正交设计优化辣椒SRAP-PCR反应体系及引物筛选

Optimization of SRAP-PCR System in Pepper Using Orthogonal Design and Selection of Primers

以辣椒基因组DNA为模板,采用L16(45)正交试验设计,对SRAP反应体系中的5种关键因素(TaqDNA聚合酶、Mg2+、dNTPs、引物、模板DNA)进行优化,结果表明,辣椒SRAP-PCR最佳反应体系为:TaqDNA聚合酶0.75 U、Mg2+0.6 mmol/L、dNTPs 0.2 mmol/L、引物0.8μmol/L、模板DNA 50 ng,总体积为10μL。运用该体系对辣椒3份种质材料进行验证,证明该体系稳定可靠,并从198个SRAP引物组合中筛选出扩增条带清晰、多态性丰富的35个引物组合。该体系的建立与多态性引物组合的筛选为SRAP标记技术在辣椒分子遗传学中的应用提供科学依据。

The L16（45） othogonal design was used to optimize SRAP-PCR system in pepper（Capsicum spp.） with five key factors（Taq DNA polymerase,Mg2＋,dNTPs,primerand template DNA,respectively）.The results showed that the optimized SRAP-PCR system for pepper was：0.75 U Taq DNA polymerase,0.6 mmol/L Mg2＋,0.2 mmol/L dNTPs,0.8 μmol/L primer,50 ng template DNA in a total of 10 μL reaction solution.The optimized SRAP-PCR system was tested on three pepper germplasms and was steady and reliable.35 primer combinations were selected with abundant polymorphism from 198 primer combinations.The optimized SRAP-PCR system and polymorphism primer combinations could be applied to research on molecular genetics in pepper.