丹参线粒体和叶绿体微卫星标记开发及多样性分析
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四川省财政创新提升工程(2016TSCY001)


Development and Diversity Analysis of Mitochondrial and Chloroplast Microsatellite Markers in Salvia miltiorrhiza
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    摘要:

    该研究通过生物信息学方法,对丹参线粒体、叶绿体基因组序列进行SSR位点搜索,利用Primer3.0软件在线设计SSR引物,通过PCR扩增,筛选出扩增效果好、多态性高的10对cpSSR引物和13对mtSSR引物,用于分析61份丹参品种的遗传多样性和鼠尾草属植物种间的通用性。结果显示:(1)从2条基因组序列中搜索到32个cpSSR位点和24个mtSSR位点,其中叶绿体单核苷酸的重复序列最多为31个,占96.9%,重复类型中以A/T形式的微卫星最为丰富。(2)23对SSR引物共检测出46个等位基因,平均每个SSR位点等位基因数是2个,有效等位基因数为1.566;预期杂合度(He)为0.346,高于双子叶植物的平均水平(He=0.190);多态信息含量(PIC)为0.278,大于0.250小于0.500;Shannons信息指数为0.519,表明所采集的丹参呈中等水平的遗传多样性。(3)聚类分析表明,61份丹参材料的遗传相似系数在0.54~0.96之间,并在遗传相似系数为0.64处将 61份丹参分为6个亚群:四川、山东和安徽省的丹参聚集在Ⅰ和Ⅱ亚群中,说明四川中江、山东和安徽地区的丹参亲缘关系较近,而来源于四川中江的丹参分布于所有亚群,表明四川中江的丹参具有丰富的多态性。(4)通用性检测结果显示,11对mtSSR引物在9种鼠尾草属中的平均通用性比率为64.63%,通用性较高;9对cpSSR引物在10种鼠尾草属中的平均通用性比率为44.66%,通用性较低;引物cp5p、cp8p和cp10p在野芝麻亚科植物属间的通用性较好。研究表明,开发的23对SSR引物在鼠尾草属内具有很好的适用性,可以为该属种间的亲缘关系和种内遗传分化研究提供分子依据。

    Abstract:

    In this test, SSR loci were searched for the mitochondrial and chloroplast genome sequences of Salvia miltiorrhiza by bioinformatics methods, and the SSR primers were designed by primer3.0 online software. 10 pairs of cpSSR primers and 13 pairs of mtSSR primers with high polymorphism were selected by PCR amplification. They were used to analyze the genetic diversity of 61 S. miltiorrhiza cultivars and the transferability of Salvia L. species.(1) Total 32 cpSSR and 24 mtSSR were identified from 2 genome sequences. There were 31 mononucleotide repeat sequences with 96.9% frequency, showing the dominant types in the cpSSR. Both A/T types were abundant among mononucleotide repeat sequences.(2) 46 alleles were detected in 23 pairs of SSR primers, the average number of alleles per SSR locus was 2, and the number of effective alleles was 1.566. The expected heterozygosities was 0.346, which was higher than the average level of dicotyledons (He=0.190); the polymorphism information content was 0.278, which was between 0.250 and 0.500; Shannons information index was 0.519. The results showed that S. miltiorrhiza had a moderate level of genetic diversity.(3) The cluster analysis on 61 S.miltiorrhiza showed that the genetic similarity coefficient was ranging from 0.54 to 0.96. It can be divided into 6 subgroups when the genetic similarity coefficient was 0.64: the S.miltiorrhiza in Sichuan, Shandong and Anhui provinces clustered in Ⅰ and Ⅱ subgroups, suggesting their close relationship. S.miltiorrhiza in Zhongjiang of Sichuan Province were distributed in all the subgroups, suggesting their rich polymorphism.(4) The results of transferability test showed that 11 pairs of mtSSR primers were successfully amplified in nine species belong to Salvia L., the transferability ratio was 64.63%, suggesting mtSSR markers showed a higher transferability. 9 pairs of cpSSR primers were successfully amplified in all samples of Salvia.L. species, and the transferability ratio was 44.66%, suggesting cpSSR markers showed a lower transferability. In addition, the primers cp5p, cp8p and cp10p had good generality among the genera of Lamioideae. These results indicated that 23 primers are suitable for SSR analysis of Salvia L. It would be useful for species relationship and study of genetic variation of Salvia L..

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沙秀芬,彭 芳,陶 珊,等.丹参线粒体和叶绿体微卫星标记开发及多样性分析[J].西北植物学报,2018,38(12):2215-2223

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  • 在线发布日期: 2019-01-24
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