白芨属SRAP引物筛选及反应体系优化

Optimization of SRAP-PCR System and Primer Screening for Bletilla

为给白芨种质遗传多样性评价、分子标记辅助育种和遗传改良研究提供基础,建立适于白芨SRAP-PCR扩增的反应体系,利用L16(45)正交试验设计对白芨SRAP-PCR反应体系中的各影响因子进行探索。结果表明:Mg2+浓度为影响白芨SRAP-PCR反应的关键因素;在优化的20μL SRAP-PCR反应体系中各组分的最适含量为10×Buffer 2.0μL,Mg2+2.5mmo1/L,dNTPs 0.5mmol/L,引物0.2μmol/L,Taq酶1.0U,模板DNA 2.0ng/μL。利用SRAP反应体系,从72对SRAP引物组合中筛选出条带清晰、扩增稳定、多态性好的引物15对。

In order to provide basis for evaluation of genetic diversity,molecular marker assisted breeding,and genetic improving for Bletilla,the key factors in SRAP-PCR reaction system were studied based on the L16（45） orthogonal experimental design.The results showed that concentration of Mg2＋ was the key factor.The optimized SRAP-PCR reaction system of Bletilla was obtained containing 2.5 mmol/L Mg2＋,0.5 mmol/L dNTPs,0.2 μmol/L each primer,1.0 U Taq DNA polymerase and 2.0 ng/μL DNA template in total 20 μL.Then 15 primer pairs with stable amplification,clear banding patterns and good polymorphism were screened out from 72 SRAP primer pair combinations.