基于抗性基因富集和第三代测序技术的簇毛麦NLRs类抗病基因的高通量发掘、克隆和功能鉴定

Haynaldia villosa (VV, 2n=14), a wild germplasm of wheat (Triticum aestivum) carrying , is one of the important gene pool for disease resistance breeding of wheat, which has been identified carrying multiple disease resistance genes or locus, including powdery mildew, stripe rust, stem rust, etc. Due to non-recombination between wheat and Haynaldia villosa homeologous chromosomes, it is difficult to isolate the important resistance genes though traditional map-based cloning from Haynaldia villosa. The high-throughput exploration of the NBS-LRRs type resistance genes (NLRs) at both whole genome and transcriptome level of Haynaldia villosa will be conducted using the specific Triticeae baits library for NLRs through the strategy combining the enrichment of resistant gene sequencing and the third generation sequencing technology. The identified NLRs will be quickly mapped to unique chromosome regions using various cytogenetic materials, including a full set of arm translocation lines and a series of small fragments inserted translocation lines, and this study will help construct a resolution physical map involved NLRs genes and help develop special markers for each chromosome arm or resistance gene loci, facilitating the breeding of disease resistant translocation lines. A database including all the full length NLRs will be construct, which will play an huge role in the comparative genomics analysis between different grasses species and facilitate exploring resistance genes by other groups from Haynaldia villosa in the future. The NLRs located to the known resistance gene loci will be cloned and functional assayed. Though our research, the resistance genetic basis of wheat can be significantly broaden, and the NLRs evolution clarifying in grass family will be dramatically promoted.

小麦近缘野生种簇毛麦（Haynaldia villosa, VV, 2n=14）是小麦抗病育种的重要基因库，携带抗白粉、条锈、秆锈等数十种病害的抗性基因或位点。由于簇毛麦与小麦染色体不能交换重组，难以通过图位克隆获得抗病基因。本项目将抗性基因富集测序和第三代测序技术（RenSeq-PacBio）相结合，利用麦类特异NLRs诱饵文库捕获长片段NLRs，从全基因组和转录组水平高通量发掘簇毛麦NLRs基因；利用一整套小麦-簇毛麦整臂易位系和若干小片段易位系等细胞遗传学材料，对NLRs进行染色体区段定位和物理图谱构建，开发各染色体臂及抗性基因位点的专化分子标记，辅助抗病易位系选育；构建簇毛麦的全长NLRs的序列库，开展与其他禾本科植物的比较基因组学分析；针对已经初步定位的抗病基因位点发掘到的NLRs基因，开展基因克隆和功能验证。本项目实施对拓宽小麦遗传基础和推进抗病基因进化研究具有重要意义。

核苷酸结合和富含亮氨酸重复序列(leucine-rich repeat, NLR)基因因其在保护植物免受病原体入侵方面的作用而受到广泛关注。SMRT-RenSeq结合抗性基因富集测序(resistance gene enrichment sequencing, RenSeq)和PacBio测序，是一种强大的选择性捕获和全长NLRs测序的方法。簇毛麦（Haynaldia villosa，2n=14，VV）是一种小麦野生近缘物种，已证明在小麦改良资源中可以提供对多种病害的抗性。本研究采用SMRT-RenSeq技术对簇毛麦全基因组NLRs进行鉴定。结果共注释了772个完整的NLRs，分为11个主要分支。NLRs在单个染色体上的物理位置与其它麦类物种的1、2、3、5和6群具有良好的同源关系，但位于4VL上的NLRs大部分位于预测的第7同源群上。NLRs在某些特定基因位点上出现聚集性的扩张。在52个NLRs中发现了15种类型的融合结构域(IDs)，其中Kelch和B3型的NLR-IDs在簇毛麦中存在扩增，DUF948、nami -associated和PRT_C是簇毛麦特有的整合域。同时，利用实验室创制的特殊细胞遗传学材料和感病突变体，成功克隆了位于簇毛麦6VS染色体上的广谱抗白粉病基因Pm21（NLR1-V）。本研究为小麦抗病育种提供了一个有价值的挖掘和转移抗病基因的资源库和一种快速分离克隆小麦野生种材料中抗病基因的新方法。