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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。
The Tianshan cherry EST sequences were searched from the NCBI database, the SSR locus was searched using SSRHunter 1.3 software, Primer 6.0 primers were designed and the SSR-PCR reaction system of Tianshan cherry was optimized by orthogonal and single factor experiments , And used the best system to validate the developed primers in order to lay a foundation for the study of Tianshan cherry genetic diversity and construction of genetic linkage map. The results showed that 30 pairs of primers were randomly selected from 58 pairs of primers, and 21 pairs of primers could amplify specific bands with an effective amplification rate of 70%. The best SSR-PCR reaction system for 20 μL Tianshan cherry was dNTP 0.2 mmol·L -1, primer 0.4 μmol·L -1, Taq enzyme 0.75U and template DNA 60 ng.