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The gastrointestinal tract is lined with a mucus layer, which is colonized by a distinct mucosal microbial population. The anaerobic gut bacterium Akkermansia muciniphila is a well-described member of the mucosal microbiota and has been shown to be a human gut symbiont. In the mucus layer this gut symbiont is likely exposed to the oxygen that diffuses from mucosal epithelial cells. We showed that A. muciniphila has an active detoxification system to cope with reactive oxygen species and can use oxygen for respiration at nanomolar oxygen concentrations, with cytochrome bd as terminal oxidase. Until now, the type strain A. muciniphila MucT was the only cultured representative of this species. We isolated and characterized six new A. muciniphila strains from faecal samples of four different human subjects. These A. muciniphila strains showed minimal genomic and physiologic divergence while retaining their mucin degrading and utilisation capacities. Apart from the human gastrointestinal tract, we detected Akkermansia species in intestinal samples of numerous mammals. An additional ten new A. muciniphila strains were isolated from seven different mammalian species and showed high genomic and physiologic similarity to type strain A. muciniphila MucT. Apart from Akkermansia species, other Verrucomicrobia were identified within the gastrointestinal tract of non-human mammals. Furthermore, we obtained an Akkermansia isolate from the reticulated python, which had a similar mucin degrading capacity as the human strain A. muciniphila MucT but showed more efficient galactose utilization. On the basis of further phylogenetic, physiological, and genomic characterisations, strain PytT was found to represent a novel species within the genus Akkermansia, for which the name Akkermansia glycaniphilus sp. nov. is proposed. Overall, A. muciniphila strains isolated from intestinal samples of human and other mammals show very limited genomic and physiologic divergence. This together with the widely-spread global presence of A. muciniphila and the dependence on mucin for optimal growth, points towards a conserved symbiosis. This conserved symbiosis might be indicative for the beneficial role of this organism in respect to the host metabolic health. This is in line with the observation that A. muciniphila has been negatively associated with obesity and its associated metabolic disorders. In mice, treatment with viable A. muciniphila cells reversed high-fat diet-induced obesity. We described a scalable workflow for the preparation and preservation of high numbers of viable cells of A. muciniphila under strict anaerobic conditions for therapeutic interventions. Moreover, we developed various quality assessment and control procedures aimed to ensure the use of viable cells of A. muciniphila at any location in the world. These viable cells were used in a pilot study in humans in which no adverse events were observed. This is promising for future applications of A. muciniphila as a new therapeutic, leading towards the potential treatment of unhealthy states of the microbiota.

A root-knot nematode, Meloidogyne floridensis n. sp., is described and illustrated from peach originally collected from Gainesville, Florida. This new species resembles M. incognita, M. christiei, M. graminicola, and M. hispanica, but with LM and SEM observations it differs from these species either by the body length, shape of head, tail and tail terminus of second-stage juveniles, body length and shape of spicules in males, and its distinctive female perineal pattern. This pattern has a high to narrowly rounded arch with coarsely broken and network-like striae in and around anal area, faint lateral lines interrupting transverse striae, a sunken vulva and anus, and large distinct phasmids. Molecular data from ribosomal IGS illustrate that M. floridensis n. sp. is different from the mitotic species M. arenaria, M. incognita, and M. javanica. Data from RAPDs confirm it and suggest that this new species lies in an intermediate phylogenetic position between the previous species and the meiotic species M. hapla, M. fallax, and M. chitwoodi. Differential host tests based on annual crops and on Prunus accessions are reported.

Some plants can tolerate and accumulate unusually high levels of toxic metals, and the analysis of such plants can provide insights into the ecology of environments that are polluted with heavy metals due to human industrial activities. The study of heavy metal hyperaccumulators such as Noccaea caerulescens can show how plants cope with excess metals and increase their fitness when growing in metalliferous environments. In this thesis, I compared the molecular mechanisms of zinc (Zn) homeostasis and cadmium (Cd) response in the hyperaccumulator species N. caerulescens and its non-accumulator relative Arabidopsis thalianaby investigating the regulation of the ZNT1/ZIP4gene that promotes Zn uptake and Zn/Cd tolerance.I also studied the ecological advantages of metal hyperaccumulators in nature and determined the DNA sequences of the N. caerulescens transcriptometo find candidate genes that control metal hyperaccumulation and provide an evolutionary perspective on the emergence of this trait. The functional characterization of the N. caerulescens and A. thaliana Zn-transporter genes NcZNT1 and AtZIP4 (and their promoters) showed how their differential expression pattern contributed to their role in metal tolerance and accumulation. The NcZNT1 gene is induced by Zn deficiency and the NcZNT1 protein is localized in the plasma membrane. Transgenic N. caerulescens roots containing a transgene for the green fluorescent protein (GFP) driven by the NcZNT1promoter revealed GFP fluorescence localized to pericycle and vascular tissues. This suggests that NcZNT1 contributes to metal loading into the xylem and long-distance metal transport. The overexpression of NcZNT1 in A. thaliana increased Zn and Cd tolerance and the capacity to accumulate these metals compared to wild-type plants. These results suggest that NcZNT1 plays an important role in Zn and Cd hypertolerance and hyperaccumulation in N. caerulescens, where it is expressed in both Zn-sufficient and Zn-excess conditions. The differential activity of the NcZNT1 and AtZIP4 promoters in N. caerulescens and A. thaliana implies that different cis-regulatory elements and trans-regulatory factors are present in both species. The ecological advantage of hyperaccumulators in metal-contaminated soils was investigated by studying a natural nas1mutant, in which the Nicotianamine Synthase1 gene is disrupted by a transposon insertion. This mutant allele was found in three natural N. caerulescens populations, which were compared to wild-type plants in terms of phenotype and adaptive advantage. Although the transposon disrupted the gene, the loss of NAS1 gene activity was compensated by enhanced expression of NAS3 and NAS4, resulting in increased nicotianamine (NA) production, which enhanced the Zn and Cd accumulation in the nas1 mutants. This increased their metal sensitivity compared to wild-type plants, but also made them more toxic towards Pieris rapae caterpillars, which developed more slowly and gained less weight when fed on mutant plants exposed to excess Zn or Cd. Therefore, the possible selective advantage of the nas1 mutant in nature is high metal accumulation and the protection of plants from herbivores. Differences in nas1 allele frequency among the three natural populations suggests that nas1 alleles experience different degrees of natural selection or may be at different stages on the route to fixation. Molecular evolutionary studies involve the identification of candidate genes that play a role in adaptation. Therefore, a comprehensive set of transcript sequences was obtained from N. caerulescens accession Ganges (GA) by 454 pyrosequencing. In total, the collected 23,836 isotigs (putative transcripts) were grouped into 20,378 isogroups and 93.2% of them could be matched to Brassicaceae protein sequences, which allowedtheir functional annotation. A total of 87 isogroups was annotated as metal homeostasis related genes, including metal transporter families, metal chelator biosynthesis families, and metal tolerance gene families, which are candidate genes for the molecular analysis of heavy metal homeostasis mechanisms. A group of genes required for the synthesis of glucosinolates, which are important secondary metabolites that protect plants against herbivores, were also shown to be expressed in N. caerulescens. The METALLOTHIONEIN3 (MT3) gene was found to have been duplicated in the N. caerulescens genome, when compared to related Brassicaceae. These transcript sequences will provide an important tool to annotate the genome sequence of the N. caerulescensGanges accession, which is in progress. This genome sequence will also be the basis of genome comparisons between the different N. caerulescens accessions that have different levels of metal accumulation and metal tolerance and which may therefore differ in terms of gene expression levels or modes of actions that affect metal homeostasis.

Some plants can tolerate and accumulate unusually high levels of toxic metals, and the analysis of such plants can provide insights into the ecology of environments that are polluted with heavy metals due to human industrial activities. The study of heavy metal hyperaccumulators such as Noccaea caerulescens can show how plants cope with excess metals and increase their fitness when growing in metalliferous environments. In this thesis, I compared the molecular mechanisms of zinc (Zn) homeostasis and cadmium (Cd) response in the hyperaccumulator species N. caerulescens and its non-accumulator relative Arabidopsis thalianaby investigating the regulation of the ZNT1/ZIP4gene that promotes Zn uptake and Zn/Cd tolerance.I also studied the ecological advantages of metal hyperaccumulators in nature and determined the DNA sequences of the N. caerulescens transcriptometo find candidate genes that control metal hyperaccumulation and provide an evolutionary perspective on the emergence of this trait. The functional characterization of the N. caerulescens and A. thaliana Zn-transporter genes NcZNT1 and AtZIP4 (and their promoters) showed how their differential expression pattern contributed to their role in metal tolerance and accumulation. The NcZNT1 gene is induced by Zn deficiency and the NcZNT1 protein is localized in the plasma membrane. Transgenic N. caerulescens roots containing a transgene for the green fluorescent protein (GFP) driven by the NcZNT1promoter revealed GFP fluorescence localized to pericycle and vascular tissues. This suggests that NcZNT1 contributes to metal loading into the xylem and long-distance metal transport. The overexpression of NcZNT1 in A. thaliana increased Zn and Cd tolerance and the capacity to accumulate these metals compared to wild-type plants. These results suggest that NcZNT1 plays an important role in Zn and Cd hypertolerance and hyperaccumulation in N. caerulescens, where it is expressed in both Zn-sufficient and Zn-excess conditions. The differential activity of the NcZNT1 and AtZIP4 promoters in N. caerulescens and A. thaliana implies that different cis-regulatory elements and trans-regulatory factors are present in both species. The ecological advantage of hyperaccumulators in metal-contaminated soils was investigated by studying a natural nas1mutant, in which the Nicotianamine Synthase1 gene is disrupted by a transposon insertion. This mutant allele was found in three natural N. caerulescens populations, which were compared to wild-type plants in terms of phenotype and adaptive advantage. Although the transposon disrupted the gene, the loss of NAS1 gene activity was compensated by enhanced expression of NAS3 and NAS4, resulting in increased nicotianamine (NA) production, which enhanced the Zn and Cd accumulation in the nas1 mutants. This increased their metal sensitivity compared to wild-type plants, but also made them more toxic towards Pieris rapae caterpillars, which developed more slowly and gained less weight when fed on mutant plants exposed to excess Zn or Cd. Therefore, the possible selective advantage of the nas1 mutant in nature is high metal accumulation and the protection of plants from herbivores. Differences in nas1 allele frequency among the three natural populations suggests that nas1 alleles experience different degrees of natural selection or may be at different stages on the route to fixation. Molecular evolutionary studies involve the identification of candidate genes that play a role in adaptation. Therefore, a comprehensive set of transcript sequences was obtained from N. caerulescens accession Ganges (GA) by 454 pyrosequencing. In total, the collected 23,836 isotigs (putative transcripts) were grouped into 20,378 isogroups and 93.2% of them could be matched to Brassicaceae protein sequences, which allowedtheir functional annotation. A total of 87 isogroups was annotated as metal homeostasis related genes, including metal transporter families, metal chelator biosynthesis families, and metal tolerance gene families, which are candidate genes for the molecular analysis of heavy metal homeostasis mechanisms. A group of genes required for the synthesis of glucosinolates, which are important secondary metabolites that protect plants against herbivores, were also shown to be expressed in N. caerulescens. The METALLOTHIONEIN3 (MT3) gene was found to have been duplicated in the N. caerulescens genome, when compared to related Brassicaceae. These transcript sequences will provide an important tool to annotate the genome sequence of the N. caerulescensGanges accession, which is in progress. This genome sequence will also be the basis of genome comparisons between the different N. caerulescens accessions that have different levels of metal accumulation and metal tolerance and which may therefore differ in terms of gene expression levels or modes of actions that affect metal homeostasis.

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

The research presented in this thesis focuses on deriving function from sequence information, with the emphasis on plant sequence data. Unravelling the impact of genomic elements, in most cases genes, on the phenotype of an organism is a major challenge in biological research and modern plant breeding. An important part of this challenge is the (functional) annotation of such genomic elements. Currently, wet lab experiments may provide high quality, but they are laborious and costly. With the advent of next generation sequencing platforms, vast amounts of sequence data are generated. This data are used in connection with the available experimental data to derive function from a bioinformatics perspective. The connection between sequence information and function was approached on the level of chromosome structure (chapter 2) and of gene families (chapter 3) using combinations of existing bioinformatics tools. The applicability of using interaction networks for function prediction was demonstrated by first markedly improving an existing method (chapter 4) and by exploring the role of network topology in function prediction (chapter 5). Taken together, the combination of methods and results presented indicate the potential as well as the current state-of-the-art of function prediction in (plant) bioinformatics. Chapter 1 introduces the basis for the approaches used and developed in this thesis. This includes the concepts of genome annotation, comparative genomics, gene function prediction and the analysis of network topology for gene function prediction. A requirement for the study of any new organism is the sequencing and annotation of its genome. Current genome annotation is divided into structural identification and functional categorization of genomic elements. The de facto standard for categorizing functional annotation is provided by the Gene Ontology. The Gene Ontology is divided into three domains, molecular function, biological process and cellular component. Approaches to predict molecular function and biological process are outlined. Accurate function prediction generally relies on existing input data, often of experimental origin, that can be transferred to unannotated genomic elements. Plants often lack such input data, which poses a big challenge for current function prediction algorithms. In unravelling the function of genomic elements, comparative genomics is an important approach. Via the comparison of multiple genomes it gives insights into evolution, function as well as genomic structure and variation. Comparative genomics has become an essential toolkit for the analysis of newly sequenced organisms. Often bioinformatics methods need to be adapted to the specific needs of plant genome research. With a focus on the commercially important crop plants tomato and potato, specific requirements of plant bioinformatics, such as the high amount of repetitive elements and the lack of experimental data, are outlined. In chapter 2, the structural homology of the long arm of chromosome 2 (2L) of tomato, potato and pepper is analyzed. Molecular organization and collinear junctions are delineated using multi-color BAC FISH analysis and comparative sequence alignment. We identify several large-scale rearrangements including inversions and segmental translocations that were not reported in previous comparative studies. Some of the structural rearrangements are specific for the tomato clade, and differentiate tomato from potato, pepper and other solanaceous species. There are many small-scale synteny perturbations, but local gene vicinity is largely preserved. The data suggests that long distance intra-chromosomal rearrangements and local gene rearrangements have evolved frequently during speciation in the Solanum genus, and that small changes are more prevalent than large-scale differences. The occurrence of transposable elements and other repeats near or at junction breaks may indicate repeat-mediated rearrangements. The ancestral 2L topology is reconstructed and the evolutionary events leading to the current topology are discussed. In chapter 3, we analyze the Snf2 gene family. As part of large protein complexes, Snf2 family ATPases are responsible for energy supply during chromatin remodeling, but the precise mechanism of action of many of these proteins is largely unknown. They influence many processes in plants, such as the response to environmental stress. The analysis is the first comprehensive study of Snf2 family ATPases in plants. Some subfamilies of the Snf2 gene family are remarkably stable in number of genes per genome, whereas others show expansion and contraction in several plants. One of these subfamilies, the plant-specific DRD1 subfamily, is non-existent in lower eukaryote genomes, yet it developed into the largest Snf2 subfamily in plant genomes. It shows the occurrence of a complex series of evolutionary events. Its expansion, notably in tomato, suggests novel functionality in processes connected to chromatin remodeling. The results underpin and extend the Snf2 subfamily classification, which could help to determine the various functional roles of Snf2 ATPases and to target environmental stress tolerance and yield in future breeding with these genes. In chapter 4, a new approach to improve the prediction of protein function in terms of biological processes is developed that is particularly attractive for sparsely annotated plant genomes. The combination of the network-based prediction method Bayesian Markov Random Field (BMRF) with the sequence-based prediction method Argot2 shows significantly improved performance compared to each of the methods separately, as well as compared to Blast2GO. The approach was applied to predict biological processes for the proteomes of rice, barrel clover, poplar, soybean and tomato. Analysis of the relationships between sequence similarity and predicted function similarity identifies numerous cases of divergence of biological processes in which proteins are involved, in spite of sequence similarity. Examples of potential divergence are identified for various biological processes, notably for processes related to cell development, regulation, and response to chemical stimulus. Such divergence in biological process annotation for proteins with similar sequences should be taken into account when analyzing plant gene and genome evolution. This way, the integration of network-based and sequence-based function prediction will strengthen the analysis of evolutionary relationships of plant genomes. In chapter 5 the influence of network topology on network-based function prediction algorithms is investigated. The analysis of biological networks using algorithms such as Bayesian Markov Random Field (BMRF) is a valuable predictor of the biological processes that proteins are involved in. The topological properties and constraints that determine prediction performance in such networks are however largely unknown. This chapter presents analyses based on network centrality measures, such as node degree, to evaluate the performance of BMRF upon progressive removal of highly connected hub nodes (pruning). Three different protein-protein interaction networks with data from Arabidopsis, human and yeast were analyzed. All three show that the average prediction performance can improve significantly. The chapter paves the way for further improvement of network-based function prediction methods based on node pruning. Chapter 6 discusses the results and methods developed in this thesis in the context of the vast amount of generated sequencing data. Sequencing or re-sequencing a (plant) genome has become fairly straightforward and affordable, but the interpretation for subsequent use of this sequence data is far from trivial. The topics addressed in this thesis, annotation of function, analysis of genome structure and identifying genomic variation, focus on this main bottleneck of biological research. Issues discussed in connection with this work and its future are data accuracy, error propagation, possible improvements and future implications for biological research in crop plants. In particular the shift of costs from sequencing to downstream analyses, with functional genome annotation as essential step, is covered. One of the biggest challenges biology and bioinformatics will face is the integration of results from such downstream analyses and other sources into a complete picture. Only this will allow understanding of complex biological systems.

The research presented in this thesis focuses on deriving function from sequence information, with the emphasis on plant sequence data. Unravelling the impact of genomic elements, in most cases genes, on the phenotype of an organism is a major challenge in biological research and modern plant breeding. An important part of this challenge is the (functional) annotation of such genomic elements. Currently, wet lab experiments may provide high quality, but they are laborious and costly. With the advent of next generation sequencing platforms, vast amounts of sequence data are generated. This data are used in connection with the available experimental data to derive function from a bioinformatics perspective. The connection between sequence information and function was approached on the level of chromosome structure (chapter 2) and of gene families (chapter 3) using combinations of existing bioinformatics tools. The applicability of using interaction networks for function prediction was demonstrated by first markedly improving an existing method (chapter 4) and by exploring the role of network topology in function prediction (chapter 5). Taken together, the combination of methods and results presented indicate the potential as well as the current state-of-the-art of function prediction in (plant) bioinformatics. Chapter 1 introduces the basis for the approaches used and developed in this thesis. This includes the concepts of genome annotation, comparative genomics, gene function prediction and the analysis of network topology for gene function prediction. A requirement for the study of any new organism is the sequencing and annotation of its genome. Current genome annotation is divided into structural identification and functional categorization of genomic elements. The de facto standard for categorizing functional annotation is provided by the Gene Ontology. The Gene Ontology is divided into three domains, molecular function, biological process and cellular component. Approaches to predict molecular function and biological process are outlined. Accurate function prediction generally relies on existing input data, often of experimental origin, that can be transferred to unannotated genomic elements. Plants often lack such input data, which poses a big challenge for current function prediction algorithms. In unravelling the function of genomic elements, comparative genomics is an important approach. Via the comparison of multiple genomes it gives insights into evolution, function as well as genomic structure and variation. Comparative genomics has become an essential toolkit for the analysis of newly sequenced organisms. Often bioinformatics methods need to be adapted to the specific needs of plant genome research. With a focus on the commercially important crop plants tomato and potato, specific requirements of plant bioinformatics, such as the high amount of repetitive elements and the lack of experimental data, are outlined. In chapter 2, the structural homology of the long arm of chromosome 2 (2L) of tomato, potato and pepper is analyzed. Molecular organization and collinear junctions are delineated using multi-color BAC FISH analysis and comparative sequence alignment. We identify several large-scale rearrangements including inversions and segmental translocations that were not reported in previous comparative studies. Some of the structural rearrangements are specific for the tomato clade, and differentiate tomato from potato, pepper and other solanaceous species. There are many small-scale synteny perturbations, but local gene vicinity is largely preserved. The data suggests that long distance intra-chromosomal rearrangements and local gene rearrangements have evolved frequently during speciation in the Solanum genus, and that small changes are more prevalent than large-scale differences. The occurrence of transposable elements and other repeats near or at junction breaks may indicate repeat-mediated rearrangements. The ancestral 2L topology is reconstructed and the evolutionary events leading to the current topology are discussed. In chapter 3, we analyze the Snf2 gene family. As part of large protein complexes, Snf2 family ATPases are responsible for energy supply during chromatin remodeling, but the precise mechanism of action of many of these proteins is largely unknown. They influence many processes in plants, such as the response to environmental stress. The analysis is the first comprehensive study of Snf2 family ATPases in plants. Some subfamilies of the Snf2 gene family are remarkably stable in number of genes per genome, whereas others show expansion and contraction in several plants. One of these subfamilies, the plant-specific DRD1 subfamily, is non-existent in lower eukaryote genomes, yet it developed into the largest Snf2 subfamily in plant genomes. It shows the occurrence of a complex series of evolutionary events. Its expansion, notably in tomato, suggests novel functionality in processes connected to chromatin remodeling. The results underpin and extend the Snf2 subfamily classification, which could help to determine the various functional roles of Snf2 ATPases and to target environmental stress tolerance and yield in future breeding with these genes. In chapter 4, a new approach to improve the prediction of protein function in terms of biological processes is developed that is particularly attractive for sparsely annotated plant genomes. The combination of the network-based prediction method Bayesian Markov Random Field (BMRF) with the sequence-based prediction method Argot2 shows significantly improved performance compared to each of the methods separately, as well as compared to Blast2GO. The approach was applied to predict biological processes for the proteomes of rice, barrel clover, poplar, soybean and tomato. Analysis of the relationships between sequence similarity and predicted function similarity identifies numerous cases of divergence of biological processes in which proteins are involved, in spite of sequence similarity. Examples of potential divergence are identified for various biological processes, notably for processes related to cell development, regulation, and response to chemical stimulus. Such divergence in biological process annotation for proteins with similar sequences should be taken into account when analyzing plant gene and genome evolution. This way, the integration of network-based and sequence-based function prediction will strengthen the analysis of evolutionary relationships of plant genomes. In chapter 5 the influence of network topology on network-based function prediction algorithms is investigated. The analysis of biological networks using algorithms such as Bayesian Markov Random Field (BMRF) is a valuable predictor of the biological processes that proteins are involved in. The topological properties and constraints that determine prediction performance in such networks are however largely unknown. This chapter presents analyses based on network centrality measures, such as node degree, to evaluate the performance of BMRF upon progressive removal of highly connected hub nodes (pruning). Three different protein-protein interaction networks with data from Arabidopsis, human and yeast were analyzed. All three show that the average prediction performance can improve significantly. The chapter paves the way for further improvement of network-based function prediction methods based on node pruning. Chapter 6 discusses the results and methods developed in this thesis in the context of the vast amount of generated sequencing data. Sequencing or re-sequencing a (plant) genome has become fairly straightforward and affordable, but the interpretation for subsequent use of this sequence data is far from trivial. The topics addressed in this thesis, annotation of function, analysis of genome structure and identifying genomic variation, focus on this main bottleneck of biological research. Issues discussed in connection with this work and its future are data accuracy, error propagation, possible improvements and future implications for biological research in crop plants. In particular the shift of costs from sequencing to downstream analyses, with functional genome annotation as essential step, is covered. One of the biggest challenges biology and bioinformatics will face is the integration of results from such downstream analyses and other sources into a complete picture. Only this will allow understanding of complex biological systems.

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

cum laude graduation (with distinction)

cum laude graduation (with distinction)

Lily (Lilium L.) is a perennial bulbous ornamental, belonging to subclass Monocotyledonae and family Liliaceae. Lily, according to statistics of Dutch auctions, is the fifth most important cut flower and the second in flower bulbs based on acreage. This species has been extensively used for cytogenetic studies, but molecular genetic studies are limited. The heterogenic nature and the very complex and huge genome (36 Gb) of lily might be the reason for this. To improve the efficiency of breeding and selection in this species, and set up the basis for genetic studies in Lilium, genomic resources are needed. Next generation sequencing (NGS) technology (454 pyro-sequencing) was used to sequence the transcriptomes (RNA-seq) of four lily cultivars: ‘Connecticut King’, ‘White Fox’, ‘Star Gazer’, and Trumpet that belong to the four most important hybrid groups: Asiatic, Longiflorum, Oriental, and Trumpet respectively. Successfully, 52,172 unigenes with an average length of 555 bp were developed and used for a wide range of genetic and genomic studies: SNP marker identification for genetic mapping, gene annotation, and comparative genomic studies. Combining NGS with SNP genotyping techniques to accelerate genetic studies is of considerable interest in different species. In this study, thousands of SNPs out of the 52,172 lily unigenes were identified. Genotyping technique KASPar (KBiosciences competitive Allele Specific PCR) was used to genotype two lily mapping populations: ‘LA (L. longiflorum ‘White Fox’ x Asiatic hybrid ‘Connecticut King’) and AA (‘Connecticut King’ x ‘Orlito’) using 225 SNP markers selected from ‘Connecticut King’ unigenes. Genotyping success rate was 75.5% (170 SNP markers worked), polymorphic SNP rate was 45% (102 SNP markers), and mapped SNP marker rate was 42% (94 SNP mapped) in LA population and 38% (85 SNP mapped) in AA population. Thus, we validated a subset of the putative SNP makers and showed the usability of this type of markers to improve genetic maps for complex genomes like that of lily. The SNP markers together with the available AFLP (amplified fragment length polymorphisms), DArT (diversity arrays technology), and NBS (nucleotide binding site) markers were used to build reference genetic maps for these two lily populations. These maps represent the first reasonably saturated maps that cover 89% of the lily genome with an average marker density of one marker per 4 cM. The availability of more SNP markers for genotyping, opens the door for further enriching these genetic maps and thus improve the marker density. The genetic maps were used to map and understand the genetic of several horticultural traits in Lilium. Fusarium oxysporum and lily mottle virus (LMoV) are considered as very serious diseases in Lilium and as such present important targets for breeding. Six putative QTLs (quantitive trait loci) were identified for Fusarium resistance in AA population, from which QTL1 was the strongest (explains ~25 % of phenotypic variation). In LA population, QTL1 was also confirmed. Thus, QTL1 is a strong and reliable QTL in both populations and it can be used to develop markers for most of the Fusarium resistance for molecular assisted breeding (MAB) applications. The LMoV was mapped as a marker on the AA genetic maps, however, no close markers to this trait (i.e. distance of the closest marker to LMoV was 9 cM) were identified yet. Several ornamental traits: lily flower color ‘carotene’ (LFCc), flower spots (lfs), stem color (LSC), antherless phenotype (lal), and flower direction (up-side facing, lfd) were phenotyped and mapped. Some of these traits showed to be recessive traits (spots, antherless, and flower direction) and controlled by a single gene. Developing markers for recessive traits is valuable since such markers allow the identification of suitable breeding parents so the presence of the recessive trait can be either enhanced or repressed. A more complex trait is flower longevity because it is a function of: the number of buds per inflorescence, the expansion and opening of the buds, the life span of the individual flowers, and also the life-span of the leaves. Moreover, senescence in Lilium is ethylene-insensitive and the regulator(s) of its vase life is not known yet. Our study showed that vase life of individual lily flowers increased significantly by the exogenous application of sugars. Abscisic acid (ABA) level, furthermore, increased dramatically in lily flowers at senescence compared with anthesis. This indicates that ABA might be the main regulator of vase life in lily. However, more experiments should be conducted to prove this conclusion. The genomic resources developed for lily together with genomic resources developed for Tulipa L., in the same way, offered a valuable source of information to conduct comparative genomic studies within and between these two genera. We initiated the first step towards linking molecular genetic maps of Lilium and Tulipa using transcriptome sequences generated by 454 pyro-sequencing. Orthologous genes between lily and tulip were identified (10,913 unigenes) based on sequence data of four lily cultivars and five tulip cultivars. Next, common SNP and EST-SSR markers between the parents of lily mapping populations (AA and LA population) and the parents of tulip mapping population (‘Kees Nelis’ (T. gesneriana) x ‘Cantata’ (T. fosteriana)) based on these orthologous sequences were generated. A total of 229 common SNP and 140 common EST-SSR markers were identified. Genotyping and mapping these markers in the populations of both genera will link the genetic maps of Lilium and Tulipa and thus allow insight into the preservation of gene order, structure, and ‘putative’ functional homology in addition to evolutionary processes. Also, these genomic resources can be used to increase the resolution of, and support for, phylogenetic trees. We selected a set of orthologous genes of Lilium (19 genes, 11,766 bp containing 433 polymorphic sites), of Tulipa (20 genes, 10,347 bp containing 216 polymorphic sites), and of the orthologous genes between the two genera (7 genes, 5,790 bp containing 587 polymorphic sites). These sets are uniquely present in the sequences and informative in estimating the genetic divergence of the two genera, thus they can be used to genotypes more species per genera to build genera and maybe family trees later on. The nucleotide polymorphism rate of Lilium was twice as high as that of Tulipa, on average one substitution per 26 bp for Lilium compared with one substitution per 48 bp for Tulipa. NGS provide a valuable source for large numbers of phylogenetic informative substitutions that might revolutionize the phylogenetic, population genetic, and biodiversity studies. However, the use of bi-allelic information from multiple loci in phylogenetic studies is still challenging and it needs to be studied further. Moreover, having such high numbers of sequence data, allows us to test some evolutionary hypotheses such as positive selection: selection during domestication/breeding processes might be imprinted in the species genome, which can be examined based on omega (dn/ds) values. The higher the omega value the stronger the indication of positive selection. Positive selection was recorded in Lilium and Tulipa genomes when this small subset of gene contigs (46) of the two genera was tested. Our hypothesis could not be confirmed, however to draw final conclusions on this matter, omega values for many more genes of the two genera have to be measured. Finally, a wealth of putative molecular markers (SNPs and SSRs) has become available that can have direct applications for breeding in these genera. SNP markers are important since they are user friendly, efficient, transferable, and co-dominant markers. Applying high throughput genotyping technology to genotype the two lily populations improved the coverage of the two genetic maps. Also, genotyping the same SNP markers in the two populations facilitated the comparisons between the linkage groups of the two populations and will allow the construction of a consensus map. Consequently, exchange of genetic knowledge (mainly QTLs) between the two populations will be easier. The thousands of SNPs identified in the genome of the four lily cultivars opens the door for combining the current linkage mapping studies with association studies which will have a direct impact on improving the resolution of mapping and on MAB applications in Lilium.

Lily (Lilium L.) is a perennial bulbous ornamental, belonging to subclass Monocotyledonae and family Liliaceae. Lily, according to statistics of Dutch auctions, is the fifth most important cut flower and the second in flower bulbs based on acreage. This species has been extensively used for cytogenetic studies, but molecular genetic studies are limited. The heterogenic nature and the very complex and huge genome (36 Gb) of lily might be the reason for this. To improve the efficiency of breeding and selection in this species, and set up the basis for genetic studies in Lilium, genomic resources are needed. Next generation sequencing (NGS) technology (454 pyro-sequencing) was used to sequence the transcriptomes (RNA-seq) of four lily cultivars: ‘Connecticut King’, ‘White Fox’, ‘Star Gazer’, and Trumpet that belong to the four most important hybrid groups: Asiatic, Longiflorum, Oriental, and Trumpet respectively. Successfully, 52,172 unigenes with an average length of 555 bp were developed and used for a wide range of genetic and genomic studies: SNP marker identification for genetic mapping, gene annotation, and comparative genomic studies. Combining NGS with SNP genotyping techniques to accelerate genetic studies is of considerable interest in different species. In this study, thousands of SNPs out of the 52,172 lily unigenes were identified. Genotyping technique KASPar (KBiosciences competitive Allele Specific PCR) was used to genotype two lily mapping populations: ‘LA (L. longiflorum ‘White Fox’ x Asiatic hybrid ‘Connecticut King’) and AA (‘Connecticut King’ x ‘Orlito’) using 225 SNP markers selected from ‘Connecticut King’ unigenes. Genotyping success rate was 75.5% (170 SNP markers worked), polymorphic SNP rate was 45% (102 SNP markers), and mapped SNP marker rate was 42% (94 SNP mapped) in LA population and 38% (85 SNP mapped) in AA population. Thus, we validated a subset of the putative SNP makers and showed the usability of this type of markers to improve genetic maps for complex genomes like that of lily. The SNP markers together with the available AFLP (amplified fragment length polymorphisms), DArT (diversity arrays technology), and NBS (nucleotide binding site) markers were used to build reference genetic maps for these two lily populations. These maps represent the first reasonably saturated maps that cover 89% of the lily genome with an average marker density of one marker per 4 cM. The availability of more SNP markers for genotyping, opens the door for further enriching these genetic maps and thus improve the marker density. The genetic maps were used to map and understand the genetic of several horticultural traits in Lilium. Fusarium oxysporum and lily mottle virus (LMoV) are considered as very serious diseases in Lilium and as such present important targets for breeding. Six putative QTLs (quantitive trait loci) were identified for Fusarium resistance in AA population, from which QTL1 was the strongest (explains ~25 % of phenotypic variation). In LA population, QTL1 was also confirmed. Thus, QTL1 is a strong and reliable QTL in both populations and it can be used to develop markers for most of the Fusarium resistance for molecular assisted breeding (MAB) applications. The LMoV was mapped as a marker on the AA genetic maps, however, no close markers to this trait (i.e. distance of the closest marker to LMoV was 9 cM) were identified yet. Several ornamental traits: lily flower color ‘carotene’ (LFCc), flower spots (lfs), stem color (LSC), antherless phenotype (lal), and flower direction (up-side facing, lfd) were phenotyped and mapped. Some of these traits showed to be recessive traits (spots, antherless, and flower direction) and controlled by a single gene. Developing markers for recessive traits is valuable since such markers allow the identification of suitable breeding parents so the presence of the recessive trait can be either enhanced or repressed. A more complex trait is flower longevity because it is a function of: the number of buds per inflorescence, the expansion and opening of the buds, the life span of the individual flowers, and also the life-span of the leaves. Moreover, senescence in Lilium is ethylene-insensitive and the regulator(s) of its vase life is not known yet. Our study showed that vase life of individual lily flowers increased significantly by the exogenous application of sugars. Abscisic acid (ABA) level, furthermore, increased dramatically in lily flowers at senescence compared with anthesis. This indicates that ABA might be the main regulator of vase life in lily. However, more experiments should be conducted to prove this conclusion. The genomic resources developed for lily together with genomic resources developed for Tulipa L., in the same way, offered a valuable source of information to conduct comparative genomic studies within and between these two genera. We initiated the first step towards linking molecular genetic maps of Lilium and Tulipa using transcriptome sequences generated by 454 pyro-sequencing. Orthologous genes between lily and tulip were identified (10,913 unigenes) based on sequence data of four lily cultivars and five tulip cultivars. Next, common SNP and EST-SSR markers between the parents of lily mapping populations (AA and LA population) and the parents of tulip mapping population (‘Kees Nelis’ (T. gesneriana) x ‘Cantata’ (T. fosteriana)) based on these orthologous sequences were generated. A total of 229 common SNP and 140 common EST-SSR markers were identified. Genotyping and mapping these markers in the populations of both genera will link the genetic maps of Lilium and Tulipa and thus allow insight into the preservation of gene order, structure, and ‘putative’ functional homology in addition to evolutionary processes. Also, these genomic resources can be used to increase the resolution of, and support for, phylogenetic trees. We selected a set of orthologous genes of Lilium (19 genes, 11,766 bp containing 433 polymorphic sites), of Tulipa (20 genes, 10,347 bp containing 216 polymorphic sites), and of the orthologous genes between the two genera (7 genes, 5,790 bp containing 587 polymorphic sites). These sets are uniquely present in the sequences and informative in estimating the genetic divergence of the two genera, thus they can be used to genotypes more species per genera to build genera and maybe family trees later on. The nucleotide polymorphism rate of Lilium was twice as high as that of Tulipa, on average one substitution per 26 bp for Lilium compared with one substitution per 48 bp for Tulipa. NGS provide a valuable source for large numbers of phylogenetic informative substitutions that might revolutionize the phylogenetic, population genetic, and biodiversity studies. However, the use of bi-allelic information from multiple loci in phylogenetic studies is still challenging and it needs to be studied further. Moreover, having such high numbers of sequence data, allows us to test some evolutionary hypotheses such as positive selection: selection during domestication/breeding processes might be imprinted in the species genome, which can be examined based on omega (dn/ds) values. The higher the omega value the stronger the indication of positive selection. Positive selection was recorded in Lilium and Tulipa genomes when this small subset of gene contigs (46) of the two genera was tested. Our hypothesis could not be confirmed, however to draw final conclusions on this matter, omega values for many more genes of the two genera have to be measured. Finally, a wealth of putative molecular markers (SNPs and SSRs) has become available that can have direct applications for breeding in these genera. SNP markers are important since they are user friendly, efficient, transferable, and co-dominant markers. Applying high throughput genotyping technology to genotype the two lily populations improved the coverage of the two genetic maps. Also, genotyping the same SNP markers in the two populations facilitated the comparisons between the linkage groups of the two populations and will allow the construction of a consensus map. Consequently, exchange of genetic knowledge (mainly QTLs) between the two populations will be easier. The thousands of SNPs identified in the genome of the four lily cultivars opens the door for combining the current linkage mapping studies with association studies which will have a direct impact on improving the resolution of mapping and on MAB applications in Lilium.

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

In the past two decades genome sequencing has developed from a laborious and costly technology employed by large international consortia to a widely used, automated and affordable tool used worldwide by many individual research groups. Genome sequences of many food animals and crop plants have been deciphered and are being exploited for fundamental research and applied to improve their breeding programs. The developments in sequencing technologies have also impacted the associated bioinformatics strategies and tools, both those that are required for data processing, management, and quality control, and those used for interpretation of the data. This thesis focuses on the application of genome sequencing, assembly and annotation to two members of the Solanaceae family, tomato and potato. Potato is the economically most important species within the Solanaceae, and its tubers contribute to dietary intake of starch, protein, antioxidants, and vitamins. Tomato fruits are the second most consumed vegetable after potato, and are a globally important dietary source of lycopene, beta-carotene, vitamin C, and fiber. The chapters in this thesis document the generation, exploitation and interpretation of genomic sequence resources for these two species and shed light on the contents, structure and evolution of their genomes. Chapter 1introduces the concepts of genome sequencing, assembly and annotation, and explains the novel genome sequencing technologies that have been developed in the past decade. These so-called Next Generation Sequencing platforms display considerable variation in chemistry and workflow, and as a consequence the throughput and data quality differs by orders of magnitude between the platforms. The currently available sequencing platforms produce a vast variety of read lengths and facilitate the generation of paired sequences with an approximately fixed distance between them. The choice of sequencing chemistry and platform combined with the type of sequencing template demands specifically adapted bioinformatics for data processing and interpretation. Irrespective of the sequencing and assembly strategy that is chosen, the resulting genome sequence, often represented by a collection of long linear strings of nucleotides, is of limited interest by itself. Interpretation of the genome can only be achieved through sequence annotation – that is, identification and classification of all functional elements in a genome sequence. Once these elements have been annotated, sequence alignments between multiple genomes of related accessions or species can be utilized to reveal the genetic variation on both the nucleotide and the structural level that underlies the difference between these species or accessions. Chapter 2describes BlastIf, a novel software tool that exploits sequence similarity searches with BLAST to provide a straightforward annotation of long nucleotide sequences. Generally, two problems are associated with the alignment of a long nucleotide sequence to a database of short gene or protein sequences: (i) the large number of similar hits that can be generated due to database redundancy; and (ii) the relationships implied between aligned segments within a hit that in fact correspond to distinct elements on the sequence such as genes. BlastIf generates a comprehensible BLAST output for long nucleotide sequences by reducing the number of similar hits while revealing most of the variation present between hits. It is a valuable tool for molecular biologists who wish to get a quick overview of the genetic elements present in a newly sequenced segment of DNA, prior to more elaborate efforts of gene structure prediction and annotation. In Chapter 3 a first genome-wide comparison between the emerging genomic sequence resources of tomato and potato is presented. Large collections of BAC end sequences from both species were annotated through repeat searches, transcript alignments and protein domain identification. In-depth comparisons of the annotated sequences revealed remarkable differences in both gene and repeat content between these closely related genomes. The tomato genome was found to be more repetitive than the potato genome, and substantial differences in the distribution of Gypsy and Copia retrotransposable elements as well as microsatellites were observed between the two genomes. A higher gene content was identified in the potato sequences, and in particular several large gene families including cytochrome P450 mono-oxygenases and serine-threonine protein kinases were significantly overrepresented in potato compared to tomato. Moreover, the cytochrome P450 gene family was found to be expanded in both tomato and potato when compared to Arabidopsis thaliana, suggesting an expanded network of secondary metabolic pathways in the Solanaceae. Together these findings present a first glimpse into the evolution of Solanaceous genomes, both within the family and relative to other plant species. Chapter 4explores the physical and genetic organization of tomato chromosome 6 through integration of BAC sequence analysis, High Information Content Fingerprinting, genetic analysis, and BAC-FISH mapping data. A collection of BACs spanning substantial parts of the short and long arm euchromatin and several dispersed regions of the pericentrometric heterochromatin were sequenced and assembled into several tiling paths spanning approximately 11 Mb. Overall, the cytogenetic order of BACs was in agreement with the order of BACs anchored to the Tomato EXPEN 2000 genetic map, although a few striking discrepancies were observed. The integration of BAC-FISH, sequence and genetic mapping data furthermore provided a clear picture of the borders between eu- and heterochromatin on chromosome 6. Annotation of the BAC sequences revealed that, although the majority of protein-coding genes were located in the euchromatin, the highly repetitive pericentromeric heterochromatin displayed an unexpectedly high gene content. Moreover, the short arm euchromatin was relatively rich in repeats, but the ratio of Gypsy and Copia retrotransposons across the different domains of the chromosome clearly distinguished euchromatin from heterochromatin. The ongoing whole-genome sequencing effort will reveal if these properties are unique for tomato chromosome 6, or a more general property of the tomato genome. Chapter 5presents the potato genome, the first genome sequence of an Asterid. To overcome the problems associated with genome assembly due tothe high level of heterozygosity that is observed in commercial tetraploid potato varieties, a homozygous doubled-monoploid potato clone was exploited to sequence and assemble 86% of the 844 Mb genome. This potato reference genome sequence was complemented with re-sequencing of aheterozygous diploid clone, revealing the form and extent of sequence polymorphism both between different genotypes and within a single heterozygous genotype. Gene presence/absence variants and other potentially deleterious mutations were found to occur frequently in potato and are a likely cause of inbreeding depression. Annotation of the genome was supported by deep transcriptome sequencing of both the doubled-monoploid and the heterozygous potato, resulting in the prediction of more than 39,000 protein coding genes. Transcriptome analysis provided evidence for the contribution of gene family expansion, tissue specific expression, and recruitment of genes to new pathways to the evolution of tuber development. The sequence of the potato genome has provided new insights into Eudicot genome evolution and has provided a solid basis for the elucidation of the evolution of tuberisation. Many traits of interest to plant breeders are quantitative in nature and the potato sequence will simplify both their characterization and deployment to generate novel cultivars. The outstanding challenges in plant genome sequencing are addressed in Chapter 6. The high concentration of repetitive elements and the heterozygosity and polyploidy of many interesting crop plant species currently pose a barrier for the efficient reconstruction of their genome sequences. Nonetheless, the completion of a large number of new genome sequences in recent years and the ongoing advances in sequencing technology provide many excitingopportunities for plant breeding and genome research. Current sequencing platforms are being continuously updated and improved, and novel technologies are being developed and implemented in third-generation sequencing platforms that sequence individual molecules without need for amplification. While these technologies create exciting opportunities for new sequencing applications, they also require robust software tools to process the data produced through them efficiently. The ever increasing amount of available genome sequences creates the need for an intuitive platform for the automated and reproducible interrogation of these data in order to formulate new biologically relevant questions on datasets spanning hundreds or thousands of genome sequences.

The requirements for alignment of capped RNA leader sequences along the viral genome during influenza transcription initiation (“cap-snatching”) have long been an enigma. Previous work on Tomato spotted wilt virus (TSWV) transcription initiation has revealed that this virus displays a preference for leaders with increasing base complementarity to the 3'-ultimate residues of the viral RNA template. Assuming that cap-snatching is a highly conserved mechanism, it is tempting to speculate that the findings for TSWV apply to all segmented negative RNA viruses. The research in this thesis aimed to analyze whether similar cap donor requirements applied for Influenza A virus transcription initiation as compared to what has been found for TSWV. Indeed, in vitro studies demonstrated that influenza transcriptase prefers multiple base-pairing capped leaders. Additionally, the occurrence of “prime-and-realign” during influenza transcription initiation was observed, as well as internal priming at the 3'-penultimate viral residue. The in vitro findings were confirmed by similar studies performed during influenza infection of cell cultures. Whereas transcription initiation of TSWV has been relatively well studied, transcription termination has not. It is postulated that transcription termination/translation is triggered by the formation of a hairpin structure. In cell experiments support a role of the TSWV hairpin structure in translation.

In the past two decades genome sequencing has developed from a laborious and costly technology employed by large international consortia to a widely used, automated and affordable tool used worldwide by many individual research groups. Genome sequences of many food animals and crop plants have been deciphered and are being exploited for fundamental research and applied to improve their breeding programs. The developments in sequencing technologies have also impacted the associated bioinformatics strategies and tools, both those that are required for data processing, management, and quality control, and those used for interpretation of the data. This thesis focuses on the application of genome sequencing, assembly and annotation to two members of the Solanaceae family, tomato and potato. Potato is the economically most important species within the Solanaceae, and its tubers contribute to dietary intake of starch, protein, antioxidants, and vitamins. Tomato fruits are the second most consumed vegetable after potato, and are a globally important dietary source of lycopene, beta-carotene, vitamin C, and fiber. The chapters in this thesis document the generation, exploitation and interpretation of genomic sequence resources for these two species and shed light on the contents, structure and evolution of their genomes. Chapter 1introduces the concepts of genome sequencing, assembly and annotation, and explains the novel genome sequencing technologies that have been developed in the past decade. These so-called Next Generation Sequencing platforms display considerable variation in chemistry and workflow, and as a consequence the throughput and data quality differs by orders of magnitude between the platforms. The currently available sequencing platforms produce a vast variety of read lengths and facilitate the generation of paired sequences with an approximately fixed distance between them. The choice of sequencing chemistry and platform combined with the type of sequencing template demands specifically adapted bioinformatics for data processing and interpretation. Irrespective of the sequencing and assembly strategy that is chosen, the resulting genome sequence, often represented by a collection of long linear strings of nucleotides, is of limited interest by itself. Interpretation of the genome can only be achieved through sequence annotation – that is, identification and classification of all functional elements in a genome sequence. Once these elements have been annotated, sequence alignments between multiple genomes of related accessions or species can be utilized to reveal the genetic variation on both the nucleotide and the structural level that underlies the difference between these species or accessions. Chapter 2describes BlastIf, a novel software tool that exploits sequence similarity searches with BLAST to provide a straightforward annotation of long nucleotide sequences. Generally, two problems are associated with the alignment of a long nucleotide sequence to a database of short gene or protein sequences: (i) the large number of similar hits that can be generated due to database redundancy; and (ii) the relationships implied between aligned segments within a hit that in fact correspond to distinct elements on the sequence such as genes. BlastIf generates a comprehensible BLAST output for long nucleotide sequences by reducing the number of similar hits while revealing most of the variation present between hits. It is a valuable tool for molecular biologists who wish to get a quick overview of the genetic elements present in a newly sequenced segment of DNA, prior to more elaborate efforts of gene structure prediction and annotation. In Chapter 3 a first genome-wide comparison between the emerging genomic sequence resources of tomato and potato is presented. Large collections of BAC end sequences from both species were annotated through repeat searches, transcript alignments and protein domain identification. In-depth comparisons of the annotated sequences revealed remarkable differences in both gene and repeat content between these closely related genomes. The tomato genome was found to be more repetitive than the potato genome, and substantial differences in the distribution of Gypsy and Copia retrotransposable elements as well as microsatellites were observed between the two genomes. A higher gene content was identified in the potato sequences, and in particular several large gene families including cytochrome P450 mono-oxygenases and serine-threonine protein kinases were significantly overrepresented in potato compared to tomato. Moreover, the cytochrome P450 gene family was found to be expanded in both tomato and potato when compared to Arabidopsis thaliana, suggesting an expanded network of secondary metabolic pathways in the Solanaceae. Together these findings present a first glimpse into the evolution of Solanaceous genomes, both within the family and relative to other plant species. Chapter 4explores the physical and genetic organization of tomato chromosome 6 through integration of BAC sequence analysis, High Information Content Fingerprinting, genetic analysis, and BAC-FISH mapping data. A collection of BACs spanning substantial parts of the short and long arm euchromatin and several dispersed regions of the pericentrometric heterochromatin were sequenced and assembled into several tiling paths spanning approximately 11 Mb. Overall, the cytogenetic order of BACs was in agreement with the order of BACs anchored to the Tomato EXPEN 2000 genetic map, although a few striking discrepancies were observed. The integration of BAC-FISH, sequence and genetic mapping data furthermore provided a clear picture of the borders between eu- and heterochromatin on chromosome 6. Annotation of the BAC sequences revealed that, although the majority of protein-coding genes were located in the euchromatin, the highly repetitive pericentromeric heterochromatin displayed an unexpectedly high gene content. Moreover, the short arm euchromatin was relatively rich in repeats, but the ratio of Gypsy and Copia retrotransposons across the different domains of the chromosome clearly distinguished euchromatin from heterochromatin. The ongoing whole-genome sequencing effort will reveal if these properties are unique for tomato chromosome 6, or a more general property of the tomato genome. Chapter 5presents the potato genome, the first genome sequence of an Asterid. To overcome the problems associated with genome assembly due tothe high level of heterozygosity that is observed in commercial tetraploid potato varieties, a homozygous doubled-monoploid potato clone was exploited to sequence and assemble 86% of the 844 Mb genome. This potato reference genome sequence was complemented with re-sequencing of aheterozygous diploid clone, revealing the form and extent of sequence polymorphism both between different genotypes and within a single heterozygous genotype. Gene presence/absence variants and other potentially deleterious mutations were found to occur frequently in potato and are a likely cause of inbreeding depression. Annotation of the genome was supported by deep transcriptome sequencing of both the doubled-monoploid and the heterozygous potato, resulting in the prediction of more than 39,000 protein coding genes. Transcriptome analysis provided evidence for the contribution of gene family expansion, tissue specific expression, and recruitment of genes to new pathways to the evolution of tuber development. The sequence of the potato genome has provided new insights into Eudicot genome evolution and has provided a solid basis for the elucidation of the evolution of tuberisation. Many traits of interest to plant breeders are quantitative in nature and the potato sequence will simplify both their characterization and deployment to generate novel cultivars. The outstanding challenges in plant genome sequencing are addressed in Chapter 6. The high concentration of repetitive elements and the heterozygosity and polyploidy of many interesting crop plant species currently pose a barrier for the efficient reconstruction of their genome sequences. Nonetheless, the completion of a large number of new genome sequences in recent years and the ongoing advances in sequencing technology provide many excitingopportunities for plant breeding and genome research. Current sequencing platforms are being continuously updated and improved, and novel technologies are being developed and implemented in third-generation sequencing platforms that sequence individual molecules without need for amplification. While these technologies create exciting opportunities for new sequencing applications, they also require robust software tools to process the data produced through them efficiently. The ever increasing amount of available genome sequences creates the need for an intuitive platform for the automated and reproducible interrogation of these data in order to formulate new biologically relevant questions on datasets spanning hundreds or thousands of genome sequences.

The requirements for alignment of capped RNA leader sequences along the viral genome during influenza transcription initiation (“cap-snatching”) have long been an enigma. Previous work on Tomato spotted wilt virus (TSWV) transcription initiation has revealed that this virus displays a preference for leaders with increasing base complementarity to the 3'-ultimate residues of the viral RNA template. Assuming that cap-snatching is a highly conserved mechanism, it is tempting to speculate that the findings for TSWV apply to all segmented negative RNA viruses. The research in this thesis aimed to analyze whether similar cap donor requirements applied for Influenza A virus transcription initiation as compared to what has been found for TSWV. Indeed, in vitro studies demonstrated that influenza transcriptase prefers multiple base-pairing capped leaders. Additionally, the occurrence of “prime-and-realign” during influenza transcription initiation was observed, as well as internal priming at the 3'-penultimate viral residue. The in vitro findings were confirmed by similar studies performed during influenza infection of cell cultures. Whereas transcription initiation of TSWV has been relatively well studied, transcription termination has not. It is postulated that transcription termination/translation is triggered by the formation of a hairpin structure. In cell experiments support a role of the TSWV hairpin structure in translation.

Plant Research International heeft een diagnostische methode ontwikkeld die toe te passen is 'in planta', en ook de meest recent beschreven (quarantaine-) soorten omvat. De methode omvat de ontwikkeling van een generieke Phytophthora-methode gevolgd door een Phytophthora-identificatie.

Current genetic marker repositories are not sufficient or even are completely lacking for most farm animals. However, genetic markers are essential for the development of a research tool facilitating discovery of genetic factors that contribute to resistance to disease and the overall welfare and performance in farm animals. By large scale identification of Single Nucleotide Polymorphisms (SNPs) and Structural Variants (SVs) we aimed to contribute to the development of a repository of genetic variants for farm animals. For this purpose bioinformatics data pipelines were designed and validated to address the challenge of the cost effective identification of genetic markers in DNA sequencing data even in absence of a fully sequenced reference genome. To find SNPs in pig, we analysed publicly available whole genome shotgun sequencing datasets by sequence alignment and clustering. Sequence clusters were assigned to genomic locations using publicly available BAC sequencing and BAC mapping data. Within the sequence clusters thousands of SNPs were detected of which the genomic location is roughly known. For turkey and duck, species that both were lacking a sufficient sequence data repository for variant discovery, we applied next-generation sequencing (NGS) on a reduced genome representation of a pooled DNA sample. For turkey a genome reference was reconstructed from our sequencing data and available public sequencing data whereas in duck the reference genome constructed by a (NGS) project was used. SNPs obtained by our cost-effective SNP detection procedure still turned out to cover, at intervals, the whole turkey and duck genomes and are of sufficient quality to be used in genotyping studies. Allele frequencies, obtained by genotyping animal panels with a subset our SNPs, correlated well with those observed during SNP detection. The availability of two external duck SNP datasets allowed for the construction of a subset of SNPs which we had in common with these sets. Genotyping turned out that this subset was of outstanding quality and can be used for benchmarking other SNPs that we identified within duck. Ongoing developments in (NGS) allowed for paired end sequencing which is an extension on sequencing analysis that provides information about which pair of reads are coming from the outer ends of one sequenced DNA fragment. We applied this technique on a reduced genome representation of four chicken breeds to detect SVs. Paired end reads were mapped to the chicken reference genome and SVs were identified as abnormally aligned read pairs that have orientation or span sizes discordant from the reference genome. SV detection parameters, to distinguish true structural variants from false positives, were designed and optimized by validation of a small representative sample of SVs using PCR and traditional capillary sequencing. To conclude: we developed SNP repositories which fulfils a requirement for SNPs to perform linkage analysis, comparative genomics QTL studies and ultimately GWA studies in a range of farm animals. We also set the first step in developing a repository for SVs in chicken, a relatively new genetic marker in animal sciences.

Current genetic marker repositories are not sufficient or even are completely lacking for most farm animals. However, genetic markers are essential for the development of a research tool facilitating discovery of genetic factors that contribute to resistance to disease and the overall welfare and performance in farm animals. By large scale identification of Single Nucleotide Polymorphisms (SNPs) and Structural Variants (SVs) we aimed to contribute to the development of a repository of genetic variants for farm animals. For this purpose bioinformatics data pipelines were designed and validated to address the challenge of the cost effective identification of genetic markers in DNA sequencing data even in absence of a fully sequenced reference genome. To find SNPs in pig, we analysed publicly available whole genome shotgun sequencing datasets by sequence alignment and clustering. Sequence clusters were assigned to genomic locations using publicly available BAC sequencing and BAC mapping data. Within the sequence clusters thousands of SNPs were detected of which the genomic location is roughly known. For turkey and duck, species that both were lacking a sufficient sequence data repository for variant discovery, we applied next-generation sequencing (NGS) on a reduced genome representation of a pooled DNA sample. For turkey a genome reference was reconstructed from our sequencing data and available public sequencing data whereas in duck the reference genome constructed by a (NGS) project was used. SNPs obtained by our cost-effective SNP detection procedure still turned out to cover, at intervals, the whole turkey and duck genomes and are of sufficient quality to be used in genotyping studies. Allele frequencies, obtained by genotyping animal panels with a subset our SNPs, correlated well with those observed during SNP detection. The availability of two external duck SNP datasets allowed for the construction of a subset of SNPs which we had in common with these sets. Genotyping turned out that this subset was of outstanding quality and can be used for benchmarking other SNPs that we identified within duck. Ongoing developments in (NGS) allowed for paired end sequencing which is an extension on sequencing analysis that provides information about which pair of reads are coming from the outer ends of one sequenced DNA fragment. We applied this technique on a reduced genome representation of four chicken breeds to detect SVs. Paired end reads were mapped to the chicken reference genome and SVs were identified as abnormally aligned read pairs that have orientation or span sizes discordant from the reference genome. SV detection parameters, to distinguish true structural variants from false positives, were designed and optimized by validation of a small representative sample of SVs using PCR and traditional capillary sequencing. To conclude: we developed SNP repositories which fulfils a requirement for SNPs to perform linkage analysis, comparative genomics QTL studies and ultimately GWA studies in a range of farm animals. We also set the first step in developing a repository for SVs in chicken, a relatively new genetic marker in animal sciences.

Lactobacillus plantarum is a lactic acid bacterium encountered in a variety of food and feed fermentations and as a natural inhabitant of human gastrointestinal tract. To survive in these niches and to maintain its capability, L. plantarum has to respond to numerous changing conditions and the cellular processes involved in these responses are frequently regulated at the transcriptional level. For this reason the transcriptome of a cell is of interest, as it contains information about the transcriptional response of individual genes. Transcriptomic approaches allow elucidation of transcriptional behaviour of all genes at once and are therefore a powerful tool to study all the transcriptional response of a bacterium to differential conditions. The complete genome of L. plantarum was sequenced and putative genes were identified and annotated, allowing design and production of a full genome amplicon based microarray. Using this array, the transcriptional response of L. plantarum to a variety of conditions could be identified. The regulon of sigma factor 54 (σ54) was elucidated and consisted of a single operon encoding a mannose phosphotransferase system (PTS). This transport system was found to be the only functional mannose transporter in L. plantarum. Additionally, a glucose transport function was established for this PTS and deletion of the PTS led to an increase of the intracellular phosphoenolpyruvate (PEP) concentration in resting cells, indicating an important role of this glucose transport system in metabolic control of carbohydrate metabolism. The mannose PTS was also found to play an important role in hydrogen peroxide caused oxidative stress survival due to its robustness to oxidative stress. The impact of mild oxidative stress caused by aerobic growth was elucidated by comparing an aerobic grown culture with an anaerobic grown culture. The results led to the identification of the underlying mechanism of an observed growth stagnation, and subsequent adjustments of fermentation conditions led to overcome the stagnation. The role of the global regulator of catabolite control (CcpA) was determined throughout the whole growth curve, showing a global role for this regulator in catabolite control. Additional studies with different carbon sources confirmed this global role of CcpA and suggested further fine-tuning of regulation by local regulators.

Lactobacillus plantarum is a lactic acid bacterium encountered in a variety of food and feed fermentations and as a natural inhabitant of human gastrointestinal tract. To survive in these niches and to maintain its capability, L. plantarum has to respond to numerous changing conditions and the cellular processes involved in these responses are frequently regulated at the transcriptional level. For this reason the transcriptome of a cell is of interest, as it contains information about the transcriptional response of individual genes. Transcriptomic approaches allow elucidation of transcriptional behaviour of all genes at once and are therefore a powerful tool to study all the transcriptional response of a bacterium to differential conditions. The complete genome of L. plantarum was sequenced and putative genes were identified and annotated, allowing design and production of a full genome amplicon based microarray. Using this array, the transcriptional response of L. plantarum to a variety of conditions could be identified. The regulon of sigma factor 54 (σ54) was elucidated and consisted of a single operon encoding a mannose phosphotransferase system (PTS). This transport system was found to be the only functional mannose transporter in L. plantarum. Additionally, a glucose transport function was established for this PTS and deletion of the PTS led to an increase of the intracellular phosphoenolpyruvate (PEP) concentration in resting cells, indicating an important role of this glucose transport system in metabolic control of carbohydrate metabolism. The mannose PTS was also found to play an important role in hydrogen peroxide caused oxidative stress survival due to its robustness to oxidative stress. The impact of mild oxidative stress caused by aerobic growth was elucidated by comparing an aerobic grown culture with an anaerobic grown culture. The results led to the identification of the underlying mechanism of an observed growth stagnation, and subsequent adjustments of fermentation conditions led to overcome the stagnation. The role of the global regulator of catabolite control (CcpA) was determined throughout the whole growth curve, showing a global role for this regulator in catabolite control. Additional studies with different carbon sources confirmed this global role of CcpA and suggested further fine-tuning of regulation by local regulators.

In this thesis we describe different approaches aiding in the utilization of the exponentially growing amount of information available in the life sciences. Briefly, we address two issues in molecular biology, on sequence analysis, and on text mining. The former issue addresses the problem how to determine remote sequence homology especially when the sequence similarity is very low. For this a visualisation tool is introduced that combines sequence alignment, domain prediction and phylogeny. The second topic on text mining centres on the question how to unambiguously formulate queries for efficient information retrieval. It tackles the problem of gene nomenclature — one in two gene symbols being ambiguous - by introducing a new text-clustering- and taxonomy-based disambiguation methodology.

In this thesis we describe different approaches aiding in the utilization of the exponentially growing amount of information available in the life sciences. Briefly, we address two issues in molecular biology, on sequence analysis, and on text mining. The former issue addresses the problem how to determine remote sequence homology especially when the sequence similarity is very low. For this a visualisation tool is introduced that combines sequence alignment, domain prediction and phylogeny. The second topic on text mining centres on the question how to unambiguously formulate queries for efficient information retrieval. It tackles the problem of gene nomenclature — one in two gene symbols being ambiguous - by introducing a new text-clustering- and taxonomy-based disambiguation methodology.

The thesis focuses on two bioinformatics research topics: the development of tools for an efficient and reliable identification of single nucleotides polymorphisms (SNPs) and polymorphic simple sequence repeats (SSRs) from expressed sequence tags (ESTs) (Chapter 2, 3 and 4), and the subsequent implementation of these tools in a pipeline for narrowing down QTL intervals to facilitate the identification of candidate genes for the QTL (Chapter 5). Chapter 1 provides an introduction to molecular markers, SNP and SSR markers, illustrated by a number of applications of molecular markers, as well as existing programs for their detection. After analysis of existing problems and programs for the detection SNPs, a new algorithm is described to reliably identify SNPs and indels in EST data from diploid and polyploid species (Chapter 2). The algorithm is implemented in a program called QualitySNP. This program uses three filters to identify reliable SNPs: filter one screens for potential SNPs by requiring at least two sequences per represented allele; filter two uses a haplotype-based strategy to filter out clusters with paralogs and false SNPs caused by sequence errors; finally, filter three calculates a confidence score for every putative SNP according to the number of occurrences of each allele in high and low quality regions. For the detection of non-synonymous SNPs (nsSNP), synonymous SNPs and SNPs in UTRs, a program was developed as well. Furthermore, these programs were implemented in a pipeline that includes the identification of SNPs and nsSNPs, as well as a storage and retrieval system. Using this pipeline, large numbers of SNPs could be identified in potato ESTs. QualitySNP is available for running on LINUX and UNIX systems; the program, user manual and examples are available at http://www.bioinformatics.nl/tools/snpweb/ . Chapter 3 describes a web-based implementation of the QualitySNP algorithm, called HaploSNPer, which is a tool for the detection of alleles and SNPs in user-specified input sequences from both diploid and polyploid species. HaploSNPer tries to find homologous sequences in user-specified sequence databases using a user-supplied seed sequence, or in a collection of input sequences. All alleles and associated SNPs are identified on clusters of these homologous sequences using QualitySNP. HaploSNPer provides a user-friendly interface for visualization of SNPs and alleles, which allows the selection of informative SNPs and allele specific makers. Currently HaploSNPer is available for nine animal and thirteen plant species, and is available from http://www.bioinformatics.nl/tools/haplosnper/ . Chapter 4 presents a new tool, called PolySSR, to identify polymorphic SSRs rather than just SSRs based on public EST sequence data derived from heterozygotes and/or different genotypes. Based on PolySSR a pipeline was developed to automatically develop primers for putatively polymorphic SSR markers, taking into account SNPs in the SSR flanking regions, thus improving the success rate of the potential markers. Furthermore, SSR positions in coding or UTR regions of genes are identified by the pipeline. The pipeline also includes a searchable database for these SSRs. The value of PolySSR was demonstrated by the fact that nearly all tested SSRs predicted to be polymorphic were indeed validated as polymorphic, and also most designed primers produced clear amplicons. Large numbers of polymorphic SSRs were identified from publicly available ESTs in potato, tomato, rice, Arabidopsis, brassica and chicken using the pipeline. They are stored into a database, which is available at http://www.bioinformatics.nl/tools/polyssr/ . PolySSR not only decreases the cost of designing and testing primers, it also brings a new approach to use the redundancy and heterozygosity of ESTs for developing SSRs that was ignored before. Analysis of the data obtained with polySSR showed that a larger percentage of short SSRs identified in the species used in the study were polymorphic than that of long SSRs. From this it is clear that in the past we have ‘forgotten’ a whole class of putatively informative markers. QualitySNP and PolySSR have been implemented into a pipeline called GeneTagger to find candidate genes underlying a QTL using the strategy of narrowing the QTL interval (Chapter 5). The pipeline first detects the syntenic region of a QTL interval in the species under study in a model species based on marker sequences linked to the QTL. Next, within the syntenic regions identified in the model species genes are identified that might have a function related to the QTL or genes within the regions that are not part of a large gene family. Based on their map position in the model species a number of genes are selected for marker development. To facilitate marker development, ESTs derived from the target species are analyzed using QualitySNP, PolySSR and other tools. Based on identified genetic variations in the selected genes, molecular markers can be developed for accurate fine-mapping of the QTL and ultimately identification of the gene underlying the QTL effect. The pipeline has been used to narrow the clubroot resistance QTL. The tool is available from the website http://www.bioinformatics.nl/tools/genetagger/ . Finally, merits and shortcomings of the tools that have been developed as well as related bioinformatics questions that arose during these studies are discussed in Chapter 6.

The thesis focuses on two bioinformatics research topics: the development of tools for an efficient and reliable identification of single nucleotides polymorphisms (SNPs) and polymorphic simple sequence repeats (SSRs) from expressed sequence tags (ESTs) (Chapter 2, 3 and 4), and the subsequent implementation of these tools in a pipeline for narrowing down QTL intervals to facilitate the identification of candidate genes for the QTL (Chapter 5). Chapter 1 provides an introduction to molecular markers, SNP and SSR markers, illustrated by a number of applications of molecular markers, as well as existing programs for their detection. After analysis of existing problems and programs for the detection SNPs, a new algorithm is described to reliably identify SNPs and indels in EST data from diploid and polyploid species (Chapter 2). The algorithm is implemented in a program called QualitySNP. This program uses three filters to identify reliable SNPs: filter one screens for potential SNPs by requiring at least two sequences per represented allele; filter two uses a haplotype-based strategy to filter out clusters with paralogs and false SNPs caused by sequence errors; finally, filter three calculates a confidence score for every putative SNP according to the number of occurrences of each allele in high and low quality regions. For the detection of non-synonymous SNPs (nsSNP), synonymous SNPs and SNPs in UTRs, a program was developed as well. Furthermore, these programs were implemented in a pipeline that includes the identification of SNPs and nsSNPs, as well as a storage and retrieval system. Using this pipeline, large numbers of SNPs could be identified in potato ESTs. QualitySNP is available for running on LINUX and UNIX systems; the program, user manual and examples are available at http://www.bioinformatics.nl/tools/snpweb/ . Chapter 3 describes a web-based implementation of the QualitySNP algorithm, called HaploSNPer, which is a tool for the detection of alleles and SNPs in user-specified input sequences from both diploid and polyploid species. HaploSNPer tries to find homologous sequences in user-specified sequence databases using a user-supplied seed sequence, or in a collection of input sequences. All alleles and associated SNPs are identified on clusters of these homologous sequences using QualitySNP. HaploSNPer provides a user-friendly interface for visualization of SNPs and alleles, which allows the selection of informative SNPs and allele specific makers. Currently HaploSNPer is available for nine animal and thirteen plant species, and is available from http://www.bioinformatics.nl/tools/haplosnper/ . Chapter 4 presents a new tool, called PolySSR, to identify polymorphic SSRs rather than just SSRs based on public EST sequence data derived from heterozygotes and/or different genotypes. Based on PolySSR a pipeline was developed to automatically develop primers for putatively polymorphic SSR markers, taking into account SNPs in the SSR flanking regions, thus improving the success rate of the potential markers. Furthermore, SSR positions in coding or UTR regions of genes are identified by the pipeline. The pipeline also includes a searchable database for these SSRs. The value of PolySSR was demonstrated by the fact that nearly all tested SSRs predicted to be polymorphic were indeed validated as polymorphic, and also most designed primers produced clear amplicons. Large numbers of polymorphic SSRs were identified from publicly available ESTs in potato, tomato, rice, Arabidopsis, brassica and chicken using the pipeline. They are stored into a database, which is available at http://www.bioinformatics.nl/tools/polyssr/ . PolySSR not only decreases the cost of designing and testing primers, it also brings a new approach to use the redundancy and heterozygosity of ESTs for developing SSRs that was ignored before. Analysis of the data obtained with polySSR showed that a larger percentage of short SSRs identified in the species used in the study were polymorphic than that of long SSRs. From this it is clear that in the past we have ‘forgotten’ a whole class of putatively informative markers. QualitySNP and PolySSR have been implemented into a pipeline called GeneTagger to find candidate genes underlying a QTL using the strategy of narrowing the QTL interval (Chapter 5). The pipeline first detects the syntenic region of a QTL interval in the species under study in a model species based on marker sequences linked to the QTL. Next, within the syntenic regions identified in the model species genes are identified that might have a function related to the QTL or genes within the regions that are not part of a large gene family. Based on their map position in the model species a number of genes are selected for marker development. To facilitate marker development, ESTs derived from the target species are analyzed using QualitySNP, PolySSR and other tools. Based on identified genetic variations in the selected genes, molecular markers can be developed for accurate fine-mapping of the QTL and ultimately identification of the gene underlying the QTL effect. The pipeline has been used to narrow the clubroot resistance QTL. The tool is available from the website http://www.bioinformatics.nl/tools/genetagger/ . Finally, merits and shortcomings of the tools that have been developed as well as related bioinformatics questions that arose during these studies are discussed in Chapter 6.

Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds- to thousands of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. In contrast to many other gel-based marker technologies DArT markers can be sequenced readily. Chapter 1 gives an overview of the most widely used genetic marker methods and their limitations. In addition some background information is provided on the wheat infecting fungal pathogen Mycosphaerella graminicola , in which we applied DArT.In Chapter 2 the DArT procedure is described in more detail. All steps in the DArT protocol are explained and discussed. In addition some adaptations to the DArT procedure with respect to the complexity reduction and use of adapter sequences are demonstrated on the basis of experiments performed in M. graminicola . Finally, the specifically developed software (DArTsoft) and the application of DArT markers in the mapping process are described.Chapter 3demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana . Restriction fragments from a genomic representation of the ecotype Landsberg erecta (L er ) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypesColumbia(Col) and L er and of individuals from an F 2 population obtained from aCol× L er cross. The scoring of markers with specialized software was highly reproducible and 107 markers could unambiguously be ordered on a genetic linkage map. The marker order on the genetic linkage map coincided with the order on the DNA sequence map. Sequencing of the L er markers and alignment with the availableColgenome sequence confirmed that the polymorphism in DArT markers is largely a result of restriction site polymorphisms.In Chapter 4DArT was used to construct two high-density genetic linkage maps of the haploid wheat pathogen M. graminicola . The isolate IPO323 that originates from a bread wheat field was crossed in planta with either another bread wheat isolate IPO94269 or the durum wheat isolate IPO95052. Host species specificity of the plant pathogenic fungus M. graminicola towards hexaploid bread wheat and tetraploid durum wheat is well-documented. Although M. graminicola has an important sexual cycle and in some parts of the world durum wheat and bread wheat are grown side by side, in nature most isolates are pathogenic to either bread wheat or durum wheat. The generation of progeny from the IPO323 x IPO95052 in planta cross was therefore unexpected as these parental isolates are avirulent to durum wheat and bread wheat, respectively. The segregation of markers was followed in the progenies of both crosses. In total 2078 markers (of which 1793 were DArT markers) could be mapped. The maps of the individual crosses showed absolute co-linearity to a constructed bridge map, with the exception of eight markers that appeared to reveal translocations between IPO95052 and IPO94269. Graphical genotyping enabled the identification of dispensable chromosomes in 15-20% of the progenies, although these chromosomes were present in both parents. We demonstrate that this is due to aberrations (partly by non-disjunction) during meiosis. The loss of chromosomes did not seem to affect viability and pathogenicity. This is the first report on detailed meiotic events using high-density mapping in a haploid filamentous fungus. We show that DArT is a highly efficient and robust marker technology for genetic mapping. The DArT markers can be readily sequenced and are currently being deployed in the assembly of the 9X M. graminicola IPO323 genome sequence.In Chapter 5 the genetic linkage map of the IPO323 x IPO95052 cross was used to map several quantitative trait loci (QTL) for host species specificity in M. graminicola .Virulence of the progeny (n=163) was tested on four bread wheat cultivars and three durum wheat cultivars. All progeny isolates grew normally in vitro,but in contrast to crosses performed with two bread wheat isolates, nearly 50% were unable to cause disease on any of the generally susceptible bread wheat and durum wheat cultivars. Interestingly, other progeny caused disease on either bread wheat cultivars or durum wheat cultivars or both wheat species. This proves that the observed host species specificity was overcome in a single generation. In addition, some progeny were able to cause disease on the bread wheat cv. Shafir even though both parents are avirulent to this cultivar. Detailed analyses allowed the identification of QTLs that determine specificity using the high-density genetic linkage map containing 1144 molecular markers, described in the previous chapter. In total, we identified nine QTLs on seven linkage groups. Remarkably, no specific locus was identified that explains the host species specificity to either durum or bread wheat. One locus with a very high LOD value had a major effect on specificity towards all tested durum wheat cultivars, but was previously identified as a locus involved in cultivar specificity. To our knowledge, this is the first comprehensive QTL analysis on virulence performed on a plant pathogenic fungus. We conclude that specificity in M. graminicola is controlled by many genes and that the long-accepted notion of species specificity among isolates of M. graminicola may in fact be a mix of genetically inherited factors, whereby the distinction between host species specificity and cultivar specificity is not clear-cut.Chapter 6shows the alignment of the DArT markers to the genome of M. graminicola that recently was sequenced by the US Department of Energy-Joint Genome Institute (DOE-JGI). Assembly (version 1.0) of the 9X shotgun sequence resulted in the identification of 129 scaffolds ranging in size from 99 kb to 6.36 Mbp and covering 41.9 Mbp. In this assembly a total of 36 telomeres were identified. Sequencing of DArT markers used for the construction of two high-density genetic linkage maps in M. graminicola provided us the opportunity to align the genetic maps with the physical (sequence) map. By performing BLAST and BLAT analysis of the sequences obtained, 1069 IPO323, 724 IPO95052 and 24 SSR markers could be positioned on the 22 largest scaffolds in the assembly allowing the most comprehensive alignment among sequenced fungal genomes. Comparison of the positions of these markers on the sequence map with the order of the markers on the genetic linkage maps resulted in a nearly complete co-linearity of the order of markers, covering >99 % of the current assembly. When a 2 cM confidence interval was applied, 99% of the markers were co-linear with the genome sequence. The alignment provided evidence that scaffolds 7 and 17, 10 and 14 and also 12 and 22 belong to one chromosome and that scaffolds 4 and 9 were wrongly assembled in the first draft. Furthermore, we identified large differences in genetic versus physical distance in certain genomic regions demonstrating that recombination frequencies increase towards the telomeres. In addition, we identified significant differences in the recombination rates in the different crosses studied. The integration of the genetic maps with the physical map will: 1) assist in finishing the genome assembly; 2) facilitate map-based cloning strategies of genes involved in pathogenesis and 3) help to understand the processes that occur during meiosis and recombination.Finally, Chapter 7 gives a general discussion. The advantages and disadvantages of the DArT marker method as well as alternative array-based technologies are discussed. We conclude that DArT is a cost effective marker technology which generates the extremely high quality data needed for high-density mapping. In addition the ease in which sequences of the markers are obtained, allow large-scale alignments between genetic and physical maps. This makes DArT the method of choice especially for species for which large genetic variation exists, limited financial resources are available or for complex polyploid genomes that may not be amenable to the whole-genome sequence approach. In addition the results obtained using the high-density genetic linkage maps from M. graminicola and the biological implications for the fungal population in respect to adaptation to the host are discussed.

A powerful method to gain biological insights in the functioning of a protein is to use data available for the protein (super) family and transfer this data to the protein of interest. Systematically correlating the sheer amount of data available is difficult and time consuming. Therefore protein superfamily-specific databases are needed. It is shown that such systems can help to predict protein interaction sites, active site residues, residues important in enzyme specificity, protein stabilizing residues, etc. These systems are therefore powerful tools in drug design and protein engineering studies, but building and keeping such systems op to date is time consuming. This thesis is on 3DM, a system that can automatically build such protein superfamily databases. The system starts with building a large accurate structure based multiple sequence alignment of a class of proteins (superfamily). Secondly, it collects and stores amino acid associated data, such as mutational information, ligand- and substrate contacts, etc. together with data derived form the alignment such as correlated mutations and conservation information. These information types are linked to the alignment making it possible to easily transfer information from well studied members of the family to the protein of interest. Navigating between the alignment and associated data is done via interactive HTML pages. Chapter 1 of this thesis is a general introduction on how 3DM databases can be used for protein engineering and drug design processes. Chapter 2 (3DM: A new generation of molecular-class-specific information systems applied to four protein super-families) describes 3DM as a method which is tested on four superfamilies. Chapter 3 (Identification of fungal oxaloacetate hydrolyase within the isocitrate lyase/PEP mutase enzyme superfamily using a sequence marker based method) describes how 3DM was used to classify a new fungal protein family (the OAH-like protein class) and how it was used to distinguish true OAH proteins from very similar paralogues proteins. In this chapter it is shown that the OAH route is the only route used by fungi to produce oxalate, which important since it was shown that oxalate production is a virulent factor used by pathogen fungi. Chapter 4 (Oxaloacetate hydrolase: The C-C bond lyase of oxalate secreting fungi) focuses on OAH. This chapter describes how the 3DM results were used to find the reaction mechanism of OAH en how this resulted in the synthesis of a compound that strongly inhibits OAH. Since oxalate production is a virulent factor used by fungi, this compound can be considered as a potential new drug. This thesis comprises all steps between data collection and the final synthesis and testing of the inhibitor. Therefore, this thesis is called: 3DM, from Data to Medicine.

Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds- to thousands of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. In contrast to many other gel-based marker technologies DArT markers can be sequenced readily. Chapter 1 gives an overview of the most widely used genetic marker methods and their limitations. In addition some background information is provided on the wheat infecting fungal pathogen Mycosphaerella graminicola , in which we applied DArT.In Chapter 2 the DArT procedure is described in more detail. All steps in the DArT protocol are explained and discussed. In addition some adaptations to the DArT procedure with respect to the complexity reduction and use of adapter sequences are demonstrated on the basis of experiments performed in M. graminicola . Finally, the specifically developed software (DArTsoft) and the application of DArT markers in the mapping process are described.Chapter 3demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana . Restriction fragments from a genomic representation of the ecotype Landsberg erecta (L er ) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypesColumbia(Col) and L er and of individuals from an F 2 population obtained from aCol× L er cross. The scoring of markers with specialized software was highly reproducible and 107 markers could unambiguously be ordered on a genetic linkage map. The marker order on the genetic linkage map coincided with the order on the DNA sequence map. Sequencing of the L er markers and alignment with the availableColgenome sequence confirmed that the polymorphism in DArT markers is largely a result of restriction site polymorphisms.In Chapter 4DArT was used to construct two high-density genetic linkage maps of the haploid wheat pathogen M. graminicola . The isolate IPO323 that originates from a bread wheat field was crossed in planta with either another bread wheat isolate IPO94269 or the durum wheat isolate IPO95052. Host species specificity of the plant pathogenic fungus M. graminicola towards hexaploid bread wheat and tetraploid durum wheat is well-documented. Although M. graminicola has an important sexual cycle and in some parts of the world durum wheat and bread wheat are grown side by side, in nature most isolates are pathogenic to either bread wheat or durum wheat. The generation of progeny from the IPO323 x IPO95052 in planta cross was therefore unexpected as these parental isolates are avirulent to durum wheat and bread wheat, respectively. The segregation of markers was followed in the progenies of both crosses. In total 2078 markers (of which 1793 were DArT markers) could be mapped. The maps of the individual crosses showed absolute co-linearity to a constructed bridge map, with the exception of eight markers that appeared to reveal translocations between IPO95052 and IPO94269. Graphical genotyping enabled the identification of dispensable chromosomes in 15-20% of the progenies, although these chromosomes were present in both parents. We demonstrate that this is due to aberrations (partly by non-disjunction) during meiosis. The loss of chromosomes did not seem to affect viability and pathogenicity. This is the first report on detailed meiotic events using high-density mapping in a haploid filamentous fungus. We show that DArT is a highly efficient and robust marker technology for genetic mapping. The DArT markers can be readily sequenced and are currently being deployed in the assembly of the 9X M. graminicola IPO323 genome sequence.In Chapter 5 the genetic linkage map of the IPO323 x IPO95052 cross was used to map several quantitative trait loci (QTL) for host species specificity in M. graminicola .Virulence of the progeny (n=163) was tested on four bread wheat cultivars and three durum wheat cultivars. All progeny isolates grew normally in vitro,but in contrast to crosses performed with two bread wheat isolates, nearly 50% were unable to cause disease on any of the generally susceptible bread wheat and durum wheat cultivars. Interestingly, other progeny caused disease on either bread wheat cultivars or durum wheat cultivars or both wheat species. This proves that the observed host species specificity was overcome in a single generation. In addition, some progeny were able to cause disease on the bread wheat cv. Shafir even though both parents are avirulent to this cultivar. Detailed analyses allowed the identification of QTLs that determine specificity using the high-density genetic linkage map containing 1144 molecular markers, described in the previous chapter. In total, we identified nine QTLs on seven linkage groups. Remarkably, no specific locus was identified that explains the host species specificity to either durum or bread wheat. One locus with a very high LOD value had a major effect on specificity towards all tested durum wheat cultivars, but was previously identified as a locus involved in cultivar specificity. To our knowledge, this is the first comprehensive QTL analysis on virulence performed on a plant pathogenic fungus. We conclude that specificity in M. graminicola is controlled by many genes and that the long-accepted notion of species specificity among isolates of M. graminicola may in fact be a mix of genetically inherited factors, whereby the distinction between host species specificity and cultivar specificity is not clear-cut.Chapter 6shows the alignment of the DArT markers to the genome of M. graminicola that recently was sequenced by the US Department of Energy-Joint Genome Institute (DOE-JGI). Assembly (version 1.0) of the 9X shotgun sequence resulted in the identification of 129 scaffolds ranging in size from 99 kb to 6.36 Mbp and covering 41.9 Mbp. In this assembly a total of 36 telomeres were identified. Sequencing of DArT markers used for the construction of two high-density genetic linkage maps in M. graminicola provided us the opportunity to align the genetic maps with the physical (sequence) map. By performing BLAST and BLAT analysis of the sequences obtained, 1069 IPO323, 724 IPO95052 and 24 SSR markers could be positioned on the 22 largest scaffolds in the assembly allowing the most comprehensive alignment among sequenced fungal genomes. Comparison of the positions of these markers on the sequence map with the order of the markers on the genetic linkage maps resulted in a nearly complete co-linearity of the order of markers, covering >99 % of the current assembly. When a 2 cM confidence interval was applied, 99% of the markers were co-linear with the genome sequence. The alignment provided evidence that scaffolds 7 and 17, 10 and 14 and also 12 and 22 belong to one chromosome and that scaffolds 4 and 9 were wrongly assembled in the first draft. Furthermore, we identified large differences in genetic versus physical distance in certain genomic regions demonstrating that recombination frequencies increase towards the telomeres. In addition, we identified significant differences in the recombination rates in the different crosses studied. The integration of the genetic maps with the physical map will: 1) assist in finishing the genome assembly; 2) facilitate map-based cloning strategies of genes involved in pathogenesis and 3) help to understand the processes that occur during meiosis and recombination.Finally, Chapter 7 gives a general discussion. The advantages and disadvantages of the DArT marker method as well as alternative array-based technologies are discussed. We conclude that DArT is a cost effective marker technology which generates the extremely high quality data needed for high-density mapping. In addition the ease in which sequences of the markers are obtained, allow large-scale alignments between genetic and physical maps. This makes DArT the method of choice especially for species for which large genetic variation exists, limited financial resources are available or for complex polyploid genomes that may not be amenable to the whole-genome sequence approach. In addition the results obtained using the high-density genetic linkage maps from M. graminicola and the biological implications for the fungal population in respect to adaptation to the host are discussed.

A powerful method to gain biological insights in the functioning of a protein is to use data available for the protein (super) family and transfer this data to the protein of interest. Systematically correlating the sheer amount of data available is difficult and time consuming. Therefore protein superfamily-specific databases are needed. It is shown that such systems can help to predict protein interaction sites, active site residues, residues important in enzyme specificity, protein stabilizing residues, etc. These systems are therefore powerful tools in drug design and protein engineering studies, but building and keeping such systems op to date is time consuming. This thesis is on 3DM, a system that can automatically build such protein superfamily databases. The system starts with building a large accurate structure based multiple sequence alignment of a class of proteins (superfamily). Secondly, it collects and stores amino acid associated data, such as mutational information, ligand- and substrate contacts, etc. together with data derived form the alignment such as correlated mutations and conservation information. These information types are linked to the alignment making it possible to easily transfer information from well studied members of the family to the protein of interest. Navigating between the alignment and associated data is done via interactive HTML pages. Chapter 1 of this thesis is a general introduction on how 3DM databases can be used for protein engineering and drug design processes. Chapter 2 (3DM: A new generation of molecular-class-specific information systems applied to four protein super-families) describes 3DM as a method which is tested on four superfamilies. Chapter 3 (Identification of fungal oxaloacetate hydrolyase within the isocitrate lyase/PEP mutase enzyme superfamily using a sequence marker based method) describes how 3DM was used to classify a new fungal protein family (the OAH-like protein class) and how it was used to distinguish true OAH proteins from very similar paralogues proteins. In this chapter it is shown that the OAH route is the only route used by fungi to produce oxalate, which important since it was shown that oxalate production is a virulent factor used by pathogen fungi. Chapter 4 (Oxaloacetate hydrolase: The C-C bond lyase of oxalate secreting fungi) focuses on OAH. This chapter describes how the 3DM results were used to find the reaction mechanism of OAH en how this resulted in the synthesis of a compound that strongly inhibits OAH. Since oxalate production is a virulent factor used by fungi, this compound can be considered as a potential new drug. This thesis comprises all steps between data collection and the final synthesis and testing of the inhibitor. Therefore, this thesis is called: 3DM, from Data to Medicine.

Abstract A root-lesion nematode, Pratylenchus dunensis sp. n., is described and illustrated from Ammophila arenaria (L.) Link, a grass occurring abundantly in coastal dunes of Atlantic Europe. The new species is characterised by medium sized (454-579 μ m) slender, vermiform, females and males having two lip annuli (sometimes three to four; incomplete incisures only visible with scanning electron microscopy), medium to robust stylet (ca 16 μ m) with robust stylet knobs slightly set off, long pharyngeal glands (ca 42 μ m), lateral field with four parallel, non-equidistant, lines, the middle ridge being narrower than the outer ones, lateral field with partial areolation and lines converging posterior to the phasmid which is located between the two inner lines of the lateral field in the posterior half of the tail, round spermatheca filled with round sperm, vulva at 78% of total body length and with protruding vulval lips, posterior uterine sac relatively short (ca 19 μ m), cylindrical tail (ca 33 μ m) narrowing in the posterior third with smooth tail tip and with conspicuous hyaline part (ca 2 μ m). Males occur abundantly and present similar characteristics except for smaller dimensions for all morphological characters, but the head region is more truncated in outline than the female, spicule length is ca 15 μ m and testis length is ca 195 μ m. Nucleotide sequences of the rDNA expansion region D2D3 differed from the morphologically similar species P. penetrans and P. brzeskii that also occur in coastal dunes. These differences are supported by PCR-RFLP of the ITS-rDNA. Pratylenchus dunensis sp. n. was also found parasitising roots of Elymus farctus Viv.

A root-knot nematode, Meloidogyne floridensis n. sp., is described and illustrated from peach originally collected from Gainesville, Florida. This new species resembles M. incognita, M. christiei, M. graminicola, and M. hispanica, but with LM and SEM observations it differs from these species either by the body length, shape of head, tail and tail terminus of second-stage juveniles, body length and shape of spicules in males, and its distinctive female perineal pattern. This pattern has a high to narrowly rounded arch with coarsely broken and network-like striae in and around anal area, faint lateral lines interrupting transverse striae, a sunken vulva and anus, and large distinct phasmids. Molecular data from ribosomal IGS illustrate that M. floridensis n. sp. is different from the mitotic species M. arenaria, M. incognita, and M. javanica. Data from RAPDs confirm it and suggest that this new species lies in an intermediate phylogenetic position between the previous species and the meiotic species M. hapla, M. fallax, and M. chitwoodi. Differential host tests based on annual crops and on Prunus accessions are reported.

This thesis describes the genomic localisation and organisation of loci that harbour resistance to the potato cyst nematode species Globodera pallida and G. rostochiensis . Resistance to the potato cyst nematodes G. pallida and G. rostochiensis is an important aspect in potato breeding. To gain insight in the structure of the genes underlying nematode resistance, it is necessary to first map nematode resistance loci, as described for the resistance loci Gpa5 and Gpa6 . Subsequently, both for map-based cloning and for marker-assisted selection, a high-resolution map of theresistance locus is required. A high-resolution map is produced for the H1 locus. To gain more insight in the genomic organisation of a complex disease resistance locus, Gpa2/Rx1 homologues derived from differenthaplotypeshave been characterised. Finally, the Gpa2/Rx1 homologues derived from fivehaplotypeshave been subjected to extensive sequence analysis to elucidate their structural diversity and evolutionary relationships.