基于SSR-HRM技术的4种药用天麻变型遗传多样性及遗传结构分析

Study on genetic diversity and genetic structure among four forms of medicinal Gastrodiae Rhizoma by SSR-HRM techniques

目的比较乌天麻Gastrodia elata f.glauca、绿天麻G.elata f.viridis、红天麻G.elata f.elata和黄天麻G.elata f.flavida 4种药用天麻变型的遗传多样性、遗传结构和遗传信息差异,为天麻新品种选育提供依据。方法从不同产地收集了40份不同天麻变型的花茎。应用简单重复序列(simple sequence repeat,SSR)分子标记与高分辨率熔解曲线(high-resolution melting,HRM)相结合技术,通过聚丙烯酰胺凝胶电泳,从80对引物中筛选出15对多态性高的SSR引物。对这些天麻花茎样品进行实时荧光定量PCR扩增,统计遗传多样性参数,分析群体遗传结构,结合40份天麻的HRM数据进行聚类分析。结果从15对SSR引物检测到114个等位基因,平均每对引物7.6个。天麻样品的观测杂合度(observed heterozygosity,Ho)、期望杂合度(expected heterozygosity,He)、和Nei’s多样性指数(Nei's diversity index,Nei)的平均值分别为0.9813、0.8019和0.8121,表明天麻的遗传多样性丰富。这4种天麻变型的遗传多样性不同,从高到低依次为乌天麻、绿天麻、红天麻和黄天麻。SSR-HRM分析将这40份天麻样品分为4个基因型,各天麻变型的大多数样品分布在不同的基因型中。这4种天麻变型之间的遗传距离不同,乌天麻与绿天麻遗传距离最近,与黄天麻遗传距离最远。结论SSR-HRM技术对天麻基因分型更准确。这4种天麻变型的遗传信息不同,但存在基因交流现象。乌天麻与绿天麻亲缘关系最近,与红天麻次之,与黄天麻亲缘关系最远。可利用不同天麻变型遗传信息差异进行新品种选育。

Objective To provide a basis for the selection of new varieties of Tianma(Gastrodiae Rhizoma)by comparing the genetic diversity,genetic structure and genetic information among the four forms of medicinal G.elata,i.e.Wutianma(G.elata Bl.f.glauca S.Chow),Lvtianma(G.elata Bl.f.viridis Makino),Hongtianma(G.elata Bl.f.elata S.Chow);Huangtianma(G.elata Bl.f.flavida S.Chow).Methods A total of 40 flowering stems of different G.elata forms were collected from different cultivating areas.15 pairs of SSR primers with high polymorphism were selected from 80 pairs of primers by polyacrylamide gel electrophoresis and combined simple sequence repeat(SSR)molecular markers with HRM(high-resolution melting)technique.Real-time fluorescence quantitative PCR amplification,counting genetic diversity parameters and analyzing population genetic structure of these flowering stems samples were performed.The cluster analysis was further conducted in combination with HRM data from the 40 Tianma samples.Results A total of 114 alleles were detected in the 15 pairs of SSR primers with an average of 7.6 alleles per pair.The mean of observed heterozygosity(Ho),expected heterozygosity(He)and Nei's diversity index(Nei)were 0.9813,0.8019 and 0.8121(n=15),respectively.It indicated that the genetic diversity of Tianma was rich.The genetic diversity of the four forms of medicinal Tianma was different with the descending order of Wutianma,Lvtianma,Hongtianma and Huangtianma.These 40 Tianma samples were classified into four genotypes based on SSR-HRM data.Most Tianma samples for specific form were distributed in same genotype.The genetic distances between the four Tianma forms were different.Wutianma was the closest genetic distance to Lvtianma and the farthest genetic distance to Huangtianma.Conclusion The accuracy of SSR-HRM technique for Tianma genotyping is better than that of SSR technique.The genetic information in the four Tianma forms is different,but gene exchange phenomenon exists among them.The relationship between Wutianma and Lvtianma is closest,next to Hongtianma,and farthest to Huangtianma.These differences of genetic information among four forms of Tianma should be used for the selecting of new varieties.