采用SLAF-seq技术开发甘蓝型油菜霜霉病抗性SNP位点

Resistance SNP development to downy mildew in Brassica napus using SLAF-seq technique

由寄生霜霉(Hyaloperonospora parasitica)引起的油菜霜霉病(downy mildew)是油菜主要病害之一。本研究通过将抗霜霉病和感病亲本油菜杂交、F_1自交,得到F_2分离群体,构建抗、感病混池,采用BSA和SLAF(specific length amplified fragment sequencing)技术开发甘蓝型油菜霜霉病抗性相关分子标记。参考已公布油菜(Brassica napus)基因组设计酶切方案,构建SLAF文库并进行双端测序,共开发出132 519个SLAF标签,再通过分析SLAF标签的多态性,检测到264 256个SNP位点。利用SNP-index方法定位关联区域,分析关联区域内的基因在两个亲本之间SNP,对这些SNP进行变异的注释,发现2个非同义突变的SNP,分别是BDsnp01和BDsnp02。经验证,这2个SNP位点是与霜霉病抗性相关的并且在亲本和F2中稳定遗传。使用SNAPER软件设计2对特异引物能够利用电泳直接检测这两个SNP。

Downy mildew of Brassica napus,caused by the oomycete Hyaloperonospora parasitica,is an important disease during the epidemic. In this study,F2 population derived from a susceptible-parent and resistant-parent was used to construct resistant and susceptible pools. In order to obtain molecular markers related to disease resistance to downy mildew,BSA and SLAF-seq technique were developed. Firstly,reference genome of B. napus was used to design marker discovery experiments in silico by simulating the number of markers produced by different enzymes. Secondly,the SLAF library was conducted and sequenced by paired-end sequencing. 132 519 SLAF labels were obtained and 264 256 SNPs were found. Thirdly,the associated region was located by SNP-index,and the SNPs locating at the associated region were analyzed between the two parents. After annotation,2 non-synonymous-coding SNPs were found. By verification,these SNPs were considered to be related to resistance to downy mildew and were genetically stable. Finally,2 specific primer pairs for each SNP were obtained by SNAPER software which designed primers generated differencs in the amplification products.