快速筛选高纯合度天麻PCR-RFLP鉴定方法  

Rapid Screening of Gastrodia elata with High Purity by PCR-RFLP Identification

作　　者：谢莹 华中一 赵玉洋 周骏辉[2] 李晓琳[2] 袁媛[2] XIE Ying;HUA Zhongyi;ZHAO Yuyang;ZHOU Junhui;LI Xiaolin;YUAN Yuan(School of Traditional Chinese Medicine,Guangdong Pharmaceutical University,Guangzhou 510006,China;State Key Laboratory Breeding Base of Dao-Di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]广东药科大学中药学院,广州510006 [2]中国中医科学院中药资源中心道地药材国家重点实验室培育基地,北京100700

出　　处：《中国实验方剂学杂志》2022年第17期113-118,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金重大项目(81891013,81891010);中国中医科学院科技创新工程重大攻关项目(CI2021A041//CI2021B014);国家科技性基础资源调查专项(2018FY100800);中央本级重大增减支项目(2060302);青年岐黄学者项目;东城区优秀人才培养项目(2021-dchrcpyzz-7)。

摘　　要：目的:建立一种快速筛选高纯合度天麻种质材料的方法,为其纯系育种和杂交育种奠定基础。方法:以本课题组前期天麻全基因组测序和群体重测序为基础,利用单核苷酸多态性(SNP)位点开发20个限制性内切酶片段长度多态性(RFLP)标记,并采用聚合酶链式反应(PCR)-RFLP法对15份天麻种质的20个RFLP标记进行限制性内切酶实验,根据20个RFLP标记位点酶切条带数目,计算15份天麻种质的纯合度,进而对天麻种质纯合度进行评估;在此基础上,利用基因组重测序技术对评估结果进行验证。结果:利用PCR-RFLP法筛选获得10份纯合度>95%的种质材料,其中3份种质材料的纯合度为95%,7份种质材料的纯合度为100%;利用基因组重测序法筛选获得9份纯合度>95%的种质材料,其中8份种质材料与PCR-RFLP法检测结果一致。结论:该文所建立的用于筛选高纯合度天麻种质的PCR-RFLP法精确度为80%,准确率为89%。该方法实验操作简便、检测效率高,且成本显著低于基因组重测序技术,为天麻纯系育种提供了技术支撑,也为其他中药材品种选育研究提供了借鉴。Objective:To establish a rapid screening method for germplasm materials of Gastrodia elata with high purity,and lay a foundation for pure line breeding and cross breeding.Method:Based on the whole genome sequencing and population resequencing of G.elata,20 restriction fragment length polymorphism(RFLP) markers were developed by single nucleotide polymorphism(SNP) sites.The polymerase chain reaction(PCR)-RFLP method was used to carry out restriction endonuclease experiments on 20 RFLP markers of 15G.elata germplasms.According to the number of enzymatic bands at 20 RFLP marker sites,the purity of 15germplasms was calculated and evaluated.On this basis,genome resequencing technology was used to verify the assessment results.Result:Ten germplasm materials with purity greater than 95% were screened out by PCRRFLP method,3 of which had 95% purity and 7 had 100% purity.Nine germplasm materials with purity greater than 95% were screened out by genome resequencing methods,and 8 of them were consistent with the results of PCR-RFLP.Conclusion:The PCR-RFLP method established in this study for screening G.elata germplasms with high purity precision of RFLP markers has 80% precision and 89% accuracy.The method is simple,efficient,and significantly less expensive than genome resequencing method,which provides technical support for pure line breeding of G.elata and references for breeding of other Chinese medicinal materials.

关 键 词：天麻 单核苷酸多态性(SNP)位点 限制性内切酶片段长度多态性(RFLP)标记 纯合度 

分 类 号：R284.2[医药卫生—中药学] R289[医药卫生—中医学] R22R2-031R33