广西莪术EST-SSR标记开发及其在遗传多样性分析中的应用

Development of EST-SSR markers and its application in genetic diversity analysis in Curcama kwangsinesis

目的:分析姜黄表达序列标签(EST)中简单重复序列(SSR)位点的分布特点,开发近缘种广西莪术EST-SSR引物,探讨EST-SSR用于进行广西莪术遗传多样性分析及分子育种的可行性。方法:下载NCBI中公布的姜黄EST 12 678条,利用SSR-FINDER搜索SSR位点,筛选符合条件的序列,采用Primer 5.0设计SSR引物,挑选30个表现型差异较大的广西莪术种质进行引物有效性及多态性检测,对50份广西莪术种质进行遗传多样性分析。结果:12 678条EST序列含有SSR位点1 243个,其中二核苷酸出现频率为50.36%,三核苷酸31.54%,四核苷酸10.30%,以AT/TA和CT/GA出现频率最高。利用Primer 5.0设计引物共325对,聚合酶链式反应(PCR)检测表明,104对引物可以扩增出理想PCR产物,在至少30份不同种质广西莪术中检测到48对SSR引物具有多态性,占设计引物的38.09%。遗传多样性分析研究,聚类分析结果表明50份广西莪术种质在相关系数0.7处,聚类为2类,遗传相似性系数变化范围较窄。结论:姜黄EST资源中含有高频率的SSR位点,且EST-SSR标记开发效率较高。本研究开发了48对广西莪术EST-SSR标记,并筛选出富含SSR位点的候选序列,用聚类图验证了植物之间的亲缘关系,为广西莪术遗传多样性分析和分子育种研究提供参考。

Objective： To analyze distribution characteristics of simple sequence repeat （ SSR ） in expressed sequence tags （ ESTs ） from turmeric （ Curcuma longa L. ）, develop relative species of Curcama kwangsinesis EST-SSR primers-, and discuss Curcama kwangsinesis in genetic diversity and molecular breeding. Methods： Download 12678 ESTs from Curcuma longa published in NCBI database; using the SSR-FINDER to search the SSR loci, screen qualified sequences, using the Primer 5.0 to design SSR primers ; choose 30 phenotype different germplasm resources of Curcama kwangsinesis to detect the effectiveness and polymorphism of primers, genetic diversity of 50 Guangxi Curcama kwangsinesis. Results： A total of 1243 microsatellite repeats were detected from 12 678 EST sequences of Curcama kwangsinesis. After redundancy elimination, the dinucleotide frequency was 50. 36%, and the trinucleotide and the tetranucleotide were 31.54% and 10. 30%, respectively. AT/TA and CT/ GA frequencies were the highest. About 325 primers were designed by Primer 5.0, the PCR detection showed that 104 primers could amplify the ideal PCR products, and 48 pairs of SSR primers were detected polymorphism in at least 30 different germplasms in Curcama kwangsinesis, accounting for 38.09% of all the designed primers. The study of the genetic diversity analysis, cluster analysis results showed that the genetic similarily coefficient range of the 50 Curcama kwangsinesis in the correlation coefficient of 0.7 was narrow for the clustering of two classes. Conclusion： The EST resources of Curcuma longa had the high frequency of SSR loci, and the development of EST-SSR markers was efficient. The study developed 48 EST-SSR markers for Curcama kwangsinesis, and selected candidate sequences that were rich in SSR loci, the clustering diagram example was used to verify the relationships between plants, and provided the reference for the genetic diversity analysis and molecular breeding researche of Curcama kwangsinesis.