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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。
OBJECTIVE: To analyze the distribution of SSR loci in EST of Guiyu gold, and to develop the EST-SSR primers of Guiyu gold so as to explore the feasibility of EST-SSR for genetic diversity of Guiyu gold varieties. Methods: 12 678 EST sequences were downloaded from the NCBI public database and searched for SSR loci by MISA software. Eligible sequences were selected and EST-SSR primers were designed by Primer 5.0 software. The characteristics of PCR amplification of these EST-SSR primers were studied by polyacrylamide gel (PAGE) electrophoresis to further verify the rationality and effectiveness of the development results. Results: Among the 12 678 EST sequences downloaded, 926 sequences contained SSR loci, accounting for 7.30% of the total EST database. Among them, the largest proportion of SSR loci were dinucleotide repeats, trinucleotide And tetranucleotide followed by 623 (50.90%), 388 (31.70%) and 125 (10.21%), respectively. A total of 165 EST-SSR primer pairs were designed based on the selected microsatellite sequences, and 24 of them were selected for synthesis. The results of PCR showed that 21 primer pairs (87.50%) could amplify the stable bands. 13 pairs of EST-SSR primers were detected in 6 accessions, accounting for 54.17% of the total primers. Thirteen pairs of validated EST-SSR primers were used to analyze the genetic relationship of 20 Osmanthus fragrans cultivars. Conclusion: The EST-SSR marker of Guiyu-Jin is highly efficient and is an important measure for the development of SSR markers of Guiyu-gold. It is of great significance for the identification, genetic diversity and breeding of Guiyu-gold varieties.