萝卜优异黑腐病抗源抗病基因的精细定位、克隆及功能分析

Black rot caused by Xanthamonas campestris pv. campestris (Xcc), is a bacterial vascular disease occurred all over the world. It is seriously harmful to the radish and other Brassicaceae crops. To breed new varieties with durable disease resistance by combining the conventional and molecular technologies is a fundamental solution to effectively prevent and control the disease. However, the serious shortage of disease-resistant genes and their mechanism study in Brassicaceae vegetables has become the bottleneck of development of molecular breeding for the disease-resistant varieties. This project will fine map and clone diverse resistant genes to black rot, understand their resistance mechanism in depth based on our previous genetic analysis and primary mapping results in radish. Firstly, two large F2 populations will be constructed using two resistant advanced inbred lines and a susceptible line with different genetic background and their resistance phenotypes to Xcc will be identified accurately; at the same time, more molecular markers of Indels and SNPs will be developed in the regions roughly mapped according to the radish genome information. The resistance of the F2 populations and parents plants will be genotyped. After that, the resistant candidate genes will be fine mapped and cloned through high-precision linkage analysis and bioinformatics analysis. Secondly, the function of the target genes will be confirmed by heterologous expression in transgenetic Brassica parachinensis Bailey. Thirdly, the Dual RNA-seq will be used for the verification of the candidate genes as well as the analysis of the resistance mechanism of the target genes interacting with Xcc. The outputs from this project will provide diverse black rot resistant genes, theoretical and technical support for the molecular design breeding of black rot-resistance genetic improvement in radish and other Brassicaceae vegetables.

黑腐病是由野油菜黄单胞菌野油菜致病变种引起的一种世界性细菌性病害，严重危害萝卜等十字花科蔬菜作物。挖掘多样性抗黑腐病基因，阐明其抗病机制，通过分子设计育种培育具有持久抗性的品种，被认为是防控黑腐病的根本途径。然而，十字花科蔬菜抗黑腐病基因的克隆及其抗病机制研究尚属空白。本项目将基于前期遗传分析和初定位的结果，利用两份具有不同抗性遗传背景的稀有抗源构建超大F2群体；根据萝卜全基因组序列信息在初定位目标区域加密开发Indel、SNP分子标记；分别对两F2群体及亲本进行抗病性表型和基因型精准鉴定；通过高精度连锁分析和生物信息分析获得抗病候选基因。克隆并通过农杆菌转化菜薹验证候选基因功能。通过双RNA测序分析再进一步确认候选基因的同时，分析抗病基因在寄主和病原菌互作中的作用机理。研究结果将为萝卜及十字花科蔬菜抗黑腐病分子设计育种提供优异基因资源、理论依据和技术支撑。

黑腐病是由野油菜黄单胞菌野油菜致病变种引起的一种世界性细菌性病害，严重危害萝卜等十字花科作物。挖掘多样性抗黑腐病基因，阐明其抗病机制，对填补相关研究空白、开展黑腐病持久抗性分子设计育种，从根本上防控黑腐病具有重要意义。.本项目首先利用抗源‘KB10Q-22’构建的120株F2群体构建了总遗传图距为1467.30cM的RAD高密度遗传图谱。QTL定位获得2个位于2号和7号染色体上的抗性QTLs（qBRR2和qBRR7），分别解释26.97%和27.06%的表型变异。进一步利用该抗源的两个F2大群体进行BSA-Seq初定位和精细定位，获得与RAD定位吻合、位于2号染色体的两个主效QTLs（qBRR2-1和qBRR2-2），其区间物理距离为12kb和125kb，分别解释19.1%和61.0%表型变异。鉴定到抗病候选基因Rsa10012735（RAR1）和Rsa10015757（NDR1/HIN1-like 25），构建RAR1过表达载体，获得转基因拟南芥T0代种子。RAR1在抗、感亲本间存在多个SNP和一个26 bp的Indel。开发Indel标记对F2群体的验证准确率达85%以上。另外，针对抗源‘KB10Q-24’F2大群体进行BSA-Seq初定位、精细定位和基因表达分析，在7号染色体15.05-22.75Mb区间发现一个QTL，并鉴定出29个抗病相关基因。综合比较发现两份抗源的抗性遗传背景不同。.比较转录组分析发现，抗源‘KB10Q-22’的差异表达基因主要富集在MAPK 信号途径、植物激素（SA和JA等）信号转导、植保素、次级代谢物生物合成、植物病原菌互作和细胞壁水解等途径。抗源‘KB10Q-24’差异表达基因主要富集在次级代谢物生物合成和植物激素信号转导等途径。二者机制不甚相同。dual RNA-seq分析发现，植物中病原菌基因表达在感病材料中随侵染时间延长而增加更多，24个DEGs富集到淀粉和蔗糖代谢途径，且多是病菌的胞外酶基因（如淀粉酶、果胶酶、内切葡聚糖酶等），它们可能在宿主中参与细胞壁降解，决定病菌能否成功侵染。.比较基因组分析构建了萝卜抗病（R）基因泛基因组，揭示了抗病基因的分布及其进化特点；分析了萝卜全基因组SPL基因家族及时空表达特点。.萝卜抗黑腐病基因挖掘及机制解析为抗病分子设计育种提供了优异基因资源、理论依据和技术支撑。