The aim of this project was to test hypotheses relating to nucleosome positioning in Arabidopsis to provide a basis for better understanding of epigenetic transcriptional regulation in plants. Prior to this study, virtually no information existed regarding nucleosome positioning in plants. Eukaryote chromosomes consist of chromatin, composed of nucleosomes separated by linker DNA of variable lengths. Nucleosomes consist of 147 bp of DNA wrapped 1.7 times around a histone octamer. Whilst no consensus nucleosome positioning DNA sequence exists, sequence preferences influence positioning, and contribute to the complex epigenetic processes which act to control transcriptional activity. These details of the underlying mechanisms are known to differ between the plant and animal kingdoms. High-throughput sequencing technologies were utilised to generate large datasets of mono- and di-nucleosome sequences from wild-type Arabidopsis. These enabled genome-wide analysis and inference of plant-specific patterns of nucleosome positioning and sequence properties. Further data were generated from a methyltransferase antisense (MET1) which is depleted in methylated CG epigenetic marks. The internal distributions of dinucleotides within Arabidopsis nucleosomes were similar to those observed in non-plant eukaryotes. A unique periodicity in the distribution of linker lengths was detected in Arabidopsis wild type chromatin. In contrast, the MET1 antisense line displayed the expected periodicity, indicating systematic differences in chromatin organisation. There was a significant increase in nucleosome occupancy within exons compared with introns. However, this difference was less marked in the MET1 antisense. Specific patterns of nucleosome phasing were observed around transcription start sites. Linker lengths within rRNA gene clusters associated with nucleolar organiser regions (NORs) differed depending on chromosome of origin, suggesting differences in higher order chromatin structure between the NORs. Comparison of the nucleosome position and DNA methylation within the rRNA gene cluster revealed interesting differences between the two regions, which may reflect interactions affecting chromatin structure and transcriptional regulation.

Self-incompatibility (SI) is the major mechanism used by flowering plants to prevent self-pollination and thus avoid inbreeding. It is a genetically controlled mechanism that encodes a highly specific recognition system that inhibits the growth of incompatible pollen, whereas compatible pollen from an unrelated plant of the same species is able to grow and effect fertilization. In Papaver rhoeas, inhibition of incompatible pollen is mediated by a signal transduction pathway that activates a complex network of intracellular events including Ca\(^{2+}\)-dependent phosphorylation of p26, an inorganic pyrophosphatase required for pollen tube growth, depolymerisation of the pollen actin cytoskeleton and ultimately programmed cell-death (PCD). To further understand the mechanisms involved in Papaver rhoeas SI it is important to identify and characterize components of the signalling network that mediate the SI response. Recent studies have revealed that p56, a mitogen-activated protein kinase (MAPK), is involved in activation of PCD during SI. MAPKs have been shown to be important signalling components in a range of cellular responses in eukaryotes. In Arabidopsis thaliana there are 20 MAPKs with roles including cell division, abiotic stress response, wounding response and hormone signalling. Preliminary studies suggested an MPK3-like gene might encode p56, as an anti-AtMPK3 antibody cross-reacts with a protein corresponding to p56. Experiments carried out here suggest this is not the case. Using a combination of bioinformatics and proteomics data it has been possible to identify and clone a candidate p56 gene from Papaver rhoeas pollen. The putative p56 gene has homology to AtMPK9 and is a member of the T-D-Y class of MAPKs. It has been designated PrMPK9-1. An antibody raised against recombinant PrMAPK9-1 protein is being used to confirm that this gene/protein does indeed correspond to p56.

During meiosis, homologous chromosomes pair and become connected by formation of the synaptonemal complex. Recombination is initiated by DNA double strand breaks (DSBs), formed by the protein SPO11-1. In Arabidopsis, ~10% of DSBs are repaired as crossovers, which are reciprocal exchanges of DNA between homologues. These physical connections ensure the correct segregation of chromosomes and generate genetic diversity. The remainder are processed as non-crossovers, important for pairing and synapsis. How these processes are integrated and controlled remains poorly understood. Telomeres are thought to play a role in pairing of homologues. The role of telomeres was investigated in Arabidopsis by treatment with colchicine, a microtubule-depolymerising drug, known to disrupt telomere clustering and pairing in rye. A mutant deficient for the telomerase reverse transcriptase TERT was studied, in which telomeres were severely shortened and showed reduced fertility. To track the movement of telomeres during meiosis the telomere binding proteins POT1a and POT1b were chosen for antibody production. Telomeres were found to be dispensable for pairing and synapsis. SPO11-1 RNA interference lines with varying reductions in DSBs were analysed, to investigate how reducing DSBs affects pairing, synapsis, and the crossover/non-crossover decision. Chromosomes showed autonomous crossover control. The synaptonemal-complex was shown to be important in preventing non-homologous interactions.

A large proportion of the genomes of species belonging to the genus Allium comprises repetitive sequence DNA, a component implicated as a cause of the large variation in C-values between even closely related species. The work presented here represents part of the first phase in the characterisation of some of these repetitive sequences in a number of Allium species. One repetitive DNA sequence family, BIOOO, isolated from the genome of A. sativum, has been characterised with respect to the genomic organisation, reiteration frequency and sequence divergence of its members within A. sativum. Sequences sharing homology with a cloned representative member of the B1OOO family have been detected in the genomes of a number of other Allium species; such sequences display quantitative and qualitative modulations in their organisation. In addition, and by contrast, the distribution and organisation of a satellite DNA family present In a number of Allium species has been investigated; the characteristics of this family differ from those of the B1OOO family in many respects. Data relating to the evolution and maintenance, functions and effects of repetitive-sequence DNA in eukaryotic genomes are reviewed and where possible the data pertaining to Allium are discussed in context with such information from other species.

The work within this thesis is concerned with the creation of a temperature-sensitive Schizosaccharomyces pombe marker protein, and the regulation of the pheromone communication system of Sz. pombe reporter strains by RGS proteins. There are a limited number of marker proteins available for use in the genetic manipulation of Sz. pombe, and the generation of a temperature-sensitive Ura4p was envisaged to expand the scope of carrying out sequential gene disruptions in the fission yeast. PCR-based mutagenesis was used to introduce mutations in the ura4 cassette, and a leucine to proline mutation identified at residue 261 in the ura4 open reading frame conferred a temperature-sensitive requirement for uracil. To demonstrate the use of the Ura4sp marker in gene disruption, the Sz. pombe irpl gene was disrupted with the ura4u cassette, and subsequently, the prkl gene was disrupted with the wild-type ura4 cassette. RGS proteins are a recently discovered family of proteins that negatively regulate G protein-coupled signalling pathways. This thesis describes the ability of mammalian RGS proteins to regulate the pheromone communication system of Sz. pombe reporter strains. Human RGS 1 and human RGS4 displayed the greatest ability to negatively regulate the Sz. pombe pheromone signalling pathway when expressed from multicopy expression vectors. Human RGS2, human RGS3, human RGS9-2 and murine RGS2 displayed lesser, varying abilities. Expression of human RGS 1 from single copy reduced signalling at low pheromone concentrations. Expression of human RGS4 from single copy was incapable of reducing pheromone-independent and pheromone-dependent signalling. This thesis also describes the search for gain-of-function RGS proteins. Two potential gain-of-function szRgslp mutants were previously identified, and these mutants were recreated. The two mutations identified (histidine to arginine at szRgslp residue 171 and valine to isoleucine at szRgslp residue 305) conferred gain-of-function szRgslp phenotypes in an sxa2:: ura4 reporter strain. Hydroxylamine treatment of the human RGS4 open reading frame resulted in the identification of a potential gain-of-function RGS4 mutant. The lysine to arginine mutation at huRGS4p residue 20 conferred a gain-of-function huRGS4p phenotype in an sxa2:: ura4 reporter strain.

Sporophytic self-incompatibility is a genetically controlled inbreeding prevention mechanism, which is prevalent in the Brassicaceae, and has been reported in a variety of high profile species. Despite the benefits of preventing self-fertilization in terms of maintaining genetic diversity, variation in the strength of self-incompatibility (SI) has also been well documented, as has a shift from SI to inbreeding at the species and population levels. An important underlying driving force behind a switch to inbreeding could be the reproductive assurance provided by not requiring an unrelated mating partner for sexual reproduction. This could be beneficial for a species undergoing rapid colonization, because only a single individual is required to begin a sexually reproducing colony after a long-distance dispersal event (Baker’s law), which is characteristic of the plight of many species after the last glacial maxima. The purpose of my thesis was to evaluate the effects of variation in mating system on post-glacial colonization, using two model species that show intraspecific variation in outcrossing rates. The first, Arabidopsis lyrata, represents an excellent model system to assess post-glacial colonization history because it exhibits broad geographical and ecological ranges, and has a recently completed genome sequence. In North America, A. lyrata has further benefits as a model system, namely it exhibits variation in the strength of SI and shift to SC at the population level, which is not observed in Europe. The second species is Arabis alpina, which also appears to show population level variation in mating system strength in Europe based on variation in FIS. This has been putatively linked to colonization history after the last glacial maxima. Unlike in A. lyrata however, its mating system has not been characterized. Mating system delimitation in A. alpina has the potential to aid the interpretation of patterns of ecological genetic diversity, which may in part be influenced by local or regional stochastic changes to mating system variation. My first objective was to identify if A. alpina had a functioning SI system based on both self-fertilization experiments, and allozyme based outcrossing rate estimations. I found strong evidence to suggest the presence of a functional barrier preventing self-fertilization in A. alpina. I identified multiple putative SRK alleles (the female determinant of self-incompatibility), suggesting that the same type of sporophytic system seen in other Brassicaceae species governs SI in this species. I also demonstrated linkage of SI phenotype to some SRK genotypes by diallel crosses, strengthening the case for a functional SI system in this species. Further to this I demonstrate variation in mating system strength between populations, and autonomous inbreeding was seen in a single population. I note that the potential changes in SI status coincide with areas suspected to differ in post-glacial history based on allozyme diversity reported in previous work. While the number of populations sampled was insufficient to link mating system variation to colonization history in A. alpina, mating system variation has been more extensively characterized in North American A. lyrata, allowing more fine-scale resolution of population structure and post-glacial colonization history; an underlying objective of my thesis. I used three molecular marker systems (cpDNA, nuclear micro-satellites and allozymes) to assess these phylogeographic questions, and present evidence of three putative colonization routes for the Great Lakes region. These putative routes are congruent with those described in other species, particularly amphibians and reptiles. Further to this I considered the possible location of glacial refugia, and likelihood that plant taxa may have survived during Pleistocene glaciation in close proximity to the Laurentide Ice Sheet, particularly in Illinois, Indiana, Wisconsin and Minnesota, which may also be true for some animal taxa. I examined patterns of population structure, and scenarios that may have influenced this, and present support for the previously documented theory of multiple breakdowns in SI in this geographic region. My final objective was to assess the suitability of the three marker systems for phylogeographic reconstruction in A. lyrata, by comparing and contrasting the patterns of population structure, and colonization history suggested by each system. Levels of variation observed between the marker systems used varied, and I explored how these patterns complemented and contradicted each other. As expected, the nuclear micro-satellite loci represent the system with the greatest genetic diversity, but do not allow meaningful conclusions to be drawn regarding colonization history because of low levels of shared variation between populations. Conversely, the allozyme loci presented much lower levels of genetic diversity, but support population structuring conclusions based on both cpDNA data and previous studies of A. lyrata and other taxa in this area. The cpDNA marker (trnF) represents a somewhat contentious system to use for phylogeography in A. lyrata, as it contains a tandem array of highly variable, but complexly evolving duplications (pseudogenes). I concluded that these structural changes could be phylogenetically informative when pseudogene evolutionary relationships can be resolved This was based on variation in patterns of diversity, and the subsequent population structure change that occurred when using different methods of assessing trnF variation.

Crop wild relatives have been identified as ecologically and economically important plant genetic resources but are often a neglected resource. The recognition of the need for their specific conservation and their value for future use has been strengthened by the Convention on Biological Diversity and the International Treaty on Plant Genetic Resources for Food and Agriculture, both of which have been ratified by the UK. This thesis provides a detailed view of the ecological, geographic and genetic background to three crop wild relative species, Trifolium dubium, T. pratense and T. repens, of which the latter two are amongst some of the most economically important legume species in the UK. Assessments of ecogeography, amplified fragment polymorphism and single nucleotide polymorphism markers were employed to investigate the distribution of variation in these species across the UK, including outlying island sites. Based on this information it was possible to look for isolation by distance in populations in UK; identify areas containing unique variation; assess the conservation importance of island sites surrounding the UK and speculate on the causes of the observed patterns of diversity. Conservation recommendations were based on the cumulative data from this research to identify how the recommendations change with an increased focus on genetic diversity. These results provide insights into the use of different types of background information when setting conservation plans in widespread species, contributing to the development of conservation strategies for widespread species in general.