葡萄SRAP反应体系优化及引物筛选

本试验利用L16（4^5）正交试验设计探寻葡萄SRAP—PCR反应体系中的关键因子，同时结合单因素试验简单、快捷的特点逐个对PCR反应体系的主要成分进行优化。充分利用两种方法的优点并降低试验工作量取得了较好的效果，建立了适于葡萄的SRAP反应体系。结果表明Mg^2＋浓度为影响葡萄SRAP—PCR反应的关键因素；优化的20μLSRAP—PCR反应体系中各组分的最适含量为：10×Buffer2．0μL，Mg^2＋2．5mmol／L，dNTPs0.3mmol／L，引物0．4μmol／L，砌DNA聚合酶1．0U，模板DNA1．0ng／μL。利用SRAP反应体系，从100对SRAP引物组合中筛选出扩增稳定，条带清晰，多态性好的引物19对。本研究建立的适于葡萄SRAP—PCR扩增的反应体系，将为葡萄种质遗传多样性评价、基因组分析、指纹图谱构建，分子标记辅助育种和遗传改良研究提供基础。

Optimization of SRAP-PCR System in Grape and Primers Screening

In this research, we firstly explored the key factors of the grape SRAP-PCR reaction system based on the L^6 （4^5） orthogonal experimental design, and then the main components were optimized one by one with the characteristics of fast and simple single factor test. By making full use of the advantages of these two methods and shortening the experiment periods, we achieved the preferable results and established a SRAP reaction system which was most suitable for grape. The results showed that the concentration of Mg^2＋ was the key factor of the grape SRAP-PCR reaction system. And the optimal content of each component in SRAP-PCR reaction system total with 20 μL containing 2.5 mmol/L Mg^2＋, 0.3 mmol/L dNTPs, 0.4 μmol/L each primer, 1.0 U Taq DNA polymerase and 1.0 ng/μL DNA template. Nineteen primer pairs were screened out from 100 SRAP primer pair combinations based on their stable amplification, clear banding patterns and good polymorphism by utilizing the SRAP reaction system. The optimal SRAP-PCR reaction system was established, which would provide the basis for evaluation of genetic diversity, genomic analysis, construction of fingerprinting, molecular marker assisted breeding and genetic improving for the grape.