黄瓜染色体片段代换系SSL508-28抗白粉病基因挖掘及功能解析

Powdery mildew (PM) is one of the main diseases in cucumber production, which severely affect the yield and fruit quality of cucumber by leaves wilting even death of whole plants. In our previous study, we developed a series of chromosome segment substitution lines (CSSLs) with PM resistance introgressed from JIN5-508 (PM tolerant, donor parent) in the genetic background of PM susceptible D8 (PM susceptible, recurrent parent) which was facilitated by marker-assisted selection with simple sequence repeat and sequence characterized amplified regions markers. After 12 generations of backcross from 2004 to 2009, one such SSL, namely SSL508-28 exhibited high resistance to the PM. A 6.81Mb substituted segment on chromosome 5 derived from donor parent were detected in SSL508-28 by SSR and whole genome resequencing technology. In screening tests over four years, the PM resistance of SSL508-28 was highly consistent and stable, indicating the substituted segment carried PM resistant gene. The objective of the present study is to identify and fine map the PM resistant locus conferring resistance to PM in SSL508-28 by KASP markers genotyping and recombinant analysis with a secondary F2 segregating population derived from a cross between SSL508-28 and the recurrent parent D8. Comparative RNA-seq profiling of SSL508-28 and D8 triggered by PM pathogen inoculation will also be conducted to help anchoring the PM resistant candidate gene. On these bases, we will verify the function of candidate gene by transgenic technique, and make clear whether it has interactive effect with previously reported PM resistant genes. The results of this study will put insight into the molecular mechanisms of cucumber resistance to PM and help for resistant germplasm innovation and new varieties breeding.

白粉病是黄瓜生产上的主要病害之一，发病时常导致叶片枯萎或死亡，严重影响黄瓜产量、品质及经济效益。本实验室前期以黄瓜高抗白粉病品系JIN5-508和易感品系D8为亲本创制了一系列染色体片段代换系。经多年抗性鉴定，片段代换系SSL508-28高抗白粉病，且能稳定遗传。用SSR标记和全基因组重测序对SSL508-28的代换片段特征进行了鉴定，在5号染色体上存在一个来源于JIN5-508的6.81Mb的代换片段。本项目旨在利用该片段代换系与D8构建次级F2群体，使用KASP标记对F2群体进行快速基因型鉴定，精细定位黄瓜抗白粉病QTL；运用RNA-seq研究SSL508-28与D8在接种白粉菌后的差异表达基因，结合精细定位与RNA-seq的结果进一步筛选候选基因；通过转基因技术对候选基因进行功能验证；研究候选基因与已鉴定的位于1号和5号染色体上的抗白粉病基因间的互作关系，最终探明黄瓜白粉病抗性机制。

黄瓜是我国栽培的主要蔬菜作物之一，具有周年生产周年供应的特点。白粉病是黄瓜生产上的主要病害之一，发病时常导致叶片失绿、枯萎甚至死亡，严重影响黄瓜产量、品质及经济效益。申请人所在实验室前期以黄瓜高抗白粉病品系JIN5-508和高感白粉病品系D8为亲本，通过杂交、连续12代回交及分子标记辅助选择，获得了一个遗传背景与D8高度相似，但高抗白粉病且能稳定遗传的染色体片段代换系SSSL508-28。通过重测序及比对发现，SSSL508-28在Chr5上带有一个来自共体亲本的6.81Mb染色体导入片段。经连续五年田间鉴定和接种鉴定，SSSL508-28白粉病抗性接近于供体亲本；用环境扫描电镜观察发现，接种白粉病48h后，SSSL508-28叶片表面几乎无白粉病分生孢子生长，而D8叶片表面则产生了大量白粉菌分生孢子，表明两者抗白粉病能力差异显著。利用RNA-seq、miRNA-seq以及iTRAQ技术比较了SSSL508-28和D8在接菌48h后全基因组在基因转录及调控水平和翻译后蛋白水平上的差异；在此基础上，以D8和SSSL508-28为亲本，构建了次级F2大群体，对SSSL508-28中抗病主效QTL进行了图位克隆，最终获得了一个编码仅含有LRR（leucine-rich repeat）结构域的抗白粉病新基因，命名为CsLRR1。序列克隆及比对分析发现，SSSL508-28和D8白粉病抗病能力差异是CsLRR1蛋白的LRR结构域上的一个非同义SNP突变导致。克隆SSSL508-28中的CsLRR1基因并转入不抗病亲本D8后，其过表达株系白粉菌抗性显著提高。综合酵母单杂、EMSA、双荧光素酶实验、酵母双杂、pull-down等一系列分子生物学实验的研究结果，推测了CsLRR1调控黄瓜白粉病抗性的分子机制如下：白粉病菌侵染黄瓜叶片后，激活水杨酸合成通路，进而诱导转录因子CsbZIP63的表达，CsbZIP63通过结合CsLRR1基因启动子上的ABRE顺式元件（ACGT）从而特异性启动CsLRR1基因的表达。CsLRR1通过蛋白-蛋白间的互作，激活CsXTH30蛋白的合成。CsXTH30通过其自身木葡聚糖内糖基转移酶活性，改进细胞初生壁纤维结构，增强细胞壁机械强度，从而抵御白粉病菌孢子的侵染。上述研究结果为今后黄瓜抗白粉病种质创新及选育高抗白粉病黄瓜新品种奠定重要的理论与技术基础。