银缕梅SSR-PCR反应体系的优化及引物筛选

Optimization and Primer Selection of SSR-PCR System of Parrotia subaequalis

为有效利用SSR-PCR技术分析国家一级重点保护植物银缕梅的遗传多样性,采用正交试验设计L16(43),即3因素4水平,对影响SSR-PCR扩增结果的因素(引物,模板DNA浓度和循环数)进行优化筛选,并在此基础上对聚丙烯酰胺凝胶上样量进行优化,建立了适合银缕梅的最佳SSR-PCR反应体系:10μL 2×PCR Mix、40 ng模板DNA、10μmol/L引物,其余用dd H_2O补齐至20μL。PCR扩增程序为:95℃预变性15 min,95℃变性30 s,57.5℃退火1.5 min,72℃延伸1 min,30个循环,循环结束后,60℃延伸30 min,扩增产物4℃保存。聚丙烯酰胺凝胶电泳上样量以1.5~2.0μL为最佳。利用优化后体系进行引物筛选,从18对引物中筛选出11对具有多态性引物,说明该反应体系具有良好的稳定性。该体系的建立可以为今后利用SSR标记对银缕梅遗传多样性分析、系统发育研究、遗传图谱构建、基因定位和分子标记辅助育种等研究提供基础。

In order to effectively use the SSR-PCR syste m to analyze the genetic diversity of the plant Parrotia subaequalis which is a national primary protection plant, an orthogonal design（L16（43）） was applied to optimize and screen the influencing factors of SSR-PCR amplification results, including primer, template DNA concentration and cycle numbers. Based on this analysis, the loading amount of polyacrylamide gel was optimized, and the optimal SSR-PCR system suitable for P. subaequalis was established as follows： 10 μL 2×PCR Mix, 40 ng template DNA,and 10 μmol/L primer, and the rest was supplemented with dd H_2O to 20 μL. The PCR amplification was performed according to the following procedure： an initial denaturation for 15 min at 95℃, denaturing for 30 s at 95℃,annealing for 1.5 min at 57.5℃, extending for 1.0 min at 72℃. After 30 cycles, a final extension at 60℃ for 30 min was applied, and the amplified DNA was stored at 4℃. The optimal loading amount of polyacrylamide gel electrophoresis was 1.5~2.0 μL. Through the optimized SSR-PCR system used for primer screening, 11 pairs of polymorphic primers were selected from 18 pairs of primers, and the result showed that the reaction system had excellent stability. Our results indicated that the establishment of SSR-PCR system would provide theoretic and technical support for genetic diversity analysis, phylogenic stud y, gen etic maps establishment, gene localizations and molecular marker assisted breeding of Parrotia subaequalis based on SSR marker.