Genomics approach to investigate the molecular control of meiosis in Triticum aestivum

Meiosis is a cell division process central to the life cycle of all sexual eukaryotic organisms. Chromosome pairing, genetic recombination and subsequent nuclear division during meiosis produces four genetically distinct haploid gametes from a single diploid cell. Allohexaploid wheat ( Triticum aestivum ) behaves meiotically as a diploid, despite the existence in the genome of three closely related ( homoeologous ) genomes, A, B and D. Chromosome pairing during prophase I of meiosis in wheat is restricted to true homologous chromosomes, the result being the formation of 21 bivalents at meiotic metaphase I. The genetic control of chromosome pairing in wheat is under the control of several pairing homoeologous ( Ph ) genes, located predominantly on chromosome groups 3 and 5. The major suppressors of homoeologous pairing are Ph1 and Ph2. Their cytogenetic effect has been intensively studied but at the molecular level little is known about their function. The isolation and characterisation of Ph genes from wheat would lead to greater understanding of chromosome pairing mechanisms in complex allopolyploids, and may enable development of effective strategies for alien gene introgression from related species to modern wheat cultivars. In this study, several genomics - based approaches were adopted to explore the expressed portion of the wheat genome in order to identify and characterise genes that could function in the molecular processes regulating meiosis. The first approach used comparative genetics to characterise the region deleted in the ph2a mutant ( a deletion mutant at Ph2 ). The rice genomic region syntenous to that deleted in the ph2a mutant was identified through comparative mapping and used in searches of wheat databases to identify ESTs with significant similarity. Southern analysis confirmed a syntenous relationship in the wheat and rice genomic regions and defined precisely the position of the breakpoint in ph2a. What seems to be a terminal deletion on 3DS is estimated to be approximately 80 Mb in length. We can tentatively predict the identification of approximately 220 genes from the region deleted in ph2a. The putative role of identified candidate Ph2 genes is discussed. The second approach explored the validity of recent proposals suggesting the presence of a meiotic gene cluster in the region of Ph2. The transcriptional characteristics of genes linked to Ph2 were investigated using data from wheat EST databases in combination with recently developed analysis software. The tissue - distribution of mRNAs derived from genes linked to Ph2 is shown to resemble that of other large chromosomal regions in the wheat genome. It is concluded that the apparently high number of genes from the Ph2 region expressed in wheat meiotic tissue is not indicative of a meiotic gene cluster in this region, but rather highlights the transcriptional complexity of meiotic anther tissue. Finally, the meiotic expression pattern of approximately 1800 wheat genes was examined using cDNA microarrays. Two approaches were taken. Firstly, the applicability of microarrays to identify differentially expressed genes between wild - type anthers and anthers of three Ph mutant genotypes was investigated. These experiments failed to reveal significant down - regulation of genes in Ph mutant anthers compared to wild - type. Possible explanations are discussed. Secondly, the expression of all microarray clones was examined from pre - meiotic interphase through to the tetrad stage of meiosis. A number of candidate wheat genes involved in meiotic and anther developmental processes have been identified and are discussed. Prior to this study, the methods available to identify wheat meiotic genes, in particular as candidates for Ph2, were limited. The recent development of genomics in plant biology provided an opportunity for a new approach towards gene discovery and genome structural analysis in relation to meiosis. This research illustrates the need for, and the effectiveness of a new approach to study meiosis, contributing to our knowledge of the structural and functional characteristics of genes linked to Ph2, and establishing a strong basis for further wheat meiotic gene characterisation.Thesis (Ph.D.)--School of Agriculture and Wine, 2003