基于SLAF-seq的辣椒根腐疫病抗性基因物理定位及候选基因克隆

Phytophthora root rot of pepper (C. annuum) caused by Phytophthora capsici (P. capsici) occurs seriously in Guangdong province with high temperature and humidity, and therefore, it is very important to mine Phytophthora root rot resistance gene. Years ago, we introduced a P. capsici-resistant accession, ‘Criollo de Morelos-334’ (CM334), with the highest resistance to P. capsici from abroad. Genetic investigation showed that the Phytophthora root rot resistance of CM334 to P. capsici race 3, the dominant physiological race of P. capsici on pepper in Guangdong province, was controlled by a single dominant gene. Additionally, based on Specific Locus Amplified Fragment sequencing (SLAF-seq), the resistance gene was mapped to an interval of 2.6 Mb at the end of the long arm of chromosome 10 of pepper. Based on these results, we will construct segregation populations with more than 2000 plants and add markers to limit the resistance gene to a region less than 100 kb and genes in this region will be analyzed by bioinformatic method and RT-qPCR. Meanwhile, we will carry out resequencing of 10399 (the male parent), the P. capsici-susceptible accession, and find out functional variations of these genes between CM334( the female parent) and 10399 by blasting to the CM334 reference genome. Considering the above factors, we will finally identify and clone resistant candidate genes. So， the smooth implementation of this project will lay a theoretical foundation for cloning, functional studies and molecular marker assisted breeding of the resistance gene.

广东地区高温高湿，辣椒根腐疫病发生严重，因此，对根腐疫病抗性基因的挖掘显得极为重要。本课题组前期从国外引进高抗疫病辣椒资源CM334，对其根腐疫病抗性遗传规律调查结果显示：在广东省辣椒疫霉菌优势生理小种Race 3侵染条件下，其根腐疫病抗性性状符合显性单基因遗传规律。随后，基于SLAF-seq技术，将抗性基因定位在辣椒第10号染色体长臂末端约2.6Mb区域内。本项目拟在此基础上构建2000株以上分离群体并加密标记，将抗性基因限定在小于100 kb的区间内并对该区域内基因进行生物信息学分析及实时荧光定量表达（RT-qPCR）分析，同时对感病材料10399重测序，结合辣椒（CM334）参考基因组序列分析该区域内基因序列差异，明确其功能性变异位点且最终确定并克隆候选基因。本研究将为抗性基因的克隆及其功能研究和辣椒抗根腐疫病分子标记辅助育种奠定理论基础。

辣椒疫病是由辣椒疫霉菌（Phytophthora capsici Leonian）引起的一种毁灭性土传病害，而广东地区因高温高湿，辣椒根腐疫病发生尤其严重，因此，对根腐疫病抗性基因的挖掘显得极为重要。本项目针对广东省辣椒疫霉菌优势生理小种Race 3，在显性单基因控制模式下，利用基于SLAF-seq简化基因组测序技术并结合BSA分析方法将辣椒根腐疫病抗性基因PhR10定位在2.6 Mb区域内。在此基础上加大定位群体（2405株）同时对感病亲本10399重测序开发InDel多态分子标记，将抗性基因PhR10进一步限定在标记Ind108和Ind119之间147kb的区间内。同时，在辣椒疫霉菌侵染条件下对抗、感亲本在接菌后0h、12h和36h进行根部转录组测序，分析结果表明在定位区间内有2个基因（CA10g20370和CA10g20470）在接菌前后基因表达存在显著差异，结合生物信息学分析结果推测富亮氨酸重复类受体蛋白激酶（LRR-RLK）基因CA10g20470为候选基因的可能性较大，将其命名为CaRLK。该基因全长（CDS序列）2655bp（抗病亲本CM334中），含有2个外显子，编码885个氨基酸。该研究结果为抗性基因的功能研究奠定了扎实的研究基础同时为辣椒抗根腐疫病育种提供了可用的紧密连锁分子标记。