甜瓜抗疫病基因的精细定位与克隆

Phytophthora blight, a soil-borne disease caused by Phytophthora spp., severely threatens the melon production in our country. Breeding and promoting disease-resistant varieties is the most economical and effective method to prevent this disease. However, the lack of resistant resources slows down the progress in genetic research of melon against Phytophthora blight, and there is no disease-resistant gene that has been identified or cloned yet, all of which seriously restrict melon resistance breeding and the study on disease-resistant mechanism. In this study, a high virulent strain of Phytophthora spp., identified as Phytophthora capsici Leonian, was isolated from the infected melon plant, and by using this strain we found a high resistant melon germplasm ZQK9. Genetic analysis showed that the resistance to P. capsici in ZQK9 was controlled by one single dominant gene. Based on the above results， whole-genome resequencing technology combined with bulked segregant analysis (BSA) will be adopted to fine map the resistant gene. In combination of melon genome information and transcriptome data, the sequence and expression differences of genes in the target region will be analyzed between the resistant and susceptible parents. Further, candidate genes will be predicted and their function will be verified by transgenic technology. This study will provide a scientific basis for melon Phytophthora blight resistance breeding and for elucidating disease-resistant mechanism.

甜瓜疫病是由疫霉菌引起的一种土传病害，对我国的甜瓜产业构成严重威胁。选育并推广抗病品种是防治疫病最经济有效的方法。但由于缺乏可靠的抗源，甜瓜抗疫病遗传研究进展缓慢，还未有抗病基因被定位或克隆，严重制约了甜瓜抗疫病分子育种和抗病机理的研究。本研究从甜瓜病株中分离得到一个高致病力疫病菌株，经鉴定为辣椒疫霉菌，利用该菌株筛选到1份高抗疫病的甜瓜材料ZQK9。遗传分析结果表明，ZQK9对疫病的抗性为单基因控制的显性遗传。本研究拟采用混合群体分离分析法和全基因组重测序技术，对ZQK9的抗疫病基因进行精细定位。结合甜瓜基因组信息和转录组测序数据，分析定位区间内基因在抗感亲本间的序列和表达差异，确定候选基因，再利用转基因技术验证候选基因的功能。该研究将为甜瓜抗疫病育种以及阐明抗疫病作用机理提供科学依据。

本项目以高感材料E31为母本，高抗材料ZQK9为父本，构建了F1、F2、BC1代群体。遗传分析显示，ZQK9对疫霉菌的抗性是受单一显性基因控制的。根据F2群体的抗性鉴定结果，利用两亲本和抗感混池进行BSA混池测序分析，将抗病基因定位在甜瓜12号染色体22,075,687-25,394,539 bp的区间内。利用在初定位区间内开发的InDel标记和含有498个单株的F2群体，将定位区间进一步缩小到52.44 kb的范围内。该区间内有8个基因，通过序列分析和表达模式分析，发现编码细胞壁相关受体激酶（WAK）的MELO3C002430基因在抗病的ZQK9接菌后前72小时内显著上调表达，但在感病材料中无显著变化，而WAKs基因在植物识别病原菌侵染并触发免疫反应的过程中具有重要作用，预测MELO3C002430可能是ZQK9的抗疫病候选基因。MELO3C002430基因的CDS序列在抗感材料间存在一个非同义突变SNP位点。利用该位点设计的CAPS标记在F2群体中的基因型和表型共分离。利用该标记对源于不同国家和地区的50份甜瓜材料（栽培种44份，野生种4份，近缘种2份）进行鉴定，除2份近缘种（PI 271337和PI 364472）的基因型与表型不一致外，其他材料表型与标记基因型完全一致，证明该标记可用于分子标记辅助育种。本研究对于揭示甜瓜抵御疫霉菌侵染的分子机制和加快抗疫病新品种选育具有重要意义。