胡麻EST-SSRs标记分布特征及PCR反应体系的优化

摘要

In this study, 286 868 expressed sequence tags (ESTs) are downloaded from the USA NCBI database. 4 310 appropriate simple sequence repeats (SSRs) are picked out with software of SSRIT. These SSRs accounted for 1.50% of the EST database and 40.09%of them is trinucleotide which is the maximum type. A total of 235 SSR types are got, 21 of them accounted for 68.63%total SSRs picked out in this study, and each of the 21 SSRs is more than 1% of the total SSRs. The optimal PCR reaction system for EST-SSR of flax is got by orthogonal design as follow:primer 0.2μmol/L;template DNA, 50 ng, dNTP, 0.10 mmol/L、Mg2+, 1.5 mmol/L、Taq DNA polymerase 0.50 U/μl、2μL 10×buffer, and at last, filling 20μL with ddH2O. The consistent result is got from single factor optimization experiment on he optimal PCR reaction system for EST-SSR of flax.%从美国NCBI数据库中下载了286868条亚麻EST，利用SSRIT软件检索出符合要求的SSR重复序列4310条，占整个EST数据库的1.50%。其中三核苷酸出现频率最高，占总SSR的40.09%； SSR重复类型总共235种，序列中占总SSR比例大于1%的重复类型共有21种，占SSR序列总数的68.63%。通过正交设计（L16）得到了胡麻EST-SSR最佳PCR反应体系：即20μL反应体系里，引物0.2μmol/L、模板DNA 50 ng、 dNTP 0.10 mmol/L、Mg2+1.5 mmol/L、 Taq DNA聚合酶0.50 U/μl、2μL 10×buffer，其余用ddH2O补齐。并通过单因素优化试验，对得到的胡麻EST-SSR最佳PCR反应体系进行了验证，结果完全一致。