小麦条锈病抗性基因YrL693的精细定位与克隆

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a highly destructive disease of wheat (Triticum aestivum) and seriously influences wheat yield in China. To develop and apply the resistance cultivars is the most economical, effective, and environmentally safe strategy to control the disease. Therefore, to fine locate the stripe rust resistance genes, to clone theses candidate genes for stripe rust resistance, and then to analyze their function would play an important role in understanding the mechanism of stripe rust occurrence and development as well as molecular designing of wheat resistant new cultivars to stripe rust. . A new wheat line named L693, with resistance to stripe rust, powdery mildew, and Fusarium head blight (FHB), was selected and developed by ourselves from the cross MY11 and YU25, and YU25 was derived from the Thinopyrum intermedium by wide cross. Firstly, it has been documented that the resistance for stripe rust was controlled by one dominant gene named YrL693, which has been primarily located on chromosome 1BS using molecular markers. Secondly, we have constructed the F7:8 recombinant inbred lines and the enough large F2:3 segregating population for fine mapping of YrL693. Thirdly, genomic in site hybrid (GISH) did not found that there is foreign chromosome segment in L693. Finally, a total of 112 expressed sequence tags (ESTs) were obtained from a suppression subtractive hybridization (SSH) cDNA library constructed from Pst-infected seedling leaves of L693 and its highly susceptible sister line L661. It is obvious that the previous achievement has paved a good way for further study YrL693. . In this study, one of our purpose is to construct the fine mapping of YrL693 using the available genetic populations, comparative genomic mapping methods, the comprehensive techniques of RNA-sequencing (RNA-Seq), bulked segregant analysis (BSA), the public ESTs, and available part wheat genomic information. The other purpose is to construct the physical mapping by screening bacterium artificial chromosome (BAC) library and to clone YrL693 candidate genes by sequencing of chromosome region in which YrL693 site and PCR technique, and then to test the functions of these candidate genes by qRT-PCR and transgene method. . The project will play a key role in wheat resistance breeding to stripe rust as well as the theoretical research about the resistance mechanism to stripe rust. Firstly, it will be helpful forward to clone YrL693. Secondly, it could identify some new molecular markers for using molecular assistant selection (MAS) to fast transfer and apply YrL693. Finally, it may also be very helpful to elucidate the resistance mechanism to wheat stripe rust.

条锈病是危害我国小麦生产的重要病害，其抗性基因的精细定位与克隆对培育抗条锈病新品种的分子设计育种以及深入理解植物抗病性的分子机理具有重要意义。L693是我们自主培育的兼抗条锈、白粉和赤霉三种小麦主要病害的新品系，前期研究表明，该品系对条锈病的抗性受一对显性基因控制，并利用分子标记将抗性基因定位于小麦的1BS染色体，暂时命名为YrL693。本研究拟采用比较基因组分析和BSA-RNA-Seq结合SNP芯片技术，开发与YrL693紧密连锁的分子标记，构建其饱和遗传图谱；结合已创制的杂交组合“L661/L693”的F7:8重组自交系群体及其F2:3大群体，对YrL693进行精细定位；通过筛选小麦BAC基因组文库和染色体步移，构建YrL693基因区域的BAC物理图谱；对该区域的BAC物理图谱测序分析，预测YrL693的候选基因；通过qRT-PCR和转基因技术对候选基因进行功能验证。

条锈病是危害我国小麦生产的重要病害，其抗性基因的精细定位与克隆对培育抗条锈新品种的分子设计育种以及深入理解植物抗病性的分子机理具有重要意义。L693使我们自主培育的兼抗条锈病、白粉和赤霉的三种小麦主要病害的新品系，前期研究表明，该品系对条锈病的抗性受一对显性基因控制，并利用分子标记将抗性基因定位于小麦的1BS染色体，暂时命名为YrL693。本研究采用比较基因组分析和BSA-RNA-Seq结合SNP芯片技术，开发与YrL693紧密连锁的分子标记，构建其饱和遗传图谱；结合已创制的杂交组合“L661/L693”的F7:8重组自交系群体及其F2:3大群体，对YrL693进行精细定位；通过筛选小麦BAC基因组文库和染色体步移，构建YrL693基因区域的BAC物理图谱；对该区域的BAC物理图谱测序分析，预测YrL693的候选基因；通过核心种质资源法对候选基因进行功能验证。结果显示小麦条锈病抗性基因YrL693位于小麦染色体C-1BL6-0.32bin，比较基因组共线性结果表明YrL693所在小麦染色体区段与水稻10号染色体、二穗短柄草3号染色体和高粱1号染色体具有良好的共线性。在构建YrL693的饱和遗传图谱后，筛选到与YrL693共分离的分子标记5个，并在此基础上建立了高效转移YrL693的分子辅助育种技术体系。通过电子克隆筛选到YrL693的候选基因重金属铜离子结合蛋白WCBP1，生物信息学结构分析表明，WCBP1基因36bp片段的缺失可能赋予L693条锈病抗性，进一步核心种质资源筛查的结果表明WCBP1基因型与核心种质材料条锈病抗性表现一致，即表明WCBP1很有可能就是YrL693基因本身。