玉米大斑病主效抗病位点qNCLB5.04的克隆和功能分析

Northern corn leaf blight (NCLB) is one of the most devastating foliar diseases in maize. Isolation of the resistance gene can play an important role in understanding the resistance molecular mechanism and utilization in maize breeding programs. In our previous studies, both genome-wide association mapping and linkage analysis were applied to dissect the genetic structure of resistance to NCLB, and a major QTL (i.e., qNCLB5.04) was identified from resistant inbred line K22. Further mapping of qNCLB5.04 has delimited the resistance interval to 16.5Mb by using near isogenic lines (NILs). In this study, gene cloning will be carried out by map-based cloning strategy. This project includes fine-mapping and cloning of qNCLB5.04 as well as functional analysis: 1) For fine-mapping qNCLB5.04, NILs segregating at qNCLB5.04 will be genotyped by molecular markers for screening recombinant lines. Combined with the phenotypic evaluation, the qNCLB5.04 region can be expected to delimit to 30Kb region, in which the candidate genes can be predicted based on the sequence analysis of positive clones screened from K22 BAC library. 2) For functional analysis of resistance gene, the expressional characteristic of target gene will be analyzed at RNA level from the resistant line K22, susceptible line By815 and NILs. Based on the re-sequencing analysis of target gene, the functional variation of target gene can be identified by candidate gene association analysis as well as the mutants analysis. The information from cloning and functional analysis of qNCLB5.04 can help to reveal the molecular resistance mechanism and facilitate molecular assisted selection in maize resistance breeding programs.

玉米大斑病是玉米生产上普遍发生的真菌性病害。鉴定和克隆主效抗病位点，对玉米抗病育种具有重要的意义。课题组前期通过连锁和关联分析，在自交系K22中鉴定出一个主效抗病位点qNCLB5.04，并利用近等基因系群体把抗病区间限定在16.5Mb的范围内。在此基础上，本项目拟采用图位克隆技术分离抗病基因。研究包括两个阶段：1、抗病基因精细定位和分离：在初步定位的16.5Mb抗病区域内开发分子标记，结合近等基因系分离群体的抗性鉴定，将qNCLB5.04定位在30Kb的范围内；通过K22的BAC文库筛选和序列分析，鉴定候选基因。2、抗病基因功能分析：采用荧光定量PCR等方法分析候选基因的时空表达特性；通过候选基因重测序，进行候选基因关联分析，鉴定抗病基因的关键功能位点；并通过感病突变体序列变异分析，对候选基因的功能进行验证。本项目的实施将为解析大斑病抗病的分子机制奠定基础，同时为抗病育种提供理论依据。

玉米大斑病是玉米生产上普遍发生的真菌性病害。课题组前期通过连锁和关联分析，在自交系K22中鉴定出一个主效抗病位点qNCLB5.04，并利用近等基因系群体把抗病区间限定在16.5Mb的范围内。本研究采用近等基因系作图群体，通过抗病区域内分子标记开发和交换单株筛选，把抗病基因限定在450Kb的范围内；在该抗病区域内，通过抗病亲本K22的序列组装和生物信息学方法对该区域进行基因预测，共鉴定出3个候选基因；结合预测基因在抗、感亲本的序列差异和时空表达分析，鉴定出Zm00001d015682是qNCLB5.04的候选基因；本项目的实施为解析玉米大斑病抗病的分子机制奠定基础，同时为抗病育种提供理论依据。