Isolation and characterisation of wheat genes with early meiotic expression.

Abstract

For all sexually reproducing organisms meiosis is an essential process. Cells undergoing meiosis go through one round of DNA replication followed by two successive rounds of chromosome segregation and cellular division, meiosis I and meiosis II. During chromosome alignment and subsequent segregation, recombination or the exchange of genetic information between homologous chromosomes takes place, which effectively maintains genetic diversity through new allelic combinations. The majority of meiotic research to date has been limited to diploids, such as yeast, mice and Arabidopsis. However, as more than 70% of all flowering plants are polyploid, exclusively using diploids could result in over simplifying the process in more complex species. A recent transcriptomics study in bread wheat conducted by Crismani and colleagues (2006) identified 1,350 transcripts which were temporally regulated during the early stages of meiosis. A number of the meiotically-regulated transcripts have annotated functions for chromatin condensation, synaptonemal complex formation, recombination and fertility; however 1,094 transcripts were deemed to have either no annotations or purely predicted annotations. The meiotic gene DMC1, previously described in several diploid species, was one of the 1,350 transcripts having meiotic regulation in wheat. Isolation of the bread wheat DMC1 homologue, and the genes encoding TaHOP2 and TaMND1 which are known to interact closely with DMC1, was successful. TaDMC1 and TaMND1 were located on chromosome group 5, while TaHOP2 was located on chromosome group 4. All three genes have significant similarity to the rice (98%, 97%, 95%, respectively) and Arabidopsis (91%, 85%, 87%, respectively) homologues. TaHOP2 and TaMND1 transcript expression was found to be highly correlated (r = 0.98) across the germline and somatic tissues examined. Using an anti-AtMND1 antibody, the TaMND1 protein localised to meiotic chromosomes during pre-meiotic (PM) interphase through to pachytene, while localisation attempts for the TaDMC1 protein with an anti-AtDMC1 antibody proved unsuccessful. The functions of the TaDMC1 and TaHOP2 proteins were found to be conserved, with these proteins having the ability to bind preferentially to single-stranded DNA. Another gene identified from the transcriptomics study, and within the 1,350 transcripts, was TaASY1 (bread wheat ASYnapsis1). While TaASY1 is known to be involved in chromosome pairing and synapsis, the mechanics of how this gene is regulated still remains somewhat unknown. To elucidate potential protein partners of the TaASY1 homologue, a yeast two-hybrid approach was conducted. Three proteins were found to interact with the full length TaASY1 protein and the HORMA domain of the ASY1 protein, along with one protein which interacted only with the isolated HORMA domain. Characterisation of two of the proteins which interact with TaASY1 revealed a Replication Protein A (RPA14) homologue and a novel plant protein. A further objective of the research conducted within this study was to isolate and characterise novel meiotic candidates to further the understanding of the process in bread wheat. From the previously identified 1,094 novel transcripts, those with either no or putative annotations underwent selective filtering. This resulted in 40 candidates being targeted for further analysis, after which eight candidates were selected for in-depth analysis. Arabidopsis homologue mutants were obtained for these candidates to determine whether a meiotic role could be assigned. Mutant plants from one candidate, AtMN29, showed altered phenotypic features that suggest this gene has a role during meiosis in Arabidopsis. Abnormalities included decreased silique length, decreased seeds per silique, decreased pollen viability and altered expression of the meiotic genes ASY1 and DMC1.