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

过去，小麦的野生近缘种已被成功地用作小麦育种计划中性状遗传变异的新来源。简而言之，小麦/外来基因渗入涉及小麦与野生近缘种杂交，然后重复回交以产生携带靶基因位于其上的野生近缘种染色体区段的小麦品系。如果小麦和野生物种染色体具有相似的基因顺序，则它们可以通过重组交换遗传物质，如在诺丁汉BBSRC小麦研究中心已经成功显示的（King等人，2017; Grewal等人，2018年）。然而，许多野生近缘种的染色体相对于小麦的染色体重排，使得通过减数分裂期间的重组进行基因转移困难，如果不是不可能的话。这大大减少了可用于小麦/外来基因渗入的野生物种的数量。我们想做的是利用特殊的基因，被称为杀配子（Gc）基因，在一些野生物种中发现，并优先传递给后代。Gc基因诱导染色体断裂，这经常导致小麦染色体和野生种染色体之间的易位或交换（King等，1991）。本研究利用一种简单、快速的方法分离出了从沙融山羊草4S染色体上渗入并易位到小麦4 B染色体上的Gc位点突变体。本研究的主要目的是：1.通过EMS诱导的Gc突变体的筛选，确定了一个可能的突变体。鉴定Gc基因座处的另外的突变体，2.通过Gc突变体的序列分析确定Gc基因座在4S片段上的位置，3.通过RNA-seq鉴定Gc突变体中差异表达的基因，和4.与美国农业部（USDA）合作开发携带Gc基因座的Bobwhite小麦品系，用于候选基因的CRISPR/Cas敲除（KO）。这项研究是对Gc功能的分子理解的关键第一步，最终将导致克隆该基因或基因复合体，用于未来的小麦育种。为了实现这些目标，学生们将首先花时间在不同小麦品系之间进行广泛的杂交，并用EMS诱变产生的种子。将用多种技术筛选后代的突变体，例如表型评分（穗育性的视觉分析）、细胞遗传学（GISH）、显微镜（花粉有丝分裂期间染色体断裂的分析）和分子标记（KASP测定; Grewal等人，2019年）。将对推定的突变体进行测序（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基因座候选基因的方法中，学生将使用RNA-seq分析Gc突变株系和非突变株系之间的差异基因表达。学生将有机会参观Rothamsted Research，我们的项目合作伙伴在植物基因组编辑技术（CRISPR/Cas）的应用方面拥有关键的专业知识。学生将学习基因克隆技术，同时为Gc位点的一些候选基因制备KO构建体。为了使下游功能研究能够使用KO构建体，我们需要携带Ae片段的Gc位点。sharonensis存在于具有小麦转化高效性的小麦背景中。因此，学生将通过杂交将该片段转移到Bobwhite小麦中，以产生准备好用KO构建体转化的基因渗入系（将由USDA的项目合作伙伴完成），这些构建体靶向Gc基因座的候选基因。等，2018年。Theor.应用遗传学131（2）389- 406 Grewal S.等，2019年。植物生物技术J. doi：10.1111/pbi.13241 King，I. P.等人1991. Genome 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