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The European oleochemical industry uses more and more renewable bio-based medium-chain fatty acids (MCFA) and longer mono-unsaturated fatty acids (MUFA) as sources for a wide range of applications such as plastics, lubricants and polymer building blocks, and can in theory completely replace the fossil-based petrochemical industry. The raw materials for these fatty acids consist of imported plant oils (e.g. palm kernel oil). The increased demand of these oils for oleochemical use, however, cannot sustainably be fulfilled by producing more palm kernel oil as such increase in production would most likely only be possible by converting tropical rainforest to plantations. The main aim of this thesis is to generate novel genetic variation by deploying CRISPR/Cas9 to camelina to in the end reduce the dependency of Europe on imported palm kernel and coconut oils and fatty acids. This should help us considerably in achieving a more sustainable bio-based economy.

The plant family Brassicaceae (or crucifers) is an economically important group that includes many food crops (e.g. cabbages and radishes), horticultural species (e.g. Draba, Iberis, Lunaria), and model plant species (particularly Arabidopsis thaliana). Because of the fundamental importance of A. thaliana to plant biology, it makes the Brassicaceae an ideal system for comparative genomics and to test wider evolutionary, ecological and speciation hypotheses. One such hypothesis is the ‘Whole Genome Duplication Radiation Lag Time’ (WGD-RLT) model for the role of polyploidy on the evolution of important plant families such as the Brassicaceae. The WGD-RLT model indicates a higher rate of diversification of a core-group compared to its sister group, due to a lag time after a whole genome duplication event that made it possible for novel traits or geo- or ecological events to increase the core groups diversification rate. Aethionema is the species-poor sister genus of the core Brassicaceae and hence is at an important comparative position to analyse trait and genomic evolution of the species-rich core group. Aethionema species occur mainly in the western Irano-Turanian region, which is concordantly the biodiversity hotspot of the Brassicaceae family. Moreover comparing Aethionema to the Brassicaceae core group can help us to understand and test the ‘WGD-RLT’ model. However to be able to do so we first need to know more about Aethionema. In this thesis, I investigated various levels of evolutionary change (from macro, to micro to trait evolution) within the genus Aethionema, with a major focus the emerging model species Aethionema arabicum. Next generation sequencing has made it possible to use the genomes of many species in a comparative framework. However, the formation of proteins and enzymes, and in the end the phenotype of the whole plant, relies on transcription from particular regions of the genome including genes. Hence, the transcriptome makes it possible to assess the functional parts of the genome. However, the functional part of the genome not only relies on the protein coding genes. Gene regulatory elements like promoters and long non-coding RNAs function as regulators of gene expression and hence are involved in increasing or decreasing transcription. In Chapter 2 I used the transcriptome of four different Aethionema species to understand the lineage specificity of these long non-coding RNAs. Moreover in a comparison with the Brassicaceae core group and Brassicaceae’s sister family the Cleomaceae I show that although the position of long non-coding RNAs can be conserved, their sequences do not have to be. Most of the Aethionema species occur in the Irano-Turanian region, a politically instable region, making it hard for scientist to collect from. However the natural history collections made throughout the last centuries are a great resource. Combing these collections with the newest sequencing techniques, e.g. next generation sequencing, have allowed me to infer the phylogeny of ~75% of the known Aethionema species in a time calibrated and historical biogeographical framework. Hence, I was able to establish that Aethionema species likely originated from the Anatolian Diagonal and that major geological events like the uplift of the Turkish and Iranian plateau have had a hand in their speciation (Chapter 3). To examine species-level processes I sequenced and analysed transcriptomes of eight Ae. arabicum accessions coming from Cyprus, Iran and Turkey to investigate population structure, genetic diversity and local adaptation (Chapter 4). The most prominent finding was a ploidy difference between the Iranian and Turkish/Cypriotic lines, whereby the former were (allo)tetraploid and the latter diploid. The tetraploid Iranian lines seem to have one set of alleles from the Turkish/Cypriotic gene-pool. However we do not know where the other alleles come from. In addition to the differences in ploidy level there are also differences in glucosinolate defence compounds between these two populations (Iranian vs Turkish/Cypriotic), with the Iranian lines lacking the diversity and concentration of indolic glucosinolates that the Turkish/Cypriotic lines have. This chapter serves as a good resource and starting point for future research in the region, maybe by using the natural history collections that are at hand. Glucosinolates (i.e. mustard oils) are mainly made by Brassicales species, with their highest structural diversity in the Brassicaceae. In Chapter 5, I examined two Ae. arabicum lines (CYP and TUR) and their recombinant inbred lines to assess glucosinolate composition in different tissues and throughout the plants development. The levels of glucosinolates in the leaves changed when Ae. arabicum went from vegetative to a reproductive state. Moreover, a major difference in glucosinolate content (up to 10-fold) between CYP and TUR indicates a likely regulatory pathway outside of the main glucosinolate biosynthesis pathway. Multi-trait and multi-environment QTL analyses based on leaves, reproductive tissues and seeds identified a single major QTL. Fine mapping this region reduced the interval to only fifteen protein coding genes, including the two most intriguing candidates: FLOWERING LOCUS C (FLC) and the sulphate transporter SULTR2;1. These findings show an interesting correlation between development and defence. Finally, Chapter 6 gives a final discussion of this thesis and its results. It brings the different topics together, put them in a bigger picture and look forward to new research possibilities.

As more and more species have been sequenced, evidence has been piling up for a fascinating phenomenon that seems to occur in all plant lineages: paleopolyploidy. Polyploidy has historically been a much observed and studied trait, but until recently it was assumed that polyploids were evolutionary dead-ends due to their sterility. However, many studies since the 1990’s have challenged this notion by finding evidence for ancient genome duplications in many genomes of current species. This lead to the observation that all seed plants share at least one ancestral polyploidy event. Another polyploidy event has been proven to lie at the base of all angiosperms, further signifying the notion that ancient polyploidy is widespread and common. These findings have led to questions regarding the apparent disadvantages that can be observed in a first generation polyploid. If these disadvantages can be overcome however, duplication of a genome also presents an enormous potential for evolutionary novelty. Duplicated copies of genes are able to acquire changes that can lead to specialization of the duplicated pair into two functions (subfunctionalization) or the development of one copy towards an entirely new function (neofunctionalization). Currently, most research towards polyploidy has focused on the economically and scientifically important Brassicaceae family containing the model plant Arabidopsis thaliana and many crops such as cabbage, rapeseed, broccoli and turnip. In this thesis, I lay the foundations for the expansion of this scope to the Cleomaceae, a widespread cosmopolitan plant family and a sister family of Brassicaceae. The species within Cleomaceae are diverse and exhibit many scientifically interesting traits. They are also in a perfect position phylogenetically to draw comparisons with the much more studied Brassicaceae. I describe the Cleomaceae and their relevance to polyploid research in more detail in the Introduction. I then describe the important first step towards setting up the genetic framework of this family with the sequencing of Tarenaya hassleriana in Chapter 1. In Chapter 2, I have studied the effects of polyploidy on the development of C4 photosynthesis by comparing the transcriptome of C3 photosynthesis based species Tarenaya hassleriana with the C4 based Gynandropsis gynandra. C4 photosynthesis is an elaboration of the more common C3 form of photosynthesis that concentrates CO2 in specific cells leading to decreased photorespiration by the RuBisCO and higher photosynthetic efficieny in low CO2 environments. I find that polyploidy has not led to sub- or neofunctionalization towards the development of this trait, but instead find evidence for another important phenomenon in postpolyploid evolution: the dosage balance hypothesis. This hypothesis states that genes which are dependent on specific dosage levels of their products will be maintained in duplicate; any change in their function would lead to dosage imbalance which would have deleterious effects on their pathway. We show that most genes involved in photosynthesis have returned to single copy in G. gynandra and that the changes leading to C4 have mostly taken place at the expression level confirming current assumptions on the development of this trait. In Chapter 3, I have studied the effects of polyploidy on an important class of plant defence compounds: glucosinolates. These compounds, sometimes referred to as ‘mustard oils’, play an important role in the defence against herbivores and have radiated widely in Brassicaceae to form many different ‘flavors’ to deter specific herbivores. I show that in Cleomaceae many genes responsible for these compounds have benefited from the three rounds of polyploidy that T. hassleriana has undergone and that many duplicated genes have been retained. We also show that more than 75% is actively expressed in the plant, proving that the majority of these duplications has an active function in the plant. Finally, in Chapter 4 I investigate a simple observation made during experiments with T. hassleriana in the greenhouse regarding the variation in flower colour between different individuals: some had pink flowers and some purple. Using LC-PDA mass spectrometry we find that the two colours are caused by different levels of two anthocyanin pigments, with cyanidin dominating in the purple flowers and pelargonidin being more abundant in pink flowers. Through sequence comparison and synteny analysis between A. thaliana and T. hassleriana we find the orthologs of the genes involved in this pathway. Using a Genotyping by Sequencing method on a cross between these two flower colours, we produce a collection of SNP markers on the reference genome. With these SNPs, we find two significant binary trait loci, one of which corresponds to the location of the F3’H ortholog which performs the conversion of a pelargonidin precursor to a cyanidin precursor. In the General Conclusion, I combine all findings of the previous chapters and explain how they establish part of a larger species framework to study ancient polyploidy in angiosperms. I then put forth what these findings can mean for possible future research and the directions that are worth to be explored further.

The plant family Brassicaceae (or crucifers) is an economically important group that includes many food crops (e.g. cabbages and radishes), horticultural species (e.g. Draba, Iberis, Lunaria), and model plant species (particularly Arabidopsis thaliana). Because of the fundamental importance of A. thaliana to plant biology, it makes the Brassicaceae an ideal system for comparative genomics and to test wider evolutionary, ecological and speciation hypotheses. One such hypothesis is the ‘Whole Genome Duplication Radiation Lag Time’ (WGD-RLT) model for the role of polyploidy on the evolution of important plant families such as the Brassicaceae. The WGD-RLT model indicates a higher rate of diversification of a core-group compared to its sister group, due to a lag time after a whole genome duplication event that made it possible for novel traits or geo- or ecological events to increase the core groups diversification rate. Aethionema is the species-poor sister genus of the core Brassicaceae and hence is at an important comparative position to analyse trait and genomic evolution of the species-rich core group. Aethionema species occur mainly in the western Irano-Turanian region, which is concordantly the biodiversity hotspot of the Brassicaceae family. Moreover comparing Aethionema to the Brassicaceae core group can help us to understand and test the ‘WGD-RLT’ model. However to be able to do so we first need to know more about Aethionema. In this thesis, I investigated various levels of evolutionary change (from macro, to micro to trait evolution) within the genus Aethionema, with a major focus the emerging model species Aethionema arabicum. Next generation sequencing has made it possible to use the genomes of many species in a comparative framework. However, the formation of proteins and enzymes, and in the end the phenotype of the whole plant, relies on transcription from particular regions of the genome including genes. Hence, the transcriptome makes it possible to assess the functional parts of the genome. However, the functional part of the genome not only relies on the protein coding genes. Gene regulatory elements like promoters and long non-coding RNAs function as regulators of gene expression and hence are involved in increasing or decreasing transcription. In Chapter 2 I used the transcriptome of four different Aethionema species to understand the lineage specificity of these long non-coding RNAs. Moreover in a comparison with the Brassicaceae core group and Brassicaceae’s sister family the Cleomaceae I show that although the position of long non-coding RNAs can be conserved, their sequences do not have to be. Most of the Aethionema species occur in the Irano-Turanian region, a politically instable region, making it hard for scientist to collect from. However the natural history collections made throughout the last centuries are a great resource. Combing these collections with the newest sequencing techniques, e.g. next generation sequencing, have allowed me to infer the phylogeny of ~75% of the known Aethionema species in a time calibrated and historical biogeographical framework. Hence, I was able to establish that Aethionema species likely originated from the Anatolian Diagonal and that major geological events like the uplift of the Turkish and Iranian plateau have had a hand in their speciation (Chapter 3). To examine species-level processes I sequenced and analysed transcriptomes of eight Ae. arabicum accessions coming from Cyprus, Iran and Turkey to investigate population structure, genetic diversity and local adaptation (Chapter 4). The most prominent finding was a ploidy difference between the Iranian and Turkish/Cypriotic lines, whereby the former were (allo)tetraploid and the latter diploid. The tetraploid Iranian lines seem to have one set of alleles from the Turkish/Cypriotic gene-pool. However we do not know where the other alleles come from. In addition to the differences in ploidy level there are also differences in glucosinolate defence compounds between these two populations (Iranian vs Turkish/Cypriotic), with the Iranian lines lacking the diversity and concentration of indolic glucosinolates that the Turkish/Cypriotic lines have. This chapter serves as a good resource and starting point for future research in the region, maybe by using the natural history collections that are at hand. Glucosinolates (i.e. mustard oils) are mainly made by Brassicales species, with their highest structural diversity in the Brassicaceae. In Chapter 5, I examined two Ae. arabicum lines (CYP and TUR) and their recombinant inbred lines to assess glucosinolate composition in different tissues and throughout the plants development. The levels of glucosinolates in the leaves changed when Ae. arabicum went from vegetative to a reproductive state. Moreover, a major difference in glucosinolate content (up to 10-fold) between CYP and TUR indicates a likely regulatory pathway outside of the main glucosinolate biosynthesis pathway. Multi-trait and multi-environment QTL analyses based on leaves, reproductive tissues and seeds identified a single major QTL. Fine mapping this region reduced the interval to only fifteen protein coding genes, including the two most intriguing candidates: FLOWERING LOCUS C (FLC) and the sulphate transporter SULTR2;1. These findings show an interesting correlation between development and defence. Finally, Chapter 6 gives a final discussion of this thesis and its results. It brings the different topics together, put them in a bigger picture and look forward to new research possibilities.

Cultivated lettuce (Lactuca sativa L.) is an important leafy vegetable worldwide. However, the phylogenetic relationships between domesticated lettuce and its wild relatives are still not clear. In this thesis, I focus on the phylogenetic relationships within Lactuca L., including an analysis of the wild Lactuca species that are endemic to Africa for the first time. The genetic variation of responses to salinity in a recombinant inbred line population, derived from a cross between the lettuce crop (L. sativa ‘Salinas’) and wild species (L. serriola), was investigated and the candidate gene in the identified QTL regions was further studied. In Chapter 1, I introduce and discuss topics related to genetic diversity and evolution in Lactuca, including an overview of lettuce cultivars and uses, its hypothesized domestication history, the taxonomic position of Lactuca, current status of molecular breeding in lettuce and mechanisms of salinity tolerance in plants, especially the High-affinity K+ Transporter (HKT) gene family. In Chapter 2, the most extensive molecular phylogenetic analysis of Lactuca was constructed based on two chloroplast genes (ndhF and trnL-F), including endemic African species for the first time. This taxon sampling covers nearly 40% of the total Lactuca species endemic to Africa and 34% of all Lactuca species. DNA sequences from all the subfamilies of Asteraceae in Genbank and those generated from Lactuca herbarium samples were used to elucidate the monophyly of Lactuca and the affiliation of Lactuca within Asteracaeae. Based on the subfamily tree, 33 ndhF sequences from 30 species and 79 trnL-F sequences from 48 species were selected to infer phylogenetic relationships within Lactuca using Randomized Axelerated Maximum Likelihood (RAxML) and Bayesian Inference (BI) analyses. In addition, biogeographical, chromosomal and morphological character states were analysed based on the Bayesian tree topology. The results showed that Lactuca contains two distinct phylogenetic clades - the crop clade and the Pterocypsela clade. Other North American, Asian and widespread species either form smaller clades or mix with the Melanoseris species in an unresolved polytomy. The newly sampled African endemic species probably should be excluded from Lactuca and treated as a new genus. In Chapter 3, twenty-seven wild Lactuca species and four outgroup species were sequenced using next generation sequencing (NGS) technology. The sampling covers 36% of total Lactuca species and all the important geographical groups in the genus. Thirty chloroplast genomes, including one complete (partial) large single copy region (LSC), one small single copy region (SSC), one inverted repeat (IR) region, and twenty-nine nuclear ribosomal DNA sequences (containing the internal transcribed spacer region ) were successfully assembled and analysed. A methodology paper for which I am co-author, but is not included in this thesis, of the sequencing pipeline was published: ‘Herbarium genomics: plastome sequence assembly from a range of herbarium specimens using an Iterative Organelle Genome Assembly (IOGA) pipeline’. These NGS data helped resolve deeper nodes in the phylogeny within Lactuca and resolved the polytomy from Chapter 2. The results showed that there are at least four main groups within Lactuca: the crop group, the Pterocypsela group, the North American group and the group containing widely-distributed species. I also confirmed that the endemic African species should be removed and treated as a new genus. In Chapter 4, quantitative trait loci (QTLs) related to salt-induced changes in Root System Architecture (RSA) and ion accumulation were determined using a recombinant inbred line population derived from a cross between cultivated lettuce and wild lettuce. I measured the components of RSA by replicated lettuce seedlings grown on vertical agar plates with different NaCl concentrations in a controlled growth chamber environment. I also quantified the concentration of sodium and potassium in replicates of greenhouse-grown plants watered with 100 mM NaCl. The results identified a total of fourteen QTLs using multi-trait linkage analysis, including three major QTLs associated with general root development (qRC9.1), root growth in salt stress condition (qRS2.1), and ion accumulation (qLS7.2). In Chapter 5, one of the identified QTL regions (qLS7.2) reported in Chapter 4 was found to contain a homolog of the HKT1 from Arabidopsis thaliana. I did a phylogenetic analysis of Lactuca HKT1-like protein sequences with other published HKT protein sequences and determined transmembrane and pore segments of lettuce HKT1;1 alleles, according to the model proposed for AtHKT1;1. Gene expression pattern and level of LsaHKT1;1 (L. sativa ‘Salinas’) and LseHKT1;1 (L. serriola) in root and shoot were investigated in plants growing hydroponically over a time-course. The measurements of Na+ and K+ contents were sampled at the same time as the samples used for gene expression test. In addition, I examined the 5’ promoter regions of the two genotypes. The results showed low expression levels of both HKT1;1 alleles in Lactuca root and relatively higher expression in shoot, probably due to the negative cis-regulatory elements of HKT1 alleles found in Lactuca promoter regions. Significant allelic differences were found in HKT1;1 expression in early stage (0-24 hours) shoots in and in late stage (2-6 days) roots. shoot HKT1;1 expression/root HKT1;1 expression was generally consistent with the ratios of Na+/K+ balance in the relevant tissues (shoot Na+/K+ divided by root Na+/K+). In Chapter 6, I summarize and discuss the results from previous chapters briefly. The implications of Chapter 2 and 3 for Lactuca phylogenetics are discussed, including some key characters for the diagnosis of species within Lactuca, the use of herbarium DNA for NGS technology, and perspectives into Lactuca phylogeny. Future perspectives of genome-wide association mapping for lettuce breeding were also discussed. Lastly, I propose to integrate phylogenetic approaches into investigations of allelic differences in lettuce, not just associated with salinity stress but also with other stressed and beneficial characters, both within and between species.

Cultivated lettuce (Lactuca sativa L.) is an important leafy vegetable worldwide. However, the phylogenetic relationships between domesticated lettuce and its wild relatives are still not clear. In this thesis, I focus on the phylogenetic relationships within Lactuca L., including an analysis of the wild Lactuca species that are endemic to Africa for the first time. The genetic variation of responses to salinity in a recombinant inbred line population, derived from a cross between the lettuce crop (L. sativa ‘Salinas’) and wild species (L. serriola), was investigated and the candidate gene in the identified QTL regions was further studied. In Chapter 1, I introduce and discuss topics related to genetic diversity and evolution in Lactuca, including an overview of lettuce cultivars and uses, its hypothesized domestication history, the taxonomic position of Lactuca, current status of molecular breeding in lettuce and mechanisms of salinity tolerance in plants, especially the High-affinity K+ Transporter (HKT) gene family. In Chapter 2, the most extensive molecular phylogenetic analysis of Lactuca was constructed based on two chloroplast genes (ndhF and trnL-F), including endemic African species for the first time. This taxon sampling covers nearly 40% of the total Lactuca species endemic to Africa and 34% of all Lactuca species. DNA sequences from all the subfamilies of Asteraceae in Genbank and those generated from Lactuca herbarium samples were used to elucidate the monophyly of Lactuca and the affiliation of Lactuca within Asteracaeae. Based on the subfamily tree, 33 ndhF sequences from 30 species and 79 trnL-F sequences from 48 species were selected to infer phylogenetic relationships within Lactuca using Randomized Axelerated Maximum Likelihood (RAxML) and Bayesian Inference (BI) analyses. In addition, biogeographical, chromosomal and morphological character states were analysed based on the Bayesian tree topology. The results showed that Lactuca contains two distinct phylogenetic clades - the crop clade and the Pterocypsela clade. Other North American, Asian and widespread species either form smaller clades or mix with the Melanoseris species in an unresolved polytomy. The newly sampled African endemic species probably should be excluded from Lactuca and treated as a new genus. In Chapter 3, twenty-seven wild Lactuca species and four outgroup species were sequenced using next generation sequencing (NGS) technology. The sampling covers 36% of total Lactuca species and all the important geographical groups in the genus. Thirty chloroplast genomes, including one complete (partial) large single copy region (LSC), one small single copy region (SSC), one inverted repeat (IR) region, and twenty-nine nuclear ribosomal DNA sequences (containing the internal transcribed spacer region ) were successfully assembled and analysed. A methodology paper for which I am co-author, but is not included in this thesis, of the sequencing pipeline was published: ‘Herbarium genomics: plastome sequence assembly from a range of herbarium specimens using an Iterative Organelle Genome Assembly (IOGA) pipeline’. These NGS data helped resolve deeper nodes in the phylogeny within Lactuca and resolved the polytomy from Chapter 2. The results showed that there are at least four main groups within Lactuca: the crop group, the Pterocypsela group, the North American group and the group containing widely-distributed species. I also confirmed that the endemic African species should be removed and treated as a new genus. In Chapter 4, quantitative trait loci (QTLs) related to salt-induced changes in Root System Architecture (RSA) and ion accumulation were determined using a recombinant inbred line population derived from a cross between cultivated lettuce and wild lettuce. I measured the components of RSA by replicated lettuce seedlings grown on vertical agar plates with different NaCl concentrations in a controlled growth chamber environment. I also quantified the concentration of sodium and potassium in replicates of greenhouse-grown plants watered with 100 mM NaCl. The results identified a total of fourteen QTLs using multi-trait linkage analysis, including three major QTLs associated with general root development (qRC9.1), root growth in salt stress condition (qRS2.1), and ion accumulation (qLS7.2). In Chapter 5, one of the identified QTL regions (qLS7.2) reported in Chapter 4 was found to contain a homolog of the HKT1 from Arabidopsis thaliana. I did a phylogenetic analysis of Lactuca HKT1-like protein sequences with other published HKT protein sequences and determined transmembrane and pore segments of lettuce HKT1;1 alleles, according to the model proposed for AtHKT1;1. Gene expression pattern and level of LsaHKT1;1 (L. sativa ‘Salinas’) and LseHKT1;1 (L. serriola) in root and shoot were investigated in plants growing hydroponically over a time-course. The measurements of Na+ and K+ contents were sampled at the same time as the samples used for gene expression test. In addition, I examined the 5’ promoter regions of the two genotypes. The results showed low expression levels of both HKT1;1 alleles in Lactuca root and relatively higher expression in shoot, probably due to the negative cis-regulatory elements of HKT1 alleles found in Lactuca promoter regions. Significant allelic differences were found in HKT1;1 expression in early stage (0-24 hours) shoots in and in late stage (2-6 days) roots. shoot HKT1;1 expression/root HKT1;1 expression was generally consistent with the ratios of Na+/K+ balance in the relevant tissues (shoot Na+/K+ divided by root Na+/K+). In Chapter 6, I summarize and discuss the results from previous chapters briefly. The implications of Chapter 2 and 3 for Lactuca phylogenetics are discussed, including some key characters for the diagnosis of species within Lactuca, the use of herbarium DNA for NGS technology, and perspectives into Lactuca phylogeny. Future perspectives of genome-wide association mapping for lettuce breeding were also discussed. Lastly, I propose to integrate phylogenetic approaches into investigations of allelic differences in lettuce, not just associated with salinity stress but also with other stressed and beneficial characters, both within and between species.

Genomics 4.0 - Syntenic Gene and Genome Duplication Drives Diversification of Plant Secondary Metabolism and Innate Immunity in Flowering Plants Johannes A. Hofberger1, 2, 3 1 Biosystematics Group, Wageningen University & Research Center, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands (August 2012 – December 2013) 2 Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands (December 2010 – July 2012) 3 Chinese Academy of Sciences/Max Planck Partner Institute for Computational Biology, 320 Yueyang Road, Shanghai 200031, PR China (January 2014 – December 2014) TWO-SENTENCE SUMMARY Large-scale comparative analysis of Big Data from next generation sequencing provides powerful means to exploit the potential of nature in context of plant breeding and biotechnology. In this thesis, we combine various computational methods for genome-wide identification of gene families involved in (a) plant innate immunity and (a) biosynthesis of defense-related plant secondary metabolites across 21 species, assess dynamics that affected evolution of underlying traits during 250 Million Years of flowering plant radiation and provide data on more than 4500 loci that can underpin crop improvement for future food and live quality. GENERAL ABSTRACT As sessile organisms, plants are permanently exposed to a plethora of potentially harmful microbes and other pests. The surprising resilience to infections observed in successful lineages is due to a complex defense network fighting off invading pathogens. Within this network, a sophisticated plant innate immune system is accompanied by a multitude of specialized biosynthetic pathways that generate more than 200,000 secondary metabolites with ecological, agricultural, energy and medicinal importance. The rapid diversification of associated genes was accompanied by a series of duplication events in virtually all plant species, including local duplication of short sequences as well as multiplication of all chromosomes due to meiotic errors (plant polyploidy). In a comparative genomics approach, we combined several bioinformatics techniques for large-scale identification of multi-domain and multi-gene families that are involved in plant innate immunity or defense-related secondary metabolite pathways across 21 representative flowering plant genomes. We introduced a framework to trace back duplicate gene copies to distinct ancient duplication events, thereby unravelling a differential impact of gene and genome duplication to molecular evolution of target genes. Comparing the genomic context among homologs within and between species in a phylogenomics perspective, we discovered orthologs conserved within genomic regions that remained structurally immobile during flowering plant radiation. In summary, we described a complex interplay of gene and genome duplication that increased genetic versatility of disease resistance and secondary metabolite pathways, thereby expanding the playground for functional diversification and thus plant trait innovation and success. Our findings give fascinating insights to evolution across lineages and can underpin crop improvement for food, fiber and biofuels production

Summary All plants are able to respond to changes in their environment by adjusting their morphology and metabolism, but large differences are observed in the effectiveness of these responses in the light of plant fitness. Between and within species large differences are observed in plant responses to drought, heat and other abiotic stresses. This natural variation is partly due to variation in the genetic composition of individuals. Within-species variation can be used to identify and study genes involved in the genetic regulation of plant performance. Growth of the world population will, in the coming years, lead to an increased demand for food, feed and other natural products. In addition, extreme weather conditions with, amongst others, more and prolonged periods of drought and heat are expected to occur due to climate change. Therefore breeders are challenged to produce stress tolerant cultivars with improved yield under sub-optimal conditions. Knowledge about the mechanisms and genes that underlie tolerance to drought, heat and other abiotic stresses will ease this challenge. The aim of this thesis was to identify and study the role of genes that are underlying natural variation in plant performance under drought, salt and heat stress. To reach this goal a genome wide association (GWA) mapping approach was taken in the model species Arabidopsis thaliana. A population of 350 natural accessions of Arabidopsis, genotyped with 215k SNPs, was grown under control and several stress conditions and plant performance was evaluated by phenotyping one or several plant traits per environment. Genes located in the genomic regions that were significantly associated with plant performance, were studied in more detail. Plant performance was first evaluated upon osmotic stress (Chapter 2). This treatment resulted not only in a reduced plant size, but also caused the colour of the rosette leaves to change from green to purple-red due to anthocyanin accumulation. The latter was visually quantified and subsequent GWA mapping revealed that a large part of the variation in anthocyanin accumulation could be explained by a small genomic region on chromosome 1. The analysis of re-sequence data allowed us to associate the second most frequent allele of MYB90 with higher anthocyanin accumulation and to identify the causal SNP. Interestingly MYB75, a close relative of MYB90, was not identified by GWA mapping, although causal sequence variation of this gene for anthocyanin accumulation was identified in the Cvi x Ler and Ler x Eri-1 RIL populations. Re-sequence data revealed that one allele of MYB75 was dominating the population and that the MYB75 alleles of Cvi and Ler were both rare, explaining the lack of association at this locus in GWA mapping. For MYB90, two alleles were present in a substantial part of the population, suggesting balancing selection between them. Next, the natural population was exposed to short-term heat stress during flowering (Chapter 3). This short-term stress has a large impact on seed set, while it hardly affects the vegetative tissues. Natural variation for tolerance against the effect of heat on seed set was evaluated by measuring the length of all siliques along the inflorescence in both heat-treated and control plants. Because the flower that opened during the treatment was tagged, we could analyse the heat response for several developmental stages separately. GWA mapping revealed that the heat response before and after anthesis involved different genes. For the heat response before anthesis strong evidence was gained that FLC, a flowering time regulator and QUL2, a gene suggested to play a role in vascular tissue development, were causal for two strong associations. Furthermore, the impact of moderate drought on plant performance was evaluated in the plant phenotyping platform PHENOPSIS. Homogeneous drought was assured by tight regulation of climate cell conditions and the robotic weighing and watering of the pots twice a day. Because plant growth is a dynamic trait it was monitored over time by top-view imaging under both moderate drought and control conditions (Chapter 4 and 5). To characterise growth it was modelled with an exponential function. GWA mapping of temporal growth data resulted in the detection of time-dependent QTLs whereas mapping of model parameters resulted in another set of QTLs related to the entire growth period. Most of these QTLs would not have been identified if plant size had only been determined on a single day. For the QTLs detected under control conditions eight candidate genes with a growth-related mutant or overexpression phenotype were identified (Chapter 4). Genes in the support window of the drought-QTLs were prioritized based on previously reported gene expression data (Chapter 5). Additional validation experiments are needed to confirm causality of the candidate genes. Next, to search for genes that determine plant size across many environments, biomass accumulation in the natural population was determined in 25 different environments (Chapter 6). Joint analysis of these data by multi-environment GWA mapping resulted in the detection of 106 strongly associated SNPs with significant effects in 7 to 16 environments. Several genes involved in starch metabolism, leaf size control and flowering time determination were located in close proximity of the associated SNPs. Two genes, RPM1 and ACD6, were located in close proximity of SNPs with significant GxE effects. For both genes, alleles have been identified that increase resistance to bacterial infection, but that reduce biomass accumulation. The sign of the allelic effect is therefore dependent on the environmental conditions. Whole genome predictions revealed that most of the GxE interactions observed at the phenotypic level were not the consequence of strong associations with strong QxE effects, but of moderate and weak associations with weak QxE effects. Finally, in Chapter 7 I discuss the usefulness of GWA mapping in the identification of genes underlying natural variation in plant performance under drought, heat stress and a number of other environments. Strong associations were observed for both environment-specific as well as common plant performance regulators. Some choices in phenotyping and experimental design were crucial for our success, like evaluation of plant performance over time and simplification of the quantification of the phenotype. It is suggested that follow-up work should focus on the functional characterization of the causal genes, because such analyses would be helpful to identify pathways in which the causal genes are involved and to understand why sequence variation results in changes at the phenotype level. Although translation of the findings to applications in crops is challenging, this thesis contributes to the understanding of the genetic regulation of stress response and therefore will likely contribute to the development of stress tolerant and stable yielding crops.

Summary All plants are able to respond to changes in their environment by adjusting their morphology and metabolism, but large differences are observed in the effectiveness of these responses in the light of plant fitness. Between and within species large differences are observed in plant responses to drought, heat and other abiotic stresses. This natural variation is partly due to variation in the genetic composition of individuals. Within-species variation can be used to identify and study genes involved in the genetic regulation of plant performance. Growth of the world population will, in the coming years, lead to an increased demand for food, feed and other natural products. In addition, extreme weather conditions with, amongst others, more and prolonged periods of drought and heat are expected to occur due to climate change. Therefore breeders are challenged to produce stress tolerant cultivars with improved yield under sub-optimal conditions. Knowledge about the mechanisms and genes that underlie tolerance to drought, heat and other abiotic stresses will ease this challenge. The aim of this thesis was to identify and study the role of genes that are underlying natural variation in plant performance under drought, salt and heat stress. To reach this goal a genome wide association (GWA) mapping approach was taken in the model species Arabidopsis thaliana. A population of 350 natural accessions of Arabidopsis, genotyped with 215k SNPs, was grown under control and several stress conditions and plant performance was evaluated by phenotyping one or several plant traits per environment. Genes located in the genomic regions that were significantly associated with plant performance, were studied in more detail. Plant performance was first evaluated upon osmotic stress (Chapter 2). This treatment resulted not only in a reduced plant size, but also caused the colour of the rosette leaves to change from green to purple-red due to anthocyanin accumulation. The latter was visually quantified and subsequent GWA mapping revealed that a large part of the variation in anthocyanin accumulation could be explained by a small genomic region on chromosome 1. The analysis of re-sequence data allowed us to associate the second most frequent allele of MYB90 with higher anthocyanin accumulation and to identify the causal SNP. Interestingly MYB75, a close relative of MYB90, was not identified by GWA mapping, although causal sequence variation of this gene for anthocyanin accumulation was identified in the Cvi x Ler and Ler x Eri-1 RIL populations. Re-sequence data revealed that one allele of MYB75 was dominating the population and that the MYB75 alleles of Cvi and Ler were both rare, explaining the lack of association at this locus in GWA mapping. For MYB90, two alleles were present in a substantial part of the population, suggesting balancing selection between them. Next, the natural population was exposed to short-term heat stress during flowering (Chapter 3). This short-term stress has a large impact on seed set, while it hardly affects the vegetative tissues. Natural variation for tolerance against the effect of heat on seed set was evaluated by measuring the length of all siliques along the inflorescence in both heat-treated and control plants. Because the flower that opened during the treatment was tagged, we could analyse the heat response for several developmental stages separately. GWA mapping revealed that the heat response before and after anthesis involved different genes. For the heat response before anthesis strong evidence was gained that FLC, a flowering time regulator and QUL2, a gene suggested to play a role in vascular tissue development, were causal for two strong associations. Furthermore, the impact of moderate drought on plant performance was evaluated in the plant phenotyping platform PHENOPSIS. Homogeneous drought was assured by tight regulation of climate cell conditions and the robotic weighing and watering of the pots twice a day. Because plant growth is a dynamic trait it was monitored over time by top-view imaging under both moderate drought and control conditions (Chapter 4 and 5). To characterise growth it was modelled with an exponential function. GWA mapping of temporal growth data resulted in the detection of time-dependent QTLs whereas mapping of model parameters resulted in another set of QTLs related to the entire growth period. Most of these QTLs would not have been identified if plant size had only been determined on a single day. For the QTLs detected under control conditions eight candidate genes with a growth-related mutant or overexpression phenotype were identified (Chapter 4). Genes in the support window of the drought-QTLs were prioritized based on previously reported gene expression data (Chapter 5). Additional validation experiments are needed to confirm causality of the candidate genes. Next, to search for genes that determine plant size across many environments, biomass accumulation in the natural population was determined in 25 different environments (Chapter 6). Joint analysis of these data by multi-environment GWA mapping resulted in the detection of 106 strongly associated SNPs with significant effects in 7 to 16 environments. Several genes involved in starch metabolism, leaf size control and flowering time determination were located in close proximity of the associated SNPs. Two genes, RPM1 and ACD6, were located in close proximity of SNPs with significant GxE effects. For both genes, alleles have been identified that increase resistance to bacterial infection, but that reduce biomass accumulation. The sign of the allelic effect is therefore dependent on the environmental conditions. Whole genome predictions revealed that most of the GxE interactions observed at the phenotypic level were not the consequence of strong associations with strong QxE effects, but of moderate and weak associations with weak QxE effects. Finally, in Chapter 7 I discuss the usefulness of GWA mapping in the identification of genes underlying natural variation in plant performance under drought, heat stress and a number of other environments. Strong associations were observed for both environment-specific as well as common plant performance regulators. Some choices in phenotyping and experimental design were crucial for our success, like evaluation of plant performance over time and simplification of the quantification of the phenotype. It is suggested that follow-up work should focus on the functional characterization of the causal genes, because such analyses would be helpful to identify pathways in which the causal genes are involved and to understand why sequence variation results in changes at the phenotype level. Although translation of the findings to applications in crops is challenging, this thesis contributes to the understanding of the genetic regulation of stress response and therefore will likely contribute to the development of stress tolerant and stable yielding crops.

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.

The evolution of rhizobium symbiosis is studied from several points of view in this thesis. The ultimate goal of the combined approaches is to unravel the genetic constrains of the symbiotic interaction. To this end the legume rhizobium symbiosis is studied in model plant species from the Papilionoideae subfamily such as Medicago truncatula and Lotus japonicus. In these model plants the genetic signaling cascade used for rhizobium symbiosis has been largely unraveled. The cascade is triggered by lipo-chitooligosaccharade-based signal molecules excreted by rhizobia, called Nod factors. In chapter 2 we make use of a whole genome duplication that has occurred at the root of the legume Papilionoideae subfamily to identify maintained paralogous gene pairs. We hypothesized that a substantial fraction of gene pairs which are maintained in distinct Papilionoideae lineages that split roughly 54 million years ago fulfill legume specific functions, among which is rhizobium symbiosis. Furthermore we argue that such approach could identify novel genes as it can also identify genes pairs that are (partially) redundant in function. With applying this approach specifically to the cytokinin phosphorelay pathway we identified a pair of type-A cytokinin Response Regulators that are involved in rhizobium symbiosis. This study provides a proof-of-principle for this strategy. It is known for over fifty years that cytokinin plays an important role in the symbiotic interaction between rhizobia and legume hosts. External application of cytokinin can even result in nodule formation. Only, never had cytokinin levels been quantified in legume root extracts upon symbiotic interaction. In chapter 3 we describe a method for extraction of both cytokinins and auxin from Medicago truncatula roots. We show that cytokinins accumulate in the root zone susceptible to symbiotic interaction upon Nod factor exposure and that this response is dependent on CCaMK; a key gene of the Nod factor signaling cascade. Furthermore, it was found that ethylene signaling has a negative effect on Nod factor induced cytokinin accumulation. The method set up to measure cytokinin as well as auxin provides a tool to further study hormone interactions in rhizobium symbiosis. Parasponia,the only non-legume that can engage the rhizobium symbiosis is also subject of study in this thesis. The genetics of the Parasponia-rhizobium symbiosis had not been studied before. It was therefore unknown whether this independently evolved rhizobium symbiosis makes use of the same symbiotic signaling cascade as legumes. In chapter 4 we provide first evidence that Parasponia indeed makes use of the same signaling cascade as found in legumes. Furthermore, we show that in Parasponia a single Nod factor-like receptor is indispensable for two symbiotic interactions; rhizobium and mycorrhiza, respectively. Therefore we conclude that the rhizobium Nod factor perception mechanism is recruited from the widespread endomycorrhizal symbiosis. Parallel to our studies in Parasponia (Chapter 4), the research team of Jean Dénarié of the French National Institute for Agricultural Research (INRA) published the structure of the signal molecule of the arbuscular endomycorrhizae; theMyc factor (Maillet et al., 2011, Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature, 469, 58-63). It appeared that Myc factors and Nod factors are structurally very similar. In chapter 5 we discuss these findings and present a more thorough phylogenetic analysis of the NFP-like LysM-type receptor kinases. Together, these results suggest that non-legumes that can engage an arbuscular endomycorrhizaesymbiosis can recognize Nod factor-like molecules as well. The last chapter is about a study on the promiscuity and effectiveness of the Parasponia-rhizobium symbiosis. Parasponia uses a single receptor to control entry of rhizobium as well as arbuscular endomycorrhizal fungi and has evolved the rhizobium symbiosis only recently. This made us to hypothesize that Parasponia Nod factor receptors did not coevolve yet with rhizobia and therefore did not diverge from mycorrhizal recognition to develop specificity for the Nod factor. This implies that Parasponia could be a very promiscuous host for rhizobium species. In chapter 6 we describe that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to 4 different genera, and therefore it is concluded that Parasponia is highly promiscuous for rhizobial engagement. There is a drawback to this high symbiotic promiscuity. Among the strains identified to nodulate Parasponia, a very inefficient rhizobium species, Rhizobium tropici WUR1, was characterized. As this species is able to make effective nodules on two different legume species it suggests that the ineffectiveness of Parasponia andersonii nodules is the result of the incompatibility between both partners. In Parasponia andersonii nodules rhizobia of the ineffective strain become embedded in a dense matrix, but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia. Therefore we argue that the Parasponia-Rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization. Parasponiahas been given little attention in the rhizobium symbiosis field over the past two decades but with our efforts renewed interest has been established. We believe that in the end, the comparison of Parasponia to its closest related non-symbiotic sister species Trema, will result in the determination of the genetic constrains of rhizobium symbiosis.

The evolution of rhizobium symbiosis is studied from several points of view in this thesis. The ultimate goal of the combined approaches is to unravel the genetic constrains of the symbiotic interaction. To this end the legume rhizobium symbiosis is studied in model plant species from the Papilionoideae subfamily such as Medicago truncatula and Lotus japonicus. In these model plants the genetic signaling cascade used for rhizobium symbiosis has been largely unraveled. The cascade is triggered by lipo-chitooligosaccharade-based signal molecules excreted by rhizobia, called Nod factors. In chapter 2 we make use of a whole genome duplication that has occurred at the root of the legume Papilionoideae subfamily to identify maintained paralogous gene pairs. We hypothesized that a substantial fraction of gene pairs which are maintained in distinct Papilionoideae lineages that split roughly 54 million years ago fulfill legume specific functions, among which is rhizobium symbiosis. Furthermore we argue that such approach could identify novel genes as it can also identify genes pairs that are (partially) redundant in function. With applying this approach specifically to the cytokinin phosphorelay pathway we identified a pair of type-A cytokinin Response Regulators that are involved in rhizobium symbiosis. This study provides a proof-of-principle for this strategy. It is known for over fifty years that cytokinin plays an important role in the symbiotic interaction between rhizobia and legume hosts. External application of cytokinin can even result in nodule formation. Only, never had cytokinin levels been quantified in legume root extracts upon symbiotic interaction. In chapter 3 we describe a method for extraction of both cytokinins and auxin from Medicago truncatula roots. We show that cytokinins accumulate in the root zone susceptible to symbiotic interaction upon Nod factor exposure and that this response is dependent on CCaMK; a key gene of the Nod factor signaling cascade. Furthermore, it was found that ethylene signaling has a negative effect on Nod factor induced cytokinin accumulation. The method set up to measure cytokinin as well as auxin provides a tool to further study hormone interactions in rhizobium symbiosis. Parasponia,the only non-legume that can engage the rhizobium symbiosis is also subject of study in this thesis. The genetics of the Parasponia-rhizobium symbiosis had not been studied before. It was therefore unknown whether this independently evolved rhizobium symbiosis makes use of the same symbiotic signaling cascade as legumes. In chapter 4 we provide first evidence that Parasponia indeed makes use of the same signaling cascade as found in legumes. Furthermore, we show that in Parasponia a single Nod factor-like receptor is indispensable for two symbiotic interactions; rhizobium and mycorrhiza, respectively. Therefore we conclude that the rhizobium Nod factor perception mechanism is recruited from the widespread endomycorrhizal symbiosis. Parallel to our studies in Parasponia (Chapter 4), the research team of Jean Dénarié of the French National Institute for Agricultural Research (INRA) published the structure of the signal molecule of the arbuscular endomycorrhizae; theMyc factor (Maillet et al., 2011, Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature, 469, 58-63). It appeared that Myc factors and Nod factors are structurally very similar. In chapter 5 we discuss these findings and present a more thorough phylogenetic analysis of the NFP-like LysM-type receptor kinases. Together, these results suggest that non-legumes that can engage an arbuscular endomycorrhizaesymbiosis can recognize Nod factor-like molecules as well. The last chapter is about a study on the promiscuity and effectiveness of the Parasponia-rhizobium symbiosis. Parasponia uses a single receptor to control entry of rhizobium as well as arbuscular endomycorrhizal fungi and has evolved the rhizobium symbiosis only recently. This made us to hypothesize that Parasponia Nod factor receptors did not coevolve yet with rhizobia and therefore did not diverge from mycorrhizal recognition to develop specificity for the Nod factor. This implies that Parasponia could be a very promiscuous host for rhizobium species. In chapter 6 we describe that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to 4 different genera, and therefore it is concluded that Parasponia is highly promiscuous for rhizobial engagement. There is a drawback to this high symbiotic promiscuity. Among the strains identified to nodulate Parasponia, a very inefficient rhizobium species, Rhizobium tropici WUR1, was characterized. As this species is able to make effective nodules on two different legume species it suggests that the ineffectiveness of Parasponia andersonii nodules is the result of the incompatibility between both partners. In Parasponia andersonii nodules rhizobia of the ineffective strain become embedded in a dense matrix, but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia. Therefore we argue that the Parasponia-Rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization. Parasponiahas been given little attention in the rhizobium symbiosis field over the past two decades but with our efforts renewed interest has been established. We believe that in the end, the comparison of Parasponia to its closest related non-symbiotic sister species Trema, will result in the determination of the genetic constrains of rhizobium symbiosis.

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.

Phytophthora infestans – the causal agent of potato late blight – secretes a plethora of effector proteins to facilitate plant infection. The central subject of this thesis is ipiO, one of the first cloned Phytophthora genes with a putative function in pathogenicity as was anticipated based on its in planta induced (ipi) expression, in particular during early stages of host infection. IPI-O contains two striking motifs: RXLR-dEER and RGD. RGD is a cell adhesion motif and was shown to be involved in binding to the extracellular lectin domain of LecRK-I.9, a lectin receptor kinase of Arabidopsis. The RXLR-dEER motif plays a role in effector trafficking into host cells and is shared by several secreted oomycete effector proteins which are known to function as race-specific avirulence (Avr) factors. In a previous study, that was aimed at identifying novel pairs of P. infestans Avr and host plant resistance (R) genes, a high-throughput effector genomics screen identified ipiO as Avr-blb1, the counterpart of the late blight R gene Rpi-blb1 which originates from the nightshade Solanum bulbocastanum. Often R genes exploited in late blight resistance breeding become rapidly ineffective as a result of adaptation of P. infestans. However, unlike most late blight R genes that interact in a gene-for-gene manner with Avr genes, Rpi-blb1 seemed to have the potential to remain its effectiveness. In section 2 we monitored the genetic variation and distribution of the ipiO family in an extensive isolate collection of P. infestans and closely related species. This resulted in the identification of 16 IPI-O variants that could be sub-divided in three distinct classes. Variants from class I and class II were shown to induce cell death when co-infiltrated with Rpi-blb1 in Nicotiana benthamiana. Class III consists solely of the highly divergent variant IPI-O4, that is not able to trigger Rpi-blb1-mediated cell death. Class I is highly diverse and represented in all P. infestans isolates analyzed so far, except in two Mexican P. infestans isolates. The latter two are capable to infect Rpi-blb1 plants, suggesting that the lack of class I variants in the genome of these strains allows them to escape recognition by Rpi-blb1 plants. We propose that profiling of the ipiO variants within P. infestans populations can predict the effectiveness of Rpi-blb1-mediated resistance in potato and, as such, can facilitate integrated disease management. Section 3 of this thesis deals with legume-like lectin receptor kinases (LecRKs), membrane-spanning proteins with potential roles in adaptive responses and cell wall integrity. We present an inventory and a phylogenetic analysis of the Arabidopsis LecRK gene family. The rationale behind this study was to gain better insight into the diversity of LecRKs and their potential roles in plant defense. A comprehensive expression analysis based on exploration of existing databases revealed that several LecRK genes are induced upon treatment with elicitors or during pathogen infection. Based on the phylogenetic analysis we have reclassified the LecRK genes and proposed a new nomenclature. LecRK-I.9, one of the clade I Arabidopsis LecRKs which binds the RGD cell adhesion motif of IPI-O, was shown to mediate adhesion between the cell wall (CW) and plasma membrane (PM). In contrast, IPI-O disrupts these adhesions by virtue of its RGD motif. We analyzed Arabidopsis LecRK-I.9 knock-out lines (lecrk-I.9) for their response to pathogen infection, in particular to Phytophthora brassicae. We also analyzed transgenic Arabidopsis lines expressing ipiO, and observed that both the ipiO-expressing lines and lecrk-I.9 lines are impaired in their resistance to oomycete pathogens. To unravel the mechanisms underlying this phenomenon we analysed callose deposition upon MAMP (i.e. flg22) treatment and investigated the strength of CW-PM adhesions under plasmolysis-inducing conditions. The results indicated that LecRK-I.9 is not only important for the maintenance of the CW-PM continuum, but also in MAMP-triggered immunity. Also here, both the ipiO-expressing lines and the lecrk-I.9 knock-outs displayed a destabilized CW-PM continuum and impaired callose deposition, and hence, they can be regarded as phenocopies. Arabidopsis plants that constitutively express LecRK-I.9 were smaller in size, and displayed increased levels of anthocyanin and lignin. Additionally, these lines were shown to exhibit enhanced resistance to P. brassicae. Furthermore, we studied transgenic potatoes that constitutively Arabidopsis LecRK-I.9. In comparison to the parental control potato line the transgenic lines were less susceptible to mild and moderately aggressive P. infestans isolates, but the increased tolerance was not sufficient to provide resistance to aggressive isolates. These results strongly suggest that LecRK-I.9 is a novel resistance component that plays a role in defense against Phytophthora. In Section 4 we describe a novel method for propagating P. brassicae zoospores on an intermediate host plant. This resulted in the production of high numbers of zoospores thereby facilitating highly reproducible small and large scale inoculation experiments. This thesis is completed with a general discussion (Section 5) addressing the current understanding of effector uptake by host cells, the subsequent recognition by cognate R proteins mediating effector-triggered immunity, and RXLR-dEER effector diversity. We also discuss the role of the RGD motif in effectors of both animal and plant pathogens, and the potential functions of LecRKs. Finally, we high-light the advantages of Arabidopsis-Phytophthora pathosystems as research object.

Phytophthora infestans – the causal agent of potato late blight – secretes a plethora of effector proteins to facilitate plant infection. The central subject of this thesis is ipiO, one of the first cloned Phytophthora genes with a putative function in pathogenicity as was anticipated based on its in planta induced (ipi) expression, in particular during early stages of host infection. IPI-O contains two striking motifs: RXLR-dEER and RGD. RGD is a cell adhesion motif and was shown to be involved in binding to the extracellular lectin domain of LecRK-I.9, a lectin receptor kinase of Arabidopsis. The RXLR-dEER motif plays a role in effector trafficking into host cells and is shared by several secreted oomycete effector proteins which are known to function as race-specific avirulence (Avr) factors. In a previous study, that was aimed at identifying novel pairs of P. infestans Avr and host plant resistance (R) genes, a high-throughput effector genomics screen identified ipiO as Avr-blb1, the counterpart of the late blight R gene Rpi-blb1 which originates from the nightshade Solanum bulbocastanum. Often R genes exploited in late blight resistance breeding become rapidly ineffective as a result of adaptation of P. infestans. However, unlike most late blight R genes that interact in a gene-for-gene manner with Avr genes, Rpi-blb1 seemed to have the potential to remain its effectiveness. In section 2 we monitored the genetic variation and distribution of the ipiO family in an extensive isolate collection of P. infestans and closely related species. This resulted in the identification of 16 IPI-O variants that could be sub-divided in three distinct classes. Variants from class I and class II were shown to induce cell death when co-infiltrated with Rpi-blb1 in Nicotiana benthamiana. Class III consists solely of the highly divergent variant IPI-O4, that is not able to trigger Rpi-blb1-mediated cell death. Class I is highly diverse and represented in all P. infestans isolates analyzed so far, except in two Mexican P. infestans isolates. The latter two are capable to infect Rpi-blb1 plants, suggesting that the lack of class I variants in the genome of these strains allows them to escape recognition by Rpi-blb1 plants. We propose that profiling of the ipiO variants within P. infestans populations can predict the effectiveness of Rpi-blb1-mediated resistance in potato and, as such, can facilitate integrated disease management. Section 3 of this thesis deals with legume-like lectin receptor kinases (LecRKs), membrane-spanning proteins with potential roles in adaptive responses and cell wall integrity. We present an inventory and a phylogenetic analysis of the Arabidopsis LecRK gene family. The rationale behind this study was to gain better insight into the diversity of LecRKs and their potential roles in plant defense. A comprehensive expression analysis based on exploration of existing databases revealed that several LecRK genes are induced upon treatment with elicitors or during pathogen infection. Based on the phylogenetic analysis we have reclassified the LecRK genes and proposed a new nomenclature. LecRK-I.9, one of the clade I Arabidopsis LecRKs which binds the RGD cell adhesion motif of IPI-O, was shown to mediate adhesion between the cell wall (CW) and plasma membrane (PM). In contrast, IPI-O disrupts these adhesions by virtue of its RGD motif. We analyzed Arabidopsis LecRK-I.9 knock-out lines (lecrk-I.9) for their response to pathogen infection, in particular to Phytophthora brassicae. We also analyzed transgenic Arabidopsis lines expressing ipiO, and observed that both the ipiO-expressing lines and lecrk-I.9 lines are impaired in their resistance to oomycete pathogens. To unravel the mechanisms underlying this phenomenon we analysed callose deposition upon MAMP (i.e. flg22) treatment and investigated the strength of CW-PM adhesions under plasmolysis-inducing conditions. The results indicated that LecRK-I.9 is not only important for the maintenance of the CW-PM continuum, but also in MAMP-triggered immunity. Also here, both the ipiO-expressing lines and the lecrk-I.9 knock-outs displayed a destabilized CW-PM continuum and impaired callose deposition, and hence, they can be regarded as phenocopies. Arabidopsis plants that constitutively express LecRK-I.9 were smaller in size, and displayed increased levels of anthocyanin and lignin. Additionally, these lines were shown to exhibit enhanced resistance to P. brassicae. Furthermore, we studied transgenic potatoes that constitutively Arabidopsis LecRK-I.9. In comparison to the parental control potato line the transgenic lines were less susceptible to mild and moderately aggressive P. infestans isolates, but the increased tolerance was not sufficient to provide resistance to aggressive isolates. These results strongly suggest that LecRK-I.9 is a novel resistance component that plays a role in defense against Phytophthora. In Section 4 we describe a novel method for propagating P. brassicae zoospores on an intermediate host plant. This resulted in the production of high numbers of zoospores thereby facilitating highly reproducible small and large scale inoculation experiments. This thesis is completed with a general discussion (Section 5) addressing the current understanding of effector uptake by host cells, the subsequent recognition by cognate R proteins mediating effector-triggered immunity, and RXLR-dEER effector diversity. We also discuss the role of the RGD motif in effectors of both animal and plant pathogens, and the potential functions of LecRKs. Finally, we high-light the advantages of Arabidopsis-Phytophthora pathosystems as research object.

Apple is generally considered as a healthy food, but 2-3% European people can not eat this fruit because it provokes allergy reaction. Four classes of apple allergen genes have been identified, they are Mal d 1, Mal d 2, Mal d 3 and Mal d 4 . This thesis focuses on the genomic characterization of these candidate genes on the apple genome by linkage mapping through sequence-specific DNA markers.In total, 26 putative Mal d genes, 25 of them were mapped on 8 linkage groups:17 for Mal d 1 , two for Mal d 2 and Mal d 3 , and four for Mal d 4 .As for the patients in northern and central Europe, the major apple allergen is Mal d 1. Genetic analysis demonstrated the Mal d 1 genes on linkage group (Chromosomes) 16 are relevant. Furthermore, a molecular marker linked to low-Mal d 1 allergenic trait was identified.

Apple is generally considered as a healthy food, but 2-3% European people can not eat this fruit because it provokes allergy reaction. Four classes of apple allergen genes have been identified, they are Mal d 1, Mal d 2, Mal d 3 and Mal d 4 . This thesis focuses on the genomic characterization of these candidate genes on the apple genome by linkage mapping through sequence-specific DNA markers.In total, 26 putative Mal d genes, 25 of them were mapped on 8 linkage groups:17 for Mal d 1 , two for Mal d 2 and Mal d 3 , and four for Mal d 4 .As for the patients in northern and central Europe, the major apple allergen is Mal d 1. Genetic analysis demonstrated the Mal d 1 genes on linkage group (Chromosomes) 16 are relevant. Furthermore, a molecular marker linked to low-Mal d 1 allergenic trait was identified.

Lactuca sativa (cultivated lettuce) is the world's most important leafy salad vegetable. Apart from L. sativa , the genus Lactuca contains ca. 75 wild species, potentially useful to improve, for example, taste, texture, and disease resistance of cultivated lettuce. The wild species L. serriola (Prickly Lettuce), L. saligna (Least Lettuce), and L. virosa (Great Lettuce) are commonly used for lettuce improvement.In preliminary experiments, we established that there is a close connection between evolutionary distances of wild species relative to cultivated lettuce, and their position in the lettuce gene pool (i.e., the possibility to hybridize them with cultivated lettuce). In the present thesis, we established evolutionary relationships among L. sativa and 22 wild species in order to predict this position.We determined that L. sativa , L. serriola , L. dregeana , and L. altaica are closely related, and can be regarded as conspecific. L. aculeata is closely related to them, but is a distinct species. L. serriola , L. dregeana , L. altaica, and L. aculeata occupy the primary gene pool of cultivated lettuce. They can be easily hybridized with cultivated lettuce, and thus are readily accessible gene sources for lettuce improvement. L. saligna and L. virosa are less closely related to L. sativa , and occupy the secondary gene pool (i.e. hybridization with L. sativa is possible, but difficult). All primary and secondary gene-pool species can be classified in Lactuca sect. Lactuca subsect. Lactuca . We found that all tertiary gene-pool species (hybridization with L. sativa only possible with radical techniques) can be classified in the remaining sections of the genus Lactuca (sections Phaenixopus , Mulgedium, and Lactucopsis ). These sections are the most promising sources of wild species for future improvement of cultivated lettuce. In the experiments, the tertiary gene-pool species were represented by L. viminea , L. tatarica , L. sibirica , and L. quercina . Surprisingly, the species classified in Lactuca sect. Lactuca subsect. Cyanicae are not evolutionary close to cultivated lettuce. They are not part of the lettuce gene pool, and should be excluded from Lactuca .To determine the evolutionary relationships among L. sativa and its wild relatives, we examined the genomes of the species at various levels, which provided additional information on genome evolution. We established, that in general the genome sizes in the group increased during evolution, while the ratio of AT/GC nucleotides decreased. Genome complexity for species with 2C DNA amounts below 8.5 pg was similar, but species with 2C DNA amounts exceeding 8.5 pg had more complex and less similar genomes. The species from the primary gene pool share a common ancestor, but the genomes of L. sativa / L. serriola , L. saligna , and L. virosa , evolved in different directions.The present thesis demonstrates that with the proper combination of techniques, a plant systematic study can provide both practically applicable results and fundamental evolutionary insights, thus bridging the gap between fundamental and applied research.

Lactuca sativa (cultivated lettuce) is the world's most important leafy salad vegetable. Apart from L. sativa , the genus Lactuca contains ca. 75 wild species, potentially useful to improve, for example, taste, texture, and disease resistance of cultivated lettuce. The wild species L. serriola (Prickly Lettuce), L. saligna (Least Lettuce), and L. virosa (Great Lettuce) are commonly used for lettuce improvement.In preliminary experiments, we established that there is a close connection between evolutionary distances of wild species relative to cultivated lettuce, and their position in the lettuce gene pool (i.e., the possibility to hybridize them with cultivated lettuce). In the present thesis, we established evolutionary relationships among L. sativa and 22 wild species in order to predict this position.We determined that L. sativa , L. serriola , L. dregeana , and L. altaica are closely related, and can be regarded as conspecific. L. aculeata is closely related to them, but is a distinct species. L. serriola , L. dregeana , L. altaica, and L. aculeata occupy the primary gene pool of cultivated lettuce. They can be easily hybridized with cultivated lettuce, and thus are readily accessible gene sources for lettuce improvement. L. saligna and L. virosa are less closely related to L. sativa , and occupy the secondary gene pool (i.e. hybridization with L. sativa is possible, but difficult). All primary and secondary gene-pool species can be classified in Lactuca sect. Lactuca subsect. Lactuca . We found that all tertiary gene-pool species (hybridization with L. sativa only possible with radical techniques) can be classified in the remaining sections of the genus Lactuca (sections Phaenixopus , Mulgedium, and Lactucopsis ). These sections are the most promising sources of wild species for future improvement of cultivated lettuce. In the experiments, the tertiary gene-pool species were represented by L. viminea , L. tatarica , L. sibirica , and L. quercina . Surprisingly, the species classified in Lactuca sect. Lactuca subsect. Cyanicae are not evolutionary close to cultivated lettuce. They are not part of the lettuce gene pool, and should be excluded from Lactuca .To determine the evolutionary relationships among L. sativa and its wild relatives, we examined the genomes of the species at various levels, which provided additional information on genome evolution. We established, that in general the genome sizes in the group increased during evolution, while the ratio of AT/GC nucleotides decreased. Genome complexity for species with 2C DNA amounts below 8.5 pg was similar, but species with 2C DNA amounts exceeding 8.5 pg had more complex and less similar genomes. The species from the primary gene pool share a common ancestor, but the genomes of L. sativa / L. serriola , L. saligna , and L. virosa , evolved in different directions.The present thesis demonstrates that with the proper combination of techniques, a plant systematic study can provide both practically applicable results and fundamental evolutionary insights, thus bridging the gap between fundamental and applied research.

To determine the usefulness of M. truncatula as intergenomic vehicle for the positional cloning of pea genes it was studied whether these legumes are microsyntenic. These studies were focused on the pea Sym2 and Nod3 genomic regions. The M. truncatula orthologous genomic regions have been cloned and it was shown that these regions of the two legumes are microsyntenic. Both Sym2 and Nod3 play a key role in the pea- Rhizobium symbiosis, controlling Nod factor-structure dependent infection and autoregulation of nodule number, respectively.A M. truncatula A17 BAC library was screened with a pea marker tightly linked to Sym2 and 11 clones were isolated. These clones formed three different contigs named C1, C2, and C3, which were extended to about twice their original size by chromosome walking resulting in contigs of 300 Kbp, 170 Kbp and 150 Kbp, respectively. Genetic and FISH mapping in M. truncatula revealed that the three contigs map on chromosome 5, and that C1 and C2 are tightly linked while C3 maps at a distance of 9 cM from C1/C2 on the same arm of this chromosome. By a combination of contig physical data and FISH it was estimated that C1 and C2 were separated by a gap of 30-40 Kbp. C1 and C2 were further linked to each other by screening an expanded version of the M. truncatula BAC library with the C1 and C2 contig-end subclones Mtg2511 and Mtg3556, respectively. Consequently, the small gap was closed by a 12 Kbp sequence linking C1/C2 which final size resulted in about 480 Kbp. Eight RFLP markers (including cDNAs and contig subclones) were isolated from C1/C2. Mapping of these markers using pea RILs and introgression lines demonstrated that C1/C2 represents the MedicagoSym2 -orthologous genomic region. Moreover, three markers showed recombinations between their pea homologous sequences and Sym2 , delimitating both the pea Sym2 region in the RILs and introgression lines, and the MedicagoSym2 -orthologous region . The MedicagoSym2 -orthologous region was delimitated to about 350 Kbp of C1/C2. In addition, by using a C2 subclone that encodes for a sequence highly homologous to the LRR-motif of the Cf4 and Cf9 tomato [ Licopersicon esculentum ] disease resistance proteins, a pea cDNA was isolated from a pea root hair library that also contains a LRR-domain highly homologous to that of Cf4 / Cf9 . The isolation of this pea RFLP marker demonstrates the use of M. truncatula as intergenomic positional-cloning vehicle of pea genes located within microsyntenic genomic regions. Furthermore, it was shown that the pea Sym2 -region is rich in Cf4 / Cf9 LRR-like sequences. The cloned C1/C2 Sym2 -orthologues candidates include receptor kinases and LRR-containing ( Cf4 / Cf9 -like, TMV -like) genes. Detailed analysis of 22 sequences from C1/C2 (including all RFLP markers) and the pea Sym2 -containing region showed that 4 of these sequences have homologues in c3, and that they are organized in clusters with a similar linear order in these contigs. This indicates that C1/C2 and C3 probably arose through duplication of a chromosomal segment.By using the RNA differential display in combination with RILs and introgression lines, the tightly linked RFLP markers dd21.5 and Psc2.6 were isolated that are linked to the hypernodulating Nod3 locus and represent the closest markers mapping to the "south" of this locus on pea linkage group I in between Eil2 and Nod3 . These markers were used to identify the M. tuncatula A17 orthologous region with the aim to start the microsynteny-based positional cloning of Nod3 . The M. truncatula A17 BAC library was screened with these two markers. BAC clone 21F22 was isolated that contains the orthologue of dd21.5 . In addition 5 BAC clones were identified that co-hybridize with both dd21.5 and Psc2.6 , demonstrating the existence of M. truncatula genomic regions microsyntenic with the pea genomic region containing these two markers. BAC 21F22 was mapped by FISH on M. truncatula chromosome 4, revealing a local disruption of synteny at the dd21.5 locus between pea linkage group 1 and M. truncatula chromosome 5 that were syntenic for other markers (i.e. the markers isolated from the Sym2 -orthologous region, and the pea Eil2 marker). This finding reveals a chromosomal translocation that took place either in pea or in M. truncatula . However, it is unknown whether this translocation extends beyond the dd21.5 locus and includes the Nod3 locus. In addition to the strong FISH signal given by 21F22 on chromosome 4, weak signals were observed close to the telomeres of chromosome 5 and the position of one of these signals is comparable to the position of the C1/C2 Sym2 -orthologous region. Thus, it is possible that in spite of the translocation, chromosome 5 contains sequences with a low homology with those of the pea linkage group I segment containing dd21.5 . It remains to be determined whether the five microsyntenic BACs map in these chromosome 5 regions or whether they map in chromosome 4 and form a contig with BAC 21F22.The data presented in this thesis set the basis for the microsynteny-based cloning of the Sym2 and Nod3 genes by using M.truncatula as intergenomic positional-cloning vehicle. In addition, the molecular resources generated in this thesis are useful to extend microsynteny studies at the Sym2 and Nod3 regions to other legume (e.g. Lotus japonicus ) and actinorhizal nodule-forming species, and also to non-nodulating related species from the Rosid I clade and non-nodulating less-related species (e.g. Arabidopsis and Brassica species that belong to the Rosid II clade). This will enable the identification of genes within these genomic regions that might be unique to nodule-forming plants engaged in symbiotic nitrogen fixation, representing a predisposition for nodulation at the ancestral root of the Rosid I clade. In other words, these studies will enable to answer one of the most interesting questions of plant biology and comparative genomics: whether the ability of legume and actinorhizal plants to establish a nodular symbioses, is given by unique properties that left their evolutionary "signatures" at the genome level.

To determine the usefulness of M. truncatula as intergenomic vehicle for the positional cloning of pea genes it was studied whether these legumes are microsyntenic. These studies were focused on the pea Sym2 and Nod3 genomic regions. The M. truncatula orthologous genomic regions have been cloned and it was shown that these regions of the two legumes are microsyntenic. Both Sym2 and Nod3 play a key role in the pea- Rhizobium symbiosis, controlling Nod factor-structure dependent infection and autoregulation of nodule number, respectively.A M. truncatula A17 BAC library was screened with a pea marker tightly linked to Sym2 and 11 clones were isolated. These clones formed three different contigs named C1, C2, and C3, which were extended to about twice their original size by chromosome walking resulting in contigs of 300 Kbp, 170 Kbp and 150 Kbp, respectively. Genetic and FISH mapping in M. truncatula revealed that the three contigs map on chromosome 5, and that C1 and C2 are tightly linked while C3 maps at a distance of 9 cM from C1/C2 on the same arm of this chromosome. By a combination of contig physical data and FISH it was estimated that C1 and C2 were separated by a gap of 30-40 Kbp. C1 and C2 were further linked to each other by screening an expanded version of the M. truncatula BAC library with the C1 and C2 contig-end subclones Mtg2511 and Mtg3556, respectively. Consequently, the small gap was closed by a 12 Kbp sequence linking C1/C2 which final size resulted in about 480 Kbp. Eight RFLP markers (including cDNAs and contig subclones) were isolated from C1/C2. Mapping of these markers using pea RILs and introgression lines demonstrated that C1/C2 represents the MedicagoSym2 -orthologous genomic region. Moreover, three markers showed recombinations between their pea homologous sequences and Sym2 , delimitating both the pea Sym2 region in the RILs and introgression lines, and the MedicagoSym2 -orthologous region . The MedicagoSym2 -orthologous region was delimitated to about 350 Kbp of C1/C2. In addition, by using a C2 subclone that encodes for a sequence highly homologous to the LRR-motif of the Cf4 and Cf9 tomato [ Licopersicon esculentum ] disease resistance proteins, a pea cDNA was isolated from a pea root hair library that also contains a LRR-domain highly homologous to that of Cf4 / Cf9 . The isolation of this pea RFLP marker demonstrates the use of M. truncatula as intergenomic positional-cloning vehicle of pea genes located within microsyntenic genomic regions. Furthermore, it was shown that the pea Sym2 -region is rich in Cf4 / Cf9 LRR-like sequences. The cloned C1/C2 Sym2 -orthologues candidates include receptor kinases and LRR-containing ( Cf4 / Cf9 -like, TMV -like) genes. Detailed analysis of 22 sequences from C1/C2 (including all RFLP markers) and the pea Sym2 -containing region showed that 4 of these sequences have homologues in c3, and that they are organized in clusters with a similar linear order in these contigs. This indicates that C1/C2 and C3 probably arose through duplication of a chromosomal segment.By using the RNA differential display in combination with RILs and introgression lines, the tightly linked RFLP markers dd21.5 and Psc2.6 were isolated that are linked to the hypernodulating Nod3 locus and represent the closest markers mapping to the "south" of this locus on pea linkage group I in between Eil2 and Nod3 . These markers were used to identify the M. tuncatula A17 orthologous region with the aim to start the microsynteny-based positional cloning of Nod3 . The M. truncatula A17 BAC library was screened with these two markers. BAC clone 21F22 was isolated that contains the orthologue of dd21.5 . In addition 5 BAC clones were identified that co-hybridize with both dd21.5 and Psc2.6 , demonstrating the existence of M. truncatula genomic regions microsyntenic with the pea genomic region containing these two markers. BAC 21F22 was mapped by FISH on M. truncatula chromosome 4, revealing a local disruption of synteny at the dd21.5 locus between pea linkage group 1 and M. truncatula chromosome 5 that were syntenic for other markers (i.e. the markers isolated from the Sym2 -orthologous region, and the pea Eil2 marker). This finding reveals a chromosomal translocation that took place either in pea or in M. truncatula . However, it is unknown whether this translocation extends beyond the dd21.5 locus and includes the Nod3 locus. In addition to the strong FISH signal given by 21F22 on chromosome 4, weak signals were observed close to the telomeres of chromosome 5 and the position of one of these signals is comparable to the position of the C1/C2 Sym2 -orthologous region. Thus, it is possible that in spite of the translocation, chromosome 5 contains sequences with a low homology with those of the pea linkage group I segment containing dd21.5 . It remains to be determined whether the five microsyntenic BACs map in these chromosome 5 regions or whether they map in chromosome 4 and form a contig with BAC 21F22.The data presented in this thesis set the basis for the microsynteny-based cloning of the Sym2 and Nod3 genes by using M.truncatula as intergenomic positional-cloning vehicle. In addition, the molecular resources generated in this thesis are useful to extend microsynteny studies at the Sym2 and Nod3 regions to other legume (e.g. Lotus japonicus ) and actinorhizal nodule-forming species, and also to non-nodulating related species from the Rosid I clade and non-nodulating less-related species (e.g. Arabidopsis and Brassica species that belong to the Rosid II clade). This will enable the identification of genes within these genomic regions that might be unique to nodule-forming plants engaged in symbiotic nitrogen fixation, representing a predisposition for nodulation at the ancestral root of the Rosid I clade. In other words, these studies will enable to answer one of the most interesting questions of plant biology and comparative genomics: whether the ability of legume and actinorhizal plants to establish a nodular symbioses, is given by unique properties that left their evolutionary "signatures" at the genome level.

The transition from the vegetative to the reproductive phase (flowering initiation) in plants has a complex regulation which is affected by environmental and internal plant factors. The understanding of this process is not only of fundamental interest but could also lead to practical applications. The research into flowering initiation has a long history. The initial emphasis on physiological and biochemical studies led to the identification of different factors that influence flowering time. During the sixties, a genetic approach was initiated in different plant species. In Arabidopsis several late flowering mutants were isolated and genetically and physiologically characterised which revealed a complex regulation of flowering time by different pathways. These are the photoperiodic promotion pathway which promotes flowering under long day light conditions, the vernalisation promotion pathway which promotes flowering by low temperatures and the autonomous promotion pathway which promotes flowering independent of the environment. Due to its favourable genetic and molecular features, research on flowering initiation became focussed on Arabidopsis. Since the beginning of the nineties, several of the genes involved in the different pathways have been cloned, providing more information about the function of these genes in the cell and their relations with each other. Despite this increasing amount of information, the picture is still far from complete.The aim of the work presented in this thesis is to increase our knowledge of flowering time regulation. It focussed on the genetic and molecular characterisation of the semi-dominant mutant fwa , which flowers late under long day light conditions and has been proposed to be part of the photoperiodic promotion pathway. One approach sought to identify additional genes that affect flowering by mutagenesis of the fwa mutant. In addition to three different intragenic revertants of fwa , this screen yielded a novel early flowering mutant.This mutant was named early flowering in short days ( efs ). Its phenotypic characterisation has shown that the main role of the wild-type EFS gene is to delay flowering in plants that have entered the adult vegetative phase, which is considered to be the phase where plants are able to respond to environmental signals in order to flower. Consistent with this, efs mutant plants do not show an early flowering phenotype when grown under environmental conditions that lead to a shortened adult vegetative phase such as long days and vernalisation. To learn more about the role of EFS in relation to other genes involved in flowering initiation, double mutants were isolated. Their characterisation showed that efs is involved in the autonomous promotion pathway. This result, together with the lack of a vernalisation response, suggests that EFS is likely to represent a new element acting at a point close to the convergence of signals from the autonomous promotion pathway and the vernalisation promotion pathway.The main topic of this thesis concerns the map based cloning of the FWA gene. By using plants which have a cross-over between FWA and surrounding markers, the FWA locus could be located in a region of about 60 Kb. Plant transformation experiments with cosmids spanning this region showed that the gene is located in the overlap of two cosmids. This overlap contained only one complete gene that encodes a homeodomain containing transcription factor. The altered expression of this gene in fwa mutants together with DNA mutations in the intragenic revertants of fwa-1 further proved that this gene is FWA .Analysis of FWA revealed several interesting characteristics. Surprisingly, the mutant and wild-type alleles had an identical DNA sequence in the FWA region, excluding DNA mutations in the gene as a cause for the mutant phenotype. Furthermore, two direct repeated sequences were found in the 5' genomic region of FWA . In wild-type plants these repeats were heavily methylated, whereas they were completely un-methylated in the mutant alleles. In contrast to fwa mutant plants, which showed a high expression of FWA at all developmental stages, wild-type plants showed only a low expression of FWA in siliques and germinating seeds. Taken together, these findings suggest that loss of methylation of the FWA repeats in the fwa mutant causes a high level of expression of the gene, leading to a late flowering phenotype. A similar correlation of late flowering, FWA overexpression and hypomethylation of FWA repeats was found in late flowering plants which were derived from the ddm1 hypomethylation mutant. The late flowering phenotype of these plants had previously been mapped to the FWA region. Nevertheless, the correlation between hypomethylation of the FWA repeats and FWA expression was not found in germinating seeds of wild-type plants which showed expression of FWA but methylation of the repeats. Although this expression might come from residual mRNA produced earlier in developing seeds, it is possible that methylation of the repeats does not always prevent expression of FWA . Perhaps a different epigenetic mechanism early in development can induce expression of methylated genes.The correlation of FWA expression with late flowering indicates that FWA is a repressor of flowering. Earlier studies had already shown that FWA does not only play a role in the initiation of flowering but also in flower meristems. However, the FWA transcript was not detected in flower buds or flowers and therefore, FWA might only affect this process when highly expressed in the fwa mutant. Possibly, FWA has no function in flowering initiation of wild-type. It might participate in a seed-specific process, as suggested by its expression in seeds. However, the lack of an obvious phenotype suggests that this role is minor or redundant with other genes.The cloning of FWA revealed that the absence of methylation in the repeating sequences in the 5' region of the FWA gene leads to an enhanced expression in the fwa mutant. However, it did not become clear whether this correlation is direct or indirect. Also the importance of the methylation in wild-type plants is still unclear. It is possible that it has a role in the expression of the gene under specific environmental conditions.The results discussed in this thesis have contributed to the existing knowledge of flowering initiation by the isolation of a mutant at a novel locus and the cloning of a previously known gene which are both involved in this process. In addition, the results suggest a possible role for DNA methylation in gene regulation of Arabidopsis.

The transition from the vegetative to the reproductive phase (flowering initiation) in plants has a complex regulation which is affected by environmental and internal plant factors. The understanding of this process is not only of fundamental interest but could also lead to practical applications. The research into flowering initiation has a long history. The initial emphasis on physiological and biochemical studies led to the identification of different factors that influence flowering time. During the sixties, a genetic approach was initiated in different plant species. In Arabidopsis several late flowering mutants were isolated and genetically and physiologically characterised which revealed a complex regulation of flowering time by different pathways. These are the photoperiodic promotion pathway which promotes flowering under long day light conditions, the vernalisation promotion pathway which promotes flowering by low temperatures and the autonomous promotion pathway which promotes flowering independent of the environment. Due to its favourable genetic and molecular features, research on flowering initiation became focussed on Arabidopsis. Since the beginning of the nineties, several of the genes involved in the different pathways have been cloned, providing more information about the function of these genes in the cell and their relations with each other. Despite this increasing amount of information, the picture is still far from complete.The aim of the work presented in this thesis is to increase our knowledge of flowering time regulation. It focussed on the genetic and molecular characterisation of the semi-dominant mutant fwa , which flowers late under long day light conditions and has been proposed to be part of the photoperiodic promotion pathway. One approach sought to identify additional genes that affect flowering by mutagenesis of the fwa mutant. In addition to three different intragenic revertants of fwa , this screen yielded a novel early flowering mutant.This mutant was named early flowering in short days ( efs ). Its phenotypic characterisation has shown that the main role of the wild-type EFS gene is to delay flowering in plants that have entered the adult vegetative phase, which is considered to be the phase where plants are able to respond to environmental signals in order to flower. Consistent with this, efs mutant plants do not show an early flowering phenotype when grown under environmental conditions that lead to a shortened adult vegetative phase such as long days and vernalisation. To learn more about the role of EFS in relation to other genes involved in flowering initiation, double mutants were isolated. Their characterisation showed that efs is involved in the autonomous promotion pathway. This result, together with the lack of a vernalisation response, suggests that EFS is likely to represent a new element acting at a point close to the convergence of signals from the autonomous promotion pathway and the vernalisation promotion pathway.The main topic of this thesis concerns the map based cloning of the FWA gene. By using plants which have a cross-over between FWA and surrounding markers, the FWA locus could be located in a region of about 60 Kb. Plant transformation experiments with cosmids spanning this region showed that the gene is located in the overlap of two cosmids. This overlap contained only one complete gene that encodes a homeodomain containing transcription factor. The altered expression of this gene in fwa mutants together with DNA mutations in the intragenic revertants of fwa-1 further proved that this gene is FWA .Analysis of FWA revealed several interesting characteristics. Surprisingly, the mutant and wild-type alleles had an identical DNA sequence in the FWA region, excluding DNA mutations in the gene as a cause for the mutant phenotype. Furthermore, two direct repeated sequences were found in the 5' genomic region of FWA . In wild-type plants these repeats were heavily methylated, whereas they were completely un-methylated in the mutant alleles. In contrast to fwa mutant plants, which showed a high expression of FWA at all developmental stages, wild-type plants showed only a low expression of FWA in siliques and germinating seeds. Taken together, these findings suggest that loss of methylation of the FWA repeats in the fwa mutant causes a high level of expression of the gene, leading to a late flowering phenotype. A similar correlation of late flowering, FWA overexpression and hypomethylation of FWA repeats was found in late flowering plants which were derived from the ddm1 hypomethylation mutant. The late flowering phenotype of these plants had previously been mapped to the FWA region. Nevertheless, the correlation between hypomethylation of the FWA repeats and FWA expression was not found in germinating seeds of wild-type plants which showed expression of FWA but methylation of the repeats. Although this expression might come from residual mRNA produced earlier in developing seeds, it is possible that methylation of the repeats does not always prevent expression of FWA . Perhaps a different epigenetic mechanism early in development can induce expression of methylated genes.The correlation of FWA expression with late flowering indicates that FWA is a repressor of flowering. Earlier studies had already shown that FWA does not only play a role in the initiation of flowering but also in flower meristems. However, the FWA transcript was not detected in flower buds or flowers and therefore, FWA might only affect this process when highly expressed in the fwa mutant. Possibly, FWA has no function in flowering initiation of wild-type. It might participate in a seed-specific process, as suggested by its expression in seeds. However, the lack of an obvious phenotype suggests that this role is minor or redundant with other genes.The cloning of FWA revealed that the absence of methylation in the repeating sequences in the 5' region of the FWA gene leads to an enhanced expression in the fwa mutant. However, it did not become clear whether this correlation is direct or indirect. Also the importance of the methylation in wild-type plants is still unclear. It is possible that it has a role in the expression of the gene under specific environmental conditions.The results discussed in this thesis have contributed to the existing knowledge of flowering initiation by the isolation of a mutant at a novel locus and the cloning of a previously known gene which are both involved in this process. In addition, the results suggest a possible role for DNA methylation in gene regulation of Arabidopsis.

The interaction between the fungal pathogen Cladosporium fulvum and tomato has been used as a model system to study the molecular basis of gene-for-gene relationships. C. fulvum is a specialized, biotrophic pathogen, which causes leaf mold on tomato. Under humid conditions conidia of C. fulvum germinate and form runner hyphae on the lower side of the leaf. If no resistance genes of the plant match any of the avirulence genes of the fungus, the interaction is compatible and infection will proceed. However, when both a resistance gene and its matching avirulence gene are present, the plant recognizes the fungus and the interaction is incompatible. In an incompatible interaction active defense responses, including the hypersensitive response (HR) are initiated, which inhibit fungal growth effectively. Avirulence genes encode lace-specific elicitors, which are present in intercellular washing fluids obtained from compatible interactions of C. fulvum and tomato (De Wit and Spikman, 1982). Injection of these intercellular washing fluids in tomato plants resistant to the C. fulvum strain from which the washing fluids were obtained, results in specific necrosis at the site of injection. The race-specific elicitor AVR9 was isolated and purified (Scholtens-Toma and de Wit, 1988). AVR9 specifically induces necrosis on tomato genotypes carrying the Cf-9 resistance gene. The encoding AVR9 gene was isolated, and it was shown that this gene specifically determines avirulence of C. fulvum on tomato plants carrying the Cf-9 resistance gene (Van den Ackerveken et al., 1992; Marmeisse et al., 1993). The Avr9 gene encodes a 63-amino acid pre-proprotein containing one potential glycosylation site (Van den Ackerveken et al., 1993). Different forms of the AVR9 elicitor were found, of which the mature AVR9 elicitor of 28 amino acids is predominantly present in C. fulvum-infected tomato plants (Van den Ackerveken et al., 1993). The global structure of the AVR9 peptide shows 3 antiparallelβ-sheets and 3 disulfide bonds that are arranged in a cystine knot (Vervoort et al., 1997).In the research project described in this thesis, we studied AVR9 elicitor perception in tomato plants that carry the Cf-9 resistance gene and compared the results to those obtained with tomato plants lacking this gene. Previously, several research groups had shown that elicitors are recognized through plants receptors, which are localized on the plasma membrane (summarized in chapter 1). To find and characterize the receptor for AVR9, the peptide was labeled with iodine-125 and binding to tomato membranes was studied, as presented in chapter 2. 125 I-AVR9 showed specific, saturable, and reversiblebinding to plasma membranes isolated from leaves of the tomato cultivar Moneymaker without Cf resistance genes (MM-Cf0) and to membranes from a near-isogenic genotype containing the Cf-9 resistance gene (MM-Cf9). Binding of AVR9 is characterized by high affinity and low receptor concentration, and thus fulfills several criteria expected for functional receptors (Hulme and Birdsall, 1992). The dissociation constant was determined at 0.07 nM, and the receptor concentration was determined at 0.8 pmol/mg microsomal membrane protein. Binding is highly influenced by pH and ionic strength of the binding buffer and by temperature, indicating the involvement of both electrostatic and hydrophobic interactions. Surprisingly, binding kinetics and binding capacity were identical for membranes of the MM-Cf0 and MM-Cf9 tomato genotype, indicating that the Cf-9 resistance gene is not required for binding of AVR9. By that time, the Cf-9 resistance gene was isolated (Jones et al., 1994). Cf-9 belongs to a gene family and homologues of the Cf-9 resistance gene are present in both resistant and susceptible tomato genotypes. Two new hypotheses were developed of which the first predicts that not only the Cf-9 resistance gene, but also homologues of the Cf-9 gene, encode the high-affinity binding site for AVR9. Only the protein encoded by Cf-9 itself, designated CF-9, would subsequently initiate the signal transduction cascade resulting in HR. The second hypothesis predicts that the AVR9 binding site is neither CF- 9 nor a homologue of CF-9. The binding site proposed in the second hypothesis would bind AVR9 and subsequently recruit the CF-9 protein to initiate HR.As described in chapters 3, 4, and 5, experiments were performed to prove or reject one of these two hypotheses. To determine whether the high-affinity binding site for AVR9 is indeed a functional receptor, we studied the correlation between binding affinity and necrosis-inducing activity of mutant AVR9 peptides. We determined structure-activity relationships of the AVR9 peptide by independently substituting each amino acid of AVR9 by alanine, using a site-directed mutagenesis approach. In addition, surfaceexposed amino acid residues of AVR9 were substituted by other amino acids. Activity of mutant Avr9 constructs was studied by expressing the constructs in MM-Cf9 tomato plants using the potato virus X (PVX) expression system, and assessing the severity of necrosis induced by each PVX::Avr9 construct. This allowed direct identification of amino acid residues of AVR9 that are essential for elicitor activity. We identified amino acid substitutions resulting in AVR9 mutants with higher, similar or lower elicitor activity compared to the wild-type AVR9 peptide. Mutants of the amino acid residues Phe21 and Leu24 had completely lost elicitor activity. Necrosis-inducing activity of isolated AVR9 peptides correlated well with the necrosis induced by the corresponding PVX::Avr9 constructs. It was concluded the PVX expression system is ideally suited to analyze necrosis-inducing activity of AVR9 peptides. We analyzed whether there is a correlation between elicitor activity of the mutant AVR9 peptides and their affinity to the binding site in membranes of tomato. Therefore, Nicotiana clevelandii plants were inoculated with a selection of PVX::Avr9 constructs and mutant AVR9 peptides were purified from these plants. In addition, some AVR9 peptides were chemically synthesized. Characterization by Electrospray Mass Spectrometry, Circular Dichroism-, and 'H-NMR- spectroscopy revealed that both the in planta produced and the synthetic mutant peptides were correctly folded. AVR9 peptides purified from PVX::Avr9-infected N. clevelandii contained one N-acetylglucosamine, although small amounts of non- glycosylated AVR9 peptides were also detected. The glycosylated AVR9 peptides showed lower affinity to the binding site than the non-glycosylated AVR9 peptides, whereas they did not differ significantly in necrosis-inducing activity. For both the non- glycosylated and glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membranelocalized binding site and their necrosis-inducing activity in MM-Cf9 tomato was found, i.e. peptides with higher affinity to the binding site showed higher necrosis-inducing activity. This correlation suggested that the characterized high-affinity binding site for AVR9 is indeed a functional receptor that initiates the AVR9- CF-9-dependent HR in MM-Cf9 plants.In chapter 5, we studied whether the Cf-9 resistance gene or (one of) its homologues code for an AVR9 binding site. We tested binding of AVR9 to microsomal membranes of a variety of solanaceous and non-solanaceous plant species and analyzed these species for the presence of Cf-9-homologues. All solanaceous species tested contain homologues of the Cf-9 resistance gene and membranes of these plants contain a highaffinity binding site for AVR9. However, a high affinity binding site for AVR9 is also present on membranes of the non-solanaceous plant species cucumber, barley and oat, which do not contain homologues of the Cf-9 resistance gene. Membranes of tobacco, transgenic for the Cf-9 resistance gene, showed no change in the number of AVR9 binding sites. Arabidopsis does not have a binding site for AVR9 and membranes of Arabidopsis, transgenic: for the Cf-9 resistance gene, also showed no AVR9 binding. From this we concluded that not only the Cf-9 resistance gene, but also its homologues are not required for high-affinity binding of AVR9. Based on the presented data, we have developed a model, explaining recognition of AVR9 in MM-Cf-9 tomato (chapter 6). This model predicts that the high-affinity binding protein either 'presents' the AVR9 elicitor to the Cf-9-encoded protein or that binding of AVR9 induces a conformational change of the high-affinity binding protein. The latter results in recruitment of Cf-9 into the AVR9- receptor complex. Subsequently, signal cascade(s) resulting in HR will be initiated.

The interaction between the fungal pathogen Cladosporium fulvum and tomato has been used as a model system to study the molecular basis of gene-for-gene relationships. C. fulvum is a specialized, biotrophic pathogen, which causes leaf mold on tomato. Under humid conditions conidia of C. fulvum germinate and form runner hyphae on the lower side of the leaf. If no resistance genes of the plant match any of the avirulence genes of the fungus, the interaction is compatible and infection will proceed. However, when both a resistance gene and its matching avirulence gene are present, the plant recognizes the fungus and the interaction is incompatible. In an incompatible interaction active defense responses, including the hypersensitive response (HR) are initiated, which inhibit fungal growth effectively. Avirulence genes encode lace-specific elicitors, which are present in intercellular washing fluids obtained from compatible interactions of C. fulvum and tomato (De Wit and Spikman, 1982). Injection of these intercellular washing fluids in tomato plants resistant to the C. fulvum strain from which the washing fluids were obtained, results in specific necrosis at the site of injection. The race-specific elicitor AVR9 was isolated and purified (Scholtens-Toma and de Wit, 1988). AVR9 specifically induces necrosis on tomato genotypes carrying the Cf-9 resistance gene. The encoding AVR9 gene was isolated, and it was shown that this gene specifically determines avirulence of C. fulvum on tomato plants carrying the Cf-9 resistance gene (Van den Ackerveken et al., 1992; Marmeisse et al., 1993). The Avr9 gene encodes a 63-amino acid pre-proprotein containing one potential glycosylation site (Van den Ackerveken et al., 1993). Different forms of the AVR9 elicitor were found, of which the mature AVR9 elicitor of 28 amino acids is predominantly present in C. fulvum-infected tomato plants (Van den Ackerveken et al., 1993). The global structure of the AVR9 peptide shows 3 antiparallelβ-sheets and 3 disulfide bonds that are arranged in a cystine knot (Vervoort et al., 1997).In the research project described in this thesis, we studied AVR9 elicitor perception in tomato plants that carry the Cf-9 resistance gene and compared the results to those obtained with tomato plants lacking this gene. Previously, several research groups had shown that elicitors are recognized through plants receptors, which are localized on the plasma membrane (summarized in chapter 1). To find and characterize the receptor for AVR9, the peptide was labeled with iodine-125 and binding to tomato membranes was studied, as presented in chapter 2. 125 I-AVR9 showed specific, saturable, and reversiblebinding to plasma membranes isolated from leaves of the tomato cultivar Moneymaker without Cf resistance genes (MM-Cf0) and to membranes from a near-isogenic genotype containing the Cf-9 resistance gene (MM-Cf9). Binding of AVR9 is characterized by high affinity and low receptor concentration, and thus fulfills several criteria expected for functional receptors (Hulme and Birdsall, 1992). The dissociation constant was determined at 0.07 nM, and the receptor concentration was determined at 0.8 pmol/mg microsomal membrane protein. Binding is highly influenced by pH and ionic strength of the binding buffer and by temperature, indicating the involvement of both electrostatic and hydrophobic interactions. Surprisingly, binding kinetics and binding capacity were identical for membranes of the MM-Cf0 and MM-Cf9 tomato genotype, indicating that the Cf-9 resistance gene is not required for binding of AVR9. By that time, the Cf-9 resistance gene was isolated (Jones et al., 1994). Cf-9 belongs to a gene family and homologues of the Cf-9 resistance gene are present in both resistant and susceptible tomato genotypes. Two new hypotheses were developed of which the first predicts that not only the Cf-9 resistance gene, but also homologues of the Cf-9 gene, encode the high-affinity binding site for AVR9. Only the protein encoded by Cf-9 itself, designated CF-9, would subsequently initiate the signal transduction cascade resulting in HR. The second hypothesis predicts that the AVR9 binding site is neither CF- 9 nor a homologue of CF-9. The binding site proposed in the second hypothesis would bind AVR9 and subsequently recruit the CF-9 protein to initiate HR.As described in chapters 3, 4, and 5, experiments were performed to prove or reject one of these two hypotheses. To determine whether the high-affinity binding site for AVR9 is indeed a functional receptor, we studied the correlation between binding affinity and necrosis-inducing activity of mutant AVR9 peptides. We determined structure-activity relationships of the AVR9 peptide by independently substituting each amino acid of AVR9 by alanine, using a site-directed mutagenesis approach. In addition, surfaceexposed amino acid residues of AVR9 were substituted by other amino acids. Activity of mutant Avr9 constructs was studied by expressing the constructs in MM-Cf9 tomato plants using the potato virus X (PVX) expression system, and assessing the severity of necrosis induced by each PVX::Avr9 construct. This allowed direct identification of amino acid residues of AVR9 that are essential for elicitor activity. We identified amino acid substitutions resulting in AVR9 mutants with higher, similar or lower elicitor activity compared to the wild-type AVR9 peptide. Mutants of the amino acid residues Phe21 and Leu24 had completely lost elicitor activity. Necrosis-inducing activity of isolated AVR9 peptides correlated well with the necrosis induced by the corresponding PVX::Avr9 constructs. It was concluded the PVX expression system is ideally suited to analyze necrosis-inducing activity of AVR9 peptides. We analyzed whether there is a correlation between elicitor activity of the mutant AVR9 peptides and their affinity to the binding site in membranes of tomato. Therefore, Nicotiana clevelandii plants were inoculated with a selection of PVX::Avr9 constructs and mutant AVR9 peptides were purified from these plants. In addition, some AVR9 peptides were chemically synthesized. Characterization by Electrospray Mass Spectrometry, Circular Dichroism-, and 'H-NMR- spectroscopy revealed that both the in planta produced and the synthetic mutant peptides were correctly folded. AVR9 peptides purified from PVX::Avr9-infected N. clevelandii contained one N-acetylglucosamine, although small amounts of non- glycosylated AVR9 peptides were also detected. The glycosylated AVR9 peptides showed lower affinity to the binding site than the non-glycosylated AVR9 peptides, whereas they did not differ significantly in necrosis-inducing activity. For both the non- glycosylated and glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membranelocalized binding site and their necrosis-inducing activity in MM-Cf9 tomato was found, i.e. peptides with higher affinity to the binding site showed higher necrosis-inducing activity. This correlation suggested that the characterized high-affinity binding site for AVR9 is indeed a functional receptor that initiates the AVR9- CF-9-dependent HR in MM-Cf9 plants.In chapter 5, we studied whether the Cf-9 resistance gene or (one of) its homologues code for an AVR9 binding site. We tested binding of AVR9 to microsomal membranes of a variety of solanaceous and non-solanaceous plant species and analyzed these species for the presence of Cf-9-homologues. All solanaceous species tested contain homologues of the Cf-9 resistance gene and membranes of these plants contain a highaffinity binding site for AVR9. However, a high affinity binding site for AVR9 is also present on membranes of the non-solanaceous plant species cucumber, barley and oat, which do not contain homologues of the Cf-9 resistance gene. Membranes of tobacco, transgenic for the Cf-9 resistance gene, showed no change in the number of AVR9 binding sites. Arabidopsis does not have a binding site for AVR9 and membranes of Arabidopsis, transgenic: for the Cf-9 resistance gene, also showed no AVR9 binding. From this we concluded that not only the Cf-9 resistance gene, but also its homologues are not required for high-affinity binding of AVR9. Based on the presented data, we have developed a model, explaining recognition of AVR9 in MM-Cf-9 tomato (chapter 6). This model predicts that the high-affinity binding protein either 'presents' the AVR9 elicitor to the Cf-9-encoded protein or that binding of AVR9 induces a conformational change of the high-affinity binding protein. The latter results in recruitment of Cf-9 into the AVR9- receptor complex. Subsequently, signal cascade(s) resulting in HR will be initiated.

Actinorhizal multilobe nodules are induced by actinomycetes of the genus Frankia on the roots of several dicotyledenous species belonging to eight different plant families. Each nodule lobe is a modified lateral root, without a root cap, with a central vascular cylinder, and with infected and uninfected cortical cells.To isolate plant genes involved in the establishment of an actinorhizal symbioses, we have differentially screened an A. glutinosa nodule cDNA library with nodule- and root cDNA, respectively. Several cDNAs, representing genes expressed at elevated levels in nodules compared to roots, as determined by RNA gel blot analysis, were isolated and sequenced. The localization of the corresponding mRNAs in the nodule was examined by in situ hybridization. Whenever necessary, model systems such as yeast and Arabidopsis were used to analyse the functions of the encoded proteins.Two of the isolated cDNA clones corresponded to sucrose synthase and enolase, enzymes involved in carbon metabolism. The corresponding genes were expressed in all plant tissues but at markedly elevated levels in nodules. In situ hybridization showed that in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the vascular bundle.Another cDNA clone, pAgthil, was shown to encode a homolog of yeast Thi4, which is involved in thiazole biosynthesis. The corresponding gene, agthil, was found to be expressed at high levels in nodules and shoot tips of A. glutinosa, while being expressed at low levels in roots, flowers, and developing fruits. In nodules, agthil mRNA was localized in the infected cortical cells and in the pericycle of the nodule vascular system. A homolog of this gene, ara6/tz was identified in Arabidopsis thaliana. ara6/tz maps in a region of chromosome 5 of Arabidopsis containing the tz locus. This is consistent with the observations that ara6/tz transcription was impaired in two out of five Arabidopsis tz mutant lines. ara6/tz is expressed at high levels in chloroplast-containing parenchymatic cells of leaves, inflorescence shoots and flowers of Arabidopsis, and at lower levels in the vascular system. The function of AgThi1 was demonstrated by yeast complementation studies, in which AgThi1 was able to rescue a yeast thi4 mutant.cDNAs encoding glutamine synthetase (GS) and acetylomithine transaminase (AOTA), both involved in nitrogen metabolism, were isolated. GS is the enzyme responsible for ammonium assimilation, while AOTA is involved in the biosynthesis of citrulline, the nitrogen transport form in Alnus. GS mRNA was found in all tissues tested with the highest levels in nodules, where it was present in the infected cells as well as in the cells of the pericycle of the vascular system. AOTA transcripts were detected at very low levels in roots and at high levels in nodules, where it was confined to the infected cells. These data suggested that in A. glutinosa nodules, ammonium assimilation takes place in both the infected cells and in the pericycle of the vascular system, and citrulline biosynthesis occurs mainly in the infected cells. Ammonium assimilation in the pericycle is likely to be related to nitrogen transport.One of the few nodule-specific genes, i.e., genes that are not expressed in roots, ag12, was shown to be expressed in nodules at the highest levels in infected cells before the onset of nitrogen fixation. Sequencing showed that ag12 encodes a serine protease of the subtilisin (EC 3.4.21.14) family. A homolog of ag12, ara12, was identified in Arabidopsis. ara12 was expressed in all organs, with the highest expression levels in the beginning of silique development. To assess the importance of this protease in other actinorhizal symbioses, the expression pattern of its homolog, cg12, was examined in nodules of Casuarina glauca and found to be similar to that of ag12. These results are discussed in view of the phylogenetical relationship of Alnus and Casuarina.

Cassava (Manihot esculenta Crantz) is a tropical crop grown for its starchy thickened roots, mainly by peasant farmers, in the tropics, for whom it is a staple food. There is an increasing demand for the use of cassava in processed food and feed products, and in the paper and textile industries amongst others. This thesis describes research on the cloning of the genes encoding ADP-glucose pyrophosphorylase small and large subunits (AGPase B and S, respectively) and granule bound starch synthase II (GBSSII). These genes and their products were extensively characterised to determine their role in starch biosynthesis in cassava. Functional verification of the genes was carried out by transforming potato and cassava followed by analysis of the starch produced by the transgenic plants.In Chapter 1 cassava production in the world in general and in Zimbabwe in particular is examined against the backdrop of new cloning and transformation strategies to improve starch quality and quantity. The development of cassava cultivars whose starches have novel physico-chemical properties by genetic modification of the process of starch biosynthesis is examined therein. The main criteria for these new cultivars to emerge are set forth as being: the availability of cloned and characterised starch biosynthesis genes, a universally applicable transformation and regeneration procedure for cassava, transfer to appropriate cassava cultivars, and biosafety analysis of transgenic cassava plants before disbursement to farmers.The cloning of the cassava starch biosynthesis genes encoding granule bound starch synthase II (GBSSII) and the large and small subunits of ADP-glucose pyrophosphorylase (AGPase) is described in Chapters 2 and 3. The cloning of GBSSII reveals that there is indeed a second isoform of this enzyme in cassava as in other plants species. While sharing very little amino acid sequence homology with cassava GBSSI the GBSSII isophorm shares high amino acid sequence homology to other GBSSII genes from pea and potato. Cassava GBSSII seems to be more important in leaf tissue where it is more highly expressed than in tuber tissue where GBSSI predominates. Mapping of GBSSII revealed that this is a single copy gene located on the male derived linkage group T of the cassava mapping population.Cloning of the cassava genes coding for the small (B) and large subunit (S) of AGPase revealed interesting aspects about the cassava enzyme. The cassava AGPase is likely to be heterotetrameric in constitution as had been found in other plant species. Comparison of the cassava AGPase sequences with those of already cloned AGPases revealed that AGPase B is more similar to small subunit genes from other plants than to cassava AGPase S coding for the large subunit (Chapter 3). Segregation analysis of a cassava mapping population revealed that AGPase S is a single copy gene that is localised on the female derived linkage group E of the cassava genetic map. Both genes are expressed in all cassava tissues but AGPase B was shown to have a higher steady state mRNA level than AGPase S especially in leaf and tuber tissue. Post-transcriptional control of small subunit polypeptide levels could be inferred from the discrepancy between AGPase B mRNA and polypeptide levels. The AGPase enzyme activity was much higher in young cassava leaves than older leaves and tubers. Cassava leaf AGPase activity was increased 3 fold by the addition of 3-PGA (3-phospho-glycerate) and inhibited by up to 90% in the presence of inorganic phosphate (Pi). The tuber enzyme was relatively unaffected by 3PGA, but was highly inhibited by Pi.In order to verify the biological role of the AGPase B gene antisense constructs were made of the cassava AGPase B behind a CaMV35S promoter (chapter 3). This was transferred into potato plants by Agrobacterium tumefaciens. While the 224 transgenic antisense AGPase B potato plants did not differ in appearance from normal potato plants, 45 transgenic plants, however, had more numerous and smaller tubers than control plants. Antisense plants with reduced AGPase B mRNA levels had 1.5 to 3 times less starch than tubers from the control plants. The levels of the soluble sugars in the antisense plants increased significantly (up to 10 times more glucose, 6 times the amount of fructose, and 5 times the amount of sucrose) when compared to those found in control plants. These results show that a heterologous gene from cassava can have an antisense effect in potato, but that the number of plants required to find plants exhibiting maximum antisense effect has to be very large. This is probably due to sequence homology differences between the cassava AGPase B and potato AGPase B genes which share only 68% amino acid sequence homology.Chapter 5 describes the further development of an efficient, time and labour saving protocol for transforming cassava based on stringent selection of the luciferase (firefly) marker gene. In addition the first reported transformation of cassava with a gene (AGPase B) other than a marker gene is described. An antisense construct was made for transforming cassava. This consisted of the cassava AGPase B gene which was placed in antisense orientation behind the CaMV35S promoter. This was then coupled to the luciferase gene driven by another CaMV35S promoter. After particle bombardment of cassava FEC transgenic tissue was selected using three different selection regimes: non stringent luciferase selection, stringent luciferase selection and combined chemical (phosphinothrycin) and luciferase selection. Stringent luciferase selection whereby luciferase positive FEC units were precisely pinpointed, isolated and cultured was found to be the most effective and time saving method. It was possible to generate cultures having more than 90% luciferase positive FEC tissue after 12 weeks of stringent LUC selection, compared to 45% and

This thesis deals with the genetic aspects of seed development in Arabidopsisthaliana. Mutants affected in several aspects of seed development and, more specifically, in seed maturation have been isolated by various selection procedures. The mutants have been analyzed genetically, physiologically, and morphologically. Some of the mutants are impaired in the biosynthesis or sensitivity to the plant hormone, abscisic acid (ABA). All ABA-related mutants show reduced seed dormancy, indicating the important role of this hormone in the establishment of dormancy. In a direct screen for reduced dormancy, two mutants (rdo) with reduced dormancy were found. These were not ABA-deficient and showed the same sensitivity to ABA, ethylene, auxin, and cytokinin as the wild-type. In contrast to this embryo-determined reduced dormancy, reduced dormancy can also originate in an altered seed coat (testa), like in the altered testa shape ( ats ) mutant. Here, the altered testa shape is caused by a defect in the development of the integuments. Extreme ABA-insensitive mutants ( abi3 ) have green seeds that fail to complete many other aspects of seed maturation, including the induction of dormancy and desiccation tolerance, and the accumulation of seed storage proteins and lipids. In addition to abi3 mutants, lec and fus mutants exhibit such a severely disturbed seed maturation as well, with dark purple seeds due to anthocyanin accumulation. The fus3 mutant shows normal ABA-sensitivity. These various seed maturation mutants indicate that specific genes, some acting dependently and some acting independently from ABA, are responsible for seed maturation programs. The seed maturation mutants were subjected to a physiological and biochemical analysis. A GA-deficient mutant was combined with these mutants. Analysis of these double mutants indicated that seeds of the abi3 and lec mutants did not require GA for germination, in contrast to fus3 seeds. This correlates with ABA-sensitivity for germination. The composition of storage proteins and carbohydrates in abi3 , lec, and fus3 mutant seeds has been compared. The abi3 , lec, and fus3 mutants all showed severely reduced storage proteins. The desiccation intolerance of these seed maturation mutants was not correlated with the lack of specific carbohydrates. Furthermore, the mutants had a higher total content of carbohydrates. This is probably a consequence of the lower levels of storage lipids and proteins.

This thesis describes the selection and evaluation of diploid potato clones (2n=2x=24) that produce unreduced or 2n-gametes with 24 chromosomes instead of the normal reduced n-gametes with 12 chromosomes. To elucidate the modes of origin of the 2n-gametes, the progenies derived from such gametes were analysed for different marker loci of one chromosome. Besides the already known mechanisms of First and Second Division Restitution (FDR and SDR) in potato, the multilocus analysis showed the additional mechanism of Post-Meiotic Restitution (PMR) resulting in completely homozygous 2n-gametes. The FDR and SDR 2n-gametes were used for gene-centromere mapping. Multilocus analysis of SDR 2n-gametes showed the map position of the centromere of chromosome 8 in relation to the RFLP-loci. These genetic analyses also demonstrated the occurrence of a single crossover per chromosome arm (high level of chiasma interference). In the light of these findings, it is possible to construct more critical genetic maps of potato.

Actinorhizal multilobe nodules are induced by actinomycetes of the genus Frankia on the roots of several dicotyledenous species belonging to eight different plant families. Each nodule lobe is a modified lateral root, without a root cap, with a central vascular cylinder, and with infected and uninfected cortical cells.To isolate plant genes involved in the establishment of an actinorhizal symbioses, we have differentially screened an A. glutinosa nodule cDNA library with nodule- and root cDNA, respectively. Several cDNAs, representing genes expressed at elevated levels in nodules compared to roots, as determined by RNA gel blot analysis, were isolated and sequenced. The localization of the corresponding mRNAs in the nodule was examined by in situ hybridization. Whenever necessary, model systems such as yeast and Arabidopsis were used to analyse the functions of the encoded proteins.Two of the isolated cDNA clones corresponded to sucrose synthase and enolase, enzymes involved in carbon metabolism. The corresponding genes were expressed in all plant tissues but at markedly elevated levels in nodules. In situ hybridization showed that in nodules, both sucrose synthase and enolase were expressed at high levels in the infected cortical cells as well as in the pericycle of the vascular bundle.Another cDNA clone, pAgthil, was shown to encode a homolog of yeast Thi4, which is involved in thiazole biosynthesis. The corresponding gene, agthil, was found to be expressed at high levels in nodules and shoot tips of A. glutinosa, while being expressed at low levels in roots, flowers, and developing fruits. In nodules, agthil mRNA was localized in the infected cortical cells and in the pericycle of the nodule vascular system. A homolog of this gene, ara6/tz was identified in Arabidopsis thaliana. ara6/tz maps in a region of chromosome 5 of Arabidopsis containing the tz locus. This is consistent with the observations that ara6/tz transcription was impaired in two out of five Arabidopsis tz mutant lines. ara6/tz is expressed at high levels in chloroplast-containing parenchymatic cells of leaves, inflorescence shoots and flowers of Arabidopsis, and at lower levels in the vascular system. The function of AgThi1 was demonstrated by yeast complementation studies, in which AgThi1 was able to rescue a yeast thi4 mutant.cDNAs encoding glutamine synthetase (GS) and acetylomithine transaminase (AOTA), both involved in nitrogen metabolism, were isolated. GS is the enzyme responsible for ammonium assimilation, while AOTA is involved in the biosynthesis of citrulline, the nitrogen transport form in Alnus. GS mRNA was found in all tissues tested with the highest levels in nodules, where it was present in the infected cells as well as in the cells of the pericycle of the vascular system. AOTA transcripts were detected at very low levels in roots and at high levels in nodules, where it was confined to the infected cells. These data suggested that in A. glutinosa nodules, ammonium assimilation takes place in both the infected cells and in the pericycle of the vascular system, and citrulline biosynthesis occurs mainly in the infected cells. Ammonium assimilation in the pericycle is likely to be related to nitrogen transport.One of the few nodule-specific genes, i.e., genes that are not expressed in roots, ag12, was shown to be expressed in nodules at the highest levels in infected cells before the onset of nitrogen fixation. Sequencing showed that ag12 encodes a serine protease of the subtilisin (EC 3.4.21.14) family. A homolog of ag12, ara12, was identified in Arabidopsis. ara12 was expressed in all organs, with the highest expression levels in the beginning of silique development. To assess the importance of this protease in other actinorhizal symbioses, the expression pattern of its homolog, cg12, was examined in nodules of Casuarina glauca and found to be similar to that of ag12. These results are discussed in view of the phylogenetical relationship of Alnus and Casuarina.

This thesis deals with the genetic aspects of seed development in Arabidopsisthaliana. Mutants affected in several aspects of seed development and, more specifically, in seed maturation have been isolated by various selection procedures. The mutants have been analyzed genetically, physiologically, and morphologically. Some of the mutants are impaired in the biosynthesis or sensitivity to the plant hormone, abscisic acid (ABA). All ABA-related mutants show reduced seed dormancy, indicating the important role of this hormone in the establishment of dormancy. In a direct screen for reduced dormancy, two mutants (rdo) with reduced dormancy were found. These were not ABA-deficient and showed the same sensitivity to ABA, ethylene, auxin, and cytokinin as the wild-type. In contrast to this embryo-determined reduced dormancy, reduced dormancy can also originate in an altered seed coat (testa), like in the altered testa shape ( ats ) mutant. Here, the altered testa shape is caused by a defect in the development of the integuments. Extreme ABA-insensitive mutants ( abi3 ) have green seeds that fail to complete many other aspects of seed maturation, including the induction of dormancy and desiccation tolerance, and the accumulation of seed storage proteins and lipids. In addition to abi3 mutants, lec and fus mutants exhibit such a severely disturbed seed maturation as well, with dark purple seeds due to anthocyanin accumulation. The fus3 mutant shows normal ABA-sensitivity. These various seed maturation mutants indicate that specific genes, some acting dependently and some acting independently from ABA, are responsible for seed maturation programs. The seed maturation mutants were subjected to a physiological and biochemical analysis. A GA-deficient mutant was combined with these mutants. Analysis of these double mutants indicated that seeds of the abi3 and lec mutants did not require GA for germination, in contrast to fus3 seeds. This correlates with ABA-sensitivity for germination. The composition of storage proteins and carbohydrates in abi3 , lec, and fus3 mutant seeds has been compared. The abi3 , lec, and fus3 mutants all showed severely reduced storage proteins. The desiccation intolerance of these seed maturation mutants was not correlated with the lack of specific carbohydrates. Furthermore, the mutants had a higher total content of carbohydrates. This is probably a consequence of the lower levels of storage lipids and proteins.

Cassava (Manihot esculenta Crantz) is a tropical crop grown for its starchy thickened roots, mainly by peasant farmers, in the tropics, for whom it is a staple food. There is an increasing demand for the use of cassava in processed food and feed products, and in the paper and textile industries amongst others. This thesis describes research on the cloning of the genes encoding ADP-glucose pyrophosphorylase small and large subunits (AGPase B and S, respectively) and granule bound starch synthase II (GBSSII). These genes and their products were extensively characterised to determine their role in starch biosynthesis in cassava. Functional verification of the genes was carried out by transforming potato and cassava followed by analysis of the starch produced by the transgenic plants.In Chapter 1 cassava production in the world in general and in Zimbabwe in particular is examined against the backdrop of new cloning and transformation strategies to improve starch quality and quantity. The development of cassava cultivars whose starches have novel physico-chemical properties by genetic modification of the process of starch biosynthesis is examined therein. The main criteria for these new cultivars to emerge are set forth as being: the availability of cloned and characterised starch biosynthesis genes, a universally applicable transformation and regeneration procedure for cassava, transfer to appropriate cassava cultivars, and biosafety analysis of transgenic cassava plants before disbursement to farmers.The cloning of the cassava starch biosynthesis genes encoding granule bound starch synthase II (GBSSII) and the large and small subunits of ADP-glucose pyrophosphorylase (AGPase) is described in Chapters 2 and 3. The cloning of GBSSII reveals that there is indeed a second isoform of this enzyme in cassava as in other plants species. While sharing very little amino acid sequence homology with cassava GBSSI the GBSSII isophorm shares high amino acid sequence homology to other GBSSII genes from pea and potato. Cassava GBSSII seems to be more important in leaf tissue where it is more highly expressed than in tuber tissue where GBSSI predominates. Mapping of GBSSII revealed that this is a single copy gene located on the male derived linkage group T of the cassava mapping population.Cloning of the cassava genes coding for the small (B) and large subunit (S) of AGPase revealed interesting aspects about the cassava enzyme. The cassava AGPase is likely to be heterotetrameric in constitution as had been found in other plant species. Comparison of the cassava AGPase sequences with those of already cloned AGPases revealed that AGPase B is more similar to small subunit genes from other plants than to cassava AGPase S coding for the large subunit (Chapter 3). Segregation analysis of a cassava mapping population revealed that AGPase S is a single copy gene that is localised on the female derived linkage group E of the cassava genetic map. Both genes are expressed in all cassava tissues but AGPase B was shown to have a higher steady state mRNA level than AGPase S especially in leaf and tuber tissue. Post-transcriptional control of small subunit polypeptide levels could be inferred from the discrepancy between AGPase B mRNA and polypeptide levels. The AGPase enzyme activity was much higher in young cassava leaves than older leaves and tubers. Cassava leaf AGPase activity was increased 3 fold by the addition of 3-PGA (3-phospho-glycerate) and inhibited by up to 90% in the presence of inorganic phosphate (Pi). The tuber enzyme was relatively unaffected by 3PGA, but was highly inhibited by Pi.In order to verify the biological role of the AGPase B gene antisense constructs were made of the cassava AGPase B behind a CaMV35S promoter (chapter 3). This was transferred into potato plants by Agrobacterium tumefaciens. While the 224 transgenic antisense AGPase B potato plants did not differ in appearance from normal potato plants, 45 transgenic plants, however, had more numerous and smaller tubers than control plants. Antisense plants with reduced AGPase B mRNA levels had 1.5 to 3 times less starch than tubers from the control plants. The levels of the soluble sugars in the antisense plants increased significantly (up to 10 times more glucose, 6 times the amount of fructose, and 5 times the amount of sucrose) when compared to those found in control plants. These results show that a heterologous gene from cassava can have an antisense effect in potato, but that the number of plants required to find plants exhibiting maximum antisense effect has to be very large. This is probably due to sequence homology differences between the cassava AGPase B and potato AGPase B genes which share only 68% amino acid sequence homology.Chapter 5 describes the further development of an efficient, time and labour saving protocol for transforming cassava based on stringent selection of the luciferase (firefly) marker gene. In addition the first reported transformation of cassava with a gene (AGPase B) other than a marker gene is described. An antisense construct was made for transforming cassava. This consisted of the cassava AGPase B gene which was placed in antisense orientation behind the CaMV35S promoter. This was then coupled to the luciferase gene driven by another CaMV35S promoter. After particle bombardment of cassava FEC transgenic tissue was selected using three different selection regimes: non stringent luciferase selection, stringent luciferase selection and combined chemical (phosphinothrycin) and luciferase selection. Stringent luciferase selection whereby luciferase positive FEC units were precisely pinpointed, isolated and cultured was found to be the most effective and time saving method. It was possible to generate cultures having more than 90% luciferase positive FEC tissue after 12 weeks of stringent LUC selection, compared to 45% and

This thesis describes the selection and evaluation of diploid potato clones (2n=2x=24) that produce unreduced or 2n-gametes with 24 chromosomes instead of the normal reduced n-gametes with 12 chromosomes. To elucidate the modes of origin of the 2n-gametes, the progenies derived from such gametes were analysed for different marker loci of one chromosome. Besides the already known mechanisms of First and Second Division Restitution (FDR and SDR) in potato, the multilocus analysis showed the additional mechanism of Post-Meiotic Restitution (PMR) resulting in completely homozygous 2n-gametes. The FDR and SDR 2n-gametes were used for gene-centromere mapping. Multilocus analysis of SDR 2n-gametes showed the map position of the centromere of chromosome 8 in relation to the RFLP-loci. These genetic analyses also demonstrated the occurrence of a single crossover per chromosome arm (high level of chiasma interference). In the light of these findings, it is possible to construct more critical genetic maps of potato.

At the start of the research described in this thesis, the main aim was to develop, study and apply an efficient En/Spm-I/dSpm based transposon tagging system in Arabidopsis thaliana to generate tagged mutants and to provide insights in the possibilities for future applications of such a transposon tagging system in studying plant gene functions. The first step was the introduction of an active En/Spm-I/dSpm system into Arabidopsis. Initially a very simple T-DNA construct was transformed, containing a nearly full En-1 element, without left and right border sequences, and with its promoter replaced by the stronger CaMV 35S promoter. As the same construct harboured a non-autonomous I/dSpm element, only one T-DNA transformation was needed. Transformation of this 'in cis two- element En/Spm-I/dSpm system' yielded one transformant with two T-DNA insertion loci, TEn2 and TEn5, each having one, respectively, five 35S -En/Spm t ransposase gene copies (Chapter 1). The transposition activity of the I/dSpm elements turned out to be surprisingly high. Instead of a germinal excision frequency, which was generally used to express the activity of heterologous transposable element systems, the term independent transposition frequency or itf was coined, as a measure accounting for the entire transposition process. Although not always easy to compare for different transposable element systems, an average itf of over 10%, as was found for this En/Spm-I/dSpm system (Chapter 1), has not been reported for any of the Ac-Ds based heterologous transposon tagging systems developed for Arabidopsis (Bancroft et al., 1992; Bhatt et al., 1996; Fedoroff and Smith, 1993; Honma et al., 1993; Long et al., 1993b; Swinburne et al., 1992). Obtaining a high transposition frequency in Ac-Ds systems is hampered by the fact that the transposase acts as an inhibitor of Ac or Ds transposition when its expression exceeds a certain level. Apparently such autoregulatory mechanism is not present in the 'in cis two-element En/Spm-I/dSpm system'.The need for a more sophisticated system diminished with the availability of this simple, but efficient En/Spm-I/dSpm transposon system and it was therefore studied in more detail to determine: 1) the ability to transpose continuously even after many plant generations; 2) the distribution of elements after transposition; 3) the ability to transpose to transcribed regions and 4) the ability to cause mutations. To start with the first issue, transposition has been studied in up to 12 generations, starting from the primary transformant. In all these generations, there was no apparent reduction in itf, demonstrating a continuous transposition of I/dSpm elements, irrespective of the generation number. The second issue, the distribution of elements after transposition, is another important aspect of a transposable element system. For maize transposons it is reported that the insertion site is preferentially physically and often genetically linked to the excision site (Dooner and Belachew, 1989; Peterson, 1970). This is not so remarkable considering that the proteins that perform the transposition steps will have a higher chance of encountering a nearby site on the genome instead of a distant DNA sequence. Like in maize (Peterson, 1970), the I/dSpm elements show a preference for insertion in genetically linked sites (Chapter 1), but the preference is not very explicit. Based on the observations of transpositions from tagged genes to sites within a few cM or only several kb away, and on the analogy to the En/Spm-I/dSpm elements in maize, the overall estimate is that about 30% of the elements transpose to sites genetically linked to the excision site. The mapped elements (Chapter 5) show a fairly even distribution over the genome, although there seems to be some clustering of elements (from different origin) to certain genomic regions, like the top of chromosome 4, the bottom of chromosome 1 and the lower half of chromosome 5.In accordance with the idea that DNA must be in an open confirmation to allow the access of transposase proteins before transposition (Zhang and Spradling, 1993), there are many indications that I/dSpm elements insert in regions of the Arabidopsis genome containing genes: a) I/dSpm flanking DNAs rarely contain repetitive sequences, but are mostly single copy sequences, as are genes (Chapters 1 and 5); b) about half of the I/dSpm elements are inserted in relatively conserved genomic regions, with no RFLPs for five restriction enzymes (Chapters 1 and 5); c) at least one third of the examined I/dSpm elements is inserted in close vicinity of transcriptionally active genomic sequences (Chapter 7). Insertion in unique, conserved and often transcribed DNA may not seem surprising for a plant species with little repetitive DNA and a small genome with a high gene density (Meyerowitz, 1989). However, a high frequency of insertion into genic regions of the genome offers the best chances for gene tagging.The most important aspect of a transposable element system is the possibility to generate tagged mutants. The En/Spm-I/dSpm system is mutagenic, with as much as 12 tagged mutants found so far. Most of these were obtained after screening for random mutant phenotypes. When screening for specific mutants, such as reduced seed dormancy, tagged alleles of the ABI3 and LEC1 genes were found (M. Koornneef et al., unpublished results). Mutants at the CER6 locus were obtained by targeted tagging, using the nearby ap1::I/dSpm allele as the I/dSpm element donor (A. Pereira, unpublished results). These selected and targeted transposon tagging experiments are illustrative examples of the feasibility to efficiently isolate tagged mutants of a special phenotypic or genotypic class.Ideally, a population saturated with different I/dSpm insertions can be made, allowing the isolation of mutants for virtually every gene. Such a 'mutation machine' can be further used for PCR based targeted gene inactivation. This novel technique, which was originally developed for Drosophila melanogaster (see O'Hare, 1990), exploits the abundance of transposons for the identification of insertions in genes with known DNA sequence, but no known mutant phenotype. In general, DNAs from multidimensional pools of individuals from a large population are used for a PCR using two primers. One primer is specific for the transposon terminus (directed outwards), the other is specific for the target gene. A fragment can only be amplified when a transposon insert is close enough to the target primer. This technique has been shown to work in Drosophila (Ballinger and Benzer, 1989; Kaiser and Goodwin, 1990), Caenorhabditis elegans (Zwaal et al., 1993), Petunia hybrida (Koes. et al., 1995) and maize (Das and Martienssen, 1995), using 'mutation machine' transposable element systems.Two I/dSpm tagged genes that have been studied in great detail are the MS2 gene (Chapters 2 and 3) and the CER1 gene (Chapter 4). Mutants for both genes display a conditional male sterile phenotype, which is the only mutant phenotype for the ms2 mutant, but for the cer1 mutant it is a pleiotropic effect of a deficiency in epicuticular wax biosynthesis. The ms2 mutants are occasionally able to self-fertilize, especially in high relative humidity and late in plant development, but seed set rarely reaches more than a few percentages of wild-type seed set. This in contrast to the cer1 mutants, which are male sterile in low relative humidity (≤50% RH) and fertile in high relative humidity (≥95% RH). Fertility cannot be completely restored by environment in the ms2 mutants due to the drastic effect of the mutation on pollen development. The MS2 gene is expressed in the tapetum around the time of microspore release from the microspore mother cells. The gene is needed for the development of a proper exine layer, protecting the microspore from harmful environmental influences. Consequently the few ms2 microspores that are produced have very feeble pollen walls, which leaves only very few pollen grains intact for fertilization.The CER1 gene acts much later in pollen development. Phenotypically cer1 and wild-type pollen cannot be distinguished, apart from a difference in gerniination ability (Chapter 4). As cer1 pollen germination is like wild type when applying CER1 pollen or by pollinating in high relative humidity, there appears to be a substance missing from the pollen coat that is required for the necessary rehydration of a pollen grain. Although essential under low relative humidity conditions, this is only a minor defect, which can be easily overcome.Besides the similarity in mutant phenotypes, the MS2 gene and the CER1 gene share the characteristic of encoding proteins with homology to enzymes in the fatty acid biosynthesis pathway. The MS2 protein has most resemblance with the wax fatty acid reductase protein from the desert shrub jojoba, which is involved in the conversion of wax fatty acids to wax alcohols (Chapter 3). The CER1 protein shares structural features of fatty acid desaturases, and it has a proposed function as a decarbonylase, converting long carbon chain aldehydes to alkanes (Chapter 4). There are more examples of a correlation between male fertility and wax biosynthesis, as also other cer mutants such as cer3, 6, 8 and 10 are known to be disturbed in male fertility. It demonstrates the general importance of fatty acid biosynthesis for male gametogenesis.The last part of this thesis has been devoted to further applications of the En/Spm-I/dSpm tagging system in Arabidopsis for the analysis of plant gene functions. The first description of transposable elements as controlling elements (McClintock, 1948), was based on the effect transposons had on the expression of maize genes. Especially (d)Spm insertions were known to cause Spm dependent or suppressible gene expression (Fedoroff, 1989). This effect is now also described for the En/Spm- I/dSpm system in Arabidopsis (Chapter 6). As in maize, an En/Spm suppressible allele contains an anti-parallel I/dSpm element insertion, which can be spliced from the mRNA. This knowledge can be further used to design an artificial gene expression system, in which an introduced gene containing an anti-parallel I/dSpm element, can be negatively controlled in the presence of an En/Spm transposase source. The reverse effect, En/Spm dependent gene expression, is now also described for Arabidopsis (Chapter 6), but the mechanism for dependence is not yet understood. The availability of the En/Spm dependent lad::I/dSpm mutant will be useful for further research.A more general way to study gene expression is the use of gene traps as detectors of gene activity. The pilot experiments described in chapter 7, have shown the possibility to adapt I/dSpm elements as gene traps. Especially their advantage in detecting genes without the need for mutation, and the possibility of studying the activity of genes which are lethal as homozygous mutants, are important additional properties of gene trap systems over "traditional" transposon tagging. In combination with its efficient transposition behaviour, an En/Spm-I/dSpm based gene trap tagging system seems an attractive alternative for the existing Ac-Ds or T-DNA based gene trap systems.Summarizing, an En/Spm-I/dSpm transposon tagging system has been well developed for Arabidopsis and many of its basic characteristics are studied and described. I/dSpm tagged mutants can be found with reasonable frequencies, either by random, selected or targeted tagging strategies. The cloning and characterization of two genes affecting male fertility has been described. Further ways to improve tagging frequencies, based on phenotypic or on genotypic selection have been discussed. In addition, the system can be exploited to study plant gene expression and gene function either by En/Spm controlled activity of I/dSpm tagged genes, or by using I/dSpm gene detector elements.

At the start of the research described in this thesis, the main aim was to develop, study and apply an efficient En/Spm-I/dSpm based transposon tagging system in Arabidopsis thaliana to generate tagged mutants and to provide insights in the possibilities for future applications of such a transposon tagging system in studying plant gene functions. The first step was the introduction of an active En/Spm-I/dSpm system into Arabidopsis. Initially a very simple T-DNA construct was transformed, containing a nearly full En-1 element, without left and right border sequences, and with its promoter replaced by the stronger CaMV 35S promoter. As the same construct harboured a non-autonomous I/dSpm element, only one T-DNA transformation was needed. Transformation of this 'in cis two- element En/Spm-I/dSpm system' yielded one transformant with two T-DNA insertion loci, TEn2 and TEn5, each having one, respectively, five 35S -En/Spm t ransposase gene copies (Chapter 1). The transposition activity of the I/dSpm elements turned out to be surprisingly high. Instead of a germinal excision frequency, which was generally used to express the activity of heterologous transposable element systems, the term independent transposition frequency or itf was coined, as a measure accounting for the entire transposition process. Although not always easy to compare for different transposable element systems, an average itf of over 10%, as was found for this En/Spm-I/dSpm system (Chapter 1), has not been reported for any of the Ac-Ds based heterologous transposon tagging systems developed for Arabidopsis (Bancroft et al., 1992; Bhatt et al., 1996; Fedoroff and Smith, 1993; Honma et al., 1993; Long et al., 1993b; Swinburne et al., 1992). Obtaining a high transposition frequency in Ac-Ds systems is hampered by the fact that the transposase acts as an inhibitor of Ac or Ds transposition when its expression exceeds a certain level. Apparently such autoregulatory mechanism is not present in the 'in cis two-element En/Spm-I/dSpm system'.The need for a more sophisticated system diminished with the availability of this simple, but efficient En/Spm-I/dSpm transposon system and it was therefore studied in more detail to determine: 1) the ability to transpose continuously even after many plant generations; 2) the distribution of elements after transposition; 3) the ability to transpose to transcribed regions and 4) the ability to cause mutations. To start with the first issue, transposition has been studied in up to 12 generations, starting from the primary transformant. In all these generations, there was no apparent reduction in itf, demonstrating a continuous transposition of I/dSpm elements, irrespective of the generation number. The second issue, the distribution of elements after transposition, is another important aspect of a transposable element system. For maize transposons it is reported that the insertion site is preferentially physically and often genetically linked to the excision site (Dooner and Belachew, 1989; Peterson, 1970). This is not so remarkable considering that the proteins that perform the transposition steps will have a higher chance of encountering a nearby site on the genome instead of a distant DNA sequence. Like in maize (Peterson, 1970), the I/dSpm elements show a preference for insertion in genetically linked sites (Chapter 1), but the preference is not very explicit. Based on the observations of transpositions from tagged genes to sites within a few cM or only several kb away, and on the analogy to the En/Spm-I/dSpm elements in maize, the overall estimate is that about 30% of the elements transpose to sites genetically linked to the excision site. The mapped elements (Chapter 5) show a fairly even distribution over the genome, although there seems to be some clustering of elements (from different origin) to certain genomic regions, like the top of chromosome 4, the bottom of chromosome 1 and the lower half of chromosome 5.In accordance with the idea that DNA must be in an open confirmation to allow the access of transposase proteins before transposition (Zhang and Spradling, 1993), there are many indications that I/dSpm elements insert in regions of the Arabidopsis genome containing genes: a) I/dSpm flanking DNAs rarely contain repetitive sequences, but are mostly single copy sequences, as are genes (Chapters 1 and 5); b) about half of the I/dSpm elements are inserted in relatively conserved genomic regions, with no RFLPs for five restriction enzymes (Chapters 1 and 5); c) at least one third of the examined I/dSpm elements is inserted in close vicinity of transcriptionally active genomic sequences (Chapter 7). Insertion in unique, conserved and often transcribed DNA may not seem surprising for a plant species with little repetitive DNA and a small genome with a high gene density (Meyerowitz, 1989). However, a high frequency of insertion into genic regions of the genome offers the best chances for gene tagging.The most important aspect of a transposable element system is the possibility to generate tagged mutants. The En/Spm-I/dSpm system is mutagenic, with as much as 12 tagged mutants found so far. Most of these were obtained after screening for random mutant phenotypes. When screening for specific mutants, such as reduced seed dormancy, tagged alleles of the ABI3 and LEC1 genes were found (M. Koornneef et al., unpublished results). Mutants at the CER6 locus were obtained by targeted tagging, using the nearby ap1::I/dSpm allele as the I/dSpm element donor (A. Pereira, unpublished results). These selected and targeted transposon tagging experiments are illustrative examples of the feasibility to efficiently isolate tagged mutants of a special phenotypic or genotypic class.Ideally, a population saturated with different I/dSpm insertions can be made, allowing the isolation of mutants for virtually every gene. Such a 'mutation machine' can be further used for PCR based targeted gene inactivation. This novel technique, which was originally developed for Drosophila melanogaster (see O'Hare, 1990), exploits the abundance of transposons for the identification of insertions in genes with known DNA sequence, but no known mutant phenotype. In general, DNAs from multidimensional pools of individuals from a large population are used for a PCR using two primers. One primer is specific for the transposon terminus (directed outwards), the other is specific for the target gene. A fragment can only be amplified when a transposon insert is close enough to the target primer. This technique has been shown to work in Drosophila (Ballinger and Benzer, 1989; Kaiser and Goodwin, 1990), Caenorhabditis elegans (Zwaal et al., 1993), Petunia hybrida (Koes. et al., 1995) and maize (Das and Martienssen, 1995), using 'mutation machine' transposable element systems.Two I/dSpm tagged genes that have been studied in great detail are the MS2 gene (Chapters 2 and 3) and the CER1 gene (Chapter 4). Mutants for both genes display a conditional male sterile phenotype, which is the only mutant phenotype for the ms2 mutant, but for the cer1 mutant it is a pleiotropic effect of a deficiency in epicuticular wax biosynthesis. The ms2 mutants are occasionally able to self-fertilize, especially in high relative humidity and late in plant development, but seed set rarely reaches more than a few percentages of wild-type seed set. This in contrast to the cer1 mutants, which are male sterile in low relative humidity (≤50% RH) and fertile in high relative humidity (≥95% RH). Fertility cannot be completely restored by environment in the ms2 mutants due to the drastic effect of the mutation on pollen development. The MS2 gene is expressed in the tapetum around the time of microspore release from the microspore mother cells. The gene is needed for the development of a proper exine layer, protecting the microspore from harmful environmental influences. Consequently the few ms2 microspores that are produced have very feeble pollen walls, which leaves only very few pollen grains intact for fertilization.The CER1 gene acts much later in pollen development. Phenotypically cer1 and wild-type pollen cannot be distinguished, apart from a difference in gerniination ability (Chapter 4). As cer1 pollen germination is like wild type when applying CER1 pollen or by pollinating in high relative humidity, there appears to be a substance missing from the pollen coat that is required for the necessary rehydration of a pollen grain. Although essential under low relative humidity conditions, this is only a minor defect, which can be easily overcome.Besides the similarity in mutant phenotypes, the MS2 gene and the CER1 gene share the characteristic of encoding proteins with homology to enzymes in the fatty acid biosynthesis pathway. The MS2 protein has most resemblance with the wax fatty acid reductase protein from the desert shrub jojoba, which is involved in the conversion of wax fatty acids to wax alcohols (Chapter 3). The CER1 protein shares structural features of fatty acid desaturases, and it has a proposed function as a decarbonylase, converting long carbon chain aldehydes to alkanes (Chapter 4). There are more examples of a correlation between male fertility and wax biosynthesis, as also other cer mutants such as cer3, 6, 8 and 10 are known to be disturbed in male fertility. It demonstrates the general importance of fatty acid biosynthesis for male gametogenesis.The last part of this thesis has been devoted to further applications of the En/Spm-I/dSpm tagging system in Arabidopsis for the analysis of plant gene functions. The first description of transposable elements as controlling elements (McClintock, 1948), was based on the effect transposons had on the expression of maize genes. Especially (d)Spm insertions were known to cause Spm dependent or suppressible gene expression (Fedoroff, 1989). This effect is now also described for the En/Spm- I/dSpm system in Arabidopsis (Chapter 6). As in maize, an En/Spm suppressible allele contains an anti-parallel I/dSpm element insertion, which can be spliced from the mRNA. This knowledge can be further used to design an artificial gene expression system, in which an introduced gene containing an anti-parallel I/dSpm element, can be negatively controlled in the presence of an En/Spm transposase source. The reverse effect, En/Spm dependent gene expression, is now also described for Arabidopsis (Chapter 6), but the mechanism for dependence is not yet understood. The availability of the En/Spm dependent lad::I/dSpm mutant will be useful for further research.A more general way to study gene expression is the use of gene traps as detectors of gene activity. The pilot experiments described in chapter 7, have shown the possibility to adapt I/dSpm elements as gene traps. Especially their advantage in detecting genes without the need for mutation, and the possibility of studying the activity of genes which are lethal as homozygous mutants, are important additional properties of gene trap systems over "traditional" transposon tagging. In combination with its efficient transposition behaviour, an En/Spm-I/dSpm based gene trap tagging system seems an attractive alternative for the existing Ac-Ds or T-DNA based gene trap systems.Summarizing, an En/Spm-I/dSpm transposon tagging system has been well developed for Arabidopsis and many of its basic characteristics are studied and described. I/dSpm tagged mutants can be found with reasonable frequencies, either by random, selected or targeted tagging strategies. The cloning and characterization of two genes affecting male fertility has been described. Further ways to improve tagging frequencies, based on phenotypic or on genotypic selection have been discussed. In addition, the system can be exploited to study plant gene expression and gene function either by En/Spm controlled activity of I/dSpm tagged genes, or by using I/dSpm gene detector elements.

In this thesis research is presented on all steps in a potato breeding program via the diploid level: dihaploid induction, selection at the diploid level and sexual polyploidization. In spite of the significant seed parent x pollinator interaction estimated, IVP 101 was found to have a significant higher dihaploid induction ability than the widely used pollinators IVP 35 and IVP 48. Significant differences in dihaploid production ability between seed parents were also found. Dihaploid populations from 31 varieties or breeding lines were evaluated for occurrence and frequency of mutant phenotypes, for tuberization, flowering, pollen stainability, 2n-pollen production and resistance to Ro-1. The expression of yield, yield components, vine maturity, under water weight and chip colour at the diploid and tetraploid level and the parental effects on these characters of 4x . 2x progenies were investigated to find an answer to the question whether at the diploid level other selection criteria should be employed than at the tetraploid level when selecting tetraploid breeding lines. For yield direct selection at the diploid level had no effect on the performance of tetraploid progeny. For under water weight more stringent and for vine maturity less stringent selection criteria were required at the diploid level than at the tetraploid level. The frequently reported superiority of FDR 2n-gametes versus SDR 2n-gametes could be confirmed for yield only. For vine maturity, under water weight and chip colour no considerable differences were found between means of FDR and SDR progenies from reciprocal 4x-2x crosses.

In this thesis research is presented on all steps in a potato breeding program via the diploid level: dihaploid induction, selection at the diploid level and sexual polyploidization. In spite of the significant seed parent x pollinator interaction estimated, IVP 101 was found to have a significant higher dihaploid induction ability than the widely used pollinators IVP 35 and IVP 48. Significant differences in dihaploid production ability between seed parents were also found. Dihaploid populations from 31 varieties or breeding lines were evaluated for occurrence and frequency of mutant phenotypes, for tuberization, flowering, pollen stainability, 2n-pollen production and resistance to Ro-1. The expression of yield, yield components, vine maturity, under water weight and chip colour at the diploid and tetraploid level and the parental effects on these characters of 4x . 2x progenies were investigated to find an answer to the question whether at the diploid level other selection criteria should be employed than at the tetraploid level when selecting tetraploid breeding lines. For yield direct selection at the diploid level had no effect on the performance of tetraploid progeny. For under water weight more stringent and for vine maturity less stringent selection criteria were required at the diploid level than at the tetraploid level. The frequently reported superiority of FDR 2n-gametes versus SDR 2n-gametes could be confirmed for yield only. For vine maturity, under water weight and chip colour no considerable differences were found between means of FDR and SDR progenies from reciprocal 4x-2x crosses.