Species of the genus Achyranthes are known to possess immense medicinal value leading to its inclusion in Ayurveda. A. aspera has been widely used in all the traditional Indian systems of medicine, while A. bidentata is mentioned in Chinese, Japanese and Korean pharmacopoeias. Molecular authentication of any medicinal plant species is a prerequisite for the success of its herbal business. Presently, DNA barcoding provides a successful tool for controlling the quality of medicinal plant materials in the trade. Here, we have evaluated four different barcode loci and their efficiency in delineating the Achyranthes species. The internal transcribed spacer 2 (ITS2) barcode proved to be the best performing marker analyzed through barcode gap estimation, BOLD and Bayesian analysis. The inter- and intra-specific genetic divergence observed among and within the Achyranthes species was validated through the Wilcoxon signed-rank test. Further secondary structure modeling of the ITS2 region was performed for better resolution at the species level. The secondary structure was generated based on the lowest free energy state deduced through the mfold web server followed by structure alignment through LocARNA web server. Analysis showed that the helix III and IV possess the maximum variability along with the central ring in differentiating the species. A total of fourteen single-nucleotide polymorphisms (SNPs) identified in the 200 aligned ITS2 dataset could be useful for the development of species-specific marker(s) in future. Through this study we recommend the use of a combination of secondary structure information with DNA barcoding for significantly improving the species identification resolution.

已知牛膝属的种类具有巨大的药用价值，使其包含在阿育吠陀中。米曲霉已被广泛用于所有传统的印度医学体系中，而中，日，韩药典中均提到了双叶曲霉。任何药用植物物种的分子认证都是其草药业务成功的前提。目前，DNA条形码技术提供了一种成功的工具来控制行业中药用植物材料的质量。在这里，我们评估了四个不同的条形码基因座及其在描绘牛膝菌种中的效率。内部转录间隔区2（ITS2）条码间隙估计，BOLD和贝叶斯分析证明，条码是性能最好的标记。通过Wilcoxon秩和检验验证了牛膝菌种之间和之内的种间和种内遗传差异。进行了ITS2区域的进一步二级结构建模，以在物种级别获得更好的分辨率。二级结构是基于通过mfold Web服务器推断出的最低自由能状态生成的，然后通过LocARNA Web服务器进行结构比对。分析表明，螺旋III和IV具有最大的变异性，同时带有中心环以区分物种。在200个比对中鉴定出总共14个单核苷酸多态性（SNP）ITS2数据集可能对将来物种特异性标记的开发有用。通过这项研究，我们建议将二级结构信息与DNA条形码结合使用，以显着提高物种识别的分辨率。