基于ITS2序列的火绒草药材分子鉴定研究

Molecular identification of Leontopodium leontopodioides (Willd.) Beauv. based on ITS2 sequence

目的 应用内转录间隔区2(ITS2)序列鉴定不同分布区、不同居群火绒草药材,明确火绒草种间、不同居群间和居群内火绒草样品遗传关系。方法 采用DNA提取试剂盒提取20个居群共220个火绒草药材基因组DNA。利用植物药DNA条形码通用引物ITS2序列对提取的DNA进行扩增并测序。将所得序列使用MEGA6.0软件进行序列对比分析,用NJ TREE法构建系统聚类树。结果 火绒草样本ITS2序列长度为222 bp, GC含量为52.25%。居群内火绒草样品ITS2序列一致,无变异位点。不同居群火绒草样品可利用72 bp处“T”碱基来区分河南产地样本。ITS2序列构建NJ系统聚类树分析显示,辽宁分布区及河北分布区火绒草聚类在一起,河南分布区火绒草与辽宁及河北分布区样本有一定的遗传距离。结论 ITS2序列可以准确、有效地鉴定不同分布区、不同居群火绒草药材,为火绒草种质资源多样性研究提供分子依据。

Objective To identify the genetic relationship of Leontopodium leontopodioides(Willd.) Beauv.in different distribution areas and populations by using the Internal Transcribed Spacer 2(ITS2) barcode, in order to clarify the genetic relationship among species, populations and within populations of L.leontopodioide.Methods Genomic DNAs were extracted from 220 samples of L.leontopodioide in 20 populations by DNA extraction kit.The extracted DNA was amplified and sequenced by using the universal primer ITS2 sequence of medicinal plant DNA bar code.The sequences were analyzed by MEGA 6.0 software, and the neighbor-joining(NJ) phylogenetic trees method was used to construct the cluster tree.Results The ITS2 sequence was 222 bp and the GC content was 52.25%.The ITS2 sequence of L.leontopodioide samples in the population were consistent, and no change of ectopic point was existed.The “T” basic group at 72 bp could be used to distinguish samples from different populations in Henan province.NJ cluster tree analysis of ITS2 sequence showed that the distribution of Liaoning and Hebei were clustered together, and there was a certain genetic distance among Henan and Liaoning and Hebei provice.Conclusion ITS2 sequence can accurately and effectively identify the medicinal materials of L.leontopodioide in different distribution areas and populations, and provide molecular basis for the study of the diversity of L.leontopodioide germplasm resources.