大豆胞囊线虫广谱型抗病位点Rscn1的精细定位、基因挖掘及功能解析

Soybean cyst nematode (SCN) widely happens in major soybean producing area in China as well worldwide and cause serious yield lost year by year. In order to construct fine map and identify resistance genes in resistant cultivar 'Dongnong L-10', 50 resistant and 50 suscetible lines out of 304 F5:10 RILs, derived from the cross of 'Dongnong L-10'（R）and 'Heinong37' (S), were selected based on their phenotypes of resistance to race 1，3，4 and 14 of SCN and were used for genome sequencing to develop polymorphic SNP markers and construct a high resolution phenotype-genotype association linkage map for redefining locus, Rscn1 with multiple SCN race resistances.Two populations of RILs derived from the crosses of 'Dongnong L-10' and susceptive cultivars were used to verify the resistance-related genomic regions. A large population of F2 including 3000 individuls derived from the cross of 'Dongnong L-10'（R）and 'Heinong37' (S) will be used for fine mapping of resistance genes. Forward and reverse genetic methods will be used to analyze the function of candidate genes from fine mapping. This study will facilitate and shorten the breeding process of SCN disease resistance variety. The results will also be the theoretical basis of molecular breeding for broad disease resistance of soybean.

大豆胞囊线虫(SCN)病是世界性土传病害，造成严重大豆减产。鉴于该病在我国大豆主产区不断扩大蔓延且又缺乏有效防控方法,以及我国大豆种质资源利用较少的局面,本项目立足于开发国内资源,在前期利用东农L-10 （多抗性）×黑农37（感病）的304个F5:10重组自交系获得的抗SCN 1, 3，4和14号小种分子标记位点Rscn1基础上, 建立抗感池小群体，进行SLAF-seq测序，开发SNP标记，构建立表型-标记关联性图谱，并通过抗感池表型与基因型关联分析将广谱型抗病位点Rscn1所在的基因组区段重新定义，利用已构建的以东农L-10为亲本的2个重组自交系群体验证该区段与抗病性的连锁关系，进而挖掘候选基因；同时利用3000个F2个体精细定位候选区段，挖掘得到候选基因；进一步利用正反向遗传学手段验证基因功能。该项目的完成可以推动和缩短我国抗SCN的育种进程,实现分子设计在大豆多小种抗病育种中的应用。

本项目借助基因组学、生物信息学、分子生物学、分子遗传学等先进技术手段，在1、3、4、14号SCN生理小种抗性基因的精细定位和抗性候选克隆及功能验证、分子标记辅助定向选育抗性品种等方面取得如下研究成果：获得3个与抗病位点重组值小于5%的分子标记，并利用标记辅助选择筛选得到12个抗多个大豆胞囊线虫生理小种的优异大豆种质；获得5个受线虫诱导上调表达的抗病候选基因，克隆并通过快速功能鉴定获得1个具有抗病功能的基因；通过正反向遗传学手段进行GmRSCN1-3功能分析，结果表明过量表达该基因可提高大豆植株对SCN的抗性，敲除该基因未显著降低大豆植株对SCN的抗性；借助荧光定量PCR技术对抗病基因的结构变异进行分析并预测其抗性作用方式，发现Rscn1位点内包括GmRSCN1-3在内的9个基因在抗感病大豆品种间存在拷贝数变异。本项目研究成果实现我国抗SCN大豆品种的分子辅助高效选育，并为抗SCN大豆育种提供基因资源。