芸薹种一个新的根肿病抗性基因Crr5的精细定位与功能分析

Clubroot (CR) caused by Plasmodiophora brassicae, is an important cruciferous disease worldwide. To breed new varieties with durable disease resistance is a fundamental solution to effectively prevent and control this disease. Our previous research indicated that a novel CR resistance gene Crr5 from ECD01-1 was identified and preliminary mapped between 0.45 and 1.95 Mb on chromosome A08 via BSA-seq. A novel CR gene Crr5, controlled by a single dominant resistance gene, was independent from previously mapped CR genes and performed resistance to the predominant pathotypes of P. brassicae (Race 4) identified in China. In this project, in order to fine map and clone Crr5 in Brassica rapa, the BC2F2:3 population will be constructed and assessed for the resistance under controlled conditions, more closer molecular markers of SNPs and InDels in the roughly mapped regions will be developed for linkage map construction. Then, the resistant candidate gene will be fine mapped and cloned through high-precision linkage analysis and bioinformatics analysis. After that, we will confirm the function of the target genes by association analysis,BSR-seq, qRT-PCR and genetic transformation. In addtion, the Dual RNA-seq will also be used for the verification of the candidate genes interacting with P. brassicae. The proposed research will provide insights into the genetic and molecular basis. The results will not only supply useful markers, diverse CR resistance genes for molecular design breeding of CR genetic improvement, but also lay a foundation for understanding of the molecular mechanisms of CR resistance, which has very important theoretical significance and application value.

根肿病是由芸薹根肿菌侵染引起的一种世界性病害，严重危害了大白菜等作物的生产。挖掘新的抗性基因并解析其抗病机制，培育聚合多个抗病基因的新品种是降低病害损失的有效途径。但目前已克隆的抗性基因不多且分子机制尚不清楚。本项目组前期在欧洲芜菁自交系ECD01-1中发现一个新的根肿病抗性基因（暂命名为Crr5），将其初定位于A8染色体顶端0.45-1.95Mb区间。该基因高抗国内根肿菌主流生理小种，与目前已克隆的抗性基因不同。本项目将在初定位目标区域开发分子标记进行精细定位，通过连锁分析、BSR-seq和生物信息学分析获得抗病候选基因。克隆并经转基因验证研究候选基因功能。通过双RNA测序分析确认候选基因，揭示抗病基因在寄主与病原菌中的互作机理。研究结果不仅为抗根肿病分子设计育种提供有价值的分子标记和优异多样性基因资源，而且从一个全新的视角诠释根肿病抗性机制，具有重要的理论意义和应用价值。

根肿病是由芸薹根肿菌侵染引起的一种世界性病害，严重危害了大白菜等作物的生产。挖掘新的抗性基因并解析其抗病机制，培育聚合多个抗病基因的新品种是降低病害损失的有效途径。本项目以欧洲芜菁ECD01为抗源，通过游离小孢子培养技术，创制了纯合的大白菜抗根肿病DH系材料DH40，对DH40进行了基因组从头组装，获得了染色体水平的高质量参考基因组。遗传分析表明DH40的抗根肿病性状由显性单基因控制，命名为Crr5。通过BSA-seq和KASP标记技术，将抗病基因Crr5精细定位在A08染色体的78.95 kb区间；候选区间有10个注释基因，功能注释和序列分析表明1EDH4000043283为一个TIR-NBS-LRR抗病蛋白，为Crr5的候选基因；序列分析发现感病亲本DH199在第三个外显子区域发生了一个T碱基的插入，该T碱基的插入导致了移码突变和蛋白翻译的提前终止。最后，本研究对抗根肿病的DH40和感病的DH199在根肿病接种前后的不同时间点（0、2、5、8、13、23 DAI）进行了转录组测序，发现了参与根肿病抗性调控的关键基因和通路；此外，构建了超表达载体获得了T0代转基因阳性植株，开发并验证了3个抗病基因Crr5的功能KASP标记。研究结果为今后大白菜抗根肿病新品种选育提供了有价值的分子标记和优异多样性基因资源，具有重要的理论意义和应用价值。