A number of morphological, floral, rhizome, root and herbage characteristics were studied in order to describe genetic variation and environmental responses in T. ambiguum. From each of six lines, 30 genotypes were clonally progagated into five diverse edaphic and altitudinal sites and grown for one season. Comparisons of growth and form were also made between T. ambiguum, T. repens cv. 'Grasslands Huia' and Lotus pedunculatus cv. 'Grasslands Maku'. The polyploid lines were found to have larger leaves than the diploids while all the lines of T. ambiguum had larger leaves than did white clover. Tetraploid lines had longer petioles and were taller and more erect than the diploids or hexaploids. Floral initiation was found to become later as ploidy level increased but the tetraploid lines exhibited a very large variation in flowering date. The cultivar Treeline was found to produce the most herbage under all conditions although not significantly more than cultivar Prairie or C.P.I. 57353. However, as nodulation was not studied it was not possible to determine whether variety differences were due to root nodulating ability or some other genetically determined parameter. None of the T. ambiguum varieties produced as much herbage as cv. Huia or cv. Maku at any site. However, all the T. ambiguum lines performed relatively better under harsher conditions. Because a large proportion of T. ambiguum was below ground the best T. ambiguum line, cultivar Treeiine, produced equivalent total plant dry weight to cv. Huia and cv. Maku at 1200 m. a.s.l., the high altitude site. Cultivar Prairie was found to have the highest proportion of rhizomes to total plant mass bu because cv. Treeline had higher total plant dry weight both cultivars produced equivalent mean rhizome dry weight. The number of rhizomes, number of daughter plants and rhizome dry weight were all highly correlated and these three characteristics showed similar trends among varieties. Rhizome length was found to increase with ploidy level, as did rhizome internode length. However, the number of nodes was found to be higher in the diploids than in the polyploids. Cultivar Treeline was found to have a high proportion of branching nodes on its rhizomes while C.P.I. 57353 and cv. Summit had the least. Rhizome production was restricted in the Wakanui silt loam soil of high bulk density. However, herbage growth and rhizome branching was increased, probably because of the higher fertility. It was shown using factor analysis on genotypic correlations, that rhizome characteristics and herbage yields were relatively independently inherited. However, morphological characters tended to be related to herbage yields. The polyploid varieties were found to be more genetically variable than the diploids . Broad sense heritabilities were calculated for all parameters measured, and in general, morphological characteristics had higher values than agronomic characteristics. As the genetic variation within each line was higher than the variation among lines, it is apparent that selection within lines should result in the largest gains. Therefore, comparison of the mean performance of the presently highly variable lines is relatively uninformative. By sacrificing some genetic diversity, large gains could be made in performance. It is suggested that cv. Prairie be used as the basis for selecting a highly rhizomatous cultivar while cv. Treeline could be used in the selection of a cultivar with higher herbage production suitable for high country conditions. In a second trial, an established stand of cv. Treeline produced up to 13250 kg per ha for one season under good growing conditions. The management required to produce this amount of herbage was to irrigate and cut to ground level every two months. The growth rate was considerably depressed when cut monthly. It was also shown that root and rhizome yield reached 12600 kg per ha, indicating a massive reserve of assimilates, particularly useful for surviving periods of stress. Seed yields were found to be adequate, reaching levels equivalent to 500-700 kg per ha. These results were discussed in relation to earlier observations on T. ambiguum by workers in Russia, Australia, U.S.A. and New Zealand. Suggestions were made for further genetic and agronomic testing.

Copies of author's previously published articles inserted. Bibliography: leaves 143-167. Temperature may affect fruit-set in grapevines through its effect on the development of the flowers up to flower opening and on pollination, be it on the germination of the pollen and the growth of the pollen tube, or on the post-fertilisation growth of the ovule.

Arabidopsis thaliana HYL1 is a nuclear doublestranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 primiRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3’ and 5’ RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1- dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5’ splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced primiRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs.

Although polyploidy is believed to be a major source of evolutionary novelty, few studies have examined the genetic basis of phenotypic variation in wild polyploids. In this thesis I have studied the genetic basis of flowering time variation in the wild tetraploid crucifer Capsella bursa-pastoris, as well as the evolutionary history of this species. First, phylogenetic methods were employed to test hypotheses on the origin of C. bursa-pastoris. Based on DNA sequences from two chloroplast DNA loci and three independent nuclear genes, we found no support for the notion of C. bursa-pastoris as an autopolyploid of the diploid C. grandiflora, or an allopolyploid of C. grandiflora and C. rubella, even though some C. bursa-pastoris accessions shared alleles with C. rubella at nuclear loci. Using divergence population genetic methods, a larger sample of accessions and data for six duplicated nuclear genes, we found that allele sharing in sympatry was better explained by introgressive hybridization than by multiple origins of the tetraploid. The genetic basis of flowering time variation was examined using three approaches. A gene expression microarray study revealed that early- and late-flowering accessions differ in circadian rhythm, as well as in the gibberellin pathway affecting flowering time. Second, two QTL (Quantitative Trait Loci) for flowering time map to duplicated linkage groups. Third, polymorphisms at the candidate genes CRYPTOCHROME1 (CRY1), in one of the QTL regions, and FLOWERING LOCUS C (FLC) are associated with natural flowering time variation. Different FLC splice site polymorphisms are associated with flowering time in samples from Western Eurasia and China. The CRY1 association is only found in Europe, where alleles introgressed from C. rubella have an effect on flowering time. In conclusion, duplicated genes, introgressive hybridization and splicing variation may all have played a role in the evolution of flowering time variation in C. bursa-pastoris.

The transition to flowering is a major developmental switch in angiosperms, the timing of which is expected to be important for fitness. In this thesis the focus has been on identification of genes affecting natural variation in flowering time in Brassica nigra. The background for this thesis is an earlier QTL-mapping study in B. nigra. The genomic area with the greatest effect on flowering time in that study contained a homolog to the CONSTANS gene, which is known to affect flowering time in A. thaliana. When studied more closely this gene did not seem to affect flowering time variation in B. nigra. Near the B.nigra CO gene (BniCOa), however, we identified a homolog to the related CONSTANS LIKE 1 (COL1) gene. In A. thaliana COL1 has not been shown to be associated with induction of flowering but since the B. nigra homolog (BniCOL1) in the QTL area showed surprising amounts of variation between early and late flowering plants we set out to test if this variation was associated with flowering time variation. In the first paper we found a significant association between flowering time and one indel (Ind2) in the coding region. Motivated by the results in paper one, we searched for evidence of selection at the BniCOL1(paper two). In paper three the aim was to validate the results from the first paper in a larger sample of populations, and to check whether the association found in paper I could reflect linkage disequilibrium with areas outside of the gene. Finally, in paper four we investigate the general evolution of three CONSTANS-LIKE genes in B. nigra, namely BniCOL1, BniCOa and BniCOb.

Photoperiodism refers to the organism’s ability to detect and respond to seasonal changes in the daily duration of light and dark and thus constitutes one of the most significant and complex examples of the interaction between the organism and its environment. This thesis attempts to describe the prevalence of variation in a photoperiodic response, its adaptive value, and its putative genetic basis in a common cruciferous weed, Capsella bursa-pastoris (Brassicaceae). Furthermore, the thesis presents a first comprehensive comparative overview of the circadian clock mechanism in an early land plant, Physcomitrella patens (Bryophyta), thus providing insights into the evolution of the plant circadian system. In an introductory survey of global gene expression changes among early- and late flowering accessions of C. bursa-pastoris we found an enrichment of genes involved in photoperiodic response and regulation of the circadian clock. Secondly, by phenotyping circadian rhythm variation in a worldwide sample of accessions with known flowering time, we detected robust latitudinal clines in flowering time and circadian period length, which constitute strong indications of local adaptation to photoperiod in the shaping of flowering time variation in this species. In an attempt to elucidate putative genetic causes for the correlated variation between circadian rhythm and flowering time, we found that sequence variation and diverged expression in components regulating light input to the clock, PHYTOCHROME B (PHYB) and DE-ETIOLATED 1 (DET1) make them strong candidate genes. Finally, we present a comparative study of circadian network topology in the moss P. patens. Phylogenetic analyses and time series expression studies of putative clock homologues indicated that several core clock genes present in vascular plants appeared to be lacking in the moss. Consequently, while the clock mechanism in higher plants constitutes at least a three-loop system of interacting components, the moss clock appears to comprise only a single loop. We conclude that C. bursa-pastoris is a highly suitable model system for the further elucidation of the molecular variation that influences adaptive change in natural plant populations. Furthermore, we believe that the continuing study of the seemingly less complex circadian network of P. patens not only can provide insights into the evolution of the plant circadian system, but also may help to clarify some of the remaining issues of the circadian clock mechanism in higher plants.

Seed coat biochemistry and colour are highly variable in common bean (Phaseolus vulgaris) (syn. dry bean). Genetic studies of dry bean seed coat chemistry and colour have important implications in breeding efforts for improving nutrition and seed quality for consumer acceptance. The results of this thesis detail the phenotypic and genotypic characterization of seed coat post harvest darkening (PHD) in parents and progeny of crosses among them as well as the phenotypic characterization of seed coat condensed tannin (CT) accumulation in five genotypes of bean.<p> Seed coat PHD represents a problem for producers and consumers of several different market classes of dry bean. There are three post harvest darkening phenotypes: (i) non-darkening (ND), (ii) slow darkening (SD) and (iii) regular darkening (RD). The inheritance of PHD was determined by evaluating 28 populations derived from crosses between RD, SD and ND genotypes. Results suggest that at least two major, unlinked genes control the PHD trait in dry bean. Recessive epistasis with three phenotypic classes best explains the segregation ratios observed in populations from crosses between SD and ND parents. One gene, J, is responsible for whether a bean will darken and seeds of plants that are jj do not darken at all. Another gene, SD, influences the rate a seed coat will darken with seed from sdsd individuals darkening more slowly that those with the dominant SD allele. Quantitative evaluation of seed coat PHD demonstrated that there was a wide range of darkening within any given PHD phenotype. Crosses made between the ND x ND cross class resulted in F2 progeny that were all ND, however, a wide range of seed coat background colours was noted in the progeny. In several of the crosses made between ND x RD and SD x RD classes the resulting F1 progeny were all RD; however, a wide range of RD phenotypes were observed in the F2 progeny. These phenotypes are not likely due to quantitative trait loci (QTL) associated with the PHD trait, but rather a result of other chemical reactions occurring in the seed coat. Condensed tannins (CT; syn. proanthocyanidins), kaempferols, polyphenol oxidase (PPO) and possibly other compounds or enzymes may be interacting and causing this quantitative range within any given genotype as a function of environmental variability, genotype and their interaction. CT have been associated with PHD but are not responsible for the major difference between RD and SD lines. They may, however be responsible for the quantitative nature of the phenotype.<p> Condensed tannins can be harmful or beneficial to human health and the environment depending on the amount present and where it is found in the plant. Manipulating the production, accumulation and form of CT in the seed coat of dry bean would be beneficial to bean producers, consumers and breeders. This experiment quantitatively and qualitatively evaluated differences in patterns of CT accumulation in the seed coats of five genotypes of dry bean which exhibited low, medium or high concentrations of CT in their seed coats at maturity. Condensed tannin content was assessed from seeds harvested every other day from 6 40 days after flowering (DAF) using a modified BuOH-HCl assay. Results illustrated that CT accumulated as early in low CT genotypes as in high CT genotypes. CT content stabilized after 14 DAF in low CT genotypes. By contrast, CT content peaked then leveled off 30 DAF in moderate and high CT genotypes. A reduction in CT content in the higher CT lines was observed in the final stages of seed development.

<p>Flowering is a critical step in the plant life cycle. If flowering occurs too early or too late, seed production suffers. Flowering is regulated through numerous flowering repressors. As long as these repressors persist, the plant will remain in a vegetative growth stage. Some plants possess two separate genetic pathways, the autonomous pathway and the vernalization pathway, that promote the transition to flowering through stable downregulation of flowering repressors. Once the plant achieves floral competence, it will flower under inductive environmental conditions.</p> <p>In <i>Arabidopsis</i>, <i>FCA</i> is a key autonomous pathway gene, acting with <i>FY</i> to promote the floral transition. Recently, gene sequences resembling <i>FCA</i> were cloned from hexaploid wheat (<i>Triticum aestivum</i>) and designated as <i>WFCA</i>. WFCA shows numerous similarities to the FCA peptide, especially regarding three key regions: two RNA Recognition Motifs and the WW domain. This study seeks to determine if <i>WFCA</i> genes function similar to <i>FCA</i> by determining if they are able to complement the <i>fca-1</i> mutant of <i>Arabidopsis thaliana</i>.</p> <p>T1 progeny from an <i>Arabidopsis fca-1</i> plant transformed with <i>WFCA</i> were grown without vernalization and assayed for the final leaf number (FLN). The late flowering <i>fca-1</i> control plants bolted with an average FLN of 14.8 while the T1 population had an average FLN of 14.3. Although the numerical difference is slight, the results are statistically significant, and suggest that <i>WFCA</i> genes may have some degree of flowering promotion activity in <i>Arabidopsis</i>. The lack of strong complementation may be due to divergence of the <i>WFCA</i> genes from their <i>Arabidopsis</i> counterparts. With increasing evidence for divergence in flowering promotion between monocot and dicot species, the development of a robust monocot model system appears to be critical to provide a good framework to assist studies of the particular nuances of the monocot flowering process.</p>

<p>Flowering is a critical step in the plant life cycle. If flowering occurs too early or too late, seed production suffers. Flowering is regulated through numerous flowering repressors. As long as these repressors persist, the plant will remain in a vegetative growth stage. Some plants possess two separate genetic pathways, the autonomous pathway and the vernalization pathway, that promote the transition to flowering through stable downregulation of flowering repressors. Once the plant achieves floral competence, it will flower under inductive environmental conditions.</p> <p>In <i>Arabidopsis</i>, <i>FCA</i> is a key autonomous pathway gene, acting with <i>FY</i> to promote the floral transition. Recently, gene sequences resembling <i>FCA</i> were cloned from hexaploid wheat (<i>Triticum aestivum</i>) and designated as <i>WFCA</i>. WFCA shows numerous similarities to the FCA peptide, especially regarding three key regions: two RNA Recognition Motifs and the WW domain. This study seeks to determine if <i>WFCA</i> genes function similar to <i>FCA</i> by determining if they are able to complement the <i>fca-1</i> mutant of <i>Arabidopsis thaliana</i>.</p> <p>T1 progeny from an <i>Arabidopsis fca-1</i> plant transformed with <i>WFCA</i> were grown without vernalization and assayed for the final leaf number (FLN). The late flowering <i>fca-1</i> control plants bolted with an average FLN of 14.8 while the T1 population had an average FLN of 14.3. Although the numerical difference is slight, the results are statistically significant, and suggest that <i>WFCA</i> genes may have some degree of flowering promotion activity in <i>Arabidopsis</i>. The lack of strong complementation may be due to divergence of the <i>WFCA</i> genes from their <i>Arabidopsis</i> counterparts. With increasing evidence for divergence in flowering promotion between monocot and dicot species, the development of a robust monocot model system appears to be critical to provide a good framework to assist studies of the particular nuances of the monocot flowering process.</p>

Seed coat biochemistry and colour are highly variable in common bean (Phaseolus vulgaris) (syn. dry bean). Genetic studies of dry bean seed coat chemistry and colour have important implications in breeding efforts for improving nutrition and seed quality for consumer acceptance. The results of this thesis detail the phenotypic and genotypic characterization of seed coat post harvest darkening (PHD) in parents and progeny of crosses among them as well as the phenotypic characterization of seed coat condensed tannin (CT) accumulation in five genotypes of bean.<p> Seed coat PHD represents a problem for producers and consumers of several different market classes of dry bean. There are three post harvest darkening phenotypes: (i) non-darkening (ND), (ii) slow darkening (SD) and (iii) regular darkening (RD). The inheritance of PHD was determined by evaluating 28 populations derived from crosses between RD, SD and ND genotypes. Results suggest that at least two major, unlinked genes control the PHD trait in dry bean. Recessive epistasis with three phenotypic classes best explains the segregation ratios observed in populations from crosses between SD and ND parents. One gene, J, is responsible for whether a bean will darken and seeds of plants that are jj do not darken at all. Another gene, SD, influences the rate a seed coat will darken with seed from sdsd individuals darkening more slowly that those with the dominant SD allele. Quantitative evaluation of seed coat PHD demonstrated that there was a wide range of darkening within any given PHD phenotype. Crosses made between the ND x ND cross class resulted in F2 progeny that were all ND, however, a wide range of seed coat background colours was noted in the progeny. In several of the crosses made between ND x RD and SD x RD classes the resulting F1 progeny were all RD; however, a wide range of RD phenotypes were observed in the F2 progeny. These phenotypes are not likely due to quantitative trait loci (QTL) associated with the PHD trait, but rather a result of other chemical reactions occurring in the seed coat. Condensed tannins (CT; syn. proanthocyanidins), kaempferols, polyphenol oxidase (PPO) and possibly other compounds or enzymes may be interacting and causing this quantitative range within any given genotype as a function of environmental variability, genotype and their interaction. CT have been associated with PHD but are not responsible for the major difference between RD and SD lines. They may, however be responsible for the quantitative nature of the phenotype.<p> Condensed tannins can be harmful or beneficial to human health and the environment depending on the amount present and where it is found in the plant. Manipulating the production, accumulation and form of CT in the seed coat of dry bean would be beneficial to bean producers, consumers and breeders. This experiment quantitatively and qualitatively evaluated differences in patterns of CT accumulation in the seed coats of five genotypes of dry bean which exhibited low, medium or high concentrations of CT in their seed coats at maturity. Condensed tannin content was assessed from seeds harvested every other day from 6 40 days after flowering (DAF) using a modified BuOH-HCl assay. Results illustrated that CT accumulated as early in low CT genotypes as in high CT genotypes. CT content stabilized after 14 DAF in low CT genotypes. By contrast, CT content peaked then leveled off 30 DAF in moderate and high CT genotypes. A reduction in CT content in the higher CT lines was observed in the final stages of seed development.