西尔斯山羊草2Ss染色体上抗白粉病新基因的精细定位与候选基因发掘

Powdery mildew of wheat, caused by Blumeria graminis f.sp. tritici (Bgt), is devastating disease worldwide and one of the three most sever diseases damaging wheat yields and production stability in China . Discovery and application of novel genes of powdery mildew resistance is always in urgent need to develop resistance varieties of wheat，and thus to prevent wheat crops from the disease economically and effectively . A novel resistant gene derived from Aegilops searsii has been identified by the applicants as the result of powdery mildew resistance assays using Bgt races popular in Henan Province. The gene was located at chromosome 2Ss of Ae. searsii and temporarily designated as Pm2Ss. On the basis of this, here propose a project to develop a series of 2Ss-2B recombinant, construct fine map and discover resistant gene candidates for the further cloning of the gene and understanding molecular mechanism of the resistance to powdery mildew. To accomplish the objectives, a specific 2Ss/2B double monosomic segregating population with homoelogous recombination inducing gene (ph1b) backgrounds will be developed to increase the recombinant efficiency of 2Ss/2B and develop a series of 2Ss-2B recombinant. Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP), powdery mildew assays by inoculation and improved genomic in situ hybridization will further be applied to the 2Ss-2B recombinant series and the segregation population for Pm2Ss fine mapping, and differential expressed gene analysis, resistant gene homology - based cloning, and colinearity or synteny analysis will also be used to discover candidates of the novel gene Pm2Ss. The accomplish of the project will provide a novel gene and co-segregated molecular markers of the gene for wheat resistance breeding, and lay the foundations for the further understanding of the molecular mechanism of the resistance to wheat powdery mildew.

小麦白粉病是一种世界性真菌病害，是严重影响我国小麦高产稳产的三大病害之一，发掘利用新抗源是培育抗病品种、经济有效地防治小麦白粉病危害的迫切需求。本申请人利用河南省白粉菌混合流行菌株接种，首次在西尔斯山羊草2Ss染色体上发现了抗白粉病新基因，暂定名为Pm2Ss。在此基础上，本项目拟用2Ss[2B]代换系和诱导部分同源染色体重组的ph1b突变体为亲本，构建ph1bph1b背景的2Ss/2B大规模分离群体，创制一系列不同2Ss片段长度和重组位点的2Ss-2B重组体；并利用KASP标记、白粉病接种鉴定和基因组原位杂交多种技术对Pm2S基因进行精细定位；然后利用差异表达基因谱、抗病基因同源序列克隆和同科异种基因共线性分析发掘其候选抗病基因。项目可以为小麦抗病育种提供新抗源和共分离分子标记，并为该基因的分离和进一步了解小麦抗白粉病的分子机理奠定基础。

小麦白粉病是严重影响小麦高产稳产的主要病害，发掘利用新抗源是培育抗病品种、经济有效地防治小麦白粉病危害的迫切需求。本项目鉴定到西尔斯山羊草2Ss#1染色体上携带有抗白粉病新基因。在此基础上，本项目利用小麦-西尔斯山羊草2Ss#1添加系与诱导部分同源染色体高频重组的ph1b突变体(CS-ph1b)杂交，构建了纯合ph1b背景的2Ss#1大规模分离群体，利用分子标记和原位杂交技术筛选鉴定出7个2Ss#1易位系，根据2Ss#1易位断点和白粉病抗性分析，将该抗白粉病基因初步定位在2Ss#1长臂FL0.75-0.87区段，并将该基因正式定名为Pm57。 利用24个来自全国各地和9个来自河南不同地区的白粉菌菌株，对小麦-西尔斯山羊草2Ss#1添加系和Pm57基因易位系进行人工苗期接种，结果Pm57对所有菌株的抗性达到高抗以上，属于广谱高抗基因。其次，以Pm57易位系89(6)88为材料，进一步创制了46个小麦-西尔斯山羊草2Ss#1小片段易位系，其中28个为抗白粉病易位系，最小片段2Ss#1只有9.88Mb。此外，开发Pm57配套诊断型PCR标记6个，通过杂交将Pm57导入到5个黄淮麦区优良小麦品种中；在Pm57基因精细定位方面，基于RNA-seq开发了79个2Ss#1特异分子标记，并分别定位在7个染色体区段。以53个2Ss#1易位系为材料，综合2Ss#1易位断点和分子标记分析、白粉病接种鉴定，将Pm57基因定位在标记X67593 (773.72Mb)和X62492 ( 778.85Mb)两标记之间，大约5.13Mb区段；在Pm57 候选基因发掘方面，采用同属异种染色体共线性分析、 转录组测序、突变体抗病基因富集测序(MutRenSeq)等多种途径发掘出21个抗病候选基因和33个CNL类抗病基因contigs。对其中7来自子RNA-seq的候选基因进行VIGS功能验证和白粉菌接种诱导表达分析，鉴定出2个Pm57候选基因(comp430422_c0和comp4364_c0)和1个抗病相关基因(comp62492_c0)。此外，发表 SCI 收录和中文核心期刊论文11篇，申请国家发明专利3项，培养研究生8人。本项目为小麦抗病育种提供了Pm57抗白粉病新种质及配套的诊断型分子标记，为后续Pm57基因的克隆和进一步了解小麦抗白粉病的分子机理奠定了基础。