of B. burgdorferi s.l. are underpinned by a complex genomic structure and gene expression, with large genetic variation among and within the genospecies. In Chapter 3, we prove that the 5S-23S rDNA intergenic spacer (IGS) is a suitable molecular marker for identification of B. burgdorferi s.l. to genospecies level, but also to characterize the genetic diversity at intragenospecies level and to detect genetic differentiation among the subpopulations of Borrelia. Consequently, we used this marker in combination with other genetic markers, in the studies addressing the genetic diversity of Borrelia in small mammals and humans.

The main transmission route of these bacteria is the interstadial one, from larvae to nymphs and from nymphs to adult ticks. Larvae of I. ricinus can become infected during a blood meal from an infected host and during a blood meal in the vicinity of an infected nymph feeding on a host, a process known as co-feeding. The infected engorged larvae then moult into infected nymphs, which can transmit the spirochaetes to new hosts. The same process is repeated in the next developmental stage – nymph to adult. Thus, the maintenance of the bacteria in enzootic cycles is dependent on various species of vertebrates and the ticks that feed on them. In order to identify the main vertebrate hosts responsible for the maintenance of B. burgdorferi in enzootic cycles, but also for feeding I. ricinus ticks, we conducted a meta-analysis on literature data (Chapter 2). Our quantification method suggests that only a few host species, which are amongst the most widespread species in the environment (rodents, thrushes and deer), feed the majority of I. ricinus individuals and that rodents infect the majority of I. ricinus larvae with B. burgdorferi s.l.. The increase in distribution and abundance of these species, could be one of the main causes for the increase in Lyme borreliosis incidence in Europe in recent decades.

While at genospecies level, there is host specificity, with B. afzelii associated with rodents and B. garinii with birds, we wanted to see if the same holds true at intragenospecies level, for the various genotypes of Borrelia. Chapter 4 focuses on the rodents, which were identified in the literature meta-analysis as being the main hosts for I. ricinus larvae as well as for Borrelia afzelii. We tested the multiple niche polymorphisms hypothesis, using IGS, dbpA and ospC as molecular markers for typing B. afzelii genotypes in fed larvae collected from rodents in various areas in The Netherlands. Despite the high genetic diversity within B. afzelii, there was no difference between wood mice and bank voles in the number and types of B. afzelii haplotypes they transmit. Additionally, we compared the quantitative role of bank voles and wood mice in B. afzelii and Neoehrlichia mikurensis maintenance, another emerging tick-borne pathogen in Europe. Neoehrlichia mikurensis prevalence was positively associated with B. afzelii. Mathematical models including tick burden and infection prevalence indicated that bank voles are better amplifiers of these two bacteria than wood mice. Our study suggests that wood mice and bank voles differ in their contribution to the dynamics of B. afzelii, and possibly other TBP, in questing ticks but not in their contribution to the genetic diversity of these microorganisms.

The density of the vertebrate hosts and the feeding preferences of the ticks should determine the prevalence of B. burgdorferi s.l. genospecies in questing ticks. We address this topic in Chapter 5, by testing 5,570 questing I. ricinus nymphs from 22 different areas in The Netherlands. We found an overall prevalence of 11.8% for B. burgdorferi s.l., with large and consistent variations among the various locations. As expected based on the results of Chapter 2, Borrelia afzelii was predominant (6.7 % of the questing ticks) among the B. burgdorferi s.l. genospecies. It was followed by B. garinii/B. bavariensis (1.5 %), B. valaisiana (1.2 %), and B. burgdorferi sensu stricto (0.2 %). We noticed that, over the usual range of questing ticks’ densities, the density of infected ticks is increasing with the overall density of questing ticks, and a downward trend might be observed only for questing tick densities of over 200/100 m2. This indicates that the density of questing nymphs is the main driver of the acarological risk of human exposure to B. burgdorferi s.l.

We also screened for the presence of other tick-borne pathogens that have previously been detected in questing ticks in The Netherlands: Rickettsia helvetica, Anaplasma phagocytophilum, Neoehrlichia mikurensis and several Babesia spp. (Chapter 5). To test whether these pathogens might share similar enzootic cycles we looked for patterns of coinfection and seasonal dynamics of infection in questing I. ricinus nymphs. One-third of the Borrelia-positive ticks were infected with at least one other pathogen. Coinfection of B. afzelii with N. mikurensis and with Babesia spp. occurred significantly more often than single infections, indicating the existence of mutual reservoir hosts. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of their coinfections with B. burgdorferi s.l. underline the need to consider them when evaluating the risks of infection and subsequently the risk of disease following a tick bite.

Chapter 6 addresses the pathogenicity of B. burgdorferi s.l. genospecies and genotypes for humans, using the eight multilocus sequence typing scheme housekeeping genes (MLST) and IGS as molecular markers. The frequency of the Borrelia spp. in humans is compared to the frequency in questing ticks to assess the infectivity of the various genospecies and genotypes. The fraction of STs that were isolated from human samples was significantly higher for the genospecies that are known to be maintained in enzootic cycles by mammals (B. afzelii, B. bavariensis, and spielmanii) than for genospecies that are maintained by birds (B. garinii and B. valaisiana) or lizards (B. lusitaniae). Just as in questing ticks, B. afzelii was the most prevalent Borrelia in in human Lyme borreliosis. Borrelia afzelii was associated with acrodermatitis chronica atrophicans, while B. garinii and B. bavariensis were associated with neuroborreliosis. Despite its high incidence in ticks and erythema migrans, in terms of disease burden (as measured by disability-adjusted life year), B. afzelii is of least concern for public health. Other Borrelia spirochaetes that are rarely found in questing I. ricinus ticks, such as B. bavariensis, seem to be responsible for most of the neuroborreliosis cases – a more severe clinical symptom of Lyme borreliosis. This implies that the prevalence of B. burgdorferi s.l. in questing ticks does not necessarily reflect the incidence of human Lyme borreliosis. We found six multilocus sequence types that were significantly associated with clinical manifestations in humans and five IGS haplotypes that were associated with the human Lyme borreliosis cases. While IGS could perform just as well as the housekeeping genes in the MLST scheme for predicting the infectivity of B. burgdorferi s.l., the advantage of MLST is that it can also capture the differential invasiveness of the various STs.

In this thesis, I have identified the most important vertebrate hosts for maintenance of B. burgdorferi s.l. in enzootic cycles. I have also shown that their density is reflected in the prevalence of B. burgdorferi s.l. in questing ticks. The comparative study of questing ticks and Lyme borreliosis indicated that some of the Borrelia genospecies have similar prevalences in the two sources. The findings in my thesis indicate, thus, that there is a link between the density of suitable hosts for ticks and Borrelia spp., the density of infected ticks and the distribution of the B. burgdorferi s.l. genospecies in Lyme borreliosis. There are exceptions, however, that cannot be explained by this simple thread line. Such a situation is the perceived association of B. bavariensis with rodents that is not reflected by its extremely low prevalence in questing ticks. Furthermore, this low prevalence cannot explain the overrepresentation of B. bavariensis in Lyme borreliosis. As result of the study of pathogenicity of the various Borrelia genospecies and genotypes, I suggest the separate hazard assignment for the Borrelia genospecies; this, in combination with the exposure (prevalence in questing ticks) would allow for individual genospecies/genotypes risk assessment. The findings in this thesis stress the importance of both ecological and clinical studies for addressing the public health issue of Lyme borreliosis.

Antibodies and longevity of dairy cattle : genetic analysis
Klerk, B. de - \ 2016
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Jan van der Poel; Bart Ducro. - Wageningen : Wageningen University - ISBN 9789462577589 - 134 p.
dairy cattle - dairy cows - antibodies - longevity - genetic analysis - breeding value - genomes - genetic improvement - animal genetics - melkvee - melkkoeien - antilichamen - gebruiksduur - genetische analyse - fokwaarde - genomen - genetische verbetering - diergenetica

The dairy sector has a big impact on food production for the growing world population and contributes substantially to the world economy. In order to produce food in a sustainable way, dairy cows need to be able to produce milk without problems and as long as possible. Therefore, breeding programs focuses on improvement of important traits for dairy cows. In order to improve desirable traits and obtain genetic gain there is a constant need for optimization of breeding programs and search for useful parameters to include within breeding programs. Over the last several decades, breeding in dairy cattle mainly focused on production and fertility traits, with less emphasis on health traits. Health problems, however, can cause substantial economic losses to the dairy industry. The economic losses, together with the rising awareness of animal welfare, increased herd size, and less attention for individual animals, have led to an increased need to focus more on health traits. Longevity is strongly related to disease resistance, since a more healthy cow will live a longer productive life (longevity). The identification of biomarkers and the detection of genes controlling health and longevity, would not only greatly enhance the understanding of such traits but also offer the opportunity to improve breeding schemes. The objectives of this thesis therefore were 1) to find an easy measurable disease resistance related biomarker in dairy cows, 2) identify the relation between antibodies and longevity, 3) identify genomic regions that are involved with antibody production/expression. In this thesis antibodies are investigated as parameter for longevity. Antibodies might be a novel parameter that enables selection of cows with an improved ability to stay healthy and to remain productive over a longer period of time. In this thesis antibodies bindiging the naive antigen keyhole limpet hemocyanin (KLH) were assumed to be natural antibodies. Antibodies binding bacteria-derived antigens lipoteichoic acid (LTA), lipopolysaccharide (LPS) and peptidoglycan (PGN) were assumed to be specific antibodies. In chapter 2 it was shown that levels of antibodies are heritable (up to h2 = 0.23). Additionally, antibody levels measured in milk and blood are genetically highly correlated (± 0.80) for the two studied isotypes (IgG and IgM). On the other hand, phenotypically, natural antibodies (from both IgG and IgM isotype) measured in milk cannot be interpreted as the same trait (phenotypic correlation = ± 0.40). In chapter 3 and 4 it was shown that levels of antibodies (both natural-and specific antibodies) showed a negative relation with longevity: first lactation cows with low IgM or IgG levels were found to have a longer productive life. When using estimated breeding values for longevity, only a significant relation was found between natural antibody level (IgM binding KLH) and longevity. Lastly chapter 5 reports on a genome-wide-association study (GWAS), to detect genes contributing to genetic variation in natural antibody level. For natural antibody isotype IgG, genomic regions with a significant association were found on chromosome 21 (BTA). These regions included genes have impact on in isotype class switching (from IgM to IgG). The gained knowledge on relations between antibodies and longevity and the gained insight on genes responsible for natural antibodies level make antibodies potential interesting biomarkers for longevity.

Genetics and regulation of combined abiotic and biotic stress tolerance in tomato
Kissoudis, C. - \ 2016
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Gerard van der Linden; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789462576568 - 212 p.
solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysis - plant breeding - salt tolerance - tomaten - ziekteresistentie - stresstolerantie - verdedigingsmechanismen - plantenziekten - abiotische beschadigingen - stressreactie - fenotypische variatie - genetische analyse - plantenveredeling - zouttolerantie

Projections on the impact of climate change on agricultural productivity foresee prolonged and/or increased stress intensities and enlargement of a significant number of pathogens habitats. This significantly raises the occurrence probability of (new) abiotic and biotic stress combinations. With stress tolerance research being mostly focused on responses to individual stresses, our understanding of plants’ ability to adapt to combined stresses is limited.

In an attempt to bridge this knowledge gap, we hierarchized in chapter 1 existing information on individual abiotic or biotic stress adaptation mechanisms taking into consideration different anatomical, physiological and molecular layers of plant stress tolerance and defense. We identified potentially crucial regulatory intersections between abiotic and biotic stress signalling pathways following the pathogenesis timeline, and emphasized the importance of subcellular to whole plant level interactions by successfully dissecting the phenotypic response to combined stress. We considered both explicit and shared adaptive responses to abiotic and biotic stress, which included amongst others R-gene and systemic acquired resistance as well as reactive oxygen species (ROS), redox and hormone signalling, and proposed breeding targets and strategies.

In chapter 3 we focused on salt stress and powdery mildew combination in tomato, a vegetable crop with a wealth of genetic resources, and started with a genetic study. S. habrochaites LYC4 was found to exhibit resistance to both salt stress and powdery mildew. A LYC4 introgression line (IL) population segregated for both salt stress tolerance and powdery mildew resistance. Introgressions contributing to salt tolerance, including Na+ and Cl- accumulation, and powdery mildew resistance were precisely pinpointed with the aid of SNP marker genotyping. Salt stress (100mM NaCl) combined with powdery mildew infection increased the susceptibility of the population to powdery mildew in an additive manner, while decreasing the phenotypic variation for this trait. Only a few overlapping QTLs for disease resistance and salt stress tolerance were identified (one on a short region at the top of Chromosome 9 where numerous receptor-like kinases reside). Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance indicating distinct genetic architectures. This enables genetic pyramiding approaches to build up combined stress tolerance.

Considering that abiotic stress in nature can be of variable intensities, we evaluated selected ILs under combined stress with salinity ranging from mild to severe (50, 100 and 150mM NaCl) in chapter 4. Mild salt stress (50mM) increased powdery mildew susceptibility and was accompanied by accelerated cell death-like senescence. On the contrary, severe salt stress (150mM) reduced the disease symptoms and this correlated with leaf Na+ and Cl- content in the leaves. The effects of salt stress on powdery mildew resistance may be dependent on resistance type and mechanisms. Near Isogenic Lines (NILs) that carry different PM resistance genes (Ol-1 (associated with slow hypersensitivity response, HR), ol-2 (an mlo mutant associated with papilla formation) and Ol-4 (an R gene associated with fast HR) indeed exhibited differential responses to combined stress. NIL-Ol-1 resembled the LYC4 ILs response, while NIL-ol-2 and NIL-Ol-4 maintained robust resistance and exhibited no senescence symptoms across all combinations, despite the observed reduction in callose deposition in NIL-ol-2. Increased susceptibility, senescence and fitness cost of NIL-Ol-1 under combined stress coincided with high induction of ethylene and jasmonate biosynthesis and response pathways, highly induced expression of cell wall invertase LsLIN6, and a reduction in the expression of genes encoding for antioxidant enzymes. These observations underlined the significance of stress intensity and mechanism of resistance to the outcome of salt stress and powdery mildew combination, underscoring the involvement of ethylene signalling to the susceptibility response under combined stress.

To examine the significance of hormone signalling in combined stress responses we evaluated crosses of tomato hormone mutants notabilis (ABA-deficient), defenseless1 (JA-deficient) and epinastic (ET overproducer) with NIL-Ol-1, NIL-ol-2 and NIL-Ol-4 in chapter 5. The highly pleiotropic epinastic mutant increased susceptibility of NIL-Ol-1, but decreased the senescence response under combined stress, and resulted in partial breakdown of NIL-ol-2 resistance, accompanied by reduced callose deposition. The effects of ET overproduction on susceptibility were more pronounced under combined stress. ABA deficiency in notabilis on the other hand greatly reduced susceptibility of NIL-Ol-1under combined stress at the expense of stronger growth reduction, and induced ROS overproduction. Partial resistance breakdown in the ol-2xnotabilis mutant accompanied by reduced callose deposition was observed, and this was restored under combined stress. Jasmonic acid deficiency phenotypic effects in defenseless mutants were subtle with modest increase in susceptibility for NIL-Ol-1 and NIL-ol-2. For NIL-ol-2 this increased susceptibility was reverted under combined stress. NIL-Ol-4 resistance remained robust across all mutant and treatment combinations. These results highlight the catalytic role of ET and ABA signalling on susceptibility and senescence under combined stress, accentuating concomitantly the importance of signalling fine tuning to minimize pleiotropic effects.

The potential of exploiting transcription factors to enhance tolerance to multiple stress factors and their combination was investigated in chapter 6 through the identification and functional characterization of tomato homologues of AtWRKYs 11, 29, 48, 70 and 72. Thirteen tomato WRKY homologues were identified, of which 9 were overexpressed (using transformation with A. tumefaciens) and 12 stably silenced via RNAi in tomato cultivar Money Maker (MM). SlWRKY11-OE and SlWRKY23-OE overexpressors and RNAi lines of SlWRKY7 and SlWRKY9 showed both increased biomass and relative salt tolerance. SlWRKY6-OE exhibited the highest relative salt stress tolerance, but had strongly decreased growth under control conditions. Exceptional phenotypes under control conditions were observed for SlWRKY10-OE (stunted growth) and SlWRKY23-RNAi (necrotic symptoms). These phenotypes were significantly restored under salt stress, and accompanied by decreased ROS production. Both lines exhibited increased resistance to powdery mildew, but this resistance was compromised under salt stress combination, indicating that these genes have important functions at the intersection of abiotic and biotic stress adaptation. SlWRKY23 appears to have a key regulatory role in the control of abiotic stress/defense and cell death control.

Experimental observations are critically discussed in the General Discussion with emphasis on potential distinctive responses in different pathosystems and abiotic and biotic stress resistance mechanisms as well as genetic manipulations for effectively achieving combined stress tolerance. This includes deployment of individual common regulators as well as pyramiding of non-(negatively) interacting components such as R-genes with abiotic stress resistance genes, and their translation potential for other abiotic and biotic stress combinations. Understanding and improving plant tolerance to stress combinations can greatly contribute to accelerating crop improvement towards sustained or even increased productivity under stress.

Identification and functional characterization of putative (a)virulence factors in the fungal wheat pathogen Zymoseptoria tritici
Mirzadi Gohari, A. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Gert Kema; Rahim Mehrabi. - Wageningen : Wageningen University - ISBN 9789462575912 - 159
triticum aestivum - wheat - plant pathogenic fungi - mycosphaerella graminicola - virulence factors - genetic analysis - pathogenesis - bioinformatics - tarwe - plantenziekteverwekkende schimmels - virulente factoren - genetische analyse - pathogenese - bio-informatica

Zymoseptoria tritici (Desm.) Quaedvlieg & Crous (previously known as Mycosphaerella graminicola) is the causal agent of septoria tritici blotch (STB), which is a devastating foliar wheat disease worldwide. It is responsible for significant yield losses occurring annually in all major wheat-growing areas and threatens global food security. Z. tritici is a hemi-biotrophic fungal pathogen that, after stomatal penetration, establishes a stealthy biotrophic and symptomless relation with its host plant that is followed by a sudden switch to a necrotrophic growth phase coinciding with chlorosis that eventually develops in large necrotic blotches containing many pycnidia producing asexual splash-borne conidia. Under natural conditions - once competent mating partners are present and conditions are conducive- pseudothecia are formed producing airborne ascospores. Disease management of STB is primarily achieved through fungicide applications and growing commercial cultivars carrying Stb resistance genes. However, the efficacy of both strategies is limited as strains resistant to fungicides frequently develop and progressively dominate natural populations, which hampers disease management; also the deployed Stb genes are often overcome by existing or newly developed isolates of the fungus. Hence, there is a need for discovery research to better understand the molecular basis of the host-pathogen interaction that enables breeders to identify and deploy new Stb genes, which will eventually contribute to more sustainable disease control.

Chapter 1 introduces the subject of the thesis and describes various aspects of the lifestyle of Z. tritici with emphasis on dissecting the various stages and physiological processes during pathogenesis on wheat. In addition, it includes a short summary and discussion of the current understanding of the role of (a)virulence factors in the Z. tritici–wheat pathosystem.

Chapter 2 describes new gateway technology-driven molecular tools comprising 22 entry constructs facilitating rapid construction of binary vectors for functional analyses of fungal genes. The entry vectors for single, double or triple gene deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, these entry vectors contain the genes encoding green fluorescent (GFP) or red fluorescent (RFP) protein in combination with the three selection markers, which enables simultaneous tagging of gene deletion mutants for microscopic analyses. The functionality of these entry vectors was validated in Z. tritici and described in Chapters 3, 4 and 5.

Chapter 3 describes the functional characterization of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans. ZtWor1 is up-regulated during initiation of colonization and fructification, and regulates expression of candidate effector genes, including one that was discovered after comparative proteome analysis of Z. tritici wild-type and ΔZtWor1 strains. Cell fusion and anastomosis occurred frequently in ΔZtWor1 strains, which is reminiscent of mutants of MgGpb1, the β-subunit of the heterotrimeric G protein. Comparative expression profiling of ΔZtWor1, ΔMgGpb1 and ΔMgTpk2 (the catalytic subunit of protein kinase A) strains, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenicity of many fungal plant pathogens.

Chapter 4 describes combined bioinformatics and expression profiling studies during pathogenesis in order to discover candidate effectors of  Z. tritici important for virulence. In addition, a genetic approach was followed to map quantitative trait loci (QTLs) in Z. tritici carrying putative effectors. Functional analysis of two top effector candidates, small-secreted proteins SSP15 and SSP18, which were selected based on their expression profile in planta, showed that they are dispensable for virulence of Z. tritici. These analyses suggest that generally adopted criteria for effector discovery, such as protein size, number of cysteine residues and up-regulated expression during pathogenesis, should be taken with caution and cannot be applied to every pathosystem, as they likely represent only a subset of effector genes.

Chapter 5 describes the functional characterization of ZtCpx1 and ZtCpx2 encoding a secreted and a cytoplasmic catalase-peroxidase (CP) in Z. tritici, respectively. Gene replacement of ZtCpx1 resulted in mutant strains that were sensitive to exogenously added H2O2 and in planta phenotyping showed they are significantly less virulent compared to wild-type. All mutant phenotypes could be restored to wild-type by complementation with the wild-type allele of ZtCpx1 driven by its native promoter. Additionally, functional analysis of ZtCpx2 confirmed that this gene encodes a secreted CP and is, however, dispensable for virulence of Z. tritici on wheat. However, we showed that both genes act synergistically, as the generated double knock-out strain showed a significantly stronger reduction in virulence than the individual single knock-out strains. Hence, both genes are required by Z. tritici for successful infection and colonization of wheat.

In Chapter 6 I discuss and summarize the genetic approaches used in this study, reflect on the major findings and bottlenecks encountered, and propose new strategies to identify effectors of Z. tritici in the future.

Non-genetic variance in pigs: genetic analysis of reproduction and production traits
Sell-Kubiak, E.B. - \ 2015
Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Piter Bijma; Herman Mulder. - Wageningen : Wageningen University - ISBN 9789462573291 - 186
varkens - dierveredeling - voortplanting - dierlijke productie - genetische analyse - genotypische variatie - genomica - fenotypische variatie - pigs - animal breeding - reproduction - animal production - genetic analysis - genetic variance - genomics - phenotypic variation

Abstract

Sell-Kubiak, E. (2015). Non-genetic variance in pigs: genetic analysis of reproduction and production traits. PhD thesis, Wageningen University, The Netherlands

The main objective of this thesis was to study the origin of random variance in reproduction and production traits of pigs. In pig breeding for many traits it is important not only to improve the reproduction and production trait itself, but also its variation. The variance of traits can be used to improve pigs’ productivity, and potentially also to improve uniformity of traits. Results presented in Chapters 2 and 3 show that the proposed approach to explore the origin of common litter variance was not successful. The impact of various sow features on growth rate and feed intake of grow-finish pigs was very small. More importantly, sow features did not explain the phenotypic variance due to common litter effects found in production traits of pigs. In Chapters 4 and 5 the residual variance of birth weight and litter size were found to have a genetic component. The genetic coefficient of variation at residual standard deviation level (GCVSDe) was proposed as a measure of expressing the potential response to selection (Chapter 4). For both traits the estimated GCVSDe was about 10%, indicating sufficient potential for response to selection. In Chapter 4 it was shown that analyzing variation in traits with Double Hierarchical Generalized Linear model (DHGLM) was highly comparable with the conventional analysis of standard deviation of a trait. The correlation between the additive genetic effects for birth weight and the residual variance was 0.6 (Chapter 4), whereas for litter size (TNB) and its residual variance (varTNB) this correlation was 0.5 (Chapter 5). Those moderate correlations are an important indication of the direction of correlated selection response in the mean of those traits. In Chapter 5 in a genome-wide association study for litter size variation, the significant SNPs explained 0.83% of total genetic variance in TNB and 1.44% in varTNB. The most significant SNP explained 0.4% of genetic variance in TNB (chromosome 11) and 0.5% in varTNB (chromosome 7). One of the possible candidate genes for varTNB on chromosome 7 is heat shock protein (HSPCB). Studying the residual variance of traits with DHGLM has a great potential to serve as an alternative to conventional analysis to study and to select for improved uniformity of various traits. Lastly, Chapter 6 focuses on discussion of the findings of this thesis and their overall importance for pig breeding, as well as highly relevant topics for breeding uniform and robust pigs (macro-micro sensitivity analysis and application of genomic selection).

One-carbon metabolism in acetogenic and sulfate-reducing bacteria
Visser, M. - \ 2015
Wageningen University. Promotor(en): Fons Stams. - Wageningen : Wageningen University - ISBN 9789462571730 - 210
anaërobe microbiologie - metabolisme - koolmonoxide - methanol - alcohol dehydrogenase - sulfaat reducerende bacteriën - genetische analyse - eiwitexpressieanalyse - anaerobic microbiology - metabolism - carbon monoxide - sulfate reducing bacteria - genetic analysis - proteomics

ABSTRACT

One-carbon metabolism in acetogenic and sulfate-reducing bacteria

Life on earth is sustained by the constant cycling of six essential elements: oxygen, hydrogen, nitrogen, sulfur, phosphorous, and carbon. The continuous cycling of these elements is due to geo-chemical processes and the combined metabolism of all life on earth. Microorganisms like bacteria and archaea play a major role in this. This is also true for the carbon cycle. In this cycle carbon dioxide and methane are two important C-1 compounds present in the atmosphere. Carbon dioxide is the highest oxidative state of carbon while methane is the highest reduced form of carbon. The art to use light to produce organic compounds and conserve energy from the highest oxidative state of carbon is called photosynthesis and is performed by plants, algae and cyanobacteria. Photosynthesis is not the only system to fix carbon from carbon dioxide. Chemolithotrophs can fix carbon from carbon dioxide using inorganic electron donors, like hydrogen. Subsequently, fixed carbon can be used by other organisms, which also makes life possible for them. Microorganisms play a major role in the degradation of complex organic matter, producing smaller compounds including C-1 compounds. C-1 compounds other than carbon dioxide are e.g. carbon monoxide (CO), methanol and formate. Bacteria and archaea can utilize these relative simple compounds in the presence and absence of oxygen, alone and in cooperation with others (syntrophy). The complex and simple carbon compounds are finally oxidized to carbon dioxide, which closes the carbon cycle.

In addition to their importance to the carbon cycle, one carbon compounds like CO, methanol and formate are important for several applications. They are used as a building block for the production of chemicals. They are also used for bioremediation purposes and for wastewater treatment. Therefore, it is important to gain insight in the one carbon metabolism of microorganisms. The research described in this thesis focuses on the proteins and encoding genes involved in anaerobic degradation of C1 compounds by using genome and proteome analysis.

In Chapter 2 the genomes of two closely related sulfate-reducing bacteria, Desulfotomaculum nigrificans and D. nigrificans strain CO-1-SRB, are compared including their CO metabolism. Both the D. nigrificans type strain and strain CO-1-SRB can grow with CO. However, there are differences. The type strain can grow with 20% CO coupled to sulfate reduction in the presence of yeast extract, while strain CO-1-SRB can grow with 100% CO in the presence of yeast extract. Moreover, strain CO-1-SRB can grow with CO in the presence and absence of sulfate. It couples the oxidation of CO to carbon dioxide to hydrogen production. This conversion, the protein complex involved, and the genes coding for these proteins have been described before in other microorganisms. The genome of strain CO-1-SRB contains the genes coding for this protein complex while the genome of the D. nigrificans type strain does not. However, the genome of the type strain contains genes encoding two other CO dehydrogenases. This indicates that one or both are necessary for the type strain to grow with 20% CO. Additional research on the different CO dehydrogenases and their regulation is essential to assess if all different CO dehydrogenases can facilitate growth and how they are linked to for example creating a proton motive force for ATP production.

The methanol metabolism of anaerobic bacteria seems to differ more from that of methanogens than initially described. Methanogens use a methanol methyltransferase system that consists of two methyltranferases, methyltransferase 1 (subunits MtaB and MtaC) and methyltransferase 2 (MtaA). The methyl group from methanol is transferred to the MtaC subunit by MtaB. Subsequently, MtaA transports the methyl group from MtaC to coenzyme M. A genome and proteome analysis of the acetogenic bacterium Sporomusa strain An4 suggests that instead of MtaA a methyl-tetrahydrofolate methyltransferase is involved in the transport of the methyl bound to MtaC to tetrahydrofolate (Chapter 3).

Research done on the methanol metabolism of the sulfate-reducing bacterium Desulfotomaculum kuznetsovii also shows differences with that of methanogens (Chapter 5). The methanol methyltransferase system is vitamin B12 and cobalt dependent. D. kuznetsovii grows with methanol and sulfate, but can do this in presence and absence of vitamin B12 and cobalt. In the absence of vitamin B12 and cobalt D. kuznetsovii grows slower and reaches a lower optical density compared to growth in the presence of vitamin B12 and cobalt. This suggests that D. kuznetsovii can use both a methyltransferase system and a vitamin B12 and cobalt independent system for the degradation of methanol. Proteome results confirm this and suggest that the vitamin B12 and cobalt independent system consists of an alcohol dehydrogenase and an aldehyde ferredoxin oxidoreductase. Moreover, the alcohol dehydrogenase seems to be involved in the oxidation of both methanol and ethanol (Chapter 5). The presence of two methanol degradation pathways give an ecological advantage to D. kuznetsovii in environments containing methanol and sulfate but limiting cobalt and vitamin B12 concentrations. Future research should elucidate if more sulfate-reducing bacteria, or perhaps even acetogenic bacteria, have two methanol degrading pathways.

Additional to the genome analysis of D. kuznetsovii to assess the genes coding for the proteins involved in the two methanol degradation pathways, the genome was also analyzed to assess genes encoding other degradation pathways (Chapter 4). This analysis shows many genes present in D. kuznetsovii are also present in Pelotomaculum thermopropionicum. P. thermopropionicum is known to degrade propionate in syntrophic interaction with a methanogen. D. kuznetsovii can also degrade propionate, but only coupled to sulfate reduction and not in syntrophy with methanogens. Moreover, P. thermopropionicum is not able to reduce sulfate. D. kuznetsovii is the only close related, non-syntrophic, propionate degrader of which the genome is available. Therefore, a genome comparison was performed between D. kuznetsovii and P. thermopropionicum to define the differences between a non-syntrophic and a syntrophic lifestyle. D. kuznetsovii misses membrane bound protein complexes like hydrogenases and an extra-cytoplasmic formate dehydrogenase. In order to expand the analysis between non-syntrophs and syntrophs, more genomes of propionate- and butyrate-degrading bacteria were included (Chapter 6). This extended analysis shows that the genomes of non-syntrophs do not contain genes coding for an extra-cytoplasmic formate dehydrogenase, in contrast to all syntrophs included in the analysis. This indicates the importance of this protein complex and the importance of formate as an interspecies electron carrier in syntrophic degradation of propionate and butyrate. Thanks to the extra cytoplasmic formate dehydrogenase the syntrophic bacteria can couple the degradation of propionate and butyrate to formate production. Subsequently, the formate is utilized by methanogens to produce methane. This keeps the formate concentration low, which is necessary for the entire process to be energetically favorable.

Genetic baculovirus determinants for pathogenicity, virulence and transmission
Serrano, A. - \ 2014
Wageningen University. Promotor(en): Just Vlak; P. Caballero, co-promotor(en): Gorben Pijlman; D. Munoz. - Wageningen : Wageningen University - ISBN 9789462571358 - 160
baculovirus - spodoptera exigua multiple nucleopolyhedrovirus - genetische analyse - genotypische variatie - pathogeniteit - virulentie - genen - biologische bestrijding - insectenplagen - genetic analysis - genetic variance - pathogenicity - virulence - genes - biological control - insect pests
The role of phosphatidylinositol-specific phospholipase-C in plant defense signaling
Abd-El-Haliem, A.M. - \ 2014
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Matthieu Joosten. - Wageningen : Wageningen University - ISBN 9789462571181 - 186
plantenziekten - ziekteresistentie - verdedigingsmechanismen - solanum lycopersicum - tomaten - signaaltransductie - fosfolipase c - plant-microbe interacties - passalora fulva - genetische analyse - nucleotidenvolgordes - plant diseases - disease resistance - defence mechanisms - tomatoes - signal transduction - phospholipase c - plant-microbe interactions - genetic analysis - nucleotide sequences

Plant innate immunity requires immune receptors that sense the presence of microbes and activate defense reactions. Phosphatidylinositol-phospholipase C (PI-PLC) activity was previously shown to be important for several types of plant defenses although its signaling mechanism is not fully understood. It is also not clear why plants possess several PI-PLC isoforms and how triggering immune receptors activates them. PI-PLC activation induces a transient release of free cytosolic Ca2+ and the turnover of specific low abundant signaling phospholipids in the plasma membrane. Both events are important signals in animals and plants. Here, the first genetic evidence linking PI-PLC signaling to plant defense against pathogens is provided. The structure of the tomato PI-PLC family was determined, the corresponding genes were cloned and the function of individual isoforms during defense was studied. We found that PLC4 and PLC6 encode active enzymes and that they have distinct roles in defense. Optimum activity requirements and substrate preferences were determined for three PI-PLC enzymes and their enzyme activity was found to be important for immune receptor-activated responses. The available information was used to draw a model explaining the role of PI-PLC signaling in immune receptor-mediated defense and resistance in plants.

Bacterial canker resistance in tomato
Sen, Y. - \ 2014
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Sjaak van Heusden. - Wageningen : Wageningen University - ISBN 9789462570764 - 140
solanum lycopersicum - tomaten - plantenziekteverwekkende bacteriën - clavibacter michiganensis subsp. michiganensis - ziekteresistentie - wilde verwanten - solanum pimpinellifolium - inteeltlijnen - terugkruisingen - genetische analyse - detectie - plant-microbe interacties - plantenveredelingsmethoden - tomatoes - plant pathogenic bacteria - disease resistance - wild relatives - inbred lines - backcrosses - genetic analysis - detection - plant-microbe interactions - plant breeding methods

Clavibacter michiganensis subsp. michiganensis (Cmm) is the pathogen causing bacterial canker in tomato. The disease was described for the first time in 1910 in Michigan, USA. Cmmis considered the most harmful bacteria threatening tomato. Disease transmission occurs via seed and symptoms become visible at least 20 days after infection. Due to its complex strategy and transmission, Cmm is under quarantine regulation in EU and other countries. There is no method to stop disease progress in plants after infection. Thus, disease management consists usually of chemical treatments as protection and by careful clean cultural practices. However, the use of resistant varieties is the most effective and environmentally friendly method. Unfortunately, there is no cultivar harboring effective resistance on the market although efforts to get resistant varieties already started in the 60s. Our aim in this thesis was to develop valuable genetic material for breeders in order to enable them to release resistant cultivars and provide comprehensive scientific knowledge for further detailed research about Cmm.

Our scientific activity in this thesis started with the identification of new Cmm resistance sources and confirmation of existent ones. In Chapter 3 we have screened a collection of wild tomatoes for resistance to Cmm. We made use of Real Time TaqMan PCR for intensive phenotyping. Using wilting and bacterial concentration as parameters for evaluation of the sources, we have identified new sources and confirmed existent ones. We have decided to continue further with one new source, S. pimpinellifolium, and one existent source, S . arcanum.

We continued our research in Chapter 4 with a genetic analysis of the new source coming from S. pimpinellifolium. A recombinant inbred line population between the resistant parent, S. pimpinellifolium, and the susceptible parent S. lycopersicum was evaluated in three different environments. Wilting, bacterial concentration, and stem discoloration were the scored parameters. Responses of resistance in different environments were determined and genomic regions responsible for different responses were mapped.

In Chapter 5, we have continued our research by fine mapping of previously identified genomic regions and developing nearly isogenic lines containing those genomic regions. By doing fine mapping, we made use of old stock DNA and recently developed different types of SNP marker technology. Previously identified Quantitative Trait Loci(QTLs)could be more precisely delimited. During isogenic line development, embryo rescue was used in order to break the genetic barrier between our S. arcanum source and tomato. Marker assisted backcrossing was applied to obtain lines with a minimum of donor parent in a faster way. By using this method we gained two generations of backcrossing.

In order to obtain comprehensive information about different Cmm isolates inTurkey, we have performed multi locus sequence analysis (MLST) analysis on a Cmm collection, which was collected in 20 years of time in different part of Turkey. In Chapter 6 a statistical analysis of this collection revealed that measurement of clonality of this collection was possible as well as it was possible to predict the virulence level of strainsusing a subset of housekeeping genes.

All knowledge gained by our experiments and knowledge coming from literature about Cmm have led a Review paper, Chapter 2, by which comprehensive information about Cmm resistance sources, genetic analysis of these sources, detection methods of Cmm, infection strategies of Cmm and interaction with its host was discussed.

In conclusion, two good Cmm resistance sources and several tools and methods are available for breeders. Genomic regions of these sources associated with resistance were determined. Wider knowledge about Cmm detection, Cmm infection and Cmm interaction with its host are available for further research.

Biogenesis and signalling requirements of plant receptor-like proteins mediating resistance to fungal pathogens
Liebrand, T.W.H. - \ 2014
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Matthieu Joosten. - Wageningen : Wageningen University - ISBN 9789461738622 - 192
solanum lycopersicum - plantenziekteverwekkende schimmels - passalora fulva - verticillium dahliae - ziekteresistentie - verdedigingsmechanismen - receptoren - pathogenesis-gerelateerde eiwitten - genetische analyse - genexpressie - plant pathogenic fungi - disease resistance - defence mechanisms - receptors - pathogenesis-related proteins - genetic analysis - gene expression - cum laude
cum laude graduation
Genetics of insect resistance to plant defence
Vermeer, K.M.C.A. - \ 2014
Wageningen University. Promotor(en): Marcel Dicke, co-promotor(en): Peter de Jong. - Wageningen : Wageningen University - ISBN 9789461738363 - 199
insect-plant relaties - co-evolutie - planten - plaagresistentie - verdedigingsmechanismen - insectenplagen - secundaire metabolieten - phyllotreta nemorum - barbarea vulgaris - genetisch bepaalde resistentie - genetische analyse - insect plant relations - coevolution - plants - pest resistance - defence mechanisms - insect pests - secondary metabolites - genetic resistance - genetic analysis

Plants are chemically defended against insect herbivory in various ways. They produce a broad range of secondary metabolites that may be toxic or deterrent to insects. Specialist insects, however, are often capable of overcoming these defences. The yellow striped flea beetle (Phyllotreta nemorumL.) is a specialist that feeds on crucifers (Brassicaceae) such as Sinapis arvensisand Barbarea vulgaris. In Denmark, two types of Barbarea vulgarisvar. arcuataare distinguished: one with pubescent leaves (P-type) and one with glabrous leaves (G-type). All individuals of P. nemorumcan feed on B. vulgarisP-type. Barbarea vulgarisG-type, on the other hand, is chemically defended against most P. nemorumindividuals during the flea beetle reproductive season. The defence compounds are hypothesized to be saponins, a class of compounds with various biological effects and insecticidal properties. Despite high levels of these saponins during summer, some flea beetles can and do feed on B. vulgaris G-type. The ability of P. nemorumto feed on B. vulgarisG-type is heritable; resistance against the defence of B. vulgarisG-type is controlled by dominant major resistance genes (R-genes). One dominant R-allele of an R-gene is enough to convert a susceptible beetle into a resistant one. Despite knowledge of the inheritance patterns of resistance in the flea beetles, which have been demonstrated to be variable, the underlying mechanism of flea beetle resistance has, so far, remained unclear. This prompted me to investigate, as an initial part of my thesis, the genetic basis of the flea beetle adaptation to the defence of B. vulgarisG-type.

The interaction between B. vulgarisand the flea beetle is a unique natural model system to study chemical defences in plants and counter-adaptations in insects. Plant and insect are both polymorphic with respect to the trait involved in resistance and hereby provide an excellent opportunity to study the geographic aspects of the evolution of the resistance trait in both interacting species. In this thesis, I focus on the resistance of the flea beetle, and take the presence of different genotypes of the plant as a given. Phyllotreta nemorumis a major pest, for example in oil seed rape. Understanding how resistance evolves in P. nemorumwill not only benefit flea beetle control, but also control of other pest insects. Understanding insect resistance includes knowledge of seasonal, geographic and genetic variation in both plant defense and herbivore adaptation.The R-gene has a remarkable distribution. Flea beetle populations living on B. vulgarisG-type consist solely of resistant individuals, but on host plant patches nearby B. vulgarisG-type lower frequencies of resistant beetles are found than one would expect with the amount of gene flow found at the neutral level between these subpopulations.

The aim of this thesis was to find the gene that is held responsible for the resistance of P. nemorumto the defences of B. vulgaris, investigate the distribution of this resistance trait and explain the distribution of this trait in natural populations. The following questions were addressed: (1) what is the genetic basis of the adaptation under study? (2) how is the resistance distributed across flea beetle populations in Denmark? and (3) which factors underlie this distribution?

In order to answer these questions, I used an integrated approach. I have combined a candidate gene approach (CHAPTER3) with an empirical approach via the study of variation in resistance in flea beetle populations (CHAPTER4), and a population genomics approach by using molecular markers to gain insight in the genomic make-up of the population and its connection with the resistance trait (CHAPTERS5 and 6). The population genomics approach is a recent advance in methods to detect the involvement of selection in the distribution of alleles at presumably adaptive loci. Using this approach one can distinguish locus-specific effects, like directional selection, from genome-wide effects, on the distribution of alleles at loci of interest.

The population genomics approach is introduced in CHAPTER2 together with the Geographic Mosaic Theory of Coevolution. I illustrate how processes underlying this theory of coevolution can be investigated with the population genomics approach. According to the geographic mosaic theory of coevolution, reciprocal selection between interacting species only happens in so-called hot-spots. Hot spots can be identified using population genomics and genetic variation found at specific loci can be attributed to locus-specific processes such as directional selection. For the B. vulgaris- flea beetle system this means that with a population genomics approach we can examine whether the distribution of resistant flea beetles on alternative host plants is only influenced by migration, or also by selection (CHAPTER5). Another valuable utility of the population genomics approach is to investigate whether a candidate gene for the R-gene is under selection, by looking whether a candidate gene is experiencing locus-specific effects beside genome-wide effects when comparing flea beetle populations living on B. vulgarisG-type with populations living on alternative host plants (CHAPTER6).

However, before using a population genomics approach to compare the resistance trait or a candidate gene with parts of the genome that only experience genome-wide effects, I have tried to identify the genetic basis of the flea beetle adaptation to the defence ofB. vulgarisG-type. In CHAPTER3, I have addressed this question by using a candidate gene approach to examine the involvement of a possible detoxifying enzyme in P. nemorum. Genes coding for β-glucosidase were a candidate for genes underlying the difference between resistant and susceptible beetles, because β-glucosidase is used as detoxifying enzyme by other organisms resistant to saponin defence. Three different β-glucosidase cDNA sequences were cloned from Danish flea beetle lines. We named them β-glucosidase A, B and C. β-glucosidase C was only found in resistant lines and not in the susceptible line. We then tested if recombinant β-glucosidase C breaks down the most abundant and most effective defence compound in B. vulgarisG-type, hederagenin cellobioside. β-glucosidase C was able to deglycosylate one glucose unit of hederagenin cellobioside, when expressed in an insect cell line. This suggests that expressed β-glucosidase C can deglycosylate antifeedant saponins and may play a role in the resistant flea beetle’s ability to overcome the defence of B. vulgaris. Next, a segregating family was created in which offspring differed in resistance genotype. Again β-glucosidase cDNA sequences were cloned to find a difference in the presence of these β-glucosidases between resistant and susceptible individuals. This time cDNA sequences of β-glucosidases A, B and C were present in both resistant and susceptible individuals although significantly fewer β-glucosidase C cDNA sequence variants were found in susceptible individuals than in resistant individuals. Thus, the genetic basis of flea beetle resistance remains unclear. Further investigation is needed to explore if the β-glucosidase C protein is also capable of inactivating hederagenin cellobioside by hydrolysizing the second glucose unit from the saponin and if there is a difference in enzyme activity of β-glucosidase C between resistant and susceptible beetles.

Subsequently, in CHAPTER4 I have investigated whether the frequency of resistant beetles decreased in populations living on other host plant patches than B. vulgarisG-type and whether the change in frequency was significant within the flea beetle season. I found that the frequency of resistant beetles varied significantly among years, but there was no evidence for a decrease in the frequency of resistant beetles, the latter being expected if selection acts against the resistance on other host plants than B. vulgarisG-type. Furthermore, I found that the frequency of resistant beetles varied significantly within a flea beetle season. This study demonstrates that relative frequencies of different resistance phenotypes of P. nemorumon other host plants than B. vulgarisG-type are highly dynamic, both within and across years. It is, therefore, important to sample season-wide when one wants to monitor the changes in frequencies of insect resistance in natural systems.

In CHAPTERS5 and 6 I took a population genomics approach to investigate if the observed geographicaldistribution of resistance of P. nemorumto chemically defendedB. vulgarisin flea beetle populations could be explained by factors that are solely associated with genome-wide effects, such as migration, or also by locus-specific factors like selection at the resistance locus. First, neutral microsatellites were used to reveal the genetic differentiation at parts of the genome that are only influenced by genome-wide processes. Next, the level of neutral genetic differentiation was compared with the genetic differentiation found for the resistance trait. The resistance trait was an outlier in pairwise comparisons between flea beetle populations on B. vulgarisand S. arvensis, meaning that the level of genetic differentiation was significantly higher than expected if the resistance trait experiences only genome-wide effects. The resistance trait was also an outlier in the pairwise comparison between populations on S. arvensis, which suggests that the resistance trait is also under directional selection on other host plants than B. vulgarisG-type.

Additionally, I examined in CHAPTER6 if the homologous β-glucosidases B and C sequences found in CHAPTER3 correspond to two alleles of the major resistance gene, because of their similarity and their presence in flea beetle lines. The sequence of β-glucosidases C had so far only been found in resistant individuals, so we hypothesized it to be the dominant resistance allele and the sequence of β-glucosidases B would then correspond to the susceptible allele. In order to find out if this hypothesized PneR-gene (Phyllotreta nemorum R-gene) is the resistance gene, we first directly compared resistance phenotypes of beetles collected from populations on B. vulgarisG-type and S. arvensiswith genotypes derived with primers developed for β-glucosidase B and C. The phenotype of the flea beetles did not match the genotype derived with the β-glucosidase primers. Additionally, the frequency of heterozygotes and homozygotes of the PneR-gene genotype was not significantly deviating from Hardy-Weinberg Equilibrium which implies that there are no locus-specific effects involved when both sequences are seen as one gene with two alleles. A population approach was taken like in CHAPTER5, this time including the genetic differentiation estimated for the candidate gene as well. The candidate gene behaved similar to the neutral loci while the resistance trait was an outlier in most pairwise comparisons between flea beetle populations. If both sequences are alleles of the same gene, then the candidate gene is not directly responsible for the flea beetle resistance to B. vulgarisG-type defence.

The results presented in this thesis show the complexity of genetic processes (either genome-wide or locus specific) affecting local adaptation and the distribution of a resistance trait in insects in natural populations. Furthermore, the present study shows that when studying coevolution between insect and host plant by means of adaptive traits, also geographical and seasonal variation in allele frequencies should be considered. A multidisciplinary approach to study adaptation in plant-insect interactions such as used in this thesis, will benefit research on plant-insect interactions, including applied research such as studying the potential of host plants as dead-end traps for pest insects and preventing/diminishing the development of resistance by pest insects to crop defences.

Breeding for quality of button mushrooms: genetically dissecting bruising sensitivity and quality-related traits of Agaricus bisporus
Gao, W. - \ 2014
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Anton Sonnenberg; Johan Baars. - Wageningen : Wageningen University - ISBN 9789461739018 - 174
agaricus bisporus - eetbare paddestoelen - plantenveredeling - kneuzen - gevoeligheid - loci voor kwantitatief kenmerk - kwaliteit - kenmerken - genetische analyse - edible fungi - plant breeding - bruising - sensitivity - quantitative trait loci - quality - traits - genetic analysis

The present-day cultivars of button mushroom are too sensitive to mechanical damage. Consequently, mushrooms for the fresh market are still handpicked, which is a main cost factor for the Dutch mushroom industry. The main objective of this thesis is to use the phenotypic variation and segregation to study the genetic determinants explaining the differences in bruising sensitivity and generate breeding stocks that can be used to construct commercial varieties that are suitable for mechanical harvest. In addition, the genetic basis of other quality-related traits was investigated. For this, segregating populations were generated and used to study in more detail the life cycle of the bisporicvariety. The availability of the whole genome sequence allowsa more detailed study of recombination frequencies generating knowledge invaluable for further mushroom breeding.

Potato genetical genomics: investigating the genetic basis of primary metabolism and its relationship to the phenotype
Carreño Quintero, N. - \ 2013
Wageningen University. Promotor(en): Harro Bouwmeester; Richard Visser, co-promotor(en): Joost Keurentjes; Christian Bachem. - Wageningen : Wageningen University - ISBN 9789461738110 - 180
solanum tuberosum - aardappelen - genomica - metabolisme - genetische analyse - metabolomica - metabolomen - fenotypen - loci voor kwantitatief kenmerk - potatoes - genomics - metabolism - genetic analysis - metabolomics - metabolomes - phenotypes - quantitative trait loci

Primary metabolism is essential for plant growth and survival and it is therefore involved in all physiological processes of the plant. In the past years the advancements in large-scale and high-throughput technologies have enhanced our ability to characterize the plant metabolome. The development of methods for the simultaneous analysis of many different plant metabolites and the necessary software for subsequent data analysis have further expanded the possibilities to investigate plant responses from a system-oriented perspective. This allows the comparison of genetic and phenotypic variation at different molecular levels, enabling us to find associations between genotype and phenotype and their intermediate levels of information transduction. Metabolomics has become increasingly important for the characterization of the metabolic status of plants under different environmental and genetic perturbations. The economic importance of potato and the increasing availability of genetic and molecular resources have stimulated research on many different aspects of the physiology of this crop and the regulation of complex traits. We used the available tools to explore the genetic basis of the composition and content of primary metabolites in a potato population. In this research, the possibilities to combine metabolite profiling with genetic information are explored to identify the genetic factors determining primary metabolism and to infer links between metabolites and agronomic phenotypes.

Analysis of Tomato spotted wilt virus effector-triggered immunity
Ronde, D. de - \ 2013
Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Richard Kormelink. - S.l. : s.n. - ISBN 9789461737212 - 190
tomatenbronsvlekkenvirus - plantenvirussen - ziekteresistentie - immuniteit - virulentie - genkartering - genetische merkers - genetische analyse - capsicum annuum - paprika's - tomato spotted wilt virus - plant viruses - disease resistance - immunity - virulence - gene mapping - genetic markers - genetic analysis - sweet peppers

ResistanceinCapsicumagainsttheTomatospottedwiltvirus(TSWV),typespeciesof the Tospovirusgenuswithinthe Bunyaviridaefamily,employsthe singledominant resistancegeneTsw.Thisresistance hasmeanwhilebeenbrokenbyresistance breaking (RB) TSWV isolates and is causing increasing problems in many different (Capsicumcultivating)countries.Theresearchdescribedhereaimedtoidentify andcharacterise theviralproteintriggeringTswresistanceandprovidefurther insightintothemechanismofTsw-mediatedresistance.Knowledgegainedfrom thegeneticandphenotypiccharacterisationofTsw-resistancebreakingisolateswas usedtodevelopdiagnosticmarkersfordetectionofTsw-breakingpathotypesin fieldcultivations.

TheNSsRNAsilencingsuppressor(RSS)proteinwasidentifiedastheavirulence determinant ofTsw-mediatedresistance(Chapter2).WhiletheNSsproteinfrom theTSWVresistanceinducer(RI)isolatewasactiveasRNAsilencingsuppressorand avirulencedeterminant,theNSsproteinfromtwodifferentTSWVRBisolateslacked bothfunctionsasevidencedfromtransientassays.Surprisingly,thecorresponding resistancebreakingvirusisolatesstillexhibitedRNAisuppressoractivity. Noneof the other viral proteins were able to aid in the transient recovery of RSS activity. Electrophoreticmobilityshift assays(EMSAs)usingplantextractscontaining transientlyexpressedNSsproteinsshowedashift ofsiRNAswithNSsRI,indicative forbinding,butnotwithNSsRB.InagreementwiththelocalleafRSSassaysusinga virusinfection,plantextractsofvirusinfectedleaveswereabletoshiftthesiRNAs, showing recovery of the RSS activityduring virus infection.

The linkage of RNAi suppression and avirulence in NSs was further investigated bymutationalanalysis(Chapter3).AlargesetofNSsmutantswasgeneratedusing alaninesubstitutions ofauthenticTSWVNSsaminoacidsandwastestedfortheir abilitytotriggerTsw-mediatedHRandabilitytosuppressRNAi.Theseassaysshowed thatthe N-terminaldomainofNSscarried mostimportantresiduesinvolvedwith bothactivities. However,singlemutationscouldbeintroducedthatdisruptedone function,whilemaintainingtheotheroneandviceversaindicatingthatRSSactivity andavirulencewerenotfunctionally linked.SwappingofdomainsbetweenNSsRI andNSsRB notonlyconfirmedtheimportanceoftheN-terminaldomainbutalso thespecificitywithintheTSWVspecies,sincedomainswapsbetweenNSsRIandNSs fromGRSV,arelatedbutdistinct Tospovirus,couldnottransfertheAvrphenotype toGRSV.MutationofaGW/WG-motifintheNterminalregionofNSsRI leadtoa lossofbothfunctionsandindicatedthatthismotif, knowntobeinvolvedinAGO1 interactionof other viral RSS, was of biological relevance for TSWV NSs.

Theputativeinteraction ofAGO1andNSswasinvestigatedbyusingdifferent approaches to co-immunoprecipitate (Co-IP) on transiently co-expressed tagged- AGO1and(His-)NSs(Chapter4).Initialindicationsforsuchinteraction were obtained,howeverfurthersupportforthisputativeinteraction willhavetocome fromcomplementaryexperiments,e.g. Yeast-2-hybrid (Y2H), FRET-FLIM or BiFC.

Severaladditional TSWVisolateswereanalysedthatbesidestheknownresistance inducing-and resistance breaking-phenotype showed a temperature-dependent phenotype(Chapter5).IsolatesclassifiedtothistypeexhibitedanRIphenotypeat standardgreenhouseconditions (~22°C)whileatelevatedtemperatures(≥28°C), butstillbelowtemperaturesthatinactivatedtheR-geneproduct(≥31°C),wereable tobreaktheresistance.Viruschallengingassaysatvariousconditionsindicatedthat inductionofTswresistanceatalower temperaturebythesesocalledtemperature dependentresistancebreakingisolates(TempRB)involveddenovosynthesisofthe avirulenceprotein,i.e.NSs,andthat proteinfoldingmight play arole. NSsproteins clonedandexpressedfromthisadditional newsetofTSWVresistanceinducing, resistancebreakingandtemperature dependentresistancebreakingisolates revealedvariableresultsregardless oftheircorrespondingvirusphenotype,when tested for their abilitytoinduceTsw-mediated HR andsuppress RNAi at normal greenhouseconditions(22°C).However,similarassaystoanalysetheiractivity attheelevatedtemperature(28°C)failedwhenusingAgrobacteriummediated transientassays.Sofar,themechanismoftemperature dependencyhasnotbeen clarified yetandneedsfurtherinvestigation.Usingtheinformationobtained,a diagnostictoolwasdevelopedtoscreenforthepotential presenceofresistance breakingisolatesofTSWVusingreversetranscription-polymerasechainreaction amplification(RT-PCR).Aprimersetwasdesignedtargetinganimportantcodon ataaposition79andshowedtobeabletodistinguishRB-isolatesfromRI-isolates. However,afewRB-isolatesstillescapedfromdetection indicatingthelimitedand conditionaluse of this tool.

In summary, NSs has been identified as Avr-determinant of Tsw-mediated resistance,butthisfunctionisnottightlylinkedtoitsRNAisuppressor-activity. Preliminarydataindicateaputativeinteraction betweenAGO1andNSs.Besides the typicalRIandRBphenotypes,athirdphenotypicclassofTSWVisolates has beenidentified thatexhibitsatemperaturedependencyontriggeringTsw- mediatedresistance andpossiblyinvolvesanalteredproteinfoldingofNSs.A diagnostic toolhasbeendevelopedtodetectresistancebreakingisolatesinthe fieldbasedonRT-PCR,butthistoolstillallowsforescapesofRBisolates.Theresults onNSsarediscussedinlightofitsroleaseffectorwithinthe‘Zig-zag-model’of planthostdefenceresponses.Finally,TSWVNSsisbriefly discussedandcompared totheanimal-infecting(NSs)paralogsoftheBunyaviridaefamily,alsoinlightof functional andstructuralhomologiesbetweenthesensorsofinnateimmunityin plant(R-genes)and animal (NLRs/TLRs) cell systems.

Genetic analysis of drought stress response in Arabidopsis thaliana and Brassica rapa
El-Soda, M. - \ 2013
Wageningen 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.

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