正交设计优化假俭草SRAP-PCR反应体系及引物筛选

Optimization of SRAP-PCR system on centipedegrass(Eremochloa ophiuroides) using orthogonal design and selection of primers

以假俭草叶片DNA为模板,采用正交试验设计,以Mg2+、dNTP、引物和Taq DNA聚合酶4种因素3个水平,对假俭草SRAP反应体系进行研究,并比较了不同浓度模板DNA对扩增效果的影响,建立了假俭草的SRAP最佳反应体系。结果表明,假俭草SRAP-PCR最佳反应体系为:2μL 10×PCR buffer、60 ng模板DNA、Mg2+1.50 mmol/L、dNTP 260μmol/L、引物0.25μmol/L、Taq DNA聚合酶0.5 U,总体积为20μL。各因素对扩增反应结果均有不同影响,其中以Mg2+浓度影响最大,Taq DNA聚合酶的影响最小。运用该体系对4份假俭草种源进行验证,证明该体系稳定可靠,并从45个SRAP引物组合中筛选出扩增条带清晰、多态性丰富的26个引物组合。这一体系的建立及多态性引物组合的筛选为今后利用SRAP标记技术进行假俭草分子遗传学研究提供了科学依据。

An orthogonal design was used to optimize a SRAP-PCR system with 4 factors （Mg^2＋, dNTP, primer and Taq polymerase） at 3 levels plus the concentration of template DNA. The optimized SRAP-PCR system for E. opiuroides was：2 μL 10×PCR buffer,60 ng template DNA, Mg^2＋ 1.50 mmol/L, dNTP 260 μmol/L, primer 0.25 μmol/L, Taq DNA polymerase 0.5 U in a total of 20 μL reaction solution. Each factor had a different effect on the results of PCR. Mg^2＋ concentration had the greatest effect and Taq DNA polymerase had the least effect. The optimized SRAP-PCR system was tested on four E. ophiuroides germplasms and shown to be steady and reliable. 26 primer combinations were selected with abundant polymorphism from 45 primer combinations. The optimized SRAP-PCR system and polymorphism primer combinations could be applied.to molecular genetics research of E. ophiuroides.