玉米抗蚜虫主效QTL的克隆与功能分析

Maize leaf aphid (Rhopalosiphum maidis Fitch) is one of the most serious pests of maize worldwide. Isolation of the resistance gene play an important role in understanding the resistance molecular mechanism and utilization in maize breeding programs. In our previous studies, both linkage mapping and genome-wide association study (GWAS) were applied to dissect the genetic architecture of resistance to R. maidis. Three QTL, named as qRrm1, qRrm2 and qRrm3, were identified for maize leaf aphid resistance and two of them were verified by NILs (near-isogenic lines) population and their candidate genes were also predicated. Base on those results, one of the two QTL will be isolated by map-based cloning strategy. This project includes two parts: 1) Fine-mapping the QTL: Combine linkage and association mapping and BSA-seq analysis results to define one of the two QTL for further studies. Map the QTL into a 100Kb region by screening and evaluation recombination lines in a big segregation population. Candidate gene was defined based on the sequence comparison analysis, transcriptome, metabolome analysis and mapping result. 2) Function analysis for resistance gene, the expressional characteristic of target gene will be analyzed at RNA level in various tissues and materials. Based on the re-sequencing analysis of target gene in GWAS population, the functional variation of target gene was identified by candidate gene association mapping analysis.The implementation of the project can help to reveal the resistance mechanism and provide the theoretical basis for resistance breeding.

玉米蚜虫是世界上发生普遍、为害严重的玉米虫害之一，鉴定和克隆主效抗性位点，对玉米抗蚜虫育种和抗性分子机理的解析有重要意义。课题组前期通过连锁分析和关联分析，定位到3个抗蚜QTL qRrm1、qRrm2和qRrm3，并利用BC2群体对前两个位点进行了初步验证和候选基因预测。在此基础上，本项目拟利用图位克隆技术对其中一个QTL进行克隆。研究包括：1）玉米抗蚜虫基因的精细定位和分离：结合连锁、关联和BSA-seq分析结果，选定1个QTL进行深入研究；利用精细定位方法把QTL定位在100Kb的范围内；再结合抗感材料间序列比对、转录组和代谢组分析，来鉴定候选基因。2）抗性基因功能分析：采用荧光定量PCR方法分析候选基因的时空表达特性；通过候选基因关联分析，鉴定功能性变异位点；开发功能分子标记用于抗性育种。本项目的实施为解析玉米抗蚜虫的分子机制和抗性育种奠定基础。

蚜虫是玉米生产上常见的虫害之一，生产上主要使用化学农药进行防治，但容易造成环境污染，克隆玉米里的原生抗蚜虫基因并鉴定其功能有利于抗虫绿色玉米新品种的培育。课题在前期初步发现2个抗蚜虫QTL的基出上，利用连锁分析和BSA-Seq方法对前期初步鉴定的QTL进行验证，并结合关联分析结果将隶属于细胞色素cP450超家族的的bx2基因为抗虫QTL qRrm1的候选基因。通过定量表达分析，发现在蚜虫侵染后候选基因bx2影应蚜虫的侵染，呈现表达量先上调后下降，在蚜虫侵染5天之后表达量趋于稳定，同时发现抗、感种质的表达量有较大差异。通过抗、感种质的序列分析发现在抗、感材料中的启动子区域内有一个插入缺失变异可能与抗性差异有关，基于KASPar高通量分子标记检测技术，开发一个功能分子标记，并验证了该变异位点与玉米抗蚜虫的表现为极显著相关。利用亚细胞定位技术将其表达作用位置定位于在细胞质中。为了进一步验证候选基因qRrm1的功能，利用同源重组方法构建了过表达载体，利用CRISPR-Cas9技术构建了基因敲除载体，并进行了遗传转化。另外，对QTL qRrm2的候选基因进行定量表达分析，发现其在玉米侵染蚜虫后表达量发生上调，初步明确其参与了玉米抗蚜虫的响应，并利用亚细胞定位技术将其表达作用位置定位于在细胞核中。项目的实施为了解玉米抗蚜虫的分子机理和抗性育种提供了理论支持。