桂郁金EST资源的SSR信息分析及EST-SSR标记开发

目的:分析桂郁金EST中SSR位点分布规律,开发桂郁金EST-SSR引物,探讨EST-SSR用于桂郁金品种遗传多样性的可行性。方法:从NCBI公共数据库下载姜黄属EST序列(expressed sequence tag,EST)12 678条,利用MISA软件对其进行SSR位点查找,选出符合条件的序列,采用Primer 5.0软件设计EST-SSR引物,利用聚丙烯酰胺凝胶(PAGE)电泳研究这些EST-SSR引物PCR扩增的特点,进一步验证开发结果的合理性与有效性。结果:下载得到的12 678条EST序列中,共有926条序列包含SSR位点,占整个EST数据库的7.30%,其中SSR位点所占比例最大的是二核苷酸重复序列,三核苷酸和四核苷酸次之,分别为623(50.90%)个,388(31.70%)个和125(10.21%)个。根据筛选得到的微卫星序列共设计了165个EST-SSR引物对,选择其中92分以上的24个合成。PCR检测表明,21个引物对(87.50%)可以扩增出稳定清晰的带型;在6份不同种质桂郁金中检测到13对EST-SSR引物有多态性,占设计引物的54.17%。利用13对验证的EST-SSR引物对20个桂郁金品种进行了亲缘关系分析。结论:桂郁金EST-SSR标记开发的效率较高,是桂郁金SSR标记开发的重要措施,对于桂郁金品种鉴定和遗传多样性分析以及育种等方面具有重要的意义。

SSR Information Analysis of Curcuma kwangsiensis EST Resources and EST-Development of SSR Markers

Objective: To analyze the distribution rules of SSR loci in Curcuma kwangsiensis EST, develop C. kwangsiensis EST-SSR primers, and explore the feasibility of EST-SSR for C. kwangsiensis species genetic diversity. Method: The 12 678 items of Curcuma EST (expressed sequence tag) sequences were downloaded from NCBI database, and MISA software was used to find SSR loci and select qualified sequences. Primer 5.0 software was used to design EST-SSR primers; polyacrylamide gel electrophoresis (PAGE) was used to analyze the PCR amplification characteristics of these EST-SSR primers, and further verify the rationality and validity of the developed results. Result: From 12 678 downloaded EST sequences, 926 sequences contained SSR loci, accounting for 7.30% of the entire EST database. The largest proportion for SSR loci was in dinucleotide repeat sequences, followed by trinucleotide and tetranucleotide sequences, 623 (50.90%), 388 (31.70%) and 125 (10.21%) respectively. A total of 165 EST-SSR primer pairs were designed according to the microsatellite sequences, and 24 of them with more than 92 points were selected for synthesis. PCR detection showed that 21 primer pairs (87.50%) could amplify stable and clear bands; in 6 different Germplasms in C. kwangsiensis, 13 pairs of EST-SSR primers showed polymorphism, accounting for 54.17% in the primer design. 13 pairs of EST-SSR primers were used to verify the phylogenetic relationship for 20 C. kwangsiensis varieties. Conclusion: C. kwangsiensis EST-SSR markers have a higher development efficiency, so it is one of the important measures for development of C. kwangsiensis SSR markers, with vital significance for the identification of C. kwangsiensis variety, genetic polymorphism analysis and breeding.