小麦抗白粉病基因Pm52的图位克隆与功能验证

The annual occurrence of wheat powdery mildew (caused by Blumeria graminis f. sp. tritici, Bgt) in covers an area of about 667 million hectares, which is a great threat to the high and stable production of wheat in China. Since it was released in 2004, Liangxing 99 has been largely grown in the Yellow-Huai Rivers Valley Facultative Wheat Zone and the Northern Winter Wheat Zone. In recent years, Liangxing 99 was also used as a promising parent to develop new wheat cultivars. Liangxing 99 was effective against over 80% of the Bgt isolates collected from different wheat producing regions at the seedling stage. It maintains its adult plant resistance to powdery mildew during the field test over the past decade. A single dominant gene, which is officially designated Pm52, has been mapped to the deletion bin 2BL2-0.36-0.50 on chromosome 2BL. Pm52 was fine-mapped in a 0.17 cM genetic interval corresponding to a 2.6 Mb physical genomic region in the Chinese Spring genome using a large mapping population with the support the previous National Natural Science Foundation of China. This project will be initiated to isolate Pm52 using the map-based cloning strategy. The physical map of Pm52 will be constructed with the BAC library screening and chromosome walking techniques. The candidate genes of Pm52 will be identified by the gene annotation of genomic sequence assembled through BAC contig sequencing. The VIGS, EMS mutants, haplotyope analysis, genetic transformation and gene expression mode will be conducted for fuctional verification of Pm52. The functional markers of Pm52 gene will be developed to be used in molecular marker assisted breeding against powdery mildew.

小麦白粉病常年发病1亿多亩，对小麦高产稳产构成巨大威胁。2004-2016年良星99已在黄淮冬麦区和北部冬麦区推广5000多万亩，近年来作为亲本培育出新品种。良星99苗期抗主产区80%以上的白粉菌菌株，而且连续10年田间接种鉴定一直保持成株期抗性。良星99的抗白粉病基因位于2BL染色体，被正式命名为Pm52。在前一项国家自然科学基金项目支持下，利用大规模作图群体将Pm52精细定位于0.17 cM的遗传区间，对应中国春基因组~2.6 Mb物理区间。本项目拟采用图位克隆的策略，借助最新释放的中国春和矮抗58基因组序列，开发Pm52连锁和共分离的多态性标记，进一步加密遗传连锁图谱，根据基因功能注释和预测确定Pm52的候选基因。利用VIGS、EMS突变体、单倍型分析、遗传转化、基因表达模式等技术进行功能验证，最终克隆Pm52基因，并进一步开发Pm52基因的功能标记，用于抗白粉病的分子标记辅助育种。

冬小麦品种良星99的抗白粉病基因位于2BL染色体。本项目的目的是克隆Pm52。我们首先利用良星99×中麦8444大规模作图群体，根据精细定位区间的参考基因组序列开发分子标记，将Pm52基因定位在191 kb物理区间，其中一个编码NLR抗病蛋白的基因受白粉菌的诱导表达。采用过表达和互补表达遗传转化、EMS突变体功能验证，证明该NLR基因为Pm52。利用Pm52功能标记检测了3029个小麦品种/品系，其中303个材料具有Pm52基因。部分品种Pm52基因DNA序列与良星99和济麦22相同。我们还利用RIL群体解析了良星99成株期白粉病抗性遗传机制，在小麦7AL染色体发现一个QTL（Qaprpm.caas.7A），控制良星99的成株期白粉病抗性。良星99的Pm52克隆以及成株期抗白粉病QTL的解析，为其在小麦抗白粉病育种上的利用提供有利的技术支撑。