芝麻SSR检测体系的优化与应用  

Optimization and Application of SSR-PCR System for Varieties Identification in Sesame(Sesamum indicum L.)

作　　者：刘红艳[1] 杨敏敏[1] 左阳[1] 舒岩[1] 赵应忠[1] 

机构地区：[1]中国农业科学院油料作物研究所/农业部油料作物生物学重点开放实验室,武汉430062

出　　处：《中国农学通报》2011年第21期138-143,共6页Chinese Agricultural Science Bulletin

基　　金：国家芝麻产业技术体系长江流域育种岗位(CARS-15-1-03);中国农业科学院油料作物研究所所长基金项目"芝麻重要性状的改良与分子标记研究"(1610172011007);国家科技支撑计划项目"特色油料抗灾与节本增效关键技术研究与示范"(2009BADA8B04)

摘　　要：为了建立一套适用于芝麻SSR标记检测的技术体系,以5个芝麻新品种为试验材料,选用35对SSR引物,从反应体系、反应程序、电泳、银染4个环节进行优化。结果表明:反应总体积为10μL,优化后的基本成分及用量分别为25mmol/L Mg2+1.2μL,10mmol/L dNTPs0.3μL,5U/μL Taq酶0.3μL,50ng/μL Primer0.9μL,10×Buffer0.7μL,25ng/μL DNA模板2.5μL,ddH2O4.1μL。反应程序为:94℃3min;94℃1min,60℃30s,72℃45s,10个循环;94℃30s,57℃30s,72℃45s,30个循环;72℃5min。PCR产物用6%变性聚丙烯酰胺凝胶电泳,硝酸银快速染色检测效果良好。筛选出多态性较强的SSR引物24对,用这些引物对20个品种进行扩增,初步分析了SSR标记应用于芝麻DNA指纹分析的潜力。A SSR marker detection system for sesame（Sesamum indicum L.） by optimizing the amplification reaction components,cycling parameters and the electrophoresis detection was established.DNA samples from five sesame cultivars were used as template for this study.The optimized PCR system contained the following components in a volume of 10 μL：25 mmol/L Mg ^2＋ 1.2 μL,10 mmol/L dNTPs 0.3 μL,5 U/μL Taq polymerase 0.3 μL,50 ng/μL Primer 0.9 μL,10×PCR reaction buffer 0.7 μL,25 ng/μL DNA template 2.5 μL,ddH 2 O 4.1 μL.PCR amplification was conducted in three phases.Firstly,DNA was denatured for 3 min at 94℃ and then 10 cycles of 94℃ 1 min,60℃ 30 s and 72℃ 45 s.Secondly,another 30 cycles of 94℃ 30 s,57℃ 30 s,72℃ 45 s were performed.Finally,the amplification was ended in 5 min at 72℃.The 6% denaturing polyacrylamide gel electrophoresis and silver staining method were employed for the detecting of banding profile.With the above detection system,35 SSR markers were screened across 5 sesame cultivars and found that 24 markers could reveal two or more polymorphic bands.The usefulness of the optimized detection system and the set of SSR markers for cultivar identification were verified in the analysis of other 20 sesame cultivars.

关 键 词：芝麻 SSR 检测体系优化 

分 类 号：S565.3[农业科学—作物学]