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Genetisch gemodificeerde gewassen (GM-gewassen) worden in Europa moeilijk geaccepteerd. De geschiedenis van de weerstand laat zien dat deze bij een klein deel van de bezwaarden te maken heeft met het modificatieproces zelf. Het overgrote deel keert zich veeleer tegen het gebruik van landbouwkundig nuttige transgenen uit virussen en bacteria, en van transgene selectiegenen die onder meer resistentie tegen een antibioticum of een herbicide geven. Al deze genen worden transgenen genoemd omdat ze uit organismen komen die niet kruisbaar zijn met de doelplant. In de beginperiode van de transgenese waren alleen deze transgenen beschikbaar. Inmiddels kan de, ook maatschappelijk belangrijke, versnelling van het klassieke plantenveredelingsproces eveneens worden gerealiseerd met cisgenen en intragenen. De huiver die nog in het publieke en politieke debat doorklinkt, krijgt daardoor een steeds meer irrationeel karakter. Een meer adequate EU-regelgeving wordt daardoor belemmerd

Cladosporium fulvum is a semi-biotrophic pathogen, which causes leaf mold of tomato (Lycopersicon spp.). In our laboratory this pathosystem serves as a model to study gene-for-gene interactions between plants and pathogenic fungi (Joosten & De Wit 1999). Many avirulence (Avr) genes and matching resistance (Cf) genes have been cloned and we are now beginning to understand how their products can induce an array of plant defense responses, including the classic hypersensitive response (HR). Here, we will discuss the latest results of our molecular studies on this interaction. These include the isolation of: (i) two new Avr genes, Avr2 and Avr4E, (ii) the determination of the specificity determinants within the Cf-4 and Cf-9 genes by artificial domain swaps and introduction of point mutations, (iii) the analysis of polymorphism occurring in AVR9-responsive Cf genes occurring in natural populations of L. pimpinellifolium, and finally (iv) the description of an efficient method to identify early HR-related genes.

Plants are chemically defended against insect herbivory in various ways. They produce a broad range of secondary metabolites that may be toxic or deterrent to insects. Specialist insects, however, are often capable of overcoming these defences. The yellow striped flea beetle (Phyllotreta nemorumL.) is a specialist that feeds on crucifers (Brassicaceae) such as Sinapis arvensisand Barbarea vulgaris. In Denmark, two types of Barbarea vulgarisvar. arcuataare distinguished: one with pubescent leaves (P-type) and one with glabrous leaves (G-type). All individuals of P. nemorumcan feed on B. vulgarisP-type. Barbarea vulgarisG-type, on the other hand, is chemically defended against most P. nemorumindividuals during the flea beetle reproductive season. The defence compounds are hypothesized to be saponins, a class of compounds with various biological effects and insecticidal properties. Despite high levels of these saponins during summer, some flea beetles can and do feed on B. vulgaris G-type. The ability of P. nemorumto feed on B. vulgarisG-type is heritable; resistance against the defence of B. vulgarisG-type is controlled by dominant major resistance genes (R-genes). One dominant R-allele of an R-gene is enough to convert a susceptible beetle into a resistant one. Despite knowledge of the inheritance patterns of resistance in the flea beetles, which have been demonstrated to be variable, the underlying mechanism of flea beetle resistance has, so far, remained unclear. This prompted me to investigate, as an initial part of my thesis, the genetic basis of the flea beetle adaptation to the defence of B. vulgarisG-type. The interaction between B. vulgarisand the flea beetle is a unique natural model system to study chemical defences in plants and counter-adaptations in insects. Plant and insect are both polymorphic with respect to the trait involved in resistance and hereby provide an excellent opportunity to study the geographic aspects of the evolution of the resistance trait in both interacting species. In this thesis, I focus on the resistance of the flea beetle, and take the presence of different genotypes of the plant as a given. Phyllotreta nemorumis a major pest, for example in oil seed rape. Understanding how resistance evolves in P. nemorumwill not only benefit flea beetle control, but also control of other pest insects. Understanding insect resistance includes knowledge of seasonal, geographic and genetic variation in both plant defense and herbivore adaptation.The R-gene has a remarkable distribution. Flea beetle populations living on B. vulgarisG-type consist solely of resistant individuals, but on host plant patches nearby B. vulgarisG-type lower frequencies of resistant beetles are found than one would expect with the amount of gene flow found at the neutral level between these subpopulations. The aim of this thesis was to find the gene that is held responsible for the resistance of P. nemorumto the defences of B. vulgaris, investigate the distribution of this resistance trait and explain the distribution of this trait in natural populations. The following questions were addressed: (1) what is the genetic basis of the adaptation under study? (2) how is the resistance distributed across flea beetle populations in Denmark? and (3) which factors underlie this distribution? In order to answer these questions, I used an integrated approach. I have combined a candidate gene approach (CHAPTER3) with an empirical approach via the study of variation in resistance in flea beetle populations (CHAPTER4), and a population genomics approach by using molecular markers to gain insight in the genomic make-up of the population and its connection with the resistance trait (CHAPTERS5 and 6). The population genomics approach is a recent advance in methods to detect the involvement of selection in the distribution of alleles at presumably adaptive loci. Using this approach one can distinguish locus-specific effects, like directional selection, from genome-wide effects, on the distribution of alleles at loci of interest. The population genomics approach is introduced in CHAPTER2 together with the Geographic Mosaic Theory of Coevolution. I illustrate how processes underlying this theory of coevolution can be investigated with the population genomics approach. According to the geographic mosaic theory of coevolution, reciprocal selection between interacting species only happens in so-called hot-spots. Hot spots can be identified using population genomics and genetic variation found at specific loci can be attributed to locus-specific processes such as directional selection. For the B. vulgaris- flea beetle system this means that with a population genomics approach we can examine whether the distribution of resistant flea beetles on alternative host plants is only influenced by migration, or also by selection (CHAPTER5). Another valuable utility of the population genomics approach is to investigate whether a candidate gene for the R-gene is under selection, by looking whether a candidate gene is experiencing locus-specific effects beside genome-wide effects when comparing flea beetle populations living on B. vulgarisG-type with populations living on alternative host plants (CHAPTER6). However, before using a population genomics approach to compare the resistance trait or a candidate gene with parts of the genome that only experience genome-wide effects, I have tried to identify the genetic basis of the flea beetle adaptation to the defence ofB. vulgarisG-type. In CHAPTER3, I have addressed this question by using a candidate gene approach to examine the involvement of a possible detoxifying enzyme in P. nemorum. Genes coding for β-glucosidase were a candidate for genes underlying the difference between resistant and susceptible beetles, because β-glucosidase is used as detoxifying enzyme by other organisms resistant to saponin defence. Three different β-glucosidase cDNA sequences were cloned from Danish flea beetle lines. We named them β-glucosidase A, B and C. β-glucosidase C was only found in resistant lines and not in the susceptible line. We then tested if recombinant β-glucosidase C breaks down the most abundant and most effective defence compound in B. vulgarisG-type, hederagenin cellobioside. β-glucosidase C was able to deglycosylate one glucose unit of hederagenin cellobioside, when expressed in an insect cell line. This suggests that expressed β-glucosidase C can deglycosylate antifeedant saponins and may play a role in the resistant flea beetle’s ability to overcome the defence of B. vulgaris. Next, a segregating family was created in which offspring differed in resistance genotype. Again β-glucosidase cDNA sequences were cloned to find a difference in the presence of these β-glucosidases between resistant and susceptible individuals. This time cDNA sequences of β-glucosidases A, B and C were present in both resistant and susceptible individuals although significantly fewer β-glucosidase C cDNA sequence variants were found in susceptible individuals than in resistant individuals. Thus, the genetic basis of flea beetle resistance remains unclear. Further investigation is needed to explore if the β-glucosidase C protein is also capable of inactivating hederagenin cellobioside by hydrolysizing the second glucose unit from the saponin and if there is a difference in enzyme activity of β-glucosidase C between resistant and susceptible beetles. Subsequently, in CHAPTER4 I have investigated whether the frequency of resistant beetles decreased in populations living on other host plant patches than B. vulgarisG-type and whether the change in frequency was significant within the flea beetle season. I found that the frequency of resistant beetles varied significantly among years, but there was no evidence for a decrease in the frequency of resistant beetles, the latter being expected if selection acts against the resistance on other host plants than B. vulgarisG-type. Furthermore, I found that the frequency of resistant beetles varied significantly within a flea beetle season. This study demonstrates that relative frequencies of different resistance phenotypes of P. nemorumon other host plants than B. vulgarisG-type are highly dynamic, both within and across years. It is, therefore, important to sample season-wide when one wants to monitor the changes in frequencies of insect resistance in natural systems. In CHAPTERS5 and 6 I took a population genomics approach to investigate if the observed geographicaldistribution of resistance of P. nemorumto chemically defendedB. vulgarisin flea beetle populations could be explained by factors that are solely associated with genome-wide effects, such as migration, or also by locus-specific factors like selection at the resistance locus. First, neutral microsatellites were used to reveal the genetic differentiation at parts of the genome that are only influenced by genome-wide processes. Next, the level of neutral genetic differentiation was compared with the genetic differentiation found for the resistance trait. The resistance trait was an outlier in pairwise comparisons between flea beetle populations on B. vulgarisand S. arvensis, meaning that the level of genetic differentiation was significantly higher than expected if the resistance trait experiences only genome-wide effects. The resistance trait was also an outlier in the pairwise comparison between populations on S. arvensis, which suggests that the resistance trait is also under directional selection on other host plants than B. vulgarisG-type. Additionally, I examined in CHAPTER6 if the homologous β-glucosidases B and C sequences found in CHAPTER3 correspond to two alleles of the major resistance gene, because of their similarity and their presence in flea beetle lines. The sequence of β-glucosidases C had so far only been found in resistant individuals, so we hypothesized it to be the dominant resistance allele and the sequence of β-glucosidases B would then correspond to the susceptible allele. In order to find out if this hypothesized PneR-gene (Phyllotreta nemorum R-gene) is the resistance gene, we first directly compared resistance phenotypes of beetles collected from populations on B. vulgarisG-type and S. arvensiswith genotypes derived with primers developed for β-glucosidase B and C. The phenotype of the flea beetles did not match the genotype derived with the β-glucosidase primers. Additionally, the frequency of heterozygotes and homozygotes of the PneR-gene genotype was not significantly deviating from Hardy-Weinberg Equilibrium which implies that there are no locus-specific effects involved when both sequences are seen as one gene with two alleles. A population approach was taken like in CHAPTER5, this time including the genetic differentiation estimated for the candidate gene as well. The candidate gene behaved similar to the neutral loci while the resistance trait was an outlier in most pairwise comparisons between flea beetle populations. If both sequences are alleles of the same gene, then the candidate gene is not directly responsible for the flea beetle resistance to B. vulgarisG-type defence. The results presented in this thesis show the complexity of genetic processes (either genome-wide or locus specific) affecting local adaptation and the distribution of a resistance trait in insects in natural populations. Furthermore, the present study shows that when studying coevolution between insect and host plant by means of adaptive traits, also geographical and seasonal variation in allele frequencies should be considered. A multidisciplinary approach to study adaptation in plant-insect interactions such as used in this thesis, will benefit research on plant-insect interactions, including applied research such as studying the potential of host plants as dead-end traps for pest insects and preventing/diminishing the development of resistance by pest insects to crop defences.

Plants are chemically defended against insect herbivory in various ways. They produce a broad range of secondary metabolites that may be toxic or deterrent to insects. Specialist insects, however, are often capable of overcoming these defences. The yellow striped flea beetle (Phyllotreta nemorumL.) is a specialist that feeds on crucifers (Brassicaceae) such as Sinapis arvensisand Barbarea vulgaris. In Denmark, two types of Barbarea vulgarisvar. arcuataare distinguished: one with pubescent leaves (P-type) and one with glabrous leaves (G-type). All individuals of P. nemorumcan feed on B. vulgarisP-type. Barbarea vulgarisG-type, on the other hand, is chemically defended against most P. nemorumindividuals during the flea beetle reproductive season. The defence compounds are hypothesized to be saponins, a class of compounds with various biological effects and insecticidal properties. Despite high levels of these saponins during summer, some flea beetles can and do feed on B. vulgaris G-type. The ability of P. nemorumto feed on B. vulgarisG-type is heritable; resistance against the defence of B. vulgarisG-type is controlled by dominant major resistance genes (R-genes). One dominant R-allele of an R-gene is enough to convert a susceptible beetle into a resistant one. Despite knowledge of the inheritance patterns of resistance in the flea beetles, which have been demonstrated to be variable, the underlying mechanism of flea beetle resistance has, so far, remained unclear. This prompted me to investigate, as an initial part of my thesis, the genetic basis of the flea beetle adaptation to the defence of B. vulgarisG-type. The interaction between B. vulgarisand the flea beetle is a unique natural model system to study chemical defences in plants and counter-adaptations in insects. Plant and insect are both polymorphic with respect to the trait involved in resistance and hereby provide an excellent opportunity to study the geographic aspects of the evolution of the resistance trait in both interacting species. In this thesis, I focus on the resistance of the flea beetle, and take the presence of different genotypes of the plant as a given. Phyllotreta nemorumis a major pest, for example in oil seed rape. Understanding how resistance evolves in P. nemorumwill not only benefit flea beetle control, but also control of other pest insects. Understanding insect resistance includes knowledge of seasonal, geographic and genetic variation in both plant defense and herbivore adaptation.The R-gene has a remarkable distribution. Flea beetle populations living on B. vulgarisG-type consist solely of resistant individuals, but on host plant patches nearby B. vulgarisG-type lower frequencies of resistant beetles are found than one would expect with the amount of gene flow found at the neutral level between these subpopulations. The aim of this thesis was to find the gene that is held responsible for the resistance of P. nemorumto the defences of B. vulgaris, investigate the distribution of this resistance trait and explain the distribution of this trait in natural populations. The following questions were addressed: (1) what is the genetic basis of the adaptation under study? (2) how is the resistance distributed across flea beetle populations in Denmark? and (3) which factors underlie this distribution? In order to answer these questions, I used an integrated approach. I have combined a candidate gene approach (CHAPTER3) with an empirical approach via the study of variation in resistance in flea beetle populations (CHAPTER4), and a population genomics approach by using molecular markers to gain insight in the genomic make-up of the population and its connection with the resistance trait (CHAPTERS5 and 6). The population genomics approach is a recent advance in methods to detect the involvement of selection in the distribution of alleles at presumably adaptive loci. Using this approach one can distinguish locus-specific effects, like directional selection, from genome-wide effects, on the distribution of alleles at loci of interest. The population genomics approach is introduced in CHAPTER2 together with the Geographic Mosaic Theory of Coevolution. I illustrate how processes underlying this theory of coevolution can be investigated with the population genomics approach. According to the geographic mosaic theory of coevolution, reciprocal selection between interacting species only happens in so-called hot-spots. Hot spots can be identified using population genomics and genetic variation found at specific loci can be attributed to locus-specific processes such as directional selection. For the B. vulgaris- flea beetle system this means that with a population genomics approach we can examine whether the distribution of resistant flea beetles on alternative host plants is only influenced by migration, or also by selection (CHAPTER5). Another valuable utility of the population genomics approach is to investigate whether a candidate gene for the R-gene is under selection, by looking whether a candidate gene is experiencing locus-specific effects beside genome-wide effects when comparing flea beetle populations living on B. vulgarisG-type with populations living on alternative host plants (CHAPTER6). However, before using a population genomics approach to compare the resistance trait or a candidate gene with parts of the genome that only experience genome-wide effects, I have tried to identify the genetic basis of the flea beetle adaptation to the defence ofB. vulgarisG-type. In CHAPTER3, I have addressed this question by using a candidate gene approach to examine the involvement of a possible detoxifying enzyme in P. nemorum. Genes coding for β-glucosidase were a candidate for genes underlying the difference between resistant and susceptible beetles, because β-glucosidase is used as detoxifying enzyme by other organisms resistant to saponin defence. Three different β-glucosidase cDNA sequences were cloned from Danish flea beetle lines. We named them β-glucosidase A, B and C. β-glucosidase C was only found in resistant lines and not in the susceptible line. We then tested if recombinant β-glucosidase C breaks down the most abundant and most effective defence compound in B. vulgarisG-type, hederagenin cellobioside. β-glucosidase C was able to deglycosylate one glucose unit of hederagenin cellobioside, when expressed in an insect cell line. This suggests that expressed β-glucosidase C can deglycosylate antifeedant saponins and may play a role in the resistant flea beetle’s ability to overcome the defence of B. vulgaris. Next, a segregating family was created in which offspring differed in resistance genotype. Again β-glucosidase cDNA sequences were cloned to find a difference in the presence of these β-glucosidases between resistant and susceptible individuals. This time cDNA sequences of β-glucosidases A, B and C were present in both resistant and susceptible individuals although significantly fewer β-glucosidase C cDNA sequence variants were found in susceptible individuals than in resistant individuals. Thus, the genetic basis of flea beetle resistance remains unclear. Further investigation is needed to explore if the β-glucosidase C protein is also capable of inactivating hederagenin cellobioside by hydrolysizing the second glucose unit from the saponin and if there is a difference in enzyme activity of β-glucosidase C between resistant and susceptible beetles. Subsequently, in CHAPTER4 I have investigated whether the frequency of resistant beetles decreased in populations living on other host plant patches than B. vulgarisG-type and whether the change in frequency was significant within the flea beetle season. I found that the frequency of resistant beetles varied significantly among years, but there was no evidence for a decrease in the frequency of resistant beetles, the latter being expected if selection acts against the resistance on other host plants than B. vulgarisG-type. Furthermore, I found that the frequency of resistant beetles varied significantly within a flea beetle season. This study demonstrates that relative frequencies of different resistance phenotypes of P. nemorumon other host plants than B. vulgarisG-type are highly dynamic, both within and across years. It is, therefore, important to sample season-wide when one wants to monitor the changes in frequencies of insect resistance in natural systems. In CHAPTERS5 and 6 I took a population genomics approach to investigate if the observed geographicaldistribution of resistance of P. nemorumto chemically defendedB. vulgarisin flea beetle populations could be explained by factors that are solely associated with genome-wide effects, such as migration, or also by locus-specific factors like selection at the resistance locus. First, neutral microsatellites were used to reveal the genetic differentiation at parts of the genome that are only influenced by genome-wide processes. Next, the level of neutral genetic differentiation was compared with the genetic differentiation found for the resistance trait. The resistance trait was an outlier in pairwise comparisons between flea beetle populations on B. vulgarisand S. arvensis, meaning that the level of genetic differentiation was significantly higher than expected if the resistance trait experiences only genome-wide effects. The resistance trait was also an outlier in the pairwise comparison between populations on S. arvensis, which suggests that the resistance trait is also under directional selection on other host plants than B. vulgarisG-type. Additionally, I examined in CHAPTER6 if the homologous β-glucosidases B and C sequences found in CHAPTER3 correspond to two alleles of the major resistance gene, because of their similarity and their presence in flea beetle lines. The sequence of β-glucosidases C had so far only been found in resistant individuals, so we hypothesized it to be the dominant resistance allele and the sequence of β-glucosidases B would then correspond to the susceptible allele. In order to find out if this hypothesized PneR-gene (Phyllotreta nemorum R-gene) is the resistance gene, we first directly compared resistance phenotypes of beetles collected from populations on B. vulgarisG-type and S. arvensiswith genotypes derived with primers developed for β-glucosidase B and C. The phenotype of the flea beetles did not match the genotype derived with the β-glucosidase primers. Additionally, the frequency of heterozygotes and homozygotes of the PneR-gene genotype was not significantly deviating from Hardy-Weinberg Equilibrium which implies that there are no locus-specific effects involved when both sequences are seen as one gene with two alleles. A population approach was taken like in CHAPTER5, this time including the genetic differentiation estimated for the candidate gene as well. The candidate gene behaved similar to the neutral loci while the resistance trait was an outlier in most pairwise comparisons between flea beetle populations. If both sequences are alleles of the same gene, then the candidate gene is not directly responsible for the flea beetle resistance to B. vulgarisG-type defence. The results presented in this thesis show the complexity of genetic processes (either genome-wide or locus specific) affecting local adaptation and the distribution of a resistance trait in insects in natural populations. Furthermore, the present study shows that when studying coevolution between insect and host plant by means of adaptive traits, also geographical and seasonal variation in allele frequencies should be considered. A multidisciplinary approach to study adaptation in plant-insect interactions such as used in this thesis, will benefit research on plant-insect interactions, including applied research such as studying the potential of host plants as dead-end traps for pest insects and preventing/diminishing the development of resistance by pest insects to crop defences.

Key words: Capsicum, Bemisia tabaci, trichome density, cuticle thickness Whitefly is economically one of the most threatening pests of pepper worldwide, which is mainly caused by its ability to transmit many different viruses. In this research, we characterized pepper germplasm to identify whitefly-resistant accessions that will form the basis for future resistance breeding. Forty-four pepper accessions representing four species (Capsicum annuum, C. frutescens, C. chinense, C. baccatum) were screened for resistance to whiteflies. Screening parameters were adult survival (AS) and oviposition rate (OR) in a no-choice test and whitefly, egg and nymphal density in free-choice tests. To combine parameters in free-choice tests, a plant resistance value was calculated. The results show that AS and OR were significantly different among accessions and were positively correlated, which was also the case for the parameters in the free-choice tests. Accessions identified as highly resistant in no-choice and free-choice tests generally were C. annuum. Whitefly density and OR correlated positively with trichome density and negatively with cuticle thickness of leaves.

Key words: Capsicum, Bemisia tabaci, trichome density, cuticle thickness Whitefly is economically one of the most threatening pests of pepper worldwide, which is mainly caused by its ability to transmit many different viruses. In this research, we characterized pepper germplasm to identify whitefly-resistant accessions that will form the basis for future resistance breeding. Forty-four pepper accessions representing four species (Capsicum annuum, C. frutescens, C. chinense, C. baccatum) were screened for resistance to whiteflies. Screening parameters were adult survival (AS) and oviposition rate (OR) in a no-choice test and whitefly, egg and nymphal density in free-choice tests. To combine parameters in free-choice tests, a plant resistance value was calculated. The results show that AS and OR were significantly different among accessions and were positively correlated, which was also the case for the parameters in the free-choice tests. Accessions identified as highly resistant in no-choice and free-choice tests generally were C. annuum. Whitefly density and OR correlated positively with trichome density and negatively with cuticle thickness of leaves.

Door het screenen van lijnen en wilde verwanten van tarwe, is een nieuw resistentiegen tegen STB (Septoria tritici blotch) gevonden.

An attempt to balance selection intensity and conservation of genetic diversity was made in the national program for breeding for scrapie resistance in the Netherlands. The ARR allele is associated with resistance to scrapie. In the Netherlands a breeding program was started on a voluntary basis in 2001 and starting 2004 the use of ARR/ARR rams was obligatory. In some rare breeds there were so few ARR/ARR rams that the risk of high inbreeding and associated loss of genetic diversity would be unacceptably high. Based on computer simulations predicting the inbreeding rates for several breeds exemptions on the obligatory breeding program were allowed. In this paper the ARR frequencies and associated inbreeding rates are analyzed as an indication of loss in genetic diversity during the years of scrapie selection in two breeds: the “Zwartbles schaap” (Black-blazed sheep) and the “Melkschaap” (Friesian-Zealand Milksheep).

Door het screenen van lijnen en wilde verwanten van tarwe, is een nieuw resistentiegen tegen STB (Septoria tritici blotch) gevonden.

An attempt to balance selection intensity and conservation of genetic diversity was made in the national program for breeding for scrapie resistance in the Netherlands. The ARR allele is associated with resistance to scrapie. In the Netherlands a breeding program was started on a voluntary basis in 2001 and starting 2004 the use of ARR/ARR rams was obligatory. In some rare breeds there were so few ARR/ARR rams that the risk of high inbreeding and associated loss of genetic diversity would be unacceptably high. Based on computer simulations predicting the inbreeding rates for several breeds exemptions on the obligatory breeding program were allowed. In this paper the ARR frequencies and associated inbreeding rates are analyzed as an indication of loss in genetic diversity during the years of scrapie selection in two breeds: the “Zwartbles schaap” (Black-blazed sheep) and the “Melkschaap” (Friesian-Zealand Milksheep).

Genetisch gemodificeerde gewassen (GM-gewassen) worden in Europa moeilijk geaccepteerd. De geschiedenis van de weerstand laat zien dat deze bij een klein deel van de bezwaarden te maken heeft met het modificatieproces zelf. Het overgrote deel keert zich veeleer tegen het gebruik van landbouwkundig nuttige transgenen uit virussen en bacteria, en van transgene selectiegenen die onder meer resistentie tegen een antibioticum of een herbicide geven. Al deze genen worden transgenen genoemd omdat ze uit organismen komen die niet kruisbaar zijn met de doelplant. In de beginperiode van de transgenese waren alleen deze transgenen beschikbaar. Inmiddels kan de, ook maatschappelijk belangrijke, versnelling van het klassieke plantenveredelingsproces eveneens worden gerealiseerd met cisgenen en intragenen. De huiver die nog in het publieke en politieke debat doorklinkt, krijgt daardoor een steeds meer irrationeel karakter. Een meer adequate EU-regelgeving wordt daardoor belemmerd

Genetisch gemodificeerde gewassen (GM-gewassen) worden in Europa moeilijk geaccepteerd. De geschiedenis van de weerstand laat zien dat deze bij een klein deel van de bezwaarden te maken heeft met het modificatieproces zelf. Het overgrote deel keert zich veeleer tegen het gebruik van landbouwkundig nuttige transgenen uit virussen en bacteria, en van transgene selectiegenen die onder meer resistentie tegen een antibioticum of een herbicide geven. Al deze genen worden transgenen genoemd omdat ze uit organismen komen die niet kruisbaar zijn met de doelplant. In de beginperiode van de transgenese waren alleen deze transgenen beschikbaar. Inmiddels kan de, ook maatschappelijk belangrijke, versnelling van het klassieke plantenveredelingsproces eveneens worden gerealiseerd met cisgenen en intragenen. De huiver die nog in het publieke en politieke debat doorklinkt, krijgt daardoor een steeds meer irrationeel karakter. Een meer adequate EU-regelgeving wordt daardoor belemmerd

Iron is fundamental to the biology of eukaryotic cells since it plays a key role in many metabolic functions. Iron concentrations are tightly regulated, for example by ferritin, because excessive iron leads to tissue damage. Iron cannot cross cellular membranes directly and most cells acquire iron from the iron transporting protein transferrin (Tf), via transferrin receptors. During nutritional immunity the body reacts with a metabolic adjustment in order to render important nutrients unavailable to invading microorganisms. However, pathogens also have evolved a range of mechanisms to acquire iron from the host (chapter 1). In the study described in this thesis, we used a natural host-parasite model of common carp (Cyprinus carpio L.) infected with Trypanoplasma borreli, a protozoan kinetoplastid, extracellular blood parasite of carp to get more insight in the competition for iron between host and parasite. Transferrin of common carp is highly polymorphic with several alleles identified according to differences in electrophoretic mobility. We studied the implications of Tf polymorphisms for iron binding and modulation of immune function. We performed a series of challenge experiments infecting five genetically different, commercially exploited carp lines with T. borreli. Our results indicated that Tf genotype may influence the susceptibility to pathogens. We observed a significant association of the DD genotype of Tf with low parasitaemia in two resistant carp lines (Polish ‘R2’ and ‘K’), but a reverse association in the most susceptible carp line ‘D’ (chapter 2). We also showed that variation in resistance to T. borreli could be controlled by sex-related genetic factors. Examination of parasite growth in vitro, in culture media supplemented with 3% serum taken from fish with different Tf genotypes, showed a faster decrease in number of parasites in media supplemented with serum from DD-typed animals (chapter 2). In general, pathogens also have mechanisms to acquire iron from the host. We developed a method for Tf depletion of carp serum using specific antibodies to carp Tf, and compared T. borreli multiplication and survival in the presence or absence of Tf in vitro. Parasites were dying in medium containing Tf-depleted serum, which clearly showed that Tf is essential for parasite growth and multiplication (chapter 3). We isolated two allelic forms of carp Tf (alleles D and G) to purity using rivanol precipitation and ion-exchange chromatography (chapter 5). We showed that parasite growth in vitro could be reconstituted by the addition of purified Tf to Tf-depleted serum (chapter 3). We observed differences in T. borreli multiplication and survival in culture media containing different sera typed differently for Tf genotypes (chapter 3). We identified four complete coding sequences for common carp Tf alleles C, D, F and G, and confirmed the overall similarity of the carp Tf three-dimensional structure to Tfs of other species. We could show that carp Tf differs significantly in critical iron-binding sites in the N-lobe of the molecule, as compared to other non-cyprinid fish species (chapter 4). The substitution of a majority of the iron-coordinating residues in the Nlobe indeed seems to affect the ability to bind iron, which may be compensated for by higher serum concentrations of Tf (chapter 7). Comparison of constitutive gene expression of two Tf alleles D and G showed a comparably high gene expression level in liver and small but consistent differences in gene expression for allele D over allele G in other immunologically important organs (chapter 4). Our data suggest that the allelic polymorphism is not related to differences in iron binding and/or binding to the host Tf receptor but could be linked with other factors, such as competition for iron with pathogens (chapter 4). Transferrin itself may also exert effects that are not directly linked with maintaining iron levels and Tf cleavage products have been shown to stimulate macrophages to produce large amounts of nitric oxide (NO). To study the induction of NO in carp head kidney-derived macrophages, we isolated two allelic forms of carp Tf (alleles D and G) to purity and showed that the level of activation of macrophages by Tf was different for the D and G allele (chapter 5). Differences in NO levels induced could be related to different cleavage forms of the two alleles D and G, as shown by Western blot, confirming that full-length Tf cannot induce NO. The D-type Tf cleavage products induced significantly higher nitric oxide (NO) production than cleavage products of Gtype Tf. (chapter 5). Transferrin uptake by trypanosome parasites involves Tf binding to a receptor. The TfR-Tf complex then is internalised and transported to lysosomes, where Tf is proteolytically degraded. We described the cloning and sequencing of a cathepsin L-like cysteine proteinase from T. borreli and production of a recombinant and biologically active enzyme (chapter 6). We demonstrated that the T. borreli cysteine proteinase is able to digest host transferrin. Likely, Tf cleavage fragments are released from the trypanosomes while iron would remain parasite-associated, possibly contributing to thepathogenicity of the parasite by inducing high amounts of NO in carp macrophages (chapter 7). Our study dealt with different aspects of Tf polymorphism, discussing the role of Tf in immunity of common carp and the influence of allelic polymorphism on competition for iron between host and pathogen (chapter 7). Further investigations should shed more light on the selective advantage of particular alleles to provide a basis for incorporating Tf as a genetic marker in marker-assisted selection programmes for increased resistance to diseases. This could contribute to improved survival of carp kept under semiintensive farming systems in ponds.

Potato late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is the most disastrous disease on potato worldwide and also the greatest threat to potato production in China. Loss of yield and quality, and the costs of chemical control of potato late blight account for multi-billion US$ annually. Using host resistance is thought to be an economical and efficient approach to control potato late blight. To combat the disease breeders have introduced late blight resistance (R) genes from various Solanum species into the cultivated potato. The proteins encoded by these R genes can recognise specific races of the pathogen. This then triggers a cascade of defence responses ultimately leading to a localized programmed cell death called the hypersensitive response (HR) that arrests growth of the pathogen. However, once the resistant potato cultivars are released into the field, the resistance based on these genes is quickly overcome due to rapid evolution and adaption of P. infestans. In the potato-P. infestans interaction race-specific recognition by R genes is based on the ‘gene-for-gene’ model which predicts that resistance is governed by the (direct or indirect) interaction of an R protein with its corresponding effector, the product of an avirulence (Avr) gene. If either the R gene or the Avr gene is absent or non-functional the interaction is compatible and the host susceptible for disease. Key to a better understanding of the molecular basis of resistance in the potato-P. infestans pathosystem is the unravelling of R protein-effector interactions and, hence, cloning of more R and Avr genes is a prerequisite to study these interactions. This thesis describes the mapping and cloning of Avr genes in P. infestans, and the phenotypic and genotypic diversity in P. infestans field isolates in Northern China. To isolate P. infestans Avr genes a positional cloning strategy was adopted. Chapter 2 presents a molecular-genetic linkage map of P. infestans that was constructed based on Single Nucleotide Polymorphism (SNP) markers and Amplified Fragment Length Polymorphism (AFLP) markers. The map was generated using a mapping population of 83 F1 progeny derived from two Dutch field isolates, NL80029 and NL88133. Of 631 markers (398 SNP and 233 AFLP markers) that segregated in this population, 534 markers were positioned on 19 linkage groups spanning a total of 1144 cM and an average distance of 2.14 cM between adjacent markers. Fourteen of the linkage groups are major linkage groups that contain markers from both parents. The others are minor linkage groups with markers of only one of the two parents. In parallel, a transcriptional profiling strategy was adopted to identify avirulence-associated transcripts (Chapter 3). cDNA-AFLP was used for comparing transcripts in P. infestans isolates with different virulence phenotypes. A large number of avirulence-associated TDFs (Transcript Derived Fragments) was cloned and sequenced, and EST and genome databases were mined to generate more sequence data. To identify promising candidates, bioinformatic predictions such as the presence of signal peptides, number of cysteine residues and putative virulence functions were used as important selection criteria. Four TDFs associated with Avr loci were identified, two for Avr4 and two for the Avr3b-Avr10-Avr11 locus. Chapter 4 describes how a combined approach of genetic mapping, transcriptional profiling and BAC marker landing resulted in isolation of the P. infestans avirulence gene Avr4. PiAvr4 encodes a 287 amino acid protein that belongs to a superfamily of effectors sharing the putative host cell targeting motif RXLR-dEER. For the functional characterization P. infestans race 4 strains were transformed with PiAvr4. This resulted in transformants that were avirulent on R4 potato plants, demonstrating that PiAvr4 is responsible for eliciting R4-mediated resistance. Expression of PiAvr4 in R4 plants using PVX-agroinfection and agroinfiltration showed that PiAvr4 itself is the effector that elicits HR on R4 plants. On potato plants lacking R4, like Bintje, there was no response. The presence of the RXLR-dEER motif suggested intracellular recognition of PiAvr4 but nevertheless a hypersensitive response was observed when PiAvr4 was targeted to the outside of the cell. Deletion of the RXLR-dEER domain neither stimulated nor prevented elicitor activity of PiAvr4. Race 4 strains have frame shift mutations in the PiAvr4 gene that result in short truncated peptides, indicating that PiAvr4 is not crucial for virulence. Chapter 5 describes Avr1-associated markers that resulted from genetic mapping, transcriptional profiling and BAC-end sequences. In silico landing of these markers on the P. infestans genome sequence narrows down a 800 kb genomic interval that carries seven genes that have the hallmarks of an oomycete Avr gene. They all encode a secreted protein with a conserved RXLR-dEER domain at the N-terminus and a divergent C-terminal region. Each of these seven could be a candidate for Avr1. The seven RXLR effector genes were further characterized by bioinformatic analyses such as HMM score of the RXLR motif, and prediction of the presence of W, Y, and L motifs in the C-terminal region. Cloning and functional analyses using transient expression assays in plants carrying the resistance gene R1 should reveal whether any of the seven candidates is Avr1. Chapter 6 describes the phenotypic and genotypic diversity of P. infestans isolates collected in Northern China between 1997 and 2003, especially in Inner Mongolia. Characterization included mating type, virulence, mitochondrial DNA (mtDNA) haplotype and DNA fingerprinting patterns based on simple sequence repeats (SSR) and amplified fragment length polymorphism (AFLP). All isolates had the A1 mating type, mtDNA haplotype IIa and an identical SSR genotype (designated as SG-01-01) that differed from the SSR genotypes found in the reference isolates, including the ones representing the ‘old’ US-1 lineage that dominated the worldwide P. infestans population prior to 1980. In contrast, the virulence spectra differed significantly and virulence to all R genes present in the standard differential set (R1 to R11) was found. AFLP analysis revealed some diversity; eight different AFLP genotypes were found that could be grouped into two major clusters. This study shows that there is very little genotypic diversity in the P. infestans population in Northern China. The occurrence of many different races within this uniform population is discussed in the framework of recently gained insights in the molecular determinants of avirulence in P. infestans and their role in the ‘gene-for-gene’ interaction with potato. Finally, in Chapter 7, the implications of the findings described in this thesis are discussed with specific emphasis on Avr gene cloning, RXLR-dEER effectors, virulence diversity and durable late blight resistance. By combining various cloning strategies it becomes feasible to speed up the cloning of putative P. infestans Avr genes. Moreover, the use of high throughput effector genomics screenings will allow the identification of the corresponding R genes. The high virulence diversity that is found in P. infestans field isolates, even within one clonal lineage, might be correlated to the observation that RXLR-dEER effector genes are the most rapidly evolving genes in the genome of P. infestans. Therefore, generating potato cultivars with durable resistance to late blight seems more challenging than anticipated.

Potato late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is the most disastrous disease on potato worldwide and also the greatest threat to potato production in China. Loss of yield and quality, and the costs of chemical control of potato late blight account for multi-billion US$ annually. Using host resistance is thought to be an economical and efficient approach to control potato late blight. To combat the disease breeders have introduced late blight resistance (R) genes from various Solanum species into the cultivated potato. The proteins encoded by these R genes can recognise specific races of the pathogen. This then triggers a cascade of defence responses ultimately leading to a localized programmed cell death called the hypersensitive response (HR) that arrests growth of the pathogen. However, once the resistant potato cultivars are released into the field, the resistance based on these genes is quickly overcome due to rapid evolution and adaption of P. infestans. In the potato-P. infestans interaction race-specific recognition by R genes is based on the ‘gene-for-gene’ model which predicts that resistance is governed by the (direct or indirect) interaction of an R protein with its corresponding effector, the product of an avirulence (Avr) gene. If either the R gene or the Avr gene is absent or non-functional the interaction is compatible and the host susceptible for disease. Key to a better understanding of the molecular basis of resistance in the potato-P. infestans pathosystem is the unravelling of R protein-effector interactions and, hence, cloning of more R and Avr genes is a prerequisite to study these interactions. This thesis describes the mapping and cloning of Avr genes in P. infestans, and the phenotypic and genotypic diversity in P. infestans field isolates in Northern China. To isolate P. infestans Avr genes a positional cloning strategy was adopted. Chapter 2 presents a molecular-genetic linkage map of P. infestans that was constructed based on Single Nucleotide Polymorphism (SNP) markers and Amplified Fragment Length Polymorphism (AFLP) markers. The map was generated using a mapping population of 83 F1 progeny derived from two Dutch field isolates, NL80029 and NL88133. Of 631 markers (398 SNP and 233 AFLP markers) that segregated in this population, 534 markers were positioned on 19 linkage groups spanning a total of 1144 cM and an average distance of 2.14 cM between adjacent markers. Fourteen of the linkage groups are major linkage groups that contain markers from both parents. The others are minor linkage groups with markers of only one of the two parents. In parallel, a transcriptional profiling strategy was adopted to identify avirulence-associated transcripts (Chapter 3). cDNA-AFLP was used for comparing transcripts in P. infestans isolates with different virulence phenotypes. A large number of avirulence-associated TDFs (Transcript Derived Fragments) was cloned and sequenced, and EST and genome databases were mined to generate more sequence data. To identify promising candidates, bioinformatic predictions such as the presence of signal peptides, number of cysteine residues and putative virulence functions were used as important selection criteria. Four TDFs associated with Avr loci were identified, two for Avr4 and two for the Avr3b-Avr10-Avr11 locus. Chapter 4 describes how a combined approach of genetic mapping, transcriptional profiling and BAC marker landing resulted in isolation of the P. infestans avirulence gene Avr4. PiAvr4 encodes a 287 amino acid protein that belongs to a superfamily of effectors sharing the putative host cell targeting motif RXLR-dEER. For the functional characterization P. infestans race 4 strains were transformed with PiAvr4. This resulted in transformants that were avirulent on R4 potato plants, demonstrating that PiAvr4 is responsible for eliciting R4-mediated resistance. Expression of PiAvr4 in R4 plants using PVX-agroinfection and agroinfiltration showed that PiAvr4 itself is the effector that elicits HR on R4 plants. On potato plants lacking R4, like Bintje, there was no response. The presence of the RXLR-dEER motif suggested intracellular recognition of PiAvr4 but nevertheless a hypersensitive response was observed when PiAvr4 was targeted to the outside of the cell. Deletion of the RXLR-dEER domain neither stimulated nor prevented elicitor activity of PiAvr4. Race 4 strains have frame shift mutations in the PiAvr4 gene that result in short truncated peptides, indicating that PiAvr4 is not crucial for virulence. Chapter 5 describes Avr1-associated markers that resulted from genetic mapping, transcriptional profiling and BAC-end sequences. In silico landing of these markers on the P. infestans genome sequence narrows down a 800 kb genomic interval that carries seven genes that have the hallmarks of an oomycete Avr gene. They all encode a secreted protein with a conserved RXLR-dEER domain at the N-terminus and a divergent C-terminal region. Each of these seven could be a candidate for Avr1. The seven RXLR effector genes were further characterized by bioinformatic analyses such as HMM score of the RXLR motif, and prediction of the presence of W, Y, and L motifs in the C-terminal region. Cloning and functional analyses using transient expression assays in plants carrying the resistance gene R1 should reveal whether any of the seven candidates is Avr1. Chapter 6 describes the phenotypic and genotypic diversity of P. infestans isolates collected in Northern China between 1997 and 2003, especially in Inner Mongolia. Characterization included mating type, virulence, mitochondrial DNA (mtDNA) haplotype and DNA fingerprinting patterns based on simple sequence repeats (SSR) and amplified fragment length polymorphism (AFLP). All isolates had the A1 mating type, mtDNA haplotype IIa and an identical SSR genotype (designated as SG-01-01) that differed from the SSR genotypes found in the reference isolates, including the ones representing the ‘old’ US-1 lineage that dominated the worldwide P. infestans population prior to 1980. In contrast, the virulence spectra differed significantly and virulence to all R genes present in the standard differential set (R1 to R11) was found. AFLP analysis revealed some diversity; eight different AFLP genotypes were found that could be grouped into two major clusters. This study shows that there is very little genotypic diversity in the P. infestans population in Northern China. The occurrence of many different races within this uniform population is discussed in the framework of recently gained insights in the molecular determinants of avirulence in P. infestans and their role in the ‘gene-for-gene’ interaction with potato. Finally, in Chapter 7, the implications of the findings described in this thesis are discussed with specific emphasis on Avr gene cloning, RXLR-dEER effectors, virulence diversity and durable late blight resistance. By combining various cloning strategies it becomes feasible to speed up the cloning of putative P. infestans Avr genes. Moreover, the use of high throughput effector genomics screenings will allow the identification of the corresponding R genes. The high virulence diversity that is found in P. infestans field isolates, even within one clonal lineage, might be correlated to the observation that RXLR-dEER effector genes are the most rapidly evolving genes in the genome of P. infestans. Therefore, generating potato cultivars with durable resistance to late blight seems more challenging than anticipated.

Iron is fundamental to the biology of eukaryotic cells since it plays a key role in many metabolic functions. Iron concentrations are tightly regulated, for example by ferritin, because excessive iron leads to tissue damage. Iron cannot cross cellular membranes directly and most cells acquire iron from the iron transporting protein transferrin (Tf), via transferrin receptors. During nutritional immunity the body reacts with a metabolic adjustment in order to render important nutrients unavailable to invading microorganisms. However, pathogens also have evolved a range of mechanisms to acquire iron from the host (chapter 1). In the study described in this thesis, we used a natural host-parasite model of common carp (Cyprinus carpio L.) infected with Trypanoplasma borreli, a protozoan kinetoplastid, extracellular blood parasite of carp to get more insight in the competition for iron between host and parasite. Transferrin of common carp is highly polymorphic with several alleles identified according to differences in electrophoretic mobility. We studied the implications of Tf polymorphisms for iron binding and modulation of immune function. We performed a series of challenge experiments infecting five genetically different, commercially exploited carp lines with T. borreli. Our results indicated that Tf genotype may influence the susceptibility to pathogens. We observed a significant association of the DD genotype of Tf with low parasitaemia in two resistant carp lines (Polish ‘R2’ and ‘K’), but a reverse association in the most susceptible carp line ‘D’ (chapter 2). We also showed that variation in resistance to T. borreli could be controlled by sex-related genetic factors. Examination of parasite growth in vitro, in culture media supplemented with 3% serum taken from fish with different Tf genotypes, showed a faster decrease in number of parasites in media supplemented with serum from DD-typed animals (chapter 2). In general, pathogens also have mechanisms to acquire iron from the host. We developed a method for Tf depletion of carp serum using specific antibodies to carp Tf, and compared T. borreli multiplication and survival in the presence or absence of Tf in vitro. Parasites were dying in medium containing Tf-depleted serum, which clearly showed that Tf is essential for parasite growth and multiplication (chapter 3). We isolated two allelic forms of carp Tf (alleles D and G) to purity using rivanol precipitation and ion-exchange chromatography (chapter 5). We showed that parasite growth in vitro could be reconstituted by the addition of purified Tf to Tf-depleted serum (chapter 3). We observed differences in T. borreli multiplication and survival in culture media containing different sera typed differently for Tf genotypes (chapter 3). We identified four complete coding sequences for common carp Tf alleles C, D, F and G, and confirmed the overall similarity of the carp Tf three-dimensional structure to Tfs of other species. We could show that carp Tf differs significantly in critical iron-binding sites in the N-lobe of the molecule, as compared to other non-cyprinid fish species (chapter 4). The substitution of a majority of the iron-coordinating residues in the Nlobe indeed seems to affect the ability to bind iron, which may be compensated for by higher serum concentrations of Tf (chapter 7). Comparison of constitutive gene expression of two Tf alleles D and G showed a comparably high gene expression level in liver and small but consistent differences in gene expression for allele D over allele G in other immunologically important organs (chapter 4). Our data suggest that the allelic polymorphism is not related to differences in iron binding and/or binding to the host Tf receptor but could be linked with other factors, such as competition for iron with pathogens (chapter 4). Transferrin itself may also exert effects that are not directly linked with maintaining iron levels and Tf cleavage products have been shown to stimulate macrophages to produce large amounts of nitric oxide (NO). To study the induction of NO in carp head kidney-derived macrophages, we isolated two allelic forms of carp Tf (alleles D and G) to purity and showed that the level of activation of macrophages by Tf was different for the D and G allele (chapter 5). Differences in NO levels induced could be related to different cleavage forms of the two alleles D and G, as shown by Western blot, confirming that full-length Tf cannot induce NO. The D-type Tf cleavage products induced significantly higher nitric oxide (NO) production than cleavage products of Gtype Tf. (chapter 5). Transferrin uptake by trypanosome parasites involves Tf binding to a receptor. The TfR-Tf complex then is internalised and transported to lysosomes, where Tf is proteolytically degraded. We described the cloning and sequencing of a cathepsin L-like cysteine proteinase from T. borreli and production of a recombinant and biologically active enzyme (chapter 6). We demonstrated that the T. borreli cysteine proteinase is able to digest host transferrin. Likely, Tf cleavage fragments are released from the trypanosomes while iron would remain parasite-associated, possibly contributing to thepathogenicity of the parasite by inducing high amounts of NO in carp macrophages (chapter 7). Our study dealt with different aspects of Tf polymorphism, discussing the role of Tf in immunity of common carp and the influence of allelic polymorphism on competition for iron between host and pathogen (chapter 7). Further investigations should shed more light on the selective advantage of particular alleles to provide a basis for incorporating Tf as a genetic marker in marker-assisted selection programmes for increased resistance to diseases. This could contribute to improved survival of carp kept under semiintensive farming systems in ponds.

Partial resistance to leaf rust ( Puccinia hordei Otth) in barley is a quantitative resistance that is not based on hypersensitivity. This resistance hampers haustorium formation and results in a long latency period in greenhouse tests. The resistance is due to genes with relatively small, quantitative effects, located on so called quantitative trait loci (QTL). A detailed chromosome map of barley, containing 3,258 molecular markers, was constructed and used as a platform to compare the genetic positions of QTLs across different mapping populations. This confirmed that partial resistance in barley to P. hordei is controlled by a high diversity of genes, each mapping population segregating for a different set of QTLs. Another consensus map was constructed that gathered together 775 barley microsatellite markers. The introgression of single QTL-allele or combination of QTL-alleles in near isogenic lines (NILs) allowed us to confirm the effect of three target QTLs ( Rphq2 , Rphq3 and Rphq4 ) in seedling disease tests performed in greenhouse compartments and in field disease tests. The use of several leaf rust isolates revealed the clear isolate-specific effect of Rphq4 . Gene Rphq2 was easy to detect in seedlings of the corresponding NILs and was located in a physical region of high recombination. The position of Rphq2was refinedto a 0.1 cM genetic interval that corresponds probably with only a relatively short stretch of DNA. This makes it feasible to pick the gene up from a bacterial DNA (BAC) library in not too distant future.

Partial resistance to leaf rust ( Puccinia hordei Otth) in barley is a quantitative resistance that is not based on hypersensitivity. This resistance hampers haustorium formation and results in a long latency period in greenhouse tests. The resistance is due to genes with relatively small, quantitative effects, located on so called quantitative trait loci (QTL). A detailed chromosome map of barley, containing 3,258 molecular markers, was constructed and used as a platform to compare the genetic positions of QTLs across different mapping populations. This confirmed that partial resistance in barley to P. hordei is controlled by a high diversity of genes, each mapping population segregating for a different set of QTLs. Another consensus map was constructed that gathered together 775 barley microsatellite markers. The introgression of single QTL-allele or combination of QTL-alleles in near isogenic lines (NILs) allowed us to confirm the effect of three target QTLs ( Rphq2 , Rphq3 and Rphq4 ) in seedling disease tests performed in greenhouse compartments and in field disease tests. The use of several leaf rust isolates revealed the clear isolate-specific effect of Rphq4 . Gene Rphq2 was easy to detect in seedlings of the corresponding NILs and was located in a physical region of high recombination. The position of Rphq2was refinedto a 0.1 cM genetic interval that corresponds probably with only a relatively short stretch of DNA. This makes it feasible to pick the gene up from a bacterial DNA (BAC) library in not too distant future.

Tomato early blight (EB) caused by the fungus Alternaria solani is a field disease with a worldwide distribution, including Indonesia. The disease is currently controlled using frequent applications of fungicides. The use of resistant cultivar would be an attractive way to reduce fungicide application. The aims of the research are to support breeding of EB resistance cultivars for the Indonesian market, by identifying EB resistance genes effective in Indonesia and developing markers for MAS.In Chapter 1 the literature on A. solani, EB and resistance is reviewed. On the fungal side pathogenicity and genetic variation have been widely investigated. However, the existence of different physiological races has not been convincingly demonstrated or disproved. Reliable methods of screening are available for use in resistance breeding. Intensive screening of tomato accessions worldwide has shown that strong resistance is not available in the cultivated tomato but only in wild species. Resistance to A. solani is expressed quantitatively and is polygenically inherited. For that reason classical breeding has not been able to achieve high levels of resistance, and undesirable traits from the donors have been introgressed as well. Recently QTLs have been mapped in a few resistance sources, which could facilitate transferring such quantitative resistance genes and circumvent the problem of unfavourable linkage drag once markers tightly linked to the QTLs have been obtained.In Chapter 2 a resistance test method involving droplet inoculation is described, which is an adaptation of an existing but hardly-used technique. This method has advantages in comparison with the more widely used spray inoculation method, including a clear distinction between lesions caused by A. solani and necroses of 1eaf loss due to other causes, and an objective measurement of damage in contrast to subjective scoring. In the droplet inoculation method leaflets of intact plants are inoculated with droplets of an A. solani conidial suspension in water or an agar solution. Lesions are assessed quantitatively by measuring the length and the perpendicular width. In this way objective and accurate assessments, which are prerequisites for QTL analysis, can be achieved. This inoculation method was used to screen a collection of 54 tomato accessions for resistance to an Indonesian A. solani isolate,S. arcanum LA2157 is highly resistant to A. solani in the glasshouse screening tests. The genetics of the resistance in this wild relative of tomato was further studied using a QTL mapping approach (Chapter 3). The mapping population consisted of 176 F2 plants. A linkage map consisting of 12 linkage groups covering 1179 cM was based on 379 markers (33 SSR, 21 SNP and 316 AFLP markers). All linkage groups could be assigned to the 12 tomato chromosomes. About half of the markers showed deviation from the expected 1:2:1 segregation ratio. The F2 population was phenotyped in a glasshouse in Netherlands with an Indonesian isolate of A. solani. EB resistance was evaluated with respect to lesion size (LS) and related parameters (relative area under the lesion expansion curve [RAULEC] and percentage of small lesions [PSmL]). The derived F3 lines (156) were tested in a field in Indonesia. Percentage of EB index (PEBI) was assessed at six times and relative area under the disease progress curve (RAUDPC) was calculated. A total of six QTLs with a range of LOD scores 3.6 to 16.4 were mapped on chromosomes 1, 2, 5, 6, 7 and 9. Three of the QTLs showed effects in both tests despite differences in experimental method and observed traits. For the first time, three QTLs for resistance affecting the development of stem lesions, another disease symptom caused by the fungus on the main and secondary stems of the plant, were identified, which completely overlapped with QTL regions for early blight resistance. Two QTLs on chromosomes 2 and 9, which explained 7 to 16% of the phenotypic variation for EB resistance and 31% for stem lesion resistance, are recommended to be used in tomato breeding programs for resistance to A. solani.

Tomato early blight (EB) caused by the fungus Alternaria solani is a field disease with a worldwide distribution, including Indonesia. The disease is currently controlled using frequent applications of fungicides. The use of resistant cultivar would be an attractive way to reduce fungicide application. The aims of the research are to support breeding of EB resistance cultivars for the Indonesian market, by identifying EB resistance genes effective in Indonesia and developing markers for MAS.In Chapter 1 the literature on A. solani, EB and resistance is reviewed. On the fungal side pathogenicity and genetic variation have been widely investigated. However, the existence of different physiological races has not been convincingly demonstrated or disproved. Reliable methods of screening are available for use in resistance breeding. Intensive screening of tomato accessions worldwide has shown that strong resistance is not available in the cultivated tomato but only in wild species. Resistance to A. solani is expressed quantitatively and is polygenically inherited. For that reason classical breeding has not been able to achieve high levels of resistance, and undesirable traits from the donors have been introgressed as well. Recently QTLs have been mapped in a few resistance sources, which could facilitate transferring such quantitative resistance genes and circumvent the problem of unfavourable linkage drag once markers tightly linked to the QTLs have been obtained.In Chapter 2 a resistance test method involving droplet inoculation is described, which is an adaptation of an existing but hardly-used technique. This method has advantages in comparison with the more widely used spray inoculation method, including a clear distinction between lesions caused by A. solani and necroses of 1eaf loss due to other causes, and an objective measurement of damage in contrast to subjective scoring. In the droplet inoculation method leaflets of intact plants are inoculated with droplets of an A. solani conidial suspension in water or an agar solution. Lesions are assessed quantitatively by measuring the length and the perpendicular width. In this way objective and accurate assessments, which are prerequisites for QTL analysis, can be achieved. This inoculation method was used to screen a collection of 54 tomato accessions for resistance to an Indonesian A. solani isolate,S. arcanum LA2157 is highly resistant to A. solani in the glasshouse screening tests. The genetics of the resistance in this wild relative of tomato was further studied using a QTL mapping approach (Chapter 3). The mapping population consisted of 176 F2 plants. A linkage map consisting of 12 linkage groups covering 1179 cM was based on 379 markers (33 SSR, 21 SNP and 316 AFLP markers). All linkage groups could be assigned to the 12 tomato chromosomes. About half of the markers showed deviation from the expected 1:2:1 segregation ratio. The F2 population was phenotyped in a glasshouse in Netherlands with an Indonesian isolate of A. solani. EB resistance was evaluated with respect to lesion size (LS) and related parameters (relative area under the lesion expansion curve [RAULEC] and percentage of small lesions [PSmL]). The derived F3 lines (156) were tested in a field in Indonesia. Percentage of EB index (PEBI) was assessed at six times and relative area under the disease progress curve (RAUDPC) was calculated. A total of six QTLs with a range of LOD scores 3.6 to 16.4 were mapped on chromosomes 1, 2, 5, 6, 7 and 9. Three of the QTLs showed effects in both tests despite differences in experimental method and observed traits. For the first time, three QTLs for resistance affecting the development of stem lesions, another disease symptom caused by the fungus on the main and secondary stems of the plant, were identified, which completely overlapped with QTL regions for early blight resistance. Two QTLs on chromosomes 2 and 9, which explained 7 to 16% of the phenotypic variation for EB resistance and 31% for stem lesion resistance, are recommended to be used in tomato breeding programs for resistance to A. solani.

Keywords:Strigahermonthica , Sorghum bicolor , selection measures, resistance, tolerance, genotypes.This thesis presents a study on the interaction between the parasitic weed Striga(S.hermonthica [Del.] Benth.) and the cereal crop sorghum ( S. bicolor [L.]Moench).Its main objective was to find suitable measures for the selection of breeding material (crop genotypes) with superior levels of resistance or superior levels of tolerance to Striga.To meet this objective the physiological background of tolerance, the relation between Striga infestation, infection and yield loss and the effect of host genotype on Striga parasitism and reproductionwere studied.These host-parasite interactions were studied with 4-10 different sorghum genotypes differing in level and mechanism of defence against Striga . Field experiments carried out inMaliwere used for yield assessments and development and validation of selection measures. Through pot and agar-gel experiments, aboveground resistance measures were validated with observations on belowground stages. Pot experimentation was also used to create infection response curves and to measure photosynthesis and chlorophyll fluorescence to develop tolerance measures.Strigaparasitism and reproduction, and the detrimental effect of Striga on crop yield can significantly be reduced through crop genotype choice. Maximum aboveground Striga number is a reliable selection measure for resistance. Striga flowerstalk dry weight can be used to identify genotypes that reduce Striga reproduction. The maximum relative yield loss is a suitable selection measure for tolerance in susceptible genotypes, while for more resistant genotypes the relative yield loss per Striga infection seems more appropriate. For these tolerance measures, yield assessment of nearby uninfected controls is indispensable. Chlorophyll fluorescence, more precisely photochemical quenching and electron transport rate, may enable screening for tolerance without this requirement.

Potato is the most important non-cereal crop in the world. Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease of potato. In the mid-191h century, P. infestans attacked the European potato fields and this resulted in a widespread famine in Ireland. Late blight remains the No.l constraint to potato production and causes a yearly multi-billion US$ loss globally. In Europe and North America, late blight con trol heavily relies on the use of chemicais, which is hardly affordable to farmers in developing countries and also raises considerabie environmental concerns in the developed countries.Use of host resistance is ecologically the most sustainable way of disease management. Disease resistance (R) genes exist in a wide range of wild species of the genera Solanum. One wild species, Solanum demissum, became the donor of most characterized R genes due to its crossability with the cultivated species S. tuberosum. However, these race-specific genes (Rl-Rll) didn't enable satisfactory protection under the current deployment scheme. Breeders either turned to other wild species or adopted a so-called 'R-gene-free' approach to explore quantitative resistance. Unfortunately, neither alternative offered a solution to late blight control and did not result in commercial release of cultivars with durable resistance.At the end of the 20th century, biological research entered the genomics era, landmarked by the Human Genome Project and by the Arabidopsis and rice genome sequencing initiatives. Genomics also became the new frontier of research in potato and P. infestans. This scientific development is deepening and broadening our understanding of the biology of the host and the pathogen and is facilitating isolation of key genes involved in the interaction. The genetically modified organism (GMO) strategy allows a much more efficient application of these genes than time-consuming conventional breeding. This thesis deals with the isolation, characterization, and deployment of host R genes with the expectation to achieve an ecologically and economically sound control of late blight.The potato-P.infestans interaction follows the gene-for-gene model, that is, resistance only occurs when a host R gene and its corresponding avirulence (Avr) gene in the pathogen are both present. Several disease testing methods have been developed for determination of the gene-for-gene interaction between potato andP. infestans. In vitro inoculation was developed as a quick, space-effective, and accurate assay (Chapter 2). The method exploits the amenability of potato for tissue culture and the suitability of the in vitro environment for late blight disease development.ltsspecificity and reliability was confirmed by comparison with the well-established detached-Ieaf assay. Currently, in vitro inoculation is routinely used in phenotyping of segregating populations, resistance testing of transformants for functional complementation, and screening of new R genes in a wide range of wild germplasms.The investigation of host resistance was focused on the R3 complex locus on the distal part of chromosome I\. The R3 complex locus segregated in a potato mapping population, which was used to construct the potato ultra-high dense (UHD) map saturated with over 10,000 amplified fragment length polymorphism (AFLP) markers. Using a population of 1748 plants, we constructed a high-resolution genetic map at the R3 complex locus. The combination of fine-mapping and accurate disease testing with specific P. infestans isolates resulted in the unexpected discovery that the R3 complex locus is composed of two functionally distinct genes, R3a and R3b, which are 0.4 cM apart and have both been introgressed from S. demissum (Chapter 3). Each gene was localized into a genetic interval of 0.25 cM, providing the starting point for map-based cloning.Plant R gene families undergo fast evolution, resulting in considerable intraand inter-specific variation. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. Comparative genomics provides a tooi to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. We were keen to investigate whether this tooi can enable new R gene cloning by a case study. The comparative study revealed that the potato late blight R3 locus and the tomato Fusarium wilt 12 locus were derived from an ancestrallocus involved in plant innate immunity. We adopted alocal RGA approach using DNA sequences of the 12 gene to isolate the R3a gene (Chapter 4).l2 and R3a share 88% and 83% identities at the DNA and protein level, respectively. R3a is a member of the R3 complex locus. Comparative physical mapping disclosed that the potato R3 complex locus underwent a significant expansion after divergence from tomato without disruption of the flanking colinearity. This expansion resulted in an increase in the number of R genes and functional diversification.lnterestingly, the differential evolution of the ancient R locus in the two closely related species is well correlated with the contrasting evolutionary potentials of the pathogens with which 12 and R3 interact.Fusarium oxysporum is a soilborne fungus with low genotype diversity, whereas the late blight pathogen P. infestans is notorious in its ability to move and mutate. An intriguing question is whyS. demissum.aspecies that appears to contain only easily-broken R genes, can display durable resistance at the population level in its natural environment. The polymorphism of parasite recognition capacity in a host population will restrict most isolates of the parasite to grow on most hosts. Allelism is an efficient way of creating recognition polymorphism. We made another unexpected discovery that the R3 complex locus has very high allelic diversity and that the R5 -R 11 resistant specificities all contain a distinct allelic version (Chapter 5). Sequence exchange between alleles and diversifying selection are the major driving forces of this allelic recogntion polymorphism to P. infestans. Remarkably, the genomic structure of the R3 complex locus favors the creation of new resistance specificity by reshuffling of elements from the two clusters (R3a and R3b). The multiple allelism of the R3 complex locus may be a natural mechanism of S. demissum to suppress late blight epidemics and should be mimicked in resistance breeding. We suggest that R-gene polyculture via the GMO approach should be the future paradigm of R gene deployment in late blight controI.Potato breeding for late blight resistance was one of the earliest mankind practices in combating plant pathogens by means of genetic improvement but the disease has not been controlled by resistance breeding so faro The non-durable nature of S. demissum R genes apparently disappointed most breeders and the blame of easily-broken R genes even led to the unsuccessful R-gene-free approach. However, from an evolutionary and ecological point of view, single R genes can never defeat pathogens such as P. infestans with extremely high evolutionary risk. We propose that the community rethinks its strategy of R gene deployment in late blight disease management. R gene monoculture is obviously not recommended. R gene pyramiding is currently practiced by breeders but this strategy is basically the same as R gene monoculture since it creates uniformity in host resistance specificity in the field, which will be eventually broken by the fast-evolving pathogen. R gene polyculture should be the strategy of the future. However, we should soberly realize that host resistance might be high enough for survival of the plant population but it alone might never offer a protection that meets the economical threshold. Integrated pest management should include R gene polyculture as the central element, cultivation measures, and limited chemical applications To provide this central element, we need to clone more R genes. Our discovery of the multiple allelism of the R3 potato late blight resistance complex and molecular characterization of one of its allelic versions will offer a possibility to a dozen R genes in the near future and then to deploy them in a potato field using marker-free GMO techniques.

Potato is the most important non-cereal crop in the world. Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease of potato. In the mid-191h century, P. infestans attacked the European potato fields and this resulted in a widespread famine in Ireland. Late blight remains the No.l constraint to potato production and causes a yearly multi-billion US$ loss globally. In Europe and North America, late blight con trol heavily relies on the use of chemicais, which is hardly affordable to farmers in developing countries and also raises considerabie environmental concerns in the developed countries.Use of host resistance is ecologically the most sustainable way of disease management. Disease resistance (R) genes exist in a wide range of wild species of the genera Solanum. One wild species, Solanum demissum, became the donor of most characterized R genes due to its crossability with the cultivated species S. tuberosum. However, these race-specific genes (Rl-Rll) didn't enable satisfactory protection under the current deployment scheme. Breeders either turned to other wild species or adopted a so-called 'R-gene-free' approach to explore quantitative resistance. Unfortunately, neither alternative offered a solution to late blight control and did not result in commercial release of cultivars with durable resistance.At the end of the 20th century, biological research entered the genomics era, landmarked by the Human Genome Project and by the Arabidopsis and rice genome sequencing initiatives. Genomics also became the new frontier of research in potato and P. infestans. This scientific development is deepening and broadening our understanding of the biology of the host and the pathogen and is facilitating isolation of key genes involved in the interaction. The genetically modified organism (GMO) strategy allows a much more efficient application of these genes than time-consuming conventional breeding. This thesis deals with the isolation, characterization, and deployment of host R genes with the expectation to achieve an ecologically and economically sound control of late blight.The potato-P.infestans interaction follows the gene-for-gene model, that is, resistance only occurs when a host R gene and its corresponding avirulence (Avr) gene in the pathogen are both present. Several disease testing methods have been developed for determination of the gene-for-gene interaction between potato andP. infestans. In vitro inoculation was developed as a quick, space-effective, and accurate assay (Chapter 2). The method exploits the amenability of potato for tissue culture and the suitability of the in vitro environment for late blight disease development.ltsspecificity and reliability was confirmed by comparison with the well-established detached-Ieaf assay. Currently, in vitro inoculation is routinely used in phenotyping of segregating populations, resistance testing of transformants for functional complementation, and screening of new R genes in a wide range of wild germplasms.The investigation of host resistance was focused on the R3 complex locus on the distal part of chromosome I\. The R3 complex locus segregated in a potato mapping population, which was used to construct the potato ultra-high dense (UHD) map saturated with over 10,000 amplified fragment length polymorphism (AFLP) markers. Using a population of 1748 plants, we constructed a high-resolution genetic map at the R3 complex locus. The combination of fine-mapping and accurate disease testing with specific P. infestans isolates resulted in the unexpected discovery that the R3 complex locus is composed of two functionally distinct genes, R3a and R3b, which are 0.4 cM apart and have both been introgressed from S. demissum (Chapter 3). Each gene was localized into a genetic interval of 0.25 cM, providing the starting point for map-based cloning.Plant R gene families undergo fast evolution, resulting in considerable intraand inter-specific variation. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. Comparative genomics provides a tooi to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. We were keen to investigate whether this tooi can enable new R gene cloning by a case study. The comparative study revealed that the potato late blight R3 locus and the tomato Fusarium wilt 12 locus were derived from an ancestrallocus involved in plant innate immunity. We adopted alocal RGA approach using DNA sequences of the 12 gene to isolate the R3a gene (Chapter 4).l2 and R3a share 88% and 83% identities at the DNA and protein level, respectively. R3a is a member of the R3 complex locus. Comparative physical mapping disclosed that the potato R3 complex locus underwent a significant expansion after divergence from tomato without disruption of the flanking colinearity. This expansion resulted in an increase in the number of R genes and functional diversification.lnterestingly, the differential evolution of the ancient R locus in the two closely related species is well correlated with the contrasting evolutionary potentials of the pathogens with which 12 and R3 interact.Fusarium oxysporum is a soilborne fungus with low genotype diversity, whereas the late blight pathogen P. infestans is notorious in its ability to move and mutate. An intriguing question is whyS. demissum.aspecies that appears to contain only easily-broken R genes, can display durable resistance at the population level in its natural environment. The polymorphism of parasite recognition capacity in a host population will restrict most isolates of the parasite to grow on most hosts. Allelism is an efficient way of creating recognition polymorphism. We made another unexpected discovery that the R3 complex locus has very high allelic diversity and that the R5 -R 11 resistant specificities all contain a distinct allelic version (Chapter 5). Sequence exchange between alleles and diversifying selection are the major driving forces of this allelic recogntion polymorphism to P. infestans. Remarkably, the genomic structure of the R3 complex locus favors the creation of new resistance specificity by reshuffling of elements from the two clusters (R3a and R3b). The multiple allelism of the R3 complex locus may be a natural mechanism of S. demissum to suppress late blight epidemics and should be mimicked in resistance breeding. We suggest that R-gene polyculture via the GMO approach should be the future paradigm of R gene deployment in late blight controI.Potato breeding for late blight resistance was one of the earliest mankind practices in combating plant pathogens by means of genetic improvement but the disease has not been controlled by resistance breeding so faro The non-durable nature of S. demissum R genes apparently disappointed most breeders and the blame of easily-broken R genes even led to the unsuccessful R-gene-free approach. However, from an evolutionary and ecological point of view, single R genes can never defeat pathogens such as P. infestans with extremely high evolutionary risk. We propose that the community rethinks its strategy of R gene deployment in late blight disease management. R gene monoculture is obviously not recommended. R gene pyramiding is currently practiced by breeders but this strategy is basically the same as R gene monoculture since it creates uniformity in host resistance specificity in the field, which will be eventually broken by the fast-evolving pathogen. R gene polyculture should be the strategy of the future. However, we should soberly realize that host resistance might be high enough for survival of the plant population but it alone might never offer a protection that meets the economical threshold. Integrated pest management should include R gene polyculture as the central element, cultivation measures, and limited chemical applications To provide this central element, we need to clone more R genes. Our discovery of the multiple allelism of the R3 potato late blight resistance complex and molecular characterization of one of its allelic versions will offer a possibility to a dozen R genes in the near future and then to deploy them in a potato field using marker-free GMO techniques.

Keywords:Strigahermonthica , Sorghum bicolor , selection measures, resistance, tolerance, genotypes.This thesis presents a study on the interaction between the parasitic weed Striga(S.hermonthica [Del.] Benth.) and the cereal crop sorghum ( S. bicolor [L.]Moench).Its main objective was to find suitable measures for the selection of breeding material (crop genotypes) with superior levels of resistance or superior levels of tolerance to Striga.To meet this objective the physiological background of tolerance, the relation between Striga infestation, infection and yield loss and the effect of host genotype on Striga parasitism and reproductionwere studied.These host-parasite interactions were studied with 4-10 different sorghum genotypes differing in level and mechanism of defence against Striga . Field experiments carried out inMaliwere used for yield assessments and development and validation of selection measures. Through pot and agar-gel experiments, aboveground resistance measures were validated with observations on belowground stages. Pot experimentation was also used to create infection response curves and to measure photosynthesis and chlorophyll fluorescence to develop tolerance measures.Strigaparasitism and reproduction, and the detrimental effect of Striga on crop yield can significantly be reduced through crop genotype choice. Maximum aboveground Striga number is a reliable selection measure for resistance. Striga flowerstalk dry weight can be used to identify genotypes that reduce Striga reproduction. The maximum relative yield loss is a suitable selection measure for tolerance in susceptible genotypes, while for more resistant genotypes the relative yield loss per Striga infection seems more appropriate. For these tolerance measures, yield assessment of nearby uninfected controls is indispensable. Chlorophyll fluorescence, more precisely photochemical quenching and electron transport rate, may enable screening for tolerance without this requirement.

Plants are continuously exposed to a wide variety of pathogens. However, all plant species are non-hosts for the majority of the potential plant pathogens. The genetic dissection of non-host resistance is hampered by the lack of segregating population from crosses between host and non-host species, since hardly any non-host is crossable with a host. We have studied the non-host resistance in Lactuca saligna (wild lettuce) to lettuce downy mildew ( Bremia lactucae ). L. saligna is one of the few examples of a non-host species that is crossable with a related host species, L. sativa (lettuce). Based on this interspecific cross, segregating populations have been developed for genetical analysis of the non-host resistance. To map the resistance, we have used two strategies in which we make use of DNA markers to genotype plants. As no accurate linkage map was available for lettuce, we started with the construction of a linkage map of L. saligna´L. sativa . In Chapter 2, the development of an integrated linkage map, based on two populations, is described. To acquire DNA markers, AFLP analyses have been performed on the F 2 populations of the crosses L. saligna CGN 5271 ´L. sativa Olof and L. saligna CGN 11341´L. sativa Norden. Based on these AFLP analyses the polymorphism rate between L. saligna and L. sativa is estimated to be 81%. A linkage map was constructed that comprises 12 SSRs and 476 AFLP markers over 854 cM in nine linkage groups (n=9). Since the markers are randomly spread over all chromosomes, we assume this map is an accurate representative of both parental genomes and very useful for Marker Assisted Selection.The first mapping strategy for downy mildew resistance is described in Chapter 3. In that study, we have performed a QTL analysis on 126 F 2 plants of a cross between the resistant L. saligna CGN 5271 and the susceptible L. sativa Olof. For this QTL analysis all 126 F 2 plants have been tested for resistance in four disease tests with two complex Bremia races (NL14 and NL16). The F 2 population showed a wide and continuous range of resistance levels from completely resistant to completely susceptible. Evidence is presented for a quantitative resistance against both Bremia races as well as for a race-specific resistance against Bremia race NL16 and not against NL14. These disease test data sets have been combined with DNA marker data of all 126 F 2 plants that had already been obtained for the construction of the linkage map. QTL mapping revealed a qualitative gene ( R39 ) explaining the race-specific resistance and three QTLs ( RBQ1 , RBQ2 and RBQ3 ) explaining the quantitative resistance. The qualitative gene R39 is a dominant gene that gives nearly complete resistance to race NL16 in L. saligna CGN 5271 and therefore it shows features similar to Dm genes (dominant race specific genes that give a complete resistance to d owny m ildew). The three QTLs explain 51% of the quantitative resistance against NL14, which indicates that probably not all QTLs have been detected in this F 2 population.In addition to this rather classical F 2 mapping strategy, we have performed an alternative mapping strategy based on the development and characterization of a set of Backcross Inbred Lines (BILs). These BILs are genetically nearly completely like L. sativa but contain a single chromosome substitution segment of L. saligna CGN 5271 (Chapter 4). Starting from an F 1 plant, BILs have been developed by four to five generations of backcrosses and one generation of selfing. All backcrosses from F 1 to BC 4 were made randomly without intentional selection. Marker Assisted Selection was started in the BC 4 generation. Finally, a set of 29 lines was obtained that covers 95% of the L. saligna genome, comprising 16 lines with a single homozygous introgression (BILs), one line with two homozygous introgressions, five lines with heterozygous single introgressions and seven lines with two or more heterozygous introgressions. Several chromosome regions showed severe distorted segregation in the F 2 population. Based on segregation ratios in backcross lines, we were able to explain distorted segregations of three chromosome regions observed in the F 2 population by genetic loci that are involved in pollen- or egg cell fitness.When seed of the first developed BILs was available, a disease test had been set up to test if the BILs, which carried QTLs as identified in the F 2 population, showed enhanced levels of quantitative resistance indeed. Nine BILs (or nearly-BILs) have been tested for resistance to Bremia race NL16. They covered together 31% of the L. saligna parental genome. Two resistance loci detected in the F 2 population ( R39 and RBQ3 ) have been confirmed in the disease test on the BILs. R39 is a dominant gene, which gives a complete resistance against Bremia race NL16. RBQ3 reduces the infection severity of the susceptible L. sativa by 49% ten days post inoculation. The quantitative effects from the resistance genes in these BILs were higher than expected from the F 2 mapping results. No conclusive comparisons of RBQ2 could have been made, as the introgression in the backcross line was not homozygous. RBQ1 has not been tested. Most exciting, the BIL method revealed a new resistance locus on Chromosome 8 with a 77% reduction on the infection severity compared to the susceptible control ten days post inoculation. We conclude that the BIL mapping method can reveal new QTLs unnoticed in the F 2 mapping method and it enables a quantification of the resistance gene effect in a L. sativa background.To extend our knowledge about the non-host resistance of L. saligna to Bremia , we have compared the genetics of non-host resistance to Bremia in L. saligna CGN 5271 with another accession L. saligna CGN 11341. The two accessions show a 39% AFLP polymorphism rate. We have analyzed the non-host resistance of L. saligna CGN 11341 by disease tests and DNA marker analyses on an F 2 and BC 1 population. Disease tests with Bremia races NL14 and NL16 showed a wide range of infection severity scores from resistant to susceptible to both races. The majority of plants had a similar resistance level to both Bremia races. These findings imply that the resistance of L. saligna is quantitatively expressed and is probably race non-specific. A few F 2 and BC 1 plants were completely resistant against Bremia race NL16 and rather susceptible to race NL14. QTL mapping revealed that a major resistance gene that was located on Chromosome 9 explains this race-specific resistance. This gene is designated R39b , as it may be different from R39 .No additional QTLs have been detected in this small F 2 population (n= 54). However, F 2 plants with L. saligna CGN 11341 alleles at loci of RBQ1 , RBQ2 , RBQ3 and RBQ4 mapped in L. saligna CGN 5271, were more resistant than F 2 plants with L. sativa alleles at these loci. In conclusion, we state that it is very likely that the same genes explain the resistances to Bremia in both L. saligna accessions. A backcross program for a set of Backcross Inbred Lines (BIL) that cover R39b and loci for putative QTLs, is in progress.In the last chapter of this thesis the basic results of the study have been discussed. We adduce that non-host resistance in L. saligna is not explained by accumulation of race-specific major resistance genes ( Dm genes) but by a resistance mechanism based on QTLs. Further, we have made a comparison for efficiency of four breeding methods to introgress the resistance genes from L. saligna . Based on this study, we conclude that twice as many resistance genes are introgressed when Marker Assisted Selection is used. Finally, several recommendations concerning research on non-host resistance and the applications of Backcross Inbred Lines have been suggested.

Cladosporium fulvum is a semi-biotrophic pathogen, which causes leaf mold of tomato (Lycopersicon spp.). In our laboratory this pathosystem serves as a model to study gene-for-gene interactions between plants and pathogenic fungi (Joosten & De Wit 1999). Many avirulence (Avr) genes and matching resistance (Cf) genes have been cloned and we are now beginning to understand how their products can induce an array of plant defense responses, including the classic hypersensitive response (HR). Here, we will discuss the latest results of our molecular studies on this interaction. These include the isolation of: (i) two new Avr genes, Avr2 and Avr4E, (ii) the determination of the specificity determinants within the Cf-4 and Cf-9 genes by artificial domain swaps and introduction of point mutations, (iii) the analysis of polymorphism occurring in AVR9-responsive Cf genes occurring in natural populations of L. pimpinellifolium, and finally (iv) the description of an efficient method to identify early HR-related genes.

Plants are continuously exposed to a wide variety of pathogens. However, all plant species are non-hosts for the majority of the potential plant pathogens. The genetic dissection of non-host resistance is hampered by the lack of segregating population from crosses between host and non-host species, since hardly any non-host is crossable with a host. We have studied the non-host resistance in Lactuca saligna (wild lettuce) to lettuce downy mildew ( Bremia lactucae ). L. saligna is one of the few examples of a non-host species that is crossable with a related host species, L. sativa (lettuce). Based on this interspecific cross, segregating populations have been developed for genetical analysis of the non-host resistance. To map the resistance, we have used two strategies in which we make use of DNA markers to genotype plants. As no accurate linkage map was available for lettuce, we started with the construction of a linkage map of L. saligna´L. sativa . In Chapter 2, the development of an integrated linkage map, based on two populations, is described. To acquire DNA markers, AFLP analyses have been performed on the F 2 populations of the crosses L. saligna CGN 5271 ´L. sativa Olof and L. saligna CGN 11341´L. sativa Norden. Based on these AFLP analyses the polymorphism rate between L. saligna and L. sativa is estimated to be 81%. A linkage map was constructed that comprises 12 SSRs and 476 AFLP markers over 854 cM in nine linkage groups (n=9). Since the markers are randomly spread over all chromosomes, we assume this map is an accurate representative of both parental genomes and very useful for Marker Assisted Selection.The first mapping strategy for downy mildew resistance is described in Chapter 3. In that study, we have performed a QTL analysis on 126 F 2 plants of a cross between the resistant L. saligna CGN 5271 and the susceptible L. sativa Olof. For this QTL analysis all 126 F 2 plants have been tested for resistance in four disease tests with two complex Bremia races (NL14 and NL16). The F 2 population showed a wide and continuous range of resistance levels from completely resistant to completely susceptible. Evidence is presented for a quantitative resistance against both Bremia races as well as for a race-specific resistance against Bremia race NL16 and not against NL14. These disease test data sets have been combined with DNA marker data of all 126 F 2 plants that had already been obtained for the construction of the linkage map. QTL mapping revealed a qualitative gene ( R39 ) explaining the race-specific resistance and three QTLs ( RBQ1 , RBQ2 and RBQ3 ) explaining the quantitative resistance. The qualitative gene R39 is a dominant gene that gives nearly complete resistance to race NL16 in L. saligna CGN 5271 and therefore it shows features similar to Dm genes (dominant race specific genes that give a complete resistance to d owny m ildew). The three QTLs explain 51% of the quantitative resistance against NL14, which indicates that probably not all QTLs have been detected in this F 2 population.In addition to this rather classical F 2 mapping strategy, we have performed an alternative mapping strategy based on the development and characterization of a set of Backcross Inbred Lines (BILs). These BILs are genetically nearly completely like L. sativa but contain a single chromosome substitution segment of L. saligna CGN 5271 (Chapter 4). Starting from an F 1 plant, BILs have been developed by four to five generations of backcrosses and one generation of selfing. All backcrosses from F 1 to BC 4 were made randomly without intentional selection. Marker Assisted Selection was started in the BC 4 generation. Finally, a set of 29 lines was obtained that covers 95% of the L. saligna genome, comprising 16 lines with a single homozygous introgression (BILs), one line with two homozygous introgressions, five lines with heterozygous single introgressions and seven lines with two or more heterozygous introgressions. Several chromosome regions showed severe distorted segregation in the F 2 population. Based on segregation ratios in backcross lines, we were able to explain distorted segregations of three chromosome regions observed in the F 2 population by genetic loci that are involved in pollen- or egg cell fitness.When seed of the first developed BILs was available, a disease test had been set up to test if the BILs, which carried QTLs as identified in the F 2 population, showed enhanced levels of quantitative resistance indeed. Nine BILs (or nearly-BILs) have been tested for resistance to Bremia race NL16. They covered together 31% of the L. saligna parental genome. Two resistance loci detected in the F 2 population ( R39 and RBQ3 ) have been confirmed in the disease test on the BILs. R39 is a dominant gene, which gives a complete resistance against Bremia race NL16. RBQ3 reduces the infection severity of the susceptible L. sativa by 49% ten days post inoculation. The quantitative effects from the resistance genes in these BILs were higher than expected from the F 2 mapping results. No conclusive comparisons of RBQ2 could have been made, as the introgression in the backcross line was not homozygous. RBQ1 has not been tested. Most exciting, the BIL method revealed a new resistance locus on Chromosome 8 with a 77% reduction on the infection severity compared to the susceptible control ten days post inoculation. We conclude that the BIL mapping method can reveal new QTLs unnoticed in the F 2 mapping method and it enables a quantification of the resistance gene effect in a L. sativa background.To extend our knowledge about the non-host resistance of L. saligna to Bremia , we have compared the genetics of non-host resistance to Bremia in L. saligna CGN 5271 with another accession L. saligna CGN 11341. The two accessions show a 39% AFLP polymorphism rate. We have analyzed the non-host resistance of L. saligna CGN 11341 by disease tests and DNA marker analyses on an F 2 and BC 1 population. Disease tests with Bremia races NL14 and NL16 showed a wide range of infection severity scores from resistant to susceptible to both races. The majority of plants had a similar resistance level to both Bremia races. These findings imply that the resistance of L. saligna is quantitatively expressed and is probably race non-specific. A few F 2 and BC 1 plants were completely resistant against Bremia race NL16 and rather susceptible to race NL14. QTL mapping revealed that a major resistance gene that was located on Chromosome 9 explains this race-specific resistance. This gene is designated R39b , as it may be different from R39 .No additional QTLs have been detected in this small F 2 population (n= 54). However, F 2 plants with L. saligna CGN 11341 alleles at loci of RBQ1 , RBQ2 , RBQ3 and RBQ4 mapped in L. saligna CGN 5271, were more resistant than F 2 plants with L. sativa alleles at these loci. In conclusion, we state that it is very likely that the same genes explain the resistances to Bremia in both L. saligna accessions. A backcross program for a set of Backcross Inbred Lines (BIL) that cover R39b and loci for putative QTLs, is in progress.In the last chapter of this thesis the basic results of the study have been discussed. We adduce that non-host resistance in L. saligna is not explained by accumulation of race-specific major resistance genes ( Dm genes) but by a resistance mechanism based on QTLs. Further, we have made a comparison for efficiency of four breeding methods to introgress the resistance genes from L. saligna . Based on this study, we conclude that twice as many resistance genes are introgressed when Marker Assisted Selection is used. Finally, several recommendations concerning research on non-host resistance and the applications of Backcross Inbred Lines have been suggested.

Door het screenen van lijnen en wilde verwanten van tarwe, is een nieuw resistentiegen tegen STB (Septoria tritici blotch) gevonden.