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Cum laude graduationAmong the factors hindering the conservation of crop genetic resources is a lack of essential information regarding this diversity. Questions include: (a) what is the status of diversity in our food systems, and where are the greatest vulnerabilities?, (b) where can genetic diversity be found that can be useful in increasing productivity and mitigating these vulnerabilities?, (c) is this genetic diversity available in the present and in the long term?, and (d) what steps are needed to improve the ability for researchers to access genetic resources critical for present and future crop improvement? This thesis aims to contribute to the knowledge required to answer these questions through an exploration of the need for, potential of, challenges and constraints regarding, and necessary steps to enhance the conservation and use of crop genetic diversity.

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

Cum laude graduationAmong the factors hindering the conservation of crop genetic resources is a lack of essential information regarding this diversity. Questions include: (a) what is the status of diversity in our food systems, and where are the greatest vulnerabilities?, (b) where can genetic diversity be found that can be useful in increasing productivity and mitigating these vulnerabilities?, (c) is this genetic diversity available in the present and in the long term?, and (d) what steps are needed to improve the ability for researchers to access genetic resources critical for present and future crop improvement? This thesis aims to contribute to the knowledge required to answer these questions through an exploration of the need for, potential of, challenges and constraints regarding, and necessary steps to enhance the conservation and use of crop genetic diversity.

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

Resistentie tegen Botrytis cinerea is gevonden in wilde verwanten van tomaat en deze resistentie was meestal kwantitatief. Het doel van dit promotieonderzoek was om kwantitatieve loci (QTLs) te identificeren die bijdragen aan resistentie tegen B. cinerea. Met behulp van DNA-merkertechnologie en een populatie van introgressie-lijnen zijn tien QTLs geïdentificeerd. Geen van de afzonderlijke QTLs resulteerde in een niveau van resistentie dat overeen kwam de resistente ouder Solanum habrochaites LYC4. Dit betekent dat QTLs gecombineerd zullen moeten worden om Botrytis cinerea-resistente tomaten te verkrijgen. Dankzij de ontwikkelde DNA-merkers kunnen de geïdentificeerde chromosoomfragmenten met resistentiegenen nu gericht ingekruist worden.

thesis Ximena Cadima Fuentes (to be defended on 8 Dec 2014): Conserving the genetic diversity of Bolivian wild potatoes The wild relatives of potatoes (Solanum sect. Petota) form the genetic reservoir for the improvement of the cultivated potato. Bolivia harbours 39 wild taxa of these wild potatoes, 21 of which are endemic species. This study aimed to evaluate to what level the current ex situ and in situ management efforts have conserved the genetic diversity of Bolivian wild potato species, and what recommendations can be formulated for improvement. The current conservation status of Bolivian endemic wild potato species was assessed using both the globally accepted IUCN criteria and a methodology developed within the framework of the UNEP/GEF-Crop Wild Relative Project (CWR Project). These two methods led to different estimates of threat status for some of the species. Spatial analysis allowed to distinguish eight priority areas for in situ conservation of the 21 Bolivian endemic wild potato species. These areas represent a high concentration of endemic species and have a relatively low level of threat, but only one of them has a conservation status. This is a first step to direct the conservation efforts for wild potato species. The genetic stability and diversity of material from different species under ex situ management was evaluated using microsatellite markers. The analysis was performed on accessions that went through a process of seed regeneration and multiplication during ex situ conservation. Genetic changes between different generations of ex situ germplasm were observed for the majority, but not all, of the investigated species. Potential causes of these changes include genetic drift and contamination resulting from human error during regeneration. The populations generated under ex situ conditions were also compared with re-collected in situ populations from the same location or area as the original collection. The results showed highly significant differences in all cases. Potential causes for these differences are changes during ex situ maintenance, sampling effects during collecting and in situ genetic change over time. The integrated conservation of Bolivian wild potatoes requires a combination of in situ and ex situ activities. The principle recommendation for the in situ conservation is to move from a passive to an active approach, where conservation areas are prioritized, conservation plans are designed according to the type of area (protected area or agro-ecosystem) and local stake holders are involved. To make sure that ex situ material provides a good representation of the in situ genetic variability, regular re-collecting of species with few accessions (and therefore less variability), endangered in situ, and with known or potential favorable traits is necessary. Gene bank management procedures should follow the FAO gene bank standards and this should be monitored by a national body responsible for genetic resources. And finally, periodic monitoring of the genetic integrity should be implemented as part of good practices during regeneration procedures in order to detect possible changes and to help combat human errors.

PGR Secure project , a collaborative project funded under the EU Seventh Framework Programme, THEME KBBE.2010.1.1-03, 'Characterization of biodiversity resources for wild crop relatives to improve crops by breeding', Grant agreement no. 266394."

PGR Secure project , a collaborative project funded under the EU Seventh Framework Programme, THEME KBBE.2010.1.1-03, 'Characterization of biodiversity resources for wild crop relatives to improve crops by breeding', Grant agreement no. 266394."

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.

Many plant species are hosts of powdery mildew fungi, including Arabidopsis and economically important crops such as wheat, barley and tomato. Resistance has been explored using induced mutagenesis and natural variation in the plant species. The isolated genes encompass loss-of-function susceptibility genes and dominantly inherited genes encoding NB-LRR proteins, receptor-like kinases or proteins that do not have typical resistance protein domains. Cultivated tomato is susceptible to powdery mildew species Oidium neolycopersici, and exploiting the resistance genes present in wild tomato species is a favourable strategy to control the disease. In chapter 2, we give an overview of all the identified resistance genes in wild tomato species and their resistance mechanisms inferred from cytological and molecular data. Furthermore, resistance genes and their mechanisms are compared between tomato and other plant species, such as dicot Arabidopsis and monocots barley and wheat. This comparison illustrates that both common and species-specific mechanisms are involved with respect to resistance to powdery mildews in different plant species. Resistance gene Ol-1 originates from wild tomato species S. habrochaites. It confers race-non-specific resistance to tomato powdery mildew. To elucidate the resistance signalling pathway, we adopted a virus induced gene silencing (VIGS) approach to suppress genes which are differentially expressed when comparing genotypes with and without the Ol-1 introgression. In chapter 3, we showed that ALS (acetolactate synthase) activity is important for Ol-1-mediated resistance, as simultaneous silencing of two ALS genes attenuated the resistance level of NIL-Ol-1. ALS is a key enzyme in the biosynthesis of branched-chain amino acids, and a target of commercial herbicides. Reducing ALS activity via herbicidal treatment did not result in altered responses to powdery mildew infection in susceptible cultivar Moneymaker and resistant line NIL-Ol-4, indicating that ALS is not involved in basal defense nor in NB-LRR gene-mediated resistance. Whether the role of ALS in Ol-1-mediated resistance is associated with amino acid homeostasis is unknown and needs further investigation. Besides tomato, Arabidopsis is a host of powdery mildew O. neolycopersici. The large collection of Arabidopsis accessions and several mutant collections are valuable resources to identify novel resistance genes. In chapter 4, we first screened 123 Arabidopsis accessions for O. neolycopersici resistance and then studied the genetic basis of theresistance by segregation analysis in 19 F2 populations. The results showed that polygenic resistance is the main form of resistance. Accession C24 displays complete resistance with polygenic nature, as shown by QTL analysis of the F2 population derived from the cross between C24 and susceptible accession Sha. The recessively inherited locus on chromosome 1 was fine-mapped by recombinant screening, and analysis of candidate genes resulted in the isolation of the gene conferring resistance. It proved to be a mutant allele of EDR1, harbouring a deletion upstream of the kinase domain resulting in a truncated protein. Previously, an induced edr1mutationin Col-0 background was obtained. However, the edr1 mutation in our C24 source (referred to as C24-W) occurred in a different position. The resistance conferred by edr1 in C24-W was not associated with constitutively expressed pathogenesis-related genes. Remarkably, we observed that although C24-W carried the edr1 mutation this mutation was absent in other C24 sources. In addition, C24-W was smaller in size than C24 from other sources. Since the edr1 mutation confers resistance to tomato powdery mildew in Arabidopsis, we investigated whether this resistance system is conserved in tomato. The results showed that individual silencing of two tomato EDR1 candidate genes in susceptible cultivar Moneymaker did not result in decreased sporulation of tomato powdery mildew. In chapter 5, we screened an activation tag Arabidopsis mutant collection. In these mutants, tagged genes are overexpressed by the strong 35S enhancers resulting in a dominant gain-of-function phenotype. One mutant line, 3221, was identified due to its resistance to powdery mildew O. neolycopersici. Additional disease tests showed that 3221 displayed resistance to the downy mildew Hyaloperonospora arabidopsidis and the aphid Myzus persicae, but susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. The mutant line 3221 also showed reduced size and serrated leaves, and the altered morphology was associated with resistance. Inverse PCR and expression analysis revealed that the gene underlying the resistance was ATHB13, a HD-Zip transcription factor. Suppression ofATHB13 in 3221 by RNAi transformation resulted in the loss of resistance and altered morphology, while overexpression of ATHB13 in wild-type plants induced resistance and altered morphology. Microarray analysis of 3221 and the parental line Ws resulted in the identification of a large number of genes showing differential expression. Analysis of these results did not give a clear indication that the resistance phenotype in 3221 is due to the activation of classical hormone pathway genes involved in resistance. The possibility of utilizing ATHB13 for engineering pathogen resistance in tomato needs to be investigated in the future. Finally, in chapter 6 the results from the previous chapters are discussed in a broader context.

Tomato (Solanum lycopersicum) is affected by a wide range of biotic stresses, of which Bemisia tabaci is one of the most important.Bemisia tabaci affects tomato directly through phloem sap feeding, and indirectly through its ability to be the vector of a large number of viruses. Different methods are available for whitefly control, and although several biological control agents are used against whiteflies in greenhouse cultivation, chemical control still is an essential component in open field tomato production. Breeding for host plant resistance is considered as one of the most promising methods in insect pest control in crop plants, and especially it is a promising alternative in whitefly control. Resistance to whiteflies was found in several wild relatives of tomato like Solanum peruvianum, S. pennellii, S. habrochaites, S. lycopersicum var. cerasiforme, S. pimpinellifolium andS. galapagense. In spite of previous breeding efforts, whiteflies are still a problem in tomato cultivation. The aim of my research was to identify and understand resistance mechanisms targeting specific stages of the whitefly life cycle in order to provide breeders with tools for developing whitefly resistant varieties. I assessed the natural variation and whitefly resistance in Solanum galapagense and S. cheesmaniae, two wild tomato species endemic to the Galapagos Islands. Previously, Solanum galapagense and S. cheesmaniae were classified as two species based on a morphological species concept, but with molecular markers no clear separation could be made. So far, only a limited number of accessions/populations of S. galapagense and S. cheesmaniae have been evaluated for insect resistance and therefore it was unknown if the insect resistance coincides with the morphological species boundaries. Neither was there any knowledge about the relation between geographical and climatic conditions today on the Galapagos and the occurrence of the two species. We characterized twelve accessions of S. galapagense, 22 of S. cheesmaniae, and as reference one of S. lycopersicum for whitefly resistance using no-choice experiments. Whitefly resistance was found in S. galapagense only and was associated with the presence of relatively high levels of acyl sugars and the presence of glandular trichomes of type I and IV.It is likely that a minimum level of acyl sugars and the presence of glandular trichomes type IV are needed to achieve an effective level of resistance. Genetic fingerprinting using 3316 polymorphic SNP markers did not show a clear differentiation between the two species endemic to the Galapagos. Acyl sugar accumulation as well as the climatic and geographical conditions at the collection sites of the accessions did not follow the morphological species boundaries. Altogether, our results suggest that S. galapagense and S. cheesmaniae might be considered as morphotypes rather than two species and that their co-existence is likely the result of selective pressure. Plants possess several resistance mechanisms acting at different time points during the interaction with herbivorous insect. Before any contact with the insects, plants emit an array of volatile organic compounds that can act as attractant or repellent of insects.Bemisia tabaci use a set of plant-derived cues in the process of host plant selection. It recognizes mainly monoterpenes (p-cymene, γ-terpinene and β-myrcene, α-phellandrene) and sesquiterpenes (7-epizingiberene and R-curcumene). Previously the line FCN93-6-2, which was derived from a cross between a susceptible tomato cultivar (Uco Plata INTA) and S. habrochaites (FCN3-5) was proved to be non-preferred by the greenhouse whitefly Trialeurodes vaporariorum. We identified chemical cues produced by FCN93-6-2 and S. habrochaites that can affect the preference of the whitefly B. tabaci as well as the potential chromosomal region(s) of S. habrochaites harbouring the genes involved in the preference. Two S. habrochaites accessions (CGN1.1561 and in FCN3-5) and the line FCN93-6-2 were non-preferred by B. tabaci when the whiteflies could get in direct contact with the plant and also when the whiteflies were offered olfactory cues only. The non-preference was independent of trichome type IV and of the presence of methyl-ketones but associated to the presence of monoterpenes in lower concentrations. Functional validation of the candidate metabolites and of the different introgressions is still needed. Once the insect has landed on a plant, another set of resistance mechanisms enter into action. We have described a 3.06 Mbp introgression on top of Chromosome 5 (OR-5) from the wild tomato species S. habrochaites (CGN1.1561). For the identification of OR-5, we went from the selection of specific F2 plants to the development of F2BC4S1 and F2BC4S2 families. This introgression was sufficient to reduce whitefly fecundity without an evident effect on whitefly survival. The identification of mechanisms exclusively affecting whitefly fecundity and independent of trichomes type IV opens new doors for resistance breeding to whiteflies that may be especially interesting in greenhouse cultivation combination with natural enemies of the whitefly. As an additional layer of defences, plants can perceive stress signals and respond to them in a specific way through induction of their immune system. This induction can also be triggered by exposing the plants to priming agents like hormones, some xenobiotic chemicals, like benzothiadiazole (BTH), β-aminobutyric acid (BABA), and sugars. Although the effect of priming agents was shown in laboratory and field studies, little is known about the effect of the genetic background of tomato on the extent of the priming, e.g. do genotypes varying in their level of resistance to insects and pathogens respond in the same way to a priming agent. We assessed the effect of selected priming agents on the effectiveness of natural defence in tomato. A set of no-choice and choice bioassays was conducted using tomato genotypes varying in their level of basal resistance to Bemisia tabaci and pathogens. We observed that whitefly survival and oviposition were not affected by the priming treatment in no-choice assays. Overall, in choice assays, fructose treated plants were more preferred by whiteflies than control plants. A genotype specific effect of priming was seen for the line FCN93-6-2. On this tomato line, JA and BABA applications decreased the number of whiteflies, e.g. making them less preferred. In this thesis, I have gone from the screening of wild relatives of tomatoes to in depth characterization of resistance mechanisms. I have identified resistance mechanisms targeting specific stages of the whitefly life cycle, thus providing new tools for breeding durable whitefly resistance in tomato.

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.

Many plant species are hosts of powdery mildew fungi, including Arabidopsis and economically important crops such as wheat, barley and tomato. Resistance has been explored using induced mutagenesis and natural variation in the plant species. The isolated genes encompass loss-of-function susceptibility genes and dominantly inherited genes encoding NB-LRR proteins, receptor-like kinases or proteins that do not have typical resistance protein domains. Cultivated tomato is susceptible to powdery mildew species Oidium neolycopersici, and exploiting the resistance genes present in wild tomato species is a favourable strategy to control the disease. In chapter 2, we give an overview of all the identified resistance genes in wild tomato species and their resistance mechanisms inferred from cytological and molecular data. Furthermore, resistance genes and their mechanisms are compared between tomato and other plant species, such as dicot Arabidopsis and monocots barley and wheat. This comparison illustrates that both common and species-specific mechanisms are involved with respect to resistance to powdery mildews in different plant species. Resistance gene Ol-1 originates from wild tomato species S. habrochaites. It confers race-non-specific resistance to tomato powdery mildew. To elucidate the resistance signalling pathway, we adopted a virus induced gene silencing (VIGS) approach to suppress genes which are differentially expressed when comparing genotypes with and without the Ol-1 introgression. In chapter 3, we showed that ALS (acetolactate synthase) activity is important for Ol-1-mediated resistance, as simultaneous silencing of two ALS genes attenuated the resistance level of NIL-Ol-1. ALS is a key enzyme in the biosynthesis of branched-chain amino acids, and a target of commercial herbicides. Reducing ALS activity via herbicidal treatment did not result in altered responses to powdery mildew infection in susceptible cultivar Moneymaker and resistant line NIL-Ol-4, indicating that ALS is not involved in basal defense nor in NB-LRR gene-mediated resistance. Whether the role of ALS in Ol-1-mediated resistance is associated with amino acid homeostasis is unknown and needs further investigation. Besides tomato, Arabidopsis is a host of powdery mildew O. neolycopersici. The large collection of Arabidopsis accessions and several mutant collections are valuable resources to identify novel resistance genes. In chapter 4, we first screened 123 Arabidopsis accessions for O. neolycopersici resistance and then studied the genetic basis of theresistance by segregation analysis in 19 F2 populations. The results showed that polygenic resistance is the main form of resistance. Accession C24 displays complete resistance with polygenic nature, as shown by QTL analysis of the F2 population derived from the cross between C24 and susceptible accession Sha. The recessively inherited locus on chromosome 1 was fine-mapped by recombinant screening, and analysis of candidate genes resulted in the isolation of the gene conferring resistance. It proved to be a mutant allele of EDR1, harbouring a deletion upstream of the kinase domain resulting in a truncated protein. Previously, an induced edr1mutationin Col-0 background was obtained. However, the edr1 mutation in our C24 source (referred to as C24-W) occurred in a different position. The resistance conferred by edr1 in C24-W was not associated with constitutively expressed pathogenesis-related genes. Remarkably, we observed that although C24-W carried the edr1 mutation this mutation was absent in other C24 sources. In addition, C24-W was smaller in size than C24 from other sources. Since the edr1 mutation confers resistance to tomato powdery mildew in Arabidopsis, we investigated whether this resistance system is conserved in tomato. The results showed that individual silencing of two tomato EDR1 candidate genes in susceptible cultivar Moneymaker did not result in decreased sporulation of tomato powdery mildew. In chapter 5, we screened an activation tag Arabidopsis mutant collection. In these mutants, tagged genes are overexpressed by the strong 35S enhancers resulting in a dominant gain-of-function phenotype. One mutant line, 3221, was identified due to its resistance to powdery mildew O. neolycopersici. Additional disease tests showed that 3221 displayed resistance to the downy mildew Hyaloperonospora arabidopsidis and the aphid Myzus persicae, but susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. The mutant line 3221 also showed reduced size and serrated leaves, and the altered morphology was associated with resistance. Inverse PCR and expression analysis revealed that the gene underlying the resistance was ATHB13, a HD-Zip transcription factor. Suppression ofATHB13 in 3221 by RNAi transformation resulted in the loss of resistance and altered morphology, while overexpression of ATHB13 in wild-type plants induced resistance and altered morphology. Microarray analysis of 3221 and the parental line Ws resulted in the identification of a large number of genes showing differential expression. Analysis of these results did not give a clear indication that the resistance phenotype in 3221 is due to the activation of classical hormone pathway genes involved in resistance. The possibility of utilizing ATHB13 for engineering pathogen resistance in tomato needs to be investigated in the future. Finally, in chapter 6 the results from the previous chapters are discussed in a broader context.

Tomato (Solanum lycopersicum) is affected by a wide range of biotic stresses, of which Bemisia tabaci is one of the most important.Bemisia tabaci affects tomato directly through phloem sap feeding, and indirectly through its ability to be the vector of a large number of viruses. Different methods are available for whitefly control, and although several biological control agents are used against whiteflies in greenhouse cultivation, chemical control still is an essential component in open field tomato production. Breeding for host plant resistance is considered as one of the most promising methods in insect pest control in crop plants, and especially it is a promising alternative in whitefly control. Resistance to whiteflies was found in several wild relatives of tomato like Solanum peruvianum, S. pennellii, S. habrochaites, S. lycopersicum var. cerasiforme, S. pimpinellifolium andS. galapagense. In spite of previous breeding efforts, whiteflies are still a problem in tomato cultivation. The aim of my research was to identify and understand resistance mechanisms targeting specific stages of the whitefly life cycle in order to provide breeders with tools for developing whitefly resistant varieties. I assessed the natural variation and whitefly resistance in Solanum galapagense and S. cheesmaniae, two wild tomato species endemic to the Galapagos Islands. Previously, Solanum galapagense and S. cheesmaniae were classified as two species based on a morphological species concept, but with molecular markers no clear separation could be made. So far, only a limited number of accessions/populations of S. galapagense and S. cheesmaniae have been evaluated for insect resistance and therefore it was unknown if the insect resistance coincides with the morphological species boundaries. Neither was there any knowledge about the relation between geographical and climatic conditions today on the Galapagos and the occurrence of the two species. We characterized twelve accessions of S. galapagense, 22 of S. cheesmaniae, and as reference one of S. lycopersicum for whitefly resistance using no-choice experiments. Whitefly resistance was found in S. galapagense only and was associated with the presence of relatively high levels of acyl sugars and the presence of glandular trichomes of type I and IV.It is likely that a minimum level of acyl sugars and the presence of glandular trichomes type IV are needed to achieve an effective level of resistance. Genetic fingerprinting using 3316 polymorphic SNP markers did not show a clear differentiation between the two species endemic to the Galapagos. Acyl sugar accumulation as well as the climatic and geographical conditions at the collection sites of the accessions did not follow the morphological species boundaries. Altogether, our results suggest that S. galapagense and S. cheesmaniae might be considered as morphotypes rather than two species and that their co-existence is likely the result of selective pressure. Plants possess several resistance mechanisms acting at different time points during the interaction with herbivorous insect. Before any contact with the insects, plants emit an array of volatile organic compounds that can act as attractant or repellent of insects.Bemisia tabaci use a set of plant-derived cues in the process of host plant selection. It recognizes mainly monoterpenes (p-cymene, γ-terpinene and β-myrcene, α-phellandrene) and sesquiterpenes (7-epizingiberene and R-curcumene). Previously the line FCN93-6-2, which was derived from a cross between a susceptible tomato cultivar (Uco Plata INTA) and S. habrochaites (FCN3-5) was proved to be non-preferred by the greenhouse whitefly Trialeurodes vaporariorum. We identified chemical cues produced by FCN93-6-2 and S. habrochaites that can affect the preference of the whitefly B. tabaci as well as the potential chromosomal region(s) of S. habrochaites harbouring the genes involved in the preference. Two S. habrochaites accessions (CGN1.1561 and in FCN3-5) and the line FCN93-6-2 were non-preferred by B. tabaci when the whiteflies could get in direct contact with the plant and also when the whiteflies were offered olfactory cues only. The non-preference was independent of trichome type IV and of the presence of methyl-ketones but associated to the presence of monoterpenes in lower concentrations. Functional validation of the candidate metabolites and of the different introgressions is still needed. Once the insect has landed on a plant, another set of resistance mechanisms enter into action. We have described a 3.06 Mbp introgression on top of Chromosome 5 (OR-5) from the wild tomato species S. habrochaites (CGN1.1561). For the identification of OR-5, we went from the selection of specific F2 plants to the development of F2BC4S1 and F2BC4S2 families. This introgression was sufficient to reduce whitefly fecundity without an evident effect on whitefly survival. The identification of mechanisms exclusively affecting whitefly fecundity and independent of trichomes type IV opens new doors for resistance breeding to whiteflies that may be especially interesting in greenhouse cultivation combination with natural enemies of the whitefly. As an additional layer of defences, plants can perceive stress signals and respond to them in a specific way through induction of their immune system. This induction can also be triggered by exposing the plants to priming agents like hormones, some xenobiotic chemicals, like benzothiadiazole (BTH), β-aminobutyric acid (BABA), and sugars. Although the effect of priming agents was shown in laboratory and field studies, little is known about the effect of the genetic background of tomato on the extent of the priming, e.g. do genotypes varying in their level of resistance to insects and pathogens respond in the same way to a priming agent. We assessed the effect of selected priming agents on the effectiveness of natural defence in tomato. A set of no-choice and choice bioassays was conducted using tomato genotypes varying in their level of basal resistance to Bemisia tabaci and pathogens. We observed that whitefly survival and oviposition were not affected by the priming treatment in no-choice assays. Overall, in choice assays, fructose treated plants were more preferred by whiteflies than control plants. A genotype specific effect of priming was seen for the line FCN93-6-2. On this tomato line, JA and BABA applications decreased the number of whiteflies, e.g. making them less preferred. In this thesis, I have gone from the screening of wild relatives of tomatoes to in depth characterization of resistance mechanisms. I have identified resistance mechanisms targeting specific stages of the whitefly life cycle, thus providing new tools for breeding durable whitefly resistance in tomato.

The silverleaf whitefly (Bemisia tabaciGenn.) poses a serious threat to tomato cultivation. A large part of the damage is done directly through heavy host plant colonization. Colonization has a negative impact on the plant, as the whitefly takes up nutrients from the phloem and induces phytotoxic responses, which result in irregular ripening of the fruits. However, most damage is done indirectly as the silverleaf whitefly vectors a broad range of plant pathogenic viruses. The silverleaf whitefly can successfully be controlled biologically in greenhouse cultivations, but control of the whitefly in the field is mainly based on the application of pesticides. The use of pesticides can have a negative effect on non-harmful or beneficial organisms in the field. Moreover, the effectiveness of pesticides can decline or even completely disappear through adaptation of the whitefly. An effective alternative for the use of pesticides could be the deployment of resistant cultivars. Nowadays, genetic factors responsible for whitefly resistance can be transferred faster and more efficiently into tomato cultivars through marker-assisted backcross breeding programs. Complete resistance against the whitefly is present in some crossable wild relatives of the cultivated tomato and the literature reports extensively about accessions with a high level of resistance against the whitefly. In this work, I have studied different populations that were developed by interspecific crosses between cultivated tomato and the tomato wild relativesS. habrochaitesLYC4 and S. pennelliiLA3791. By integrating datasets from different research disciplines, I have studied the background of whitefly resistance in these populations. Furthermore, these data were used to identify the chromosomal loci in the wild tomato relatives that harbor genes responsible for the resistance and that can be bred into cultivated tomato. The mechanisms underlying the resistance in S. pennelliiLA3791 were studied through phenotypic resistance assays that demonstrated that survival and oviposition of the whitefly were not possible on this wild relative. Through removal of glandular cells, present on the leaf trichomes, the resistance was almost completely lost and only adult survival was still significantly different from the wild type. This result led to the hypothesis that glandular trichomes play an important role in the resistance. This was confirmed in a segregating population based on a cross between S. pennelliiLA3791 and a susceptible cultivated tomato. Plants that lacked glandular trichomes type I and IV, had the same resistance level as the susceptible parent. Further analyses of the segregating population showed that the presence of glandular trichomes was not the only factor determining resistance, but that the composition and quantity of the metabolites in the glandular trichomes also played an important role. To gain more knowledge on the role of individual metabolites on whitefly resistance and susceptibility, we analyzed the total metabolite content of extreme phenotypes of the F2 population. Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography Time-Of-Flight Mass Spectrometry (LC-TOF-MS) were employed for the analyses of the total metabolite content. Analyses revealed that on basis of the total metabolite profiles the extreme phenotypes (susceptible versus resistant for the silverleaf whitefly) could be discriminated into two groups that were correlated with resistance or susceptibility. A number of these metabolites could be annotated, but for the majority of the components this was not possible on the basis of available literature and databases. Subsequently, I have studied the genetic basis of the phenotypic resistance parameters as well as the genetic basis of the metabolites from the GC-MS and LC-TOF-MS analyses. A genetic linkage map of the F2 mapping population was developed using DNA markers (Amplification Fragment Length Polymorphisms,AFLPs and Single Nucleotide Polymorphisms, SNPs). QTLs (Quantitative Trait Loci) were identified between the majority of the metabolites and the genetic markers (>90%) and also we found genetic linkages between whitefly resistance parameters and markers. The QTLs for metabolites and phenotypic parameters partly co-localized at the same positions on the genetic map. Several metabolite QTLs (mQTLs) co-localized with each other in so-called ‘hotspots’. Remarkably, the results of the individual phenotypic QTLs (phQTLs) for adult survival and oviposition as well as the mQTLs for the individual components did not give high explained variances (

The silverleaf whitefly (Bemisia tabaciGenn.) poses a serious threat to tomato cultivation. A large part of the damage is done directly through heavy host plant colonization. Colonization has a negative impact on the plant, as the whitefly takes up nutrients from the phloem and induces phytotoxic responses, which result in irregular ripening of the fruits. However, most damage is done indirectly as the silverleaf whitefly vectors a broad range of plant pathogenic viruses. The silverleaf whitefly can successfully be controlled biologically in greenhouse cultivations, but control of the whitefly in the field is mainly based on the application of pesticides. The use of pesticides can have a negative effect on non-harmful or beneficial organisms in the field. Moreover, the effectiveness of pesticides can decline or even completely disappear through adaptation of the whitefly. An effective alternative for the use of pesticides could be the deployment of resistant cultivars. Nowadays, genetic factors responsible for whitefly resistance can be transferred faster and more efficiently into tomato cultivars through marker-assisted backcross breeding programs. Complete resistance against the whitefly is present in some crossable wild relatives of the cultivated tomato and the literature reports extensively about accessions with a high level of resistance against the whitefly. In this work, I have studied different populations that were developed by interspecific crosses between cultivated tomato and the tomato wild relativesS. habrochaitesLYC4 and S. pennelliiLA3791. By integrating datasets from different research disciplines, I have studied the background of whitefly resistance in these populations. Furthermore, these data were used to identify the chromosomal loci in the wild tomato relatives that harbor genes responsible for the resistance and that can be bred into cultivated tomato. The mechanisms underlying the resistance in S. pennelliiLA3791 were studied through phenotypic resistance assays that demonstrated that survival and oviposition of the whitefly were not possible on this wild relative. Through removal of glandular cells, present on the leaf trichomes, the resistance was almost completely lost and only adult survival was still significantly different from the wild type. This result led to the hypothesis that glandular trichomes play an important role in the resistance. This was confirmed in a segregating population based on a cross between S. pennelliiLA3791 and a susceptible cultivated tomato. Plants that lacked glandular trichomes type I and IV, had the same resistance level as the susceptible parent. Further analyses of the segregating population showed that the presence of glandular trichomes was not the only factor determining resistance, but that the composition and quantity of the metabolites in the glandular trichomes also played an important role. To gain more knowledge on the role of individual metabolites on whitefly resistance and susceptibility, we analyzed the total metabolite content of extreme phenotypes of the F2 population. Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography Time-Of-Flight Mass Spectrometry (LC-TOF-MS) were employed for the analyses of the total metabolite content. Analyses revealed that on basis of the total metabolite profiles the extreme phenotypes (susceptible versus resistant for the silverleaf whitefly) could be discriminated into two groups that were correlated with resistance or susceptibility. A number of these metabolites could be annotated, but for the majority of the components this was not possible on the basis of available literature and databases. Subsequently, I have studied the genetic basis of the phenotypic resistance parameters as well as the genetic basis of the metabolites from the GC-MS and LC-TOF-MS analyses. A genetic linkage map of the F2 mapping population was developed using DNA markers (Amplification Fragment Length Polymorphisms,AFLPs and Single Nucleotide Polymorphisms, SNPs). QTLs (Quantitative Trait Loci) were identified between the majority of the metabolites and the genetic markers (>90%) and also we found genetic linkages between whitefly resistance parameters and markers. The QTLs for metabolites and phenotypic parameters partly co-localized at the same positions on the genetic map. Several metabolite QTLs (mQTLs) co-localized with each other in so-called ‘hotspots’. Remarkably, the results of the individual phenotypic QTLs (phQTLs) for adult survival and oviposition as well as the mQTLs for the individual components did not give high explained variances (

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.

Honey bees are important pollinators in agricultural crops, home gardens, orchards and wildlife habitats. As they fly from flower to flower in search of nectar and pollen, they transfer pollen from plant to plant, thus fertilizing the plants and enabling them to bear fruit. In light of this, honey bees could be a factor in spreading pollen grains derived from genetically modified (GM) plants in field trials. The extent to which pollen dispersal occurs and the distances achieved depends on many factors. Knowledge of these factors may be important for (future) risk assessments of GM plants. An overview of relevant information concerning the relationship between honey bees and pollen is presented, based on a literature survey, a database of pollen composition of Dutch honeys and a concise laboratory experiment.

Honey bees are important pollinators in agricultural crops, home gardens, orchards and wildlife habitats. As they fly from flower to flower in search of nectar and pollen, they transfer pollen from plant to plant, thus fertilizing the plants and enabling them to bear fruit. In light of this, honey bees could be a factor in spreading pollen grains derived from genetically modified (GM) plants in field trials. The extent to which pollen dispersal occurs and the distances achieved depends on many factors. Knowledge of these factors may be important for (future) risk assessments of GM plants. An overview of relevant information concerning the relationship between honey bees and pollen is presented, based on a literature survey, a database of pollen composition of Dutch honeys and a concise laboratory experiment.

The apple is the most common and culturally important fruit crop of temperate areas. The elucidation of its origin and domestication history is therefore of great interest. The wild Central Asian species Malus sieversii has previously been identified as the main contributor to the genome of the cultivated apple (Malus domestica), on the basis of morphological, molecular, and historical evidence. The possible contribution of other wild species present along the Silk Route running from Asia to Western Europe remains a matter of debate, particularly with respect to the contribution of the European wild apple. We used microsatellite markers and an unprecedented large sampling of five Malus species throughout Eurasia (839 accessions from China to Spain) to show that multiple species have contributed to the genetic makeup of domesticated apples. The wild European crabapple M. sylvestris, in particular, was a major secondary contributor. Bidirectional gene flow between the domesticated apple and the European crabapple resulted in the current M. domestica being genetically more closely related to this species than to its Central Asian progenitor, M. sieversii. We found no evidence of a domestication bottleneck or clonal population structure in apples, despite the use of vegetative propagation by grafting. We show that the evolution of domesticated apples occurred over a long time period and involved more than one wild species. Our results support the view that self-incompatibility, a long lifespan, and cultural practices such as selection from open-pollinated seeds have facilitated introgression from wild relatives and the maintenance of genetic variation during domestication. This combination of processes may account for the diversification of several long-lived perennial crops, yielding domestication patterns different from those observed for annual species., Author Summary The apple, one of the most ubiquitous and culturally important temperate fruit crops, provides us with a unique opportunity to study the process of domestication in trees. The number and identity of the progenitors of the domesticated apple and the erosion of genetic diversity associated with the domestication process remain debated. The Central Asian wild apple has been identified as the main progenitor, but other closely related species along the Silk Route running from Asia to Western Europe may have contributed to the genome of the domesticated crop. Using rapidly evolving genetic markers to make inferences about the recent evolutionary history of the domesticated apple, we found that the European crabapple has made an unexpectedly large contribution to the genome of the domesticated apple. Bidirectional gene flow between the domesticated apple and the European crabapple resulted in the domesticated apple being currently more similar genetically to this secondary genepool than to the ancestral progenitor, the Central Asian wild apple. We found that domesticated apples have evolved over long time scales, with contributions from at least two wild species in different geographic areas, with no significant erosion of genetic diversity. This process of domestication and diversification may be common to other fruit trees and contrasts with the models documented for annual crops.

Many plant species can hybridise and produce fertile offspring. Hybridization between cultivated species and their wild relatives has raised concerns with regard to GM crops, as it constitutes a possible route along which the transgene could disperse from crops into related wild species, establish itself in the natural population, and persist under natural conditions. This may cause unintended ecological consequences such as the formation of more invasive weeds and genetic erosion. After crop-wild hybridization, the persistence of the hybrids and of the crop genes (including the transgenes) in later generations depends on their genetic make-up, which consists of specific combinations of wild and crop genomic segments, and on the environmental conditions. Therefore, knowledge on the dynamics of crop-wild hybridization and introgression using conventional crop varieties is needed as it constitutes the baseline for putting into perspective the effects of transgene introgression under natural conditions. This study focused on understanding the genetic process of hybridization and introgression from cultivated to wild relative species using cultivated lettuce (Lactuca sativa L.) and its closest wild relative, prickly lettuce (L. serriola L.) as a crop-wild complex model. Natural populations of prickly lettuce in Europe were tested for the occurrence of crop-wild hybrids using Bayesian-based programmes (Structure, InStruct and NewHybrids) which returned a crop-wild hybrid occurrence of 7%. However, the geographical location of the hybrids led to the conclusion that crop-wild hybridization is not the cause of the recent northward spread of L. serriola in Europe. To test for the importance of the crop genomic segments to the performance of lettuce crop-wild hybrids, three hybrid generations were created (F2 as a selfing generation, and BC1 and BC2, backcrossed to the wild genotype to mimick the introgression process) by crossing L. sativa (cv. Dynamite) with L. serriola collected from Eys (the Netherlands). The three populations were genotyped with Single Nucleotide Polymorphism markers for genetic analysis. Because of the importance of the abiotic stresses as selection factors under natural conditions and the prospective of generating GM crop varieties with enhanced abiotic stress tolerance, the three populations were evaluated for vigour at the rosette stage under greenhouse conditions of non-stress, drought, salinity and nutrient deficiency. The BC1 population was also evaluated under field conditions at two locations (Wageningen and Sijbekarspel, the Netherlands) for hybrid germination, vigour, survival and reproduction. Based on the location and allelic effect of the QTLs for germination, vigour, survival and reproductive traits in the current lettuce crop-wild cross, genomic regions were suggested where transgenes could be or could not be located in order to mitigate their persistence in crop-wild hybrids through genetic hitchhiking and background selection.

This thesis aims to fill the gap of information on the potato landrace diversity present in farmer fields of Ecuador. Passport data from previous collections (1970’s and 1980’s) were used to identify Carchi, Chimborazo and Loja as representative areas of potato diversity. The status of on-farm conservation in these three selected areas is covered in Chapter 2. Microsatellites (SSRs) helped us to describe the genetic relationships among the landraces found in these areas (Chapter 3). The characterization of potato landraces with respect to late blight resistance (Chapter 4) and quality traits (Chapter 5) complement the description. Previous reports suggested loss of potato diversity (genetic erosion) in Ecuadorian farmer fields, but our collection of a total of 174 landraces showed that these areas still hold a substantial amount of potato landrace diversity (Chapter 2). More potato landraces were found in Chimborazo and Loja than previously sampled in the 70’s and 80’s. A comparison between the two collections, in each of the three areas, indicated only a small overlap in landrace names suggesting that the sampling of local landraces was far from exhaustive, both during the 70’s and 80’s and during the present collection trips. This is further supported by the fact that the diversity fair, which was organized after our collection trips in Chimborazo, resulted in many new landraces. Surveys and farmer meetings in the study areas were used to describe the landrace-holders and the characteristics of the farming system they use. Mostly elderly people and small-scale farmers are currently maintaining potato landraces. These farmers look for income alternatives besides agriculture, resulting in migration. The vulnerability of the potato conservation varies among our study areas. In Carchi younger farmers demonstrate a lack of interest in cropping potato landraces. In Loja farming is not seen as the only sustainable source of income and there is a perceived lack of support from the government for the activities necessary to maintain local landraces. In Chimborazo farmers are culturally more attached to their land and see agriculture as a family activity, rendering the potato landrace conservation less vulnerable. Externally driven on-farm conservation interventions, such as diversity fairs or re-introduction of landraces, were highly appreciated by the farmers and could help to conserve the potatoes. Diploid, triploid and tetraploid potato landraces are found in farmers fields. The material sampled at the three areas shows a high allelic diversity. At the tetraploid level (the most abundant) this was comparable to the variation present in an European collection of more than 800 varieties. More alleles are expected to be found when more material from other areas will be screened. There was no clear grouping of material collected according to study region, suggesting extensive movement of seed potatoes all over Ecuador. A comparison of the application of variety names with the genetic relationships among potato landraces can result in either under- or over-estimation of the variability present in farmer fields (Chapter 3). In a number of cases landraces with identical common names proved to be genetically different or individual collection samples were actually a mixture of two landraces, pointing at under-estimation of diversity present. On the other hand, cases that might lead to over-estimation were also evident, e.g. genetically identical material was present under different names. Our sampling of genetically different landraces for late blight (LB) resistance characterization (Chapter 4) confirmed that there was some variation for this trait among the landraces. Most of the landraces were susceptible to moderately resistant, but also some landraces with field resistance were identified. The observed field resistance was comparable to that in the widespread improved variety Fripapa. Possible strategies to improve late blight resistance in potato in Ecuador could include the identification of accessions with resistance among the local landraces, although only a few accessions may be expected to present field resistance. The introduction of new sources of resistance from other origins is a more viable alternative. One could attempt to introduce novel R-genes in material that already contains some level of quantitative resistance. We found varying levels of dry matter, total polyphenol and total carotenoid contents among Ecuadorian potato landraces, some were comparable to the improved varieties. Based on the dry matter content most of the Ecuadorian landraces evaluated were suitable for processing as French fries or chips. The total polyphenol content of these potatoes were quite similar to those reported by the International Potato Center (Peru) for a set of accessions representing more than 60% of the variability in their potato collection. The total carotenoid content values of the Ecuadorian potatoes included in our study were similar or lower compared to previous studies on improved or Andean potatoes. The identified outstanding potato materials could be used to develop new potato varieties through plant breeding. In Chimborazo and Loja farmers select landraces mainly based on their nutritional characteristics. However, in Carchi farmers prefer commercial improved varieties. Farmers´ preferences include empirical valuation of potato-quality rather than specific knowledge on nutritional characteristics of these potatoes. This thesis provides important knowledge about the potato landraces in Ecuador. Our results can serve as the basis for further description and use of Ecuadorian native potatoes by breeders and local communities.

Tomato, Solanum lycopersicum, is a host for Oidium neolycopersici, the cause of powdery mildew (PM). Though cultivated tomatoes are susceptible to PM, resistance is reported in wild Solanum species. By screening wild tomato species, nine loci conferring resistance to PM have been identified, namely Ol-1, ol-2, Ol-3, Ol-4, Ol-5, Ol-6, Ol-qtl1, Ol-qtl2, andOl-qtl3. These genes are located on different chromosomes and mediate different levels of resistance by different mechanisms. In this thesis we mainly focused on the Ol genes located on chromosome 6 (Ol-1, Ol-4, Ol-5 and Ol-6) with the aim to fine-map and eventually clone these genes. In addition, we studied the contribution of different phytohormone pathways to the resistance mediated by Ol-1, ol-2, Ol-4 and Ol-qtls. We first focused on the Ol genes on the short arm of tomato chromosome 6, Ol-4 originating from S. peruvianum LA2172 and Ol-6 with unknown origin (Chapter 2). We showed that Ol-4 and Ol-6 are homologues of the Mi-1 gene. On the short arm of tomato chromosome 6, the Mi-1gene cluster is about 400 Kb in size and consists of several other genes besides the Mi-1 homologues. There are transport inhibitor responses-like (TIR-like) genes embedded in this cluster. Interestingly, the copy number of these TIR-like genes in the nematode-resistant tomatoes is less than that in nematode-susceptible ones (Chapter 3). Furthermore, lower expression of these TIR-like genes was observed in roots, but not in leaves, of nematode-resistant plants compared to nematode-susceptible plants. These observations prompted us to suggest and to discuss two different scenarios explaining how TIR-like genes could play a role in the plant response to root-knot nematodes. Then, we studied the Ol-1 and Ol-5, which are located on the long arm of chromosome 6, and originated from different S. habrochaites accessions. Ol-1 is closely linked to Ol-5. With fine-mapping, we narrowed down this locus to a 73 Kb interval which contains at least 10 putative genes. Interestingly we observed an interaction between chromosome regions harboring Ol-1 and Ol-5, indicating that the interaction between Ol-1 and Ol-5 is needed to confer PM resistance. Both Ol-1 and Ol-5 trigger delayed cell death that is distinguishable from hypersensitive response (HR), the hallmark of R gene response to biotrophic pathogens. The delayed cell death associated with Ol-1 and Ol-5 resembles the autophagic PCD. We observed that Ol-1 and Ol-5 were both required for on-time and effective cell death to stop PM. If one of these two genes was not present, cell deathcould not happen or not be effective enough to stop pathogen growth. Finally, we investigated the involvement of phytohormone pathways in PM resistance conferred by the Olgenes, including Ol-1, ol-2, Ol-4 and Ol-qtls (Chapter 5). There is overwhelming evidence implicating plant hormones in plant responses to pathogens. In this experiment we, in addition to Ol-1 and Ol-4, included other resistance loci for PM resistance in tomato. The first one is ol-2, a homologue of the barley mlo gene and derived from S. lycopersicum var cerasiforme LA1230. This gene confers resistance to PM by triggering callose deposition and, thereby, cell wall fortification. The other is Ol-qtls, a combination of three QTLs for PM resistance associated with both delayed cell deathand callose deposition. NILs carrying Ol-1, ol-2, Ol-4 and Ol-qtls, plus the background of these NILs (S. lycopersicum cv Moneymaker, MM), provided us the possibility to compare the involvement of hormonal pathways in different kinds of tomato responses. These responses include basal defense, cell wall fortification, delayed cell death,and HR. We quantified the expression of marker genes for the pathways of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and ethylene (ET) over a time-course after inoculation with PM. As a complementary approach, we crossed our NILs with tomato mutants for JA, ET and ABA. Our results suggested that Ol-4-mediated resistance probably relies on the SA pathway. Ol-1 and Ol-qtls require ET to promote the delayed cell deathfor PM resistance. JA deficiency can compromise resistance mediated by ol-2. Our results also suggested that ABA is required for those interactions demanding callose deposition, resistance associated with ol-2 and Ol-qtls. These results present a nice example of the involvement of different phytohormones in different phases of resistance against PM in tomato. Altogether, this thesis describes different tomato resistance mechanisms triggered by different resistance genes in the same pathosystem, underscoring the plant ability to adopt diverse molecular mechanisms to defense itself against intruders.

The powdery mildew disease, caused by obligate biotrophic fungi belonging to the Ascomycete order of Erysiphales, is common among higher plants and represents one of the most important threats for the cultivation of many crop species. Although powdery mildew resistance is usually a dominant trait, recessively inherited resistance has been reported to occur in Arabidopsis, barley, tomato and pea. In Chapter 1 of this thesis, we provide a state of the art on the understanding of mechanisms underlying plant immunity and review monogenic dominant and recessive sources used in breeding in order to develop resistant cultivars. In Chapter 2 and 3, we describe a successful homology-based cloning approach for the isolation of the recessive ol-2 gene, naturally occurring in a tomato accession collected in Ecuador and conferring broad-spectrum resistance to the powdery mildew fungus Oidium neolycopersici. We realized that ol-2 resistance shares striking similarities with well-known barley and Arabidopsis mlo powdery mildew resistance, originating from loss-of-function mutations of genes encoding for specific seven transmembrane domains MLO isoforms. The following chain of evidence was provided demonstrating that ol-2 resistance is due to the loss of the tomato MLO homolog SlMLO1: a) Ol-2 and SlMLO1 loci share the same genetic and cytogenetic position; b) resistant ol-2/ol-2 lines are homozygous for a loss-of-function deletion in the sequence of SlMLO1; c) a molecular marker developed on the mutation site co-segregates with resistant individuals in an F2population; d) SlMLO1 transgenic expression in ol-2/ol-2 individuals results in disease susceptibility; e) SlMLO1 virus-induced silencing in Ol-2/Ol-2 individuals is associated to increased powdery mildew resistance. In Chapter 4, we first illustrate a chemical mutagenesis program allowing the identification of a pea line showing recessive resistance towards the powdery mildew fungus Erysiphe pisi. Histological and genetic analyses revealed that the mutated gene is allelic to er1, commonly used in pea breeding for the development of resistant cultivars. As defense mechanisms associated to er1 resistance are reminiscent of mlo immunity, we sequenced the pea MLO homolog PsMLO1 and found a loss-of-function point mutation characterizing the resistant line. A polymorphic CAPS marker was developed on the mutation site and found to be fully co-segregating with resistance in a large F2population. Finally, PsMLO1 sequencing in three er1 resistant cultivars also resulted in the identification of aberrant alleles, further substantiating the identification of another case of mlo-based immunity. In Chapter 5, we report the identification (in vitro and in silico) of a series of MLO homolog sequences in five cultivated Solanaceae species affected by the powdery mildew disease. Comparative analyses using a dataset of several dicot MLO proteins allowed the identification of candidate isoforms for disease susceptibility and the detection of cluster-specific transmembrane amino acid motifs. In Chapter 6, we look at disease resistance as a condition due to the lack of susceptibility genes like MLO. We review several susceptibility genes isolated in crop species and in Arabidopsis, with respect to their molecular characterization, their role in plant-pathogen interactions and the resistant phenotype deriving from their loss-of-function mutations/silencing. A breeding strategy based on the lack of plant susceptibility genes is discussed.

Chris Kik van het Centrum voor Genetische Bronnen Nederland (CGN) keerde afgelopen weekend terug van een eenmansexpeditie door Azerbeidzjan, Georgië en Armenië. Resultaat van de reis: een koffer volgepropt met 53 witte linnen zakjes zaad van wilde en lokaal geteelde spinazie. Dat materiaal gaat dienen als vers bloed voor de veredelingsbedrijven, maar, zo benadrukt Kik, het verzamelen ervan is ook belangrijk voor behoud van de biodiversteit.

Tomato, Solanum lycopersicum, is a host for Oidium neolycopersici, the cause of powdery mildew (PM). Though cultivated tomatoes are susceptible to PM, resistance is reported in wild Solanum species. By screening wild tomato species, nine loci conferring resistance to PM have been identified, namely Ol-1, ol-2, Ol-3, Ol-4, Ol-5, Ol-6, Ol-qtl1, Ol-qtl2, andOl-qtl3. These genes are located on different chromosomes and mediate different levels of resistance by different mechanisms. In this thesis we mainly focused on the Ol genes located on chromosome 6 (Ol-1, Ol-4, Ol-5 and Ol-6) with the aim to fine-map and eventually clone these genes. In addition, we studied the contribution of different phytohormone pathways to the resistance mediated by Ol-1, ol-2, Ol-4 and Ol-qtls. We first focused on the Ol genes on the short arm of tomato chromosome 6, Ol-4 originating from S. peruvianum LA2172 and Ol-6 with unknown origin (Chapter 2). We showed that Ol-4 and Ol-6 are homologues of the Mi-1 gene. On the short arm of tomato chromosome 6, the Mi-1gene cluster is about 400 Kb in size and consists of several other genes besides the Mi-1 homologues. There are transport inhibitor responses-like (TIR-like) genes embedded in this cluster. Interestingly, the copy number of these TIR-like genes in the nematode-resistant tomatoes is less than that in nematode-susceptible ones (Chapter 3). Furthermore, lower expression of these TIR-like genes was observed in roots, but not in leaves, of nematode-resistant plants compared to nematode-susceptible plants. These observations prompted us to suggest and to discuss two different scenarios explaining how TIR-like genes could play a role in the plant response to root-knot nematodes. Then, we studied the Ol-1 and Ol-5, which are located on the long arm of chromosome 6, and originated from different S. habrochaites accessions. Ol-1 is closely linked to Ol-5. With fine-mapping, we narrowed down this locus to a 73 Kb interval which contains at least 10 putative genes. Interestingly we observed an interaction between chromosome regions harboring Ol-1 and Ol-5, indicating that the interaction between Ol-1 and Ol-5 is needed to confer PM resistance. Both Ol-1 and Ol-5 trigger delayed cell death that is distinguishable from hypersensitive response (HR), the hallmark of R gene response to biotrophic pathogens. The delayed cell death associated with Ol-1 and Ol-5 resembles the autophagic PCD. We observed that Ol-1 and Ol-5 were both required for on-time and effective cell death to stop PM. If one of these two genes was not present, cell deathcould not happen or not be effective enough to stop pathogen growth. Finally, we investigated the involvement of phytohormone pathways in PM resistance conferred by the Olgenes, including Ol-1, ol-2, Ol-4 and Ol-qtls (Chapter 5). There is overwhelming evidence implicating plant hormones in plant responses to pathogens. In this experiment we, in addition to Ol-1 and Ol-4, included other resistance loci for PM resistance in tomato. The first one is ol-2, a homologue of the barley mlo gene and derived from S. lycopersicum var cerasiforme LA1230. This gene confers resistance to PM by triggering callose deposition and, thereby, cell wall fortification. The other is Ol-qtls, a combination of three QTLs for PM resistance associated with both delayed cell deathand callose deposition. NILs carrying Ol-1, ol-2, Ol-4 and Ol-qtls, plus the background of these NILs (S. lycopersicum cv Moneymaker, MM), provided us the possibility to compare the involvement of hormonal pathways in different kinds of tomato responses. These responses include basal defense, cell wall fortification, delayed cell death,and HR. We quantified the expression of marker genes for the pathways of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and ethylene (ET) over a time-course after inoculation with PM. As a complementary approach, we crossed our NILs with tomato mutants for JA, ET and ABA. Our results suggested that Ol-4-mediated resistance probably relies on the SA pathway. Ol-1 and Ol-qtls require ET to promote the delayed cell deathfor PM resistance. JA deficiency can compromise resistance mediated by ol-2. Our results also suggested that ABA is required for those interactions demanding callose deposition, resistance associated with ol-2 and Ol-qtls. These results present a nice example of the involvement of different phytohormones in different phases of resistance against PM in tomato. Altogether, this thesis describes different tomato resistance mechanisms triggered by different resistance genes in the same pathosystem, underscoring the plant ability to adopt diverse molecular mechanisms to defense itself against intruders.

The powdery mildew disease, caused by obligate biotrophic fungi belonging to the Ascomycete order of Erysiphales, is common among higher plants and represents one of the most important threats for the cultivation of many crop species. Although powdery mildew resistance is usually a dominant trait, recessively inherited resistance has been reported to occur in Arabidopsis, barley, tomato and pea. In Chapter 1 of this thesis, we provide a state of the art on the understanding of mechanisms underlying plant immunity and review monogenic dominant and recessive sources used in breeding in order to develop resistant cultivars. In Chapter 2 and 3, we describe a successful homology-based cloning approach for the isolation of the recessive ol-2 gene, naturally occurring in a tomato accession collected in Ecuador and conferring broad-spectrum resistance to the powdery mildew fungus Oidium neolycopersici. We realized that ol-2 resistance shares striking similarities with well-known barley and Arabidopsis mlo powdery mildew resistance, originating from loss-of-function mutations of genes encoding for specific seven transmembrane domains MLO isoforms. The following chain of evidence was provided demonstrating that ol-2 resistance is due to the loss of the tomato MLO homolog SlMLO1: a) Ol-2 and SlMLO1 loci share the same genetic and cytogenetic position; b) resistant ol-2/ol-2 lines are homozygous for a loss-of-function deletion in the sequence of SlMLO1; c) a molecular marker developed on the mutation site co-segregates with resistant individuals in an F2population; d) SlMLO1 transgenic expression in ol-2/ol-2 individuals results in disease susceptibility; e) SlMLO1 virus-induced silencing in Ol-2/Ol-2 individuals is associated to increased powdery mildew resistance. In Chapter 4, we first illustrate a chemical mutagenesis program allowing the identification of a pea line showing recessive resistance towards the powdery mildew fungus Erysiphe pisi. Histological and genetic analyses revealed that the mutated gene is allelic to er1, commonly used in pea breeding for the development of resistant cultivars. As defense mechanisms associated to er1 resistance are reminiscent of mlo immunity, we sequenced the pea MLO homolog PsMLO1 and found a loss-of-function point mutation characterizing the resistant line. A polymorphic CAPS marker was developed on the mutation site and found to be fully co-segregating with resistance in a large F2population. Finally, PsMLO1 sequencing in three er1 resistant cultivars also resulted in the identification of aberrant alleles, further substantiating the identification of another case of mlo-based immunity. In Chapter 5, we report the identification (in vitro and in silico) of a series of MLO homolog sequences in five cultivated Solanaceae species affected by the powdery mildew disease. Comparative analyses using a dataset of several dicot MLO proteins allowed the identification of candidate isoforms for disease susceptibility and the detection of cluster-specific transmembrane amino acid motifs. In Chapter 6, we look at disease resistance as a condition due to the lack of susceptibility genes like MLO. We review several susceptibility genes isolated in crop species and in Arabidopsis, with respect to their molecular characterization, their role in plant-pathogen interactions and the resistant phenotype deriving from their loss-of-function mutations/silencing. A breeding strategy based on the lack of plant susceptibility genes is discussed.

This thesis aims to fill the gap of information on the potato landrace diversity present in farmer fields of Ecuador. Passport data from previous collections (1970’s and 1980’s) were used to identify Carchi, Chimborazo and Loja as representative areas of potato diversity. The status of on-farm conservation in these three selected areas is covered in Chapter 2. Microsatellites (SSRs) helped us to describe the genetic relationships among the landraces found in these areas (Chapter 3). The characterization of potato landraces with respect to late blight resistance (Chapter 4) and quality traits (Chapter 5) complement the description. Previous reports suggested loss of potato diversity (genetic erosion) in Ecuadorian farmer fields, but our collection of a total of 174 landraces showed that these areas still hold a substantial amount of potato landrace diversity (Chapter 2). More potato landraces were found in Chimborazo and Loja than previously sampled in the 70’s and 80’s. A comparison between the two collections, in each of the three areas, indicated only a small overlap in landrace names suggesting that the sampling of local landraces was far from exhaustive, both during the 70’s and 80’s and during the present collection trips. This is further supported by the fact that the diversity fair, which was organized after our collection trips in Chimborazo, resulted in many new landraces. Surveys and farmer meetings in the study areas were used to describe the landrace-holders and the characteristics of the farming system they use. Mostly elderly people and small-scale farmers are currently maintaining potato landraces. These farmers look for income alternatives besides agriculture, resulting in migration. The vulnerability of the potato conservation varies among our study areas. In Carchi younger farmers demonstrate a lack of interest in cropping potato landraces. In Loja farming is not seen as the only sustainable source of income and there is a perceived lack of support from the government for the activities necessary to maintain local landraces. In Chimborazo farmers are culturally more attached to their land and see agriculture as a family activity, rendering the potato landrace conservation less vulnerable. Externally driven on-farm conservation interventions, such as diversity fairs or re-introduction of landraces, were highly appreciated by the farmers and could help to conserve the potatoes. Diploid, triploid and tetraploid potato landraces are found in farmers fields. The material sampled at the three areas shows a high allelic diversity. At the tetraploid level (the most abundant) this was comparable to the variation present in an European collection of more than 800 varieties. More alleles are expected to be found when more material from other areas will be screened. There was no clear grouping of material collected according to study region, suggesting extensive movement of seed potatoes all over Ecuador. A comparison of the application of variety names with the genetic relationships among potato landraces can result in either under- or over-estimation of the variability present in farmer fields (Chapter 3). In a number of cases landraces with identical common names proved to be genetically different or individual collection samples were actually a mixture of two landraces, pointing at under-estimation of diversity present. On the other hand, cases that might lead to over-estimation were also evident, e.g. genetically identical material was present under different names. Our sampling of genetically different landraces for late blight (LB) resistance characterization (Chapter 4) confirmed that there was some variation for this trait among the landraces. Most of the landraces were susceptible to moderately resistant, but also some landraces with field resistance were identified. The observed field resistance was comparable to that in the widespread improved variety Fripapa. Possible strategies to improve late blight resistance in potato in Ecuador could include the identification of accessions with resistance among the local landraces, although only a few accessions may be expected to present field resistance. The introduction of new sources of resistance from other origins is a more viable alternative. One could attempt to introduce novel R-genes in material that already contains some level of quantitative resistance. We found varying levels of dry matter, total polyphenol and total carotenoid contents among Ecuadorian potato landraces, some were comparable to the improved varieties. Based on the dry matter content most of the Ecuadorian landraces evaluated were suitable for processing as French fries or chips. The total polyphenol content of these potatoes were quite similar to those reported by the International Potato Center (Peru) for a set of accessions representing more than 60% of the variability in their potato collection. The total carotenoid content values of the Ecuadorian potatoes included in our study were similar or lower compared to previous studies on improved or Andean potatoes. The identified outstanding potato materials could be used to develop new potato varieties through plant breeding. In Chimborazo and Loja farmers select landraces mainly based on their nutritional characteristics. However, in Carchi farmers prefer commercial improved varieties. Farmers´ preferences include empirical valuation of potato-quality rather than specific knowledge on nutritional characteristics of these potatoes. This thesis provides important knowledge about the potato landraces in Ecuador. Our results can serve as the basis for further description and use of Ecuadorian native potatoes by breeders and local communities.

Many plant species can hybridise and produce fertile offspring. Hybridization between cultivated species and their wild relatives has raised concerns with regard to GM crops, as it constitutes a possible route along which the transgene could disperse from crops into related wild species, establish itself in the natural population, and persist under natural conditions. This may cause unintended ecological consequences such as the formation of more invasive weeds and genetic erosion. After crop-wild hybridization, the persistence of the hybrids and of the crop genes (including the transgenes) in later generations depends on their genetic make-up, which consists of specific combinations of wild and crop genomic segments, and on the environmental conditions. Therefore, knowledge on the dynamics of crop-wild hybridization and introgression using conventional crop varieties is needed as it constitutes the baseline for putting into perspective the effects of transgene introgression under natural conditions. This study focused on understanding the genetic process of hybridization and introgression from cultivated to wild relative species using cultivated lettuce (Lactuca sativa L.) and its closest wild relative, prickly lettuce (L. serriola L.) as a crop-wild complex model. Natural populations of prickly lettuce in Europe were tested for the occurrence of crop-wild hybrids using Bayesian-based programmes (Structure, InStruct and NewHybrids) which returned a crop-wild hybrid occurrence of 7%. However, the geographical location of the hybrids led to the conclusion that crop-wild hybridization is not the cause of the recent northward spread of L. serriola in Europe. To test for the importance of the crop genomic segments to the performance of lettuce crop-wild hybrids, three hybrid generations were created (F2 as a selfing generation, and BC1 and BC2, backcrossed to the wild genotype to mimick the introgression process) by crossing L. sativa (cv. Dynamite) with L. serriola collected from Eys (the Netherlands). The three populations were genotyped with Single Nucleotide Polymorphism markers for genetic analysis. Because of the importance of the abiotic stresses as selection factors under natural conditions and the prospective of generating GM crop varieties with enhanced abiotic stress tolerance, the three populations were evaluated for vigour at the rosette stage under greenhouse conditions of non-stress, drought, salinity and nutrient deficiency. The BC1 population was also evaluated under field conditions at two locations (Wageningen and Sijbekarspel, the Netherlands) for hybrid germination, vigour, survival and reproduction. Based on the location and allelic effect of the QTLs for germination, vigour, survival and reproductive traits in the current lettuce crop-wild cross, genomic regions were suggested where transgenes could be or could not be located in order to mitigate their persistence in crop-wild hybrids through genetic hitchhiking and background selection.

Resistentie tegen Botrytis cinerea is gevonden in wilde verwanten van tomaat en deze resistentie was meestal kwantitatief. Het doel van dit promotieonderzoek was om kwantitatieve loci (QTLs) te identificeren die bijdragen aan resistentie tegen B. cinerea. Met behulp van DNA-merkertechnologie en een populatie van introgressie-lijnen zijn tien QTLs geïdentificeerd. Geen van de afzonderlijke QTLs resulteerde in een niveau van resistentie dat overeen kwam de resistente ouder Solanum habrochaites LYC4. Dit betekent dat QTLs gecombineerd zullen moeten worden om Botrytis cinerea-resistente tomaten te verkrijgen. Dankzij de ontwikkelde DNA-merkers kunnen de geïdentificeerde chromosoomfragmenten met resistentiegenen nu gericht ingekruist worden.

Potato (Solanum tuberosum L.) is nowadays the most important non-cereal food crop in the world. It is prone to huge annual losses due to late blight, the disease caused by the oomycete pathogen Phytophthora infestans. Modern management of late blight necessitates the use of multiple resistance (R) genes, which requires efficient pipelines for identification, isolation and characterization of R genes. This thesis employs effectoromics, i.e. the use of effectors (pathogenic secreted protein) to probe corresponding R gene(s) in a host plant and sort out their functional redundancy and specificity. Using cytoplasmic RXLR effectors of P. infestans to probe resistant Solanum germplasm for late blight R genes, we were able to: (i) assess the biodiversity of Avr-blb1, characterize the genomic structure of virulent P. infestans isolates on Rpi-blb1 plants and thus provide a technical solution for long-term disease management; (ii) identify the centre of origin of R3a, characterize R3a gene homologues and a functional R gene (Rpi-sto2), and (iii) uncover the potential co-evolution at both R and Avr side for the R2/PiAvr2-PexRD11 interactions, providing more diversity and specificity of R2 homologues, which may be valuable for potato breeding

Among the different abiotic and biotic stresses, Botrytis cinerea, Phytophthora infestans and high salt concentrations are world-wide the most destructive. Several wild relatives of tomato were identified as source for tolerance to these stresses. Three introgression line (IL) populations derived from S. habrochaites LA1777, S. pennellii LA716 and S. lycopersicoides LA2951 were employed to identify quantitative trait loci (QTL). For B. cinerea resistance twenty four QTLs were identified in S. habrochaites LA1777 and S. lycopersicoides LA2951. These QTLs resulted in reduced lesion size (LS) and disease incidence (DI) in leaves, stem or fruits. Five QTLs were found in S. habrochaites LA1777 for reduced LS in the interaction between tomato and P. infestans. For salt tolerance in the seedling stage ten QTLs were identified in S. pennellii LA716 and five in S. lycopersicoides LA2951. Some QTLs were semi-dominant with a non-additive or even epistatic effect. Many QTLs co-localized indicating that cross talk between coordinating pathways for abiotic and biotic stress might exist. The results provide the basis to combine QTLs with tolerance to abiotic and biotic stresses and for further narrowing down the size of the introgressions. The introgressions from these wild relatives which are involved in tolerance to multiple stresses are of interest for tomato breeders.

The potato late blight disease, caused by the oomycete Phytophthora infestans, is a major threat for potato production worldwide. To breed potato varieties with durable resistance against P. infestans, it is necessary to combine two or more resistance (R) genes. Single R genes are easily overcome by the rapidly evolving pathogen, whereas the presence of several R genes could probably prevent gain of virulence from a single mutation in the pathogen. The large gene pool available within wild potato species offers sufficient possibilities to identify new and diverse R genes conferring resistance to P. infestans (Rpi). Map-based cloning is the most suitable strategy to isolate such new Rpi genes. The objective of this research was mapping, if possible, followed by cloning of Rpi genes from wild Solanum species. Resistance to P. infestans occurring in four different wild Solanum species were mapped in a major R gene cluster on chromosome 11. Natural stacking of three R genes located on different chromosomes was identified in a natural hybrid. In addition, we initiated studies on another type of defense system that is not based on the typical R genes, namely the response of Solanum to INF1 elicitin.

Among the different abiotic and biotic stresses, Botrytis cinerea, Phytophthora infestans and high salt concentrations are world-wide the most destructive. Several wild relatives of tomato were identified as source for tolerance to these stresses. Three introgression line (IL) populations derived from S. habrochaites LA1777, S. pennellii LA716 and S. lycopersicoides LA2951 were employed to identify quantitative trait loci (QTL). For B. cinerea resistance twenty four QTLs were identified in S. habrochaites LA1777 and S. lycopersicoides LA2951. These QTLs resulted in reduced lesion size (LS) and disease incidence (DI) in leaves, stem or fruits. Five QTLs were found in S. habrochaites LA1777 for reduced LS in the interaction between tomato and P. infestans. For salt tolerance in the seedling stage ten QTLs were identified in S. pennellii LA716 and five in S. lycopersicoides LA2951. Some QTLs were semi-dominant with a non-additive or even epistatic effect. Many QTLs co-localized indicating that cross talk between coordinating pathways for abiotic and biotic stress might exist. The results provide the basis to combine QTLs with tolerance to abiotic and biotic stresses and for further narrowing down the size of the introgressions. The introgressions from these wild relatives which are involved in tolerance to multiple stresses are of interest for tomato breeders.

The potato late blight disease, caused by the oomycete Phytophthora infestans, is a major threat for potato production worldwide. To breed potato varieties with durable resistance against P. infestans, it is necessary to combine two or more resistance (R) genes. Single R genes are easily overcome by the rapidly evolving pathogen, whereas the presence of several R genes could probably prevent gain of virulence from a single mutation in the pathogen. The large gene pool available within wild potato species offers sufficient possibilities to identify new and diverse R genes conferring resistance to P. infestans (Rpi). Map-based cloning is the most suitable strategy to isolate such new Rpi genes. The objective of this research was mapping, if possible, followed by cloning of Rpi genes from wild Solanum species. Resistance to P. infestans occurring in four different wild Solanum species were mapped in a major R gene cluster on chromosome 11. Natural stacking of three R genes located on different chromosomes was identified in a natural hybrid. In addition, we initiated studies on another type of defense system that is not based on the typical R genes, namely the response of Solanum to INF1 elicitin.

Potato (Solanum tuberosum L.) is nowadays the most important non-cereal food crop in the world. It is prone to huge annual losses due to late blight, the disease caused by the oomycete pathogen Phytophthora infestans. Modern management of late blight necessitates the use of multiple resistance (R) genes, which requires efficient pipelines for identification, isolation and characterization of R genes. This thesis employs effectoromics, i.e. the use of effectors (pathogenic secreted protein) to probe corresponding R gene(s) in a host plant and sort out their functional redundancy and specificity. Using cytoplasmic RXLR effectors of P. infestans to probe resistant Solanum germplasm for late blight R genes, we were able to: (i) assess the biodiversity of Avr-blb1, characterize the genomic structure of virulent P. infestans isolates on Rpi-blb1 plants and thus provide a technical solution for long-term disease management; (ii) identify the centre of origin of R3a, characterize R3a gene homologues and a functional R gene (Rpi-sto2), and (iii) uncover the potential co-evolution at both R and Avr side for the R2/PiAvr2-PexRD11 interactions, providing more diversity and specificity of R2 homologues, which may be valuable for potato breeding