菊科藏药材及其近缘物种的分子鉴别

目的 评估ITS2(internal transcribed spacer 2)序列作为DNA条码鉴定菊科藏药材及其近缘物种的能力。方法 于西藏自治区等地采集菊科植物样本50份,共26个物种,分布于8个属,它们大部分可以用作藏药材。采用遗传距离比较法和系统聚类树法评估ITS2序列的鉴定能力。首先,采用试剂盒法提取基因组DNA,PCR扩增ITS2序列并双向测序。从GenBank数据库中下载26种菊科植物的rDNA序列(包含完整的ITS2区域)。上述序列经过CodonCode Aligner v.9.0.1软件拼接后,用MEGA 11.0.10软件构建NJ系统聚类树,用TaxonGap v.2.4.1软件分析种内和种间变异。结果 采用系统聚类树法和遗传距离比较法,ITS2序列可以从3种蒿属植物中鉴别出大籽蒿(Artemisia sieversiana),6种紫菀属植物中鉴别出灰枝紫菀(Aster poliothamnus),2种垂头菊属植物中鉴别出车前状垂头菊(Cremanthodium ellisii)和条叶垂头菊(Cremanthodium lineare),3种风毛菊属植物中鉴别出美丽风毛菊(Saussurea superba),并且可以将小苦荬属的中华小苦荬(Ixeridium chinense)和窄叶小苦荬(Ixeridium gramineum)区分开,可以将毛香火绒草(Leontopodium stracheyi)、三角叶蟹甲草(Parasenecio deltophyllus)与其他物种区分开。ITS2序列可以成功地从26个菊科物种中鉴别出9个物种,鉴定效率为34.6%。结论 ITS2序列可以用于快速、准确地识别和鉴定部分菊科藏药材。

Molecular Identification of Tibetan Herbs and Their Closely Related Species in Family Compositae

OBJECTIVE To evaluate the ability of ITS2(internal transcribed spacer 2) sequence as a DNA barcode to identify Tibetan herbs and their closely related species in family Compositae.METHODS A total of 50 plant samples of 26 species distributed in 8 genera of family Compositae were collected from Tibet Autonomous Region and other places.Most of these plant species were Tibetan herbs.The Distance-Based Method and Tree-Based Method were used to evaluate the identification ability of ITS2 sequence.First,the genomic DNAs were extracted by kit.The ITS2 sequences were amplified and bi-directional sequenced.The rDNA sequences containing the ITS2 regions of 26 species were downloaded from the GenBank database.After these sequences were spliced and assembled by CodonCode Aligner v.9.0.1 software,MEGA 11.0.10 software was used to construct the NJ phylogenetic tree,and TaxonGap v.2.4.1 software was used to analyze the intra-species and inter-species variation.RESULTS Using the Distance-Based Method and the Tree-Based Method,ITS2 sequence could successfully identify Artemisia sieversiana from 3 species in Artemisia genus,Aster poliothamnus from 6 species in Aster genus,Cremanthodium ellisii and Cremanthodium lineare from 2 species in Cremanthodium genus,Saussurea superba from 3 species in Saussurea genus.It could also distinguish Ixeridium chinense from Ixeridium gramineum,identify Leontopodium stracheyi and Parasenecio deltophyllus from other species.ITS2 sequence could distinguish 9 species from 26 Compositae species with 34.6% identification efficiency.CONCLUSION ITS2 can be used for the rapid and accurate identification of Tibetan herbs and related species in family Compositae.