千里香杂合度PCR-RFLP快速评估方法的建立  

A Rapid PCR-RFLP Method for Assessing Heterozygosity of Murraya paniculata Germplasm

作　　者：王博铖 陈梓媛 华中一 田慧[1] 谢文波 袁媛[2] WANG Bocheng;CHEN Ziyuan;HUA Zhongyi;TIAN Hui;XIE Wenbo;YUAN Yuan(Guangxi University of Chinese Medicine,Nanning 530200,China;National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;China Resources Sanjiu Medical&Pharmaceutical Co.Ltd.,Shenzhen 518029,China)

机构地区：[1]广西中医药大学,南宁530200 [2]中国中医科学院中药资源中心,北京100700 [3]华润三九医药股份有限公司,广东深圳518029

出　　处：《中国实验方剂学杂志》2024年第4期29-34,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家科技性基础资源调查项目(2018FY100800);中国中医科学院科技创新工程重大攻关项目(C12021A041);青年岐黄学者项目;中央级公益性科研院所基本科研业务费专项(ZZXT202001)。

摘　　要：目的:建立一种快速评估千里香种质材料杂合度的方法,为制定千里香的优异种质繁育策略和促进种质创新提供依据。方法:以65株千里香重测序数据为基础,检测并筛选单核苷酸多态性(SNPs),利用其中20个SNP位点,通过自编脚本将其转化为限制性内切酶片段长度多态性(RFLP)标记;采用聚合酶链式反应-限制性内切酶长度多态性(PCR-RFLP)法对12份千里香种质的20个RFLP标记进行检测,根据RFLP标记位点酶切片段数目计算千里香种质的杂合度;采用plink软件计算12份千里香种质的全基因组杂合度,并比较不同种质杂合度评估方法得到的结果。结果:PCR-RFLP法和基因组重测序法计算的杂合度间差异无统计学意义。利用PCR-RFLP法筛选获得了8份杂合度<30%的种质材料,利用基因组重测序法筛选获得9份杂合度<30%的种质材料,2种方法计算杂合度均<30%的种质材料共7份。结论:该文建立了千里香种质杂合度评估的PCR-RFLP法,其精确度为87.5%,准确率为77.8%,该法可为其他药用植物种质杂合度评估方法研究提供借鉴。Objective:To establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M.paniculata.Method:Single nucleotide polymorphisms(SNPs)were screened from the genome resequencing data of 65 plants of M.paniculata.A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism(RFLP)markers.Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)was employed to detect the 20 RFLP markers in 12 M.paniculata germplasm accessions,and the heterozygosity of M.paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites.Plink was used to calculate the whole genome heterozygosity of 12 M.paniculata germplasm accessions,and the results obtained with different methods were compared.Result:There was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method.The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions,respectively,with a heterozygosity level less than 30%.Seven germplasm accessions with heterozygosity less than 30.00%were calculated by both methods.Conclusion:The PCR-RFLP method established in this study for evaluating the heterozygosity of M.paniculata germplasm demonstrates the precision of 87.5%and the accuracy of 77.8%.This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.

关 键 词：千里香 聚合酶链式反应-限制性内切酶长度多态性(PCR-RFLP) 单核苷酸多态性(SNPs) 杂合度 全基因组重测序 

分 类 号：R284.2[医药卫生—中药学] R285[医药卫生—中医学] R289R287R22R2-031R33R24