天山樱桃EST-SSR引物开发及PCR反应体系优化

Development of EST-SSR Markers and Optimization of SSR System in Cerasus tianschanica

以新疆天山樱桃叶片为试材,从NCBI数据库中搜索天山樱桃EST序列,利用SSRHunter 1.3软件查找SSR位点,结合Primer 6.0设计引物,通过正交实验和单因素试验优化天山樱桃SSR-PCR反应体系,并用最佳体系对所开发的引物进行验证,以期为研究天山樱桃遗传多样性、构建遗传连锁图谱奠定基础。结果表明:从58对备选引物中随机挑选30对引物进行试验,有21对引物能够扩增出特异性条带,有效扩增率为70%。20μL天山樱桃SSR-PCR最佳反应体系为dNTP 0.2mmol·L^(-1)、引物0.4μmol·L^(-1)、Taq酶0.75U、模板DNA 60ng。

Leaves of Cerasus tianschanica were used as materials. The EST sequences of Cerasus tianschanica were obtained from NCBI, SSR motifs were screened by using SSRHunter 1.3 software from these sequences, and specific primers were designed with the software Primer 6.0. Besides, the SSR-PCR reaction system of Cerasus tianschanica was optimized by orthogonal test and single factor experiment to verify the developed primers in order to lay the foundation for genetic diversity and construction of genetic map. The results showed that 21 pairs of primers could amplify specific bands, and the effective amplification rate was 70 %. Moreover,the 20μL. reaction system contained 0. 2 mmol L-1 dNTPs, 0.4 μmol L-1 primer, 0.75 U Taq polymerase and 60 ng template DNA.