多花黄精转录组SSR位点分析及分子标记开发

Analysis on SSR loci in transcriptome and development of molecular markers in Polygonatum cyrtonema

目的基于转录组测序数据鉴别和分析多花黄精EST-SSR位点,开发可用于多花黄精种质资源评价与利用的SSR标记。方法利用MISA工具从多花黄精转录组126544条Unigenes中鉴定SSR位点并进行分析;利用Primer 3.0设计SSR引物,随机选择50对SSR引物进行有效性验证。结果从9982条Unigenes中鉴定出12317个包含2~6个核苷酸重复类型的SSR位点,SSR的分布频率为9.73%,发生频率为1/5.91 kb;SSR位点中的主导类型是二核苷酸重复,占总SSR的53.14%,其次是三核苷酸重复,占33.31%。SSR引物的有效性筛选显示,50个SSR引物对中有29个(58%)可从多花黄精中扩增出预期大小的SSR条带。SSR荧光标记的毛细管电泳检测显示在多花黄精的7个SSR位点共鉴定出了9种基因型,进一步验证了SSR引物的有效性。结论多花黄精转录组SSR位点出现频率高,重复类型丰富,多态性较高,这将为多花黄精遗传多样性分析和遗传图谱构建提供大量有价值的候选标记,也可为黄精属内种间物种的分子鉴别、多花黄精优良品种的分子辅助育种提供技术手段。

Objective EST-SSR loci were identified and analyzed based on the transcriptome sequencing data in Polygonatum cyrtonema,in order to develop SSR markers suitable for evaluation and application of germplasm resources on P.cyrtonema.Methods SSR loci were identified and analyzed in all of 126544 Unigenes by using MISA tool.SSR primers were designed by using Primer 3.0 software and 50 pairs of SSR primers were randomly selected for validation test.Results A total of 12317 SSR loci,including the types of 2—6 nucleotide repeats with occurring frequency of 1/5.91 kb,were identified from 9982 Unigenes in P.cyrtonema transcriptome.The distribution frequency of SSRs was 9.73%.Dinucleotide repeat was the main type,accounting for as much as 53.14%of all SSRs,followed by trinucleotide repeat(33.31%).The validation test on 50 pairs of SSR primers showed that 29 of them(58%)generated fragments with expected molecular size from P.cyrtonema.The capillary electrophoresis using fluorescence-labeled SSR markers showed that nine genotypes were identified at seven SSR loci in P.cyrtonema,which further demonstrated the validity of these SSR primers.Conclusion There are numerous SSRs in P.cyrtonema transcriptome with high frequency,rich repeat types and relatively high polymorphic,which will provide abundant valuable candidate markers for genetic diversity analysis and genetic mapping construction in P.cyrtonema,also can be used as a technical tool for molecular identification among Polygonatum species and for molecular marker assistant breeding in superior cultivars of P.cyrtonema.