唐古特大黄SRAP反应体系优化及引物筛选

Establishment of SRAP Amplification System and Primer Selection in Rheum tanguticum

SRAP-PCR反应体系的优化是SRAP分子标记的基础,为建立高效稳定的唐古特大黄SRAP反应体系,进一步对唐古特大黄进行遗传多样性、种质资源分析,本研究在单因素试验基础上,对唐古特大黄SRAP-PCR反应体系主要影响因素Mg^(2+)、d NTPs、引物、Taq酶进行正交试验L9(34)。结果表明,唐古特大黄最佳SRAP-PCR反应体系为:Mg^(2+)0.5 mmol·L^(-1),d NTPs 0.4 mmol·L^(-1),引物0.2μmol·L^(-1),Taq聚合酶0.06 U·μL^(-1),总体积25μL。利用最优体系进行引物筛选,从58对SRAP引物中获得了30对条带清晰、多态性好的引物对组合。SRAP-PCR优化反应体系的建立为利用SRAP技术进行唐古特大黄种质资源分析、遗传多样性研究等奠定了基础。

The optimization of SRAP-PCR system is a critical step for SRAP molecular markers. In order to establish a high efficient and stable SRAP reaction system for future study of genetic diversity and germplasm resources analysis in Rheum tanguticum. The study carried out orthogonal design to optimize a SRAP-PCR system for Rheum tanguticum,this system included 4 factors（ Mg2＋,d NTPs,primer and Taq polymerase） from 3 different levels based on the single factor analysis experiment. The result showed that the optimized SRAP-PCR system for Rheum tanguticum was as following：Mg2＋0. 5 mmol·L-1,d NTPs 0. 4 mmol·L-1,each primer 0. 2 μmol·L-1,Taq polymerase 0. 06 U·μL-1. Based on the optimized system,30 SRAP primers out of 58 primers were found with abundant polymorphism in Rh. tanguticum population. This study will provide a technology basis for the genetic analysis of Rh. tanguticum in future.