芝麻SSR检测体系的优化与应用

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10 μL，优化后的基本成分及用量分别为25 mmol/L Mg2+ 1.2 μL，10 mmol/L dNTPs 0.3 μL，5 U/μL Taq酶0.3 μL，50 ng/μL Primer 0.9 μL，10×Buffer 0.7 μL，25 ng/μL DNA模板2.5 μL，ddH2O 4.1 μL。反应程序为：94℃ 3 min；94℃ 1 min，60℃ 30 s，72℃ 45 s，10个循环；94℃ 30 s，57℃ 30 s，72℃ 45 s，30个循环；72℃ 5 min。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。

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

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.