description
- In the past, the wild relatives of wheat have been successfully exploited as a novel source of genetic variation for traits in wheat breeding programmes. In brief, wheat/alien introgression involves the hybridisation of wheat with a wild relative followed by repeated backcrossing to generate lines of wheat carrying a wild relative chromosome segment on which a target gene is located. If the wheat and wild species chromosomes have similar gene order they can exchange genetic material through recombination, as has been shown successfully at the Nottingham BBSRC Wheat Research Centre (King et al., 2017; Grewal et al., 2018). However, many wild relatives have rearranged their chromosomes relative to that of wheat making gene transfer difficult, if not impossible, via recombination during meiosis. This reduces significantly the numbers of wild species that can be exploited for wheat/alien introgression. What we want to do is to exploit special genes, known as gametocidal (Gc) genes that are found in some wild species and transmit preferentially to the offspring. Gc genes induce chromosomal breakages which frequently result in translocations, or exchanges, between the chromosomes of wheat and those of the wild species (King et al, 1991). This strategy provides an alternative route for the transfer of genes from chromosomes of wild species into wheat. We have identified a simple and rapid assay to isolate mutants at the Gc locus that was introgressed from chromosome 4S of Aegilops sharonensis and translocated to wheat chromosome 4B. One putative EMS (ethyl methansulfonate)-induced Gc mutant was identified. The specific objectives of the proposed research are to: 1. identify additional mutants at the Gc locus, 2. determine the location of the Gc locus on the 4S segment through sequence analysis of the Gc mutant(s), 3. identify differentially expressed genes in the Gc mutant(s) via RNA-seq, and 4. develop Bobwhite wheat lines carrying the Gc locus for CRISPR/Cas knockout (KO) of candidate genes in collaboration with US Department of Agriculture (USDA). The proposed research is a crucial first step towards the molecular understanding of Gc function, which eventually will lead to the cloning of this gene or gene complex for future use in wheat breeding. To achieve these objectives, the students will initially spend time making wide crosses between different wheat lines and mutagenizing the resulting seeds with EMS. The progeny will be screened for mutants with a variety of techniques such as phenotypic scoring (visual analysis of spike fertility), cytogenetics (GISH), microscopy (analysis of chromosomal breakage during pollen mitosis) and molecular markers (KASP assays; Grewal et al., 2019). The putative mutants will be sequenced (in collaboration with USDA) and common genes with mutations will be identified through Next-Gen sequencing (NGS) bioinformatics. In another approach to identify candidate genes for the Gc locus, the student will use RNA-seq to analyse differential gene expression between the Gc mutant and non-mutant lines. There will be an opportunity to visit Rothamsted Research where our project partner has key expertise in applying genome editing technology (CRISPR/Cas) in plants. The student will learn gene cloning techniques while making KO constructs for a few candidate genes for the Gc locus. To enable downstream functional studies, using the KO constructs, we need the Gc locus carrying segment from Ae. sharonensis to be present in a wheat background that has high-efficiency of wheat transformation. As such, the student will transfer this segment, via crossing, into Bobwhite wheat to produce introgression lines ready for transformation with KO constructs (will be done by project partners in USDA) targeting candidate genes for Gc locus. Grewal S. et al. 2018. Theor. Appl. Genet. 131 (2) 389-406 Grewal S. et al. 2019. Plant Biotechnol. J. doi: 10.1111/pbi.13241 King, I.P. et al. 1991. Genome 34:944-9