Whole genome approaches to identify genes involved in early meiosis.

Meiosis is a process which occurs in sexually reproducing organisms to halve the genetic complement prior to fertilisation. During meiosis a single round of DNA replication is followed by two successive rounds of chromosome segregation and cell division. The meiotic pathway in plants is complex from multiple perspectives. From a mechanical view; prior to the first meiotic division the chromosomes must replicate during meiotic interphase, then while retaining sister chromatid cohesion the homologous chromosomes must align, physically synapse and also concomitantly recombine (with the majority of sites being non-randomly positioned). Further complexities arise in allopolyploids such as bread wheat, which contains three very similar genomes from slightly diverged progenitors. Despite having homoeologous chromosomes present in the same nucleus, bread wheat displays diploid-like behaviour during meiosis I. Such an involved physical process as meiosis also has complexity reflected in the transcriptome and proteome, whether the organism be a simple eukaryote such as yeast, or a more complex eukaryote such as bread wheat. Initially, this study utilised whole genome approaches to identify novel genes that could be involved in early meiosis, focusing on bread wheat in particular. Analysis of the wheat meiotic transcriptome over seven stages of anther development identified at least 1,350 transcripts which displayed meiotic regulation. The expression profiles of a subset of selected transcripts were analysed with Q-PCR and found to correlate strongly to those obtained in the microarray. Available meiotic transcriptome data from rice was compared to the wheat data, which enabled the identification of similar sequences, many previously unidentified, which also displayed meiotic regulation. Selected candidate genes from the microarray study were also mapped in bread wheat. This data was combined with available literature and approximately 70% of candidate meiotic loci were located on chromosome group 3 or 5, which historically has been shown to contain multiple loci involved in chromosome pairing control. One of the candidates located on chromosome group 3, a plant-specific mismatch repair gene, Triticum aestivum MSH7 (TaMSH7), has previously been speculated to suppress homoeologous chromosome associations. Independent transgenic wheat plants produced using RNA interference (RNAi) were functionally characterised to ascertain a greater understanding of the role TaMSH7 has during early meiosis in bread wheat. Localisation of a synaptonemal complex-associated protein (TaASY1) displayed subtle abnormalities in these mutants when compared to wild-type. Feulgen staining of meiotic chromosomes at metaphase I in these mutants revealed some interlocking and multivalent associations. These results suggest that TaMSH7 may be linked to the mechanism underlying the phenotype that is observed in the ph2a/ph2b mutant, however further research still needs to be conducted to conclusively demonstrate that this is the case. A component of the research presented in this study was performed in the model plant Arabidopsis thaliana due to the limitations of bread wheat. Extensive mutant banks and a sequenced genome have aided a decade of meiotic research in Arabidopsis and the identification of close to 50 meiotic genes. One of these, AtMER3, has been shown to control the non-random location of well above half of the recombination events that occur in many species. AtMER3 was localised in meiotic nuclei in wild-type Arabidopsis and found to form foci on freshly synapsed regions of chromosomes in quantities far in excess of the average number of crossovers, indicating that AtMER3 does not localise exclusively to sites of crossovers. AtMER3 localisation was also analysed in several mutant backgrounds and found to act in an AtSPO11-dependent manner. However, AtMER3 loading onto meiotic chromosomes was not affected in Atrad51, Atdmc1 or Atmsh5 mutant backgrounds.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 200