DNA barcoding provides distinction between Radix Astragali and its adulterants

Based on variable nuclear and/or organellar DNA sequences among vastly divergent species as well as morphologically indistinguishable species, DNA barcoding is widely applicable in species identification, biodiversity studies, forensic analyses, and authentication of medicinal plants. The roots of Astragalus membranaceus and A. membranaceus var. mongholica are commonly used as Radix Astragali in several Asian countries, including China, Japan, and Korea. However, in addition to the two species recorded in the Chinese Pharmacopoeia, there are twenty-three species from different genera including Astragalus, Oxytropis, Hedysarum, and Glycyrrhiza, which have been used as adulterants not only in trading markets but also by the herbal medicine industry. Therefore, a simple, reliable, and accurate classification method is important for distinguishing authentic Radix Astragali from its adulterants. In this study, we acquired data for 37 samples from four related genera within the family Fabaceae. Then we compared four candidate DNA barcoding markers using ITS, matK, rbcL, and coxI sequences from nuclear, chloroplast, and mitochondrial genomes, all commonly used for plants to identify genetic variations among genera, intraspecies, and interspecies. We observed higher divergences among genera and interspecies for ITS, which have the average Kimura 2-parameter distances of 4.5% and 14.1%, respectively, whereas matK was found to have sufficient divergence at the intraspecific level. Moreover, two indels detected in the matK sequence are useful for PCR studies in distinguishing Radix Astragali from its adulterants. This study suggests that the combined barcoding regions of ITS and matK are superior barcodes for Radix Astragali and further studies should focus on evaluating the applicability and accuracy of such combined markers for a wide range of traditional Chinese herbs.     

应用matK基因鉴别青天葵及其常见混伪品

分析了青天葵及其混伪品matK序列,以期在分子水平建立青天葵及其常见混伪品的鉴别方法。采用一对通用引物对matK基因进行PCR扩增并测序,所得序列用DNAMAN、MEGA等软件进行分析。获得青天葵及其混伪品的matK基因序列长度为587 bp,平均GC含量为32.8%。青天葵的种内遗传距离为0,与混伪品种间遗传距离范围为0.016～0.375。基于matK基因序列构建的NJ聚类树能明显地区别青天葵及其混伪品。因此,应用matK基因序列可以有效鉴别青天葵及其混伪品。     

中药材蛤蚧的特异性PCR鉴定

以线粒体Cytb、COI、12SrRNA、16SrRNA基因序列为基础,探讨特异性PCR技术鉴定蛤蚧及伪品的可行性。本研究以所扩增的16条COI序列为引物设计依据序列设计了1对位点特异性引物COISF,COISR,同时从Gen-Bank上下载30条序列,设计了CytbSF,CytbSR;16S rRNASF,16S rRNASR;12S rRNASF,12S rRNASR另外3对位点特异性引物,因此共用4对位点特异性引物分别扩增蛤蚧及伪品实验样本。结果表明在复性温度为65℃时,4对引物都出现了理想的结果,即蛤蚧正品出现了扩增条带,而伪品没有扩增条带。同时对市售的蛤蚧商品进行了检测,在所供的7号标本中,有4号为蛤蚧正品,其余3号为蛤蚧伪品。本研究所设计的位点特异性引物可快捷、准确的鉴定蛤蚧及伪品,且在药检工作中具有极大的应用前景。