Wheat non-specific Lipid Transfer Proteins: identification and characterisation of nuclear-encoded fertility restorer genes

Abstract

Plant non-specific lipid transfer proteins (nsLTPs) constitute a large protein family found in all land plants. NsLTPs are involved in a wide range of biological processes, however, only few members have been functionally characterised. The aim of this project was to investigate the biological function of TaMs1, a wheat glycosylphosphatidylinositol (GPI)-anchored nsLTP. TaMs1 was identified as a dominant wheat fertility gene necessary for pollen exine development and proposed for use in the efficient bulking of male-sterile lines required for hybrid wheat seed production. Additionally, we identified and analysed nsLTP members in bread wheat on a genome-wide scale, providing a valuable resource aimed at elucidating the function of these genes in wheat development. Firstly, to investigate the biological function of TaMs1, we conducted expression analysis of its three homeoalleles (A, B, and D sub-genomes) on a series of wheat tissues collected at different developmental stages. TaMs1 transcripts were detected exclusively in anthers during early microspore development, and we only observed expression of the B-genome-derived TaMs1 (Chapter 3). Additionally, we observed that previously reported genes deemed necessary for pollen exine formation were not differentially regulated in ms1 deletion mutants (ms1c, cv. Cornerstone) relative to Wild-Type, suggesting an independent function for TaMs1 from these genes during pollen development. Moreover, we showed that the encoded protein TaMs1 is targeted to the plasma membrane. For a commercially viable hybrid wheat production platform, complete penetrance of the ms1-induced male sterility is critical. Here, we observed male sterility penetrance to be variable depending upon ms1 mutation type and genotypic background (Chapter 5). The single nucleotide polymorphism (SNP mutant) ms1d (cv. Chris), showed near-complete male sterility in various backgrounds, whilst partial to full fertility (incomplete penetrance) was observed for the homozygous ms1c deletion mutant. To identify possible loci involved with this incomplete penetrance, we used a genotyping-by-sequencing (GbS)-SNP-based linkage map followed by analysis of quantitative trait loci (QTL) for selfed-seed set in the homozygous mutant ms1c. Two QTLs, on chromosome 4AL and 2BS respectively, were identified to be associated with selfed-seed set. The 4AL QTL spans 36 cM and contains three nsLTPs, including the TaMs1-A homeologue, whereas the 2BS QTL spans 42.5 cM and contains four nsLTPs. Finally, to study the evolution of nsLTPs we conducted a genome-wide identification of this gene family in wheat (Chapter 4). A total of 461 putative nsLTPs were identified from the wheat genome (TaLTPs) (cv. Chinese Spring). The evolutionary relationships of the retrieved TaLTPs with rice and A.thaliana nsLTPs revealed an expansion of this family in the wheat genome, emerging mainly from tandem duplications. We showed that wheat nsLTP transcripts were detected in most tissues and stages of wheat development, which is in accordance with the diverse reported roles of this gene family. We further refined the expression profile of anther-expressed nsLTPs to provide additional male fertility gene sequences that can be used for the generation of alternative male-sterile wheat mutants. The present work contributes towards an understanding of the biological role for TaMs1 in male fertility, providing valuable information to help the development of large-scale and cost-effective multiplication of ms1 male-steriles. Furthermore, this project provides groundwork for future fundamental research in wheat focussing on the role of nsLTPs in wheat development.