大白菜简单序列重复(SSR)和插入／缺失(InDel)标记的开发及通用性分析  

Development of Simple Sequence Repeat(SSR) and Insertion/Deletion(InDel) Markers in Chinese Cabbage(Brassica rapa ssp. pekinesis) and Analysis of Their Transferability

作　　者：仪泽会[1] 卢有飞[1] 郭晓芹[1] 惠麦侠[1] 张鲁刚[1] 张明科[1] 

机构地区：[1]西北农林科技大学园艺学院、农业部西北地区园艺作物生物学与种质创制重点实验室,杨凌712100

出　　处：《农业生物技术学报》2012年第12期1398-1406,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31272164);西北农林科技大学青年学术骨干项目(No.Z111020902);高校基本科研业务费科技创新专项(重点项目)(No.QN2011057)

摘　　要：为探讨大白菜基因组序列中SSR位点的分布规律并开发SSR引物,利用SSRHunter软件对大白菜A10(16899818～17299817)的DNA序列进行简单序列重复(SSR)位点查找,共得到394个SSRs,平均每1.02kb出现1个SSR。二核苷酸和三核苷酸重复是最主要的SSR类型,分别占79.44%和18.78%。为了提高SSR标记开发的准确性和通用性,对检索得到的含SSR位点的序列进行了同源比对,选取符合条件的15条SSR序列并设计引物;依据Blast过程中发现的在SSR位点不存在差异而在其侧翼序列中存在插入/缺失(InDel)差异的序列,设计了19条InDel引物。用34对SSR及InDel引物在6个大白菜(Brassica rapassp.pekinesis)材料中进行多态性研究,发现28对引物能扩增出理想的PCR产物,有效扩增率为82.35%,其中27对引物具有多态性,多态性比率为79.41%。为验证SSR引物的真实性,随机对4对SSR引物的部分白菜扩增片段进行了测序,发现100%的片段具有相应的SSR位点。28对SSR和InDel引物在甘蓝(B.oleracea)、油菜(B.napus)和萝卜(Raphanus sativus)品种的有效扩增率分别为85.71%、100%和77.78%,说明新开发的SSR和InDel标记具有较好的多态性和通用性。利用6对引物分析了48份十字花科种质的遗传多样性,结果表明48份材料被明显地区分成白菜和甘蓝组、萝卜组、油菜组3大类群,与传统分类一致。大白菜SSR和InDel标记的开发对于十字花科种质亲缘关系及遗传多样性分析具有重要的应用价值。In order to analyze the SSR distribution in genomic sequence of Brassica rapa and develop new SSR markers, the DNA sequences of Chinese cabbage A10 （16899818-17299817） were screened using SSRHunter software and 394 Simple sequence repeats （SSRs） were mined with an average distance of 1.02 kb.Dinucleotide and trinucleotide repeat SSRs were the dominant types, accounting for 79.44% and 18.78%, respectively, of the SSR obtained. In order to improve the accuracy and to test the transferability of the developed SSRs, the mined SSRs were blasted and 15 SSR sequences were selected and primers were designed. Morever, some differences of Insertion/Deletion（inDels） rather than SSRs were found in some SSRs sequences and 19 InDels primers were designed. The 34 resultant primer pairs were used for polymorphism analysis in 6 genotypes of Chinese cabbage （Brassica rapa ssp. pekinesis）. Twenty eight primer pairs had expected PCR products, accounting for 82.35% of designed primers, and 27 primer pairs showed polymorphisms, accounting for 79.41% of the designed primers. PCR products from 4 random SSR primer pairs were used for sequence analysis. The results showed that 100% of the fragments contained target SSRs. The percentage of available SSRs and InDels amplified in cabbage（B, oleracea）, rapeseed（B, napus） and radish（Raphanus sativus） samples were 85.71%, 100% and 77.78%, respectively. It showed that these SSRs and InDels had good polymorphisms and transferability among Brassicaceae. A subset of 6 validated primers was also used to assess genetic diversity in a collection of 48 elite Brassieaeeae germplasms. The dendrogram revealed that all germplasms were obviously classified into 3 groups, including Brassiea rapa and Brassica oleracea, Raphanus sativus and Brassica napus, and the result accorded with the conventional taxonomy. The SSR and InDel markers can be used in genetic analysis ofBrassica ropa.

关 键 词：白菜 SSR INDEL 通用性 

分 类 号：S813[农业科学—畜牧学]