应用种子形态及DNA条形码技术鉴定黄芪及混伪品种子  

Identification of Astragalus membranaceus seeds and its adulterants by morphology and DNA barcoding technique

作　　者：李军[1] 张召雷 王晓敏[3] 刘立轩[1] 冷晓红[1] LI Jun;ZHANG Zhao-lei;WANG Xiao-min;LIU Li-xuan;LENG Xiao-hong(Ningxia Research Center for Development and Utilization of Chinese Medicinal Materials,Ningxia Polytechnic,Yinchuan 750021,China;Hebei Key Laboratory of Research and Development of Chinese Medicine,Chengde Medical University,Chengde 067000,China;School of Agriculture,Ningxia University,Yinchuan 750021,China)

机构地区：[1]宁夏职业技术学院宁夏中药材开发与利用工程技术研究中心,宁夏银川750021 [2]承德医学院河北省中药研究与开发重点实验室,河北承德067000 [3]宁夏大学农学院,宁夏银川750021

出　　处：《中草药》2022年第24期7871-7879,共9页Chinese Traditional and Herbal Drugs

基　　金：宁夏回族自治区教育厅高等学校科学研究项目资助(NGY2020132)。

摘　　要：目的采用传统形态鉴定方法结合DNA条形码分子检测技术对黄芪及混伪品种子进行鉴定。方法收集黄芪及混伪品种子、市售黄芪种子样品共58份,利用体视显微镜和游标卡尺进行种子外观形态特征的观察和记录,测定千粒重;提取单粒种子的基因组DNA,PCR扩增、双向测序获得ITS2及psb A-trn H序列。使用邻接法(neighbor joining,NJ)构建系统发育树,kimura二参数(kimura two-parameter,K2P)模型计算遗传距离,进行物种鉴定分析。结果黄芪及混伪品种子在颜色、形状、长、宽、厚度及千粒重等方面存在细微差别;ITS2的测序成功率为100%,黄芪种间最小遗传距离均大于种内最大遗传距离;ITS2序列构建的NJ系统进化树将蒙古黄芪A.mongholicus var.mongholicus聚为独立一支,并将蒙古黄芪Astragalus membranaceus、紫花苜蓿Medicago sativa、锦鸡儿Caragana sinica、蜀葵Althaea rosea及黄芪属其他物种分开,可进行黄芪及其混伪品种子的鉴定。基于外观形态和ITS2条形码序列鉴定发现12份市售黄芪种子中有1份为斜茎黄芪。结论基于种子形态鉴定和ITS2条形码相结合的方法可以准确鉴定黄芪及混伪品种子,为规范黄芪种源及种植生产,进而保障黄芪药材质量提供科学依据。Objective Using the traditional morphological identification method in combination with DNA barcoding technique,Astragalus mongholicus seeds and the adulterants were authenticated.Methods A total of 58 samples including cultivated,marketed A.mongholicus seeds and the adulterants were collected.Seeds morphological characters were determined using stereomicroscope and vernier caliper observations,along with calculation of their 1000-grain weights.Genomic DNA was extracted from the single seed.PCR and bidirectional sequencing yielded the ITS2 and psb A-trn H sequences.Species identification were emoloyed,using kimura two-parameter model(K2P)genetic distance method and neighbor joining(NJ) phylogenetic analysis.Results Slight differences in color,shape,length,width,thinkness and 1000-grain weight among A.mongholicus seeds samples and the adulterants could be observed.The ITS2 sequencing success rate was100%.The minimum genetic distance between species of A.mongholicus was greater than the maximum genetic distance within species.The NJ dendrogram of ITS2 sequences indicated that all the A.membranaceus var.mongholicus seeds could form independent branches,which could be distinguished from Medicago sativa,Caragana sinica,Althaea rosea and other species of Astragalus in the collected samples,thus possessing the ability to authenticate A.mongholicus seeds from the adulterants.Based on seed morphology and ITS2 barcoding identification strategy,one Astragalus laxmannii sample was identified from the 12 marketed A.mongholicus seeds samples.Conclusion Based on the combination of seed morphology and ITS2 sequence barcoding identification,the A.mongholicus seeds and the adulterants can be accurately identified,which provides scientific bases for standardizing the source and cultivation of A.mongholicus,ensuring the quality of Chinese medicinal materials Astragalus Radix.

关 键 词：黄芪 种子 蒙古黄芪 扁茎黄芪 斜茎黄芪 紫云英 外观形态 ITS2 DNA条形码 系统发育分析 

分 类 号：R286.12[医药卫生—中药学]