Mapping the Breaker Element of the Cuckoo Chromosome 4SL of Wheat Wild Relative Aegilops Sharonensis

过去，小麦的野生近缘已被成功地利用为小麦育种计划中性状遗传变异的新来源。简而言之，小麦/外源渐渗包括小麦与野生亲缘杂交，然后反复回交，产生携带目标基因所在的野生亲缘染色体片段的小麦品系。如果小麦和野生物种的染色体具有相似的基因顺序，它们可以通过重组交换遗传物质，诺丁汉BBSRC小麦研究中心已经成功证明了这一点（King et al., 2017; Grewal et al., 2018）。然而，许多野生近缘种在减数分裂期间通过重组将染色体重新排列，使基因转移变得困难，如果不是不可能的话。这大大减少了可用于小麦/外来入侵的野生物种的数量。我们想要做的是利用在一些野生物种中发现的特殊基因，即所谓的“杀配子基因”，并优先传递给后代。Gc基因诱导染色体断裂，经常导致小麦和野生物种染色体之间的易位或交换（King et al, 1991）。这一策略为基因从野生种的染色体转移到小麦中提供了另一种途径。我们建立了一种简单、快速的方法来分离从沙龙山羊第4S染色体渗入并易位到小麦第4B染色体上的Gc位点突变体。鉴定了一种假定的EMS（甲磺酸乙酯）诱导的Gc突变体。拟定研究的具体目标是：1。确定Gc位点的其他突变体；2 .通过Gc突变体(s)的序列分析，确定Gc位点在4S片段上的位置；3 .通过RNA-seq鉴定Gc突变体中的差异表达基因；与美国农业部（USDA）合作，开发携带候选基因CRISPR/Cas敲除（KO） Gc位点的山齿白小麦品系。这项研究是对Gc功能的分子理解的关键的第一步，最终将导致该基因或基因复合物的克隆，用于未来的小麦育种。为了实现这些目标，学生们最初将花时间在不同小麦品系之间进行宽杂交，并用EMS诱变产生的种子。后代将通过各种技术筛选突变体，如表型评分（穗育性视觉分析）、细胞遗传学（GISH）、显微镜（花粉有丝分裂期间染色体断裂分析）和分子标记（KASP测定；Grewal等人，2019）。将对假定的突变进行测序（与美国农业部合作），并通过下一代测序（NGS）生物信息学鉴定具有突变的常见基因。在另一种鉴定Gc位点候选基因的方法中，学生将使用RNA-seq分析Gc突变系和非突变系之间的差异基因表达。将有机会访问洛桑研究所，我们的项目合作伙伴在植物基因组编辑技术（CRISPR/Cas）应用方面拥有关键专业知识。学生将学习基因克隆技术，同时为Gc位点的一些候选基因构建KO结构。为了使用KO结构进行下游功能研究，我们需要Ae的Gc基因座携带片段。沙龙菌在小麦转化效率高的背景下存在。因此，该学生将通过杂交将该片段转移到山齿白小麦中，以产生针对Gc位点候选基因的KO结构（将由美国农业部的项目合作伙伴完成）准备转化的渗入系。Grewal S. et al. 2018。定理。达成。中国生物医学工程学报，2016,31(2):389-406。生物科技植物》。[j] . doi: 10.1111/pbi。[13]王志强等。1991。基因组34:944-9

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 ofgenes 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) 389-406Grewal S. et al. 2019. Plant Biotechnol. J. doi: 10.1111/pbi.13241King, I.P. et al. 1991. Genome 34:944-9