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Circadian clocks are endogenous, persistent, temperature-compensating timekeepers which provide temporal organization of biological processes from cyanobacteria to man. Within plants, the circadian clock plays an important role in controlling desirable agronomic traits, integrating a number of abiotic stress signalling pathways. Much of our current understanding of the plant clock has come from the model organism Arabidopsis thaliana, with the vast majority of studies using null mutants and transgenic luciferase reporters. However, whilst our models of the transcriptional-translational feedback loops underlying the clock have become increasingly complex in the last 20 years, the regulation of several key components of the clock on the transcriptional level remains unknown. There is a need for new components in the model to fill in these gaps. Many known components of the plant clock are transcription factors. It was hypothesised therefore that the systematic screening and analysis of putative transcription factors would reveal novel circadian phenotypes. To this effect, a library of 338 hormone inducible transcription factor over-expression lines was screened with the novel delayed fluorescence assay to identify transcription factors giving circadian period phenotypes. Over-expression allows the investigation of partially redundant genes, and of those normally expressed within a hard-to-assay tissue. Twenty-one such genes were identified. Through the use of in silico tools to identify circadian expression patterns and promoter motifs, this shortlist was analysed for predicted interactions and modes of regulation. Traditional pCAB2:LUC+ luciferase markers were used to further phenotype the TF lines, providing a deeper understanding of the effect of the gene on the circadian clock, whilst knock out libraries and hypocotyl elongation assays under monochromatic light were employed to further characterise the genes in question. In this way, the longlist was reduced to three putative transcription factors which gave a circadian phenotypes of interest: INDOLE-3-ACETIC ACID INDUCIBLE 11, the freezing-response regulator MYBC1, and B-BOX DOMAIN PROTEIN/CONSTANS-LIKE 15 (BBX13/COL15).

Rocket species are increasing in popularity with consumers, and in the last ten years scientific interest has also increased due to the potential health benefits of consuming leaves. They are known for pungent and bitter taste components, and the chemical compounds largely responsible for these sensations are also health beneficial. These compounds are called isothiocyanates (ITCs), and they are ubiquitous in the plant family Brassicaceae. Precursor compounds called glucosinolates (GSLs) are converted to ITCs via the action of myrosinase enzyme. This thesis presents data relating to numerous aspects of rocket species, such as differences in GSLs and ITCs. Other phytochemical constituents (flavonols, volatile organic chemicals (VOCs), free amino acids, free sugars, polyatomic ion content, and organic acids) are explored to determine their impacts on human sensory perceptions and consumer acceptance. The data presented highlight significant differences between ‘wild’ accessions of rocket and commercially available varieties, in terms of flavonol and GSL content and sensory attributes. There is great potential to develop underutilised genetic resources in breeding programs, and through collaboration with a breeding company (Elsoms Seeds Ltd., Spalding, UK) and a commercial salad supplier (Bakkavör Group Ltd., Spalding, UK), several accessions were selected for detailed analyses. Analysis of VOC profiles further demonstrated the differences between the selected cultivars, and by combining these data with sensory and consumer studies, it was observed that the diversity of phytochemical components fundamentally underpins taste, flavour, and consumer acceptance. The same accessions of rocket were also tested under commercial growth, processing and storage conditions. It was hypothesised that this would negatively impact GSL and ITC content of leaves, but in fact increased concentrations up to five fold from the point of harvest in all accessions analysed. We also observed a previously undocumented link between GSL, ITC and free amino acid content with bacterial load.

Tilia L. is a temperate-forest tree genus with a wide northern hemisphere distribution. Several species within the genus are affected by forest fragmentation. Three species were the focus of this study, T. cordata Mill. (small-leaved lime) and T. platyphyllos Scop. (large-leaved lime) from the UK, Austria, Poland, and western Siberia and T. sibirica Bayer (Siberian lime) from southern Siberia. Tilia specific microsatellite markers were used to assess various population genetics indices. Genetic diversity and structure of UK T. cordata and T. platyphyllos populations were estimated. To determine the genetic and demographic history of T. sibirica and T. cordata, Approximate Bayesian Computation (ABC) analyses were used. An investigation into the clonal architecture of the three species was carried out to assess the level of clonality and the impact of clonal reproduction on genetic diversity. In addition, Next Generation Sequencing of the Tilia leaf transcriptome was carried out using direct RNA sequencing Results confirm that the three species are diploid and outcrossing. Although hybridisation occurs among T. cordata and T. platyphyllos, the two are distinct biological units with high genetic diversity and intra-specific population structure. Significant genetic differentiation was observed between T. sibirica and T. cordata and low genetic diversity in the Siberian lime was revealed. ABC analysis suggests a relatively recent (Early Holocene) divergence between the Siberian lime and the small-leaved lime. The Holocene split coincided with a westerly migration of Tilia genotypes that may have contributed to the recolonization of T. cordata in Europe. Fewer clones were observed in T. platyphyllos than the other two species and range-edge populations experience greater clonality than central European populations. Clonal occurrence does not appear to have had a negative effect on genetic diversity. A method for the de novo assembly and annotation of the leaf transcriptome from T. cordata and T. platyphyllos is provided. Potentially thousands of simple sequence repeats (SSRs) from each species have been identified.

Primula vulgaris is a model species for the study of heterostyly, and displays two floral morphologies, pin and thrum, which show a reciprocal arrangement of the anthers and stigma. The differences in floral morphology are controlled by the S locus, which consists of several closely linked genes. One of the first genes to be identified as part of the S locus was GLOT, a paralogue of GLOBOSA. Preliminary results have shown that GLOT expression is confined to the second and third floral whorls of thrum flowers. In pin flowers, where GLOT is not expressed, the anthers are lowered. This study involves characterisation of the expression dynamics of GLOT in comparison to its paralogue GLO, in the context of the recent discovery that the S locus is hemizygous and not heterozygous as previously thought. The selection of normalisation genes for qPCR was conducted, and the temporal expression of both GLO and GLOT was measured across bud development; the genes showed different expression patterns. RNA in situ hybridisation was then used to assess spatial expression of both genes in floral meristems, with GLO showing defined localisation within the developing second and third whorls and GLOT showing more dispersed expression. The interactions of P. vulgaris GLO and GLOT proteins with A. thaliana MADS box proteins were tested in Yeast 2-Hybrid experiments, and while GLO showed interactions with the orthologue of its partner, AP3, GLOT did not show interactions with any of the other proteins tested. Antibodies were designed against peptide sequences to assess protein localisation for use in future experiments. This work has furthered knowledge on the expression patterns of these genes, the divergence of GLOT from GLO, and has generated tools that will enable further analyses of the differences between these two genes.

The study focuses on the herbaceous perennial plant genus Aubrieta (Brassicaceae). Distributed from Armenia through the Levant and Anatolia to Greece, the Balkans and Italy, the species have proved difficult to distinguish. About 12 species are currently recognised, although estimates range from only one up to about 20 or more. Furthermore, their evolutionary relationships are unknown. In order to remedy this situation molecular and cytogenetical studies were conducted. Next Generation Sequencing (NGS) data were produced to generate a complete chloroplast genome for four species of Aubrieta in order to confirm the phylogenetic position of the genus in the family. Earlier suggestions that it belongs in tribe Arabdieae were confirmed. Details of plastome structure were analysed in A. gracilis, which was shown to have 88 protein-encoding, 37 transfer-RNA and eight ribosomal-RNA genes. A phylogenetic analysis of the genus was conducted based on chloroplast and nuclear sequences as well as mitochondrial RFLPs. Five chloroplast regions (matK, trnD-trnT, ycf6-psbM, rps11-rpl36 and trnH-psbA) and two nuclear genes (duo1 and rbp2) were amplified and sequenced successfully. Six mitochondrial gene regions (Orf114, Nad9-ccmFN2, Orf25, matR, ccm FC, trnK-rps3) were studied by means of restriction enzymes. Data analyses show that Aubrieta comprises the annual, pan-Mediterranean Arabis verna plus perennial taxa that fall into one of five geographically delimited gene pools: i) Near East (Levant, Iraq and Iran); ii) Anatolia; iii) Aegean Basin; iv) Greece, Albania, Bulgaria (Pindus Mts and associated ranges); and v) Trans-Adriatic Sea and Sicily. There was some disagreement between the plastid and nuclear trees, which was attributed to hybridisation, chiefly affecting the taxa occupying the Aegean Basin. Evolution in the genus appears to have proceeded largely at the diploid level (2n=2x=16). In order to see what changes at the chromosome level have accompanied speciation, fluorescence in situ hybridization (FISH) studies were conducted. The probes pTa71 and pTa794 were used to locate the position of 45S and 5S rDNA sites on the chromosomes. The number of 45S rDNA sites are 2, 4, 5, or 6, localized on short-arms, the centromere and on long-arms. The number of 5S rDNA sites is a constant two, located either on short-arms or long-arms. These rDNA sites (45S and 5S) are either located on different chromosomes or shared by one or two chromosomes. Speciation is accompanied (promoted?) by translocations and duplications. Hybridisation was confirmed in the genus. The timing of the various bifurcations in the evolutionary tree were estimated from a study of the concatenated chloroplast sequences, but the major split into an annual lineage (Arabis verna) and a perennial lineage appears to date from 1.4 Mya. The hybridisation events involving the Aegean Basin taxa appear to date from the early to mid-Pleistocene (ca 600-800 Kya), a time when considerable parts of the Aegean were above sea level. The taxonomy of the genus is still problematic, it being impossible to diagnose the five geographical genepools by means of morphological characters. Instead, a splitting approach is adopted whereby regional or local phenotypes are recognised as species. This can be unsatisfactory in some cases where there is considerable morphological, but not geographical, overlap. A total of 21 species, including Arabis verna which is recombined into Aubrieta, is recognised.

Whole genomic survey sequences were obtained for Taraxacum obtusifrons Markl. (O978); T. stridulum Trávniček ined. (S3); and T. amplum Markl. (A978), three apomictic triploid (2n=3x=24) dandelions from the T. officinale agg. (Asteraceae) Retroelement-based markers and chloroplast data showed that S3 and O978 are genetically the most similar microspecies. Genomic diversity in Taraxacum and also Hieracium was high, discriminating species but not showing phylogeny; major groups of retroelements were abundant in both genera. The chloroplast genomes of accessions O978 and S3 were identical. Repetitive DNA including transposable elements (TEs) are dynamically evolving in genomes, but their variability and abundance make them challenging to study using molecular biology. In the current study, we used the whole genomic sequences to investigate the repetitive structure, diversity and components of the three closely related Taraxacum accessions. Analysis of about 45Gb sequence (10x to 20× genome coverage) of three closely related Taraxacum microspecies, were analysed by graph-based clustering of the raw reads (using the program RepeatExplorer) and frequency analysis of all DNA motifs possible for various motif lengths (k-mer analysis). Different DNA motif lengths were evaluated and complemented the graph-based results. Graph-based clustering showed that many of the Taraxacum microspecies repeats consist of Ty1-copia (13-16%) and Ty3-gypsy (10-14%) family retroelements, while DNA transposons were rare. Unclassified repetitive DNA sequence clusters were investigated. In situ hybridization was used to localize major repetitive DNA families on chromosomes. Apart from 5S and 45S rDNA and telomere sequences, few tandemly repeated DNA motifs were found, although a 49bp repeat was found at some centromeres. There were differences between the three Taraxacum microspecies in genomic proportions and locations for repetitive DNA types suggesting many sequence motifs are evolving rapidly with increasing or decreasing copy numbers. A class of repetitive DNA has been recognized as Passively Amplified DNA Sequences, PADS.

No description available.

No description available.