轮叶党参EST-SSR标记的开发及在党参属中的应用  

Development of EST-SSR markers in Codonopsis lanceolata and its application in Codonopsis

作　　者：刘新星[1] 石有太[1] 罗俊杰[1] 欧巧明[1] 陈子萱[1] 李忠旺[1] LIU Xin-xing;SHI You-tai;LUO Jun-jie;OU Qiao-ming;CHEN Zi-xuan;LI Zhong-wang(Biotechnology Institute,Gansu Academy of Agricultural Sciences,Lanzhou 730070,Chin)

机构地区：[1]甘肃省农业科学院生物技术研究所,甘肃兰州730070

出　　处：《中草药》2018年第13期3110-3115,3121,共7页Chinese Traditional and Herbal Drugs

基　　金：甘肃省农业科学院中青年基金项目(2016GAAS52);甘肃省农业科学院科技支撑计划(2016GAAS59);甘肃省农业科学院科技创新工程学科团队项目(2015GAAS02)

摘　　要：目的开发EST-SSR标记,分析其在党参属及近缘属种间的通用性。方法利用MISA软件对轮叶党参在NCBI公布的EST序列进行SSR位点查找,Primer 3.0软件设计引物,经PCR扩增和琼脂糖凝胶电泳对引物进行初步筛选,聚丙烯酰胺凝胶电泳对所筛选的引物进行多态性检测。结果共查找到204个EST-SSR位点,总长5 263 bp,平均长度25.80 bp,出现频率为22.97%,其中单核苷酸、二核苷酸和六核苷酸重复占比最多,分别为36.8%、24.8%和15.3%。单核苷酸、二核苷酸、六核苷酸中的主要重复基元类型是A、TG和CAGCTC/GTGGCA。共设计引物112对,以党参为模板,能够扩增出清晰稳定条带的引物有67对,有效引物比率59.82%,其中27对能扩增出多态性条带,多态引物比率24.11%。随机选取5对引物对党参属及桔梗共12份材料进行PCR扩增,聚类结果将党参属和桔梗属分为2大类。结论研究筛选出的EST-SSR标记能够在党参属及近缘属内进行区分鉴别,研究结果为党参种质鉴别、遗传多样性分析及资源的评价利用等提供了重要技术手段。Objective To develop EST-SSR markers and analyze its universality in Codonopsis and its related genera. Methods EST-SSR detection was performed by Perl program MISA. The primers were designed by Primer 3.0 software, followed by agarose gel electrophoresis for preliminary screening after PCR amplification, and polyacrylamide gel electrophoresis was used to detect the polymorphism of the selected primers. Results A total of 204 SSRs were mined from 888 ESTs with a frequency of 22.97 %. The whole length was 5 263 bp and the average length of EST-SSR was 25.80 bp. Mononucleotide, dinucleotide, and hexanucleotide nucleotide EST-SSR were dominant, which accounted for 36.8%, 24.8%, and 15.3%, respectively. Among them, A, TG, and CAGCTC/GTGGCA were highly abundant in mononucleotide, dinucleotide, and hexanucleotide nucleotide repeats. Among 112 designed primer pairs, 67 primer pairs showed ideal amplifications and the effective amplification rate was 59.82%. Moreover, 27 primer pairs displayed polymorphic, which accounts for 24.11% of the total primers. Five pairs of primers were selected randomly to mark test materials, and the UPGMA result showed that 12 materials were mainly divided into two categories. Conclusion The Codonopsis and its related genera can be identified by EST-SSR markers selected from our study, they could be used in germplasm identification, genetic diversity analysis, and resources evaluation.

关 键 词：轮叶党参 EST-SSR 党参属 分子标记 鉴别 

分 类 号：R282.12[医药卫生—中药学]