DNA barcoding coupled with secondary structure information enhances Achyranthes species resolution

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.