Distinguishing Dioscorea and Smilax species as potential Hua-Khao-Yen using DNA barcoding coupled with high resolution melting analysis (Bar-HRM)

• Bar-HRM using HKYndhF(1) and HKYrbcL(1) distinguished Dioscorea spp. but Smilax spp. had similar melting profiles. • Bar-HRM using HKYndhF(1) and HKYrbcL(1) requires a minimal DNA concentration at 0.001 ng per reaction. • Bar-HRM authenticated HKY-Nuer as Smilax spp. and HKY-Tai as Premna herbacea . Hua-Khao-Yen (HKY) refers to a group of Thai medicinal plants valued for their pharmaceutical properties, commonly obtained from the rhizomes of five species: Dioscorea birmanica, Dioscorea membranacea, Smilax corbularia, Smilax glabra , and Premna herbacea . However, Traditional Thai HKY suggests that HKY-Nuer (red rhizome) might be S. corbularia and HKY-Tai (white rhizome) might be S. glabra , contributing to challenges in species differentiation due to morphological similarities resulting in misidentification and misuse. This research investigated the species authentication of Thai medicinal plants, HKY-Nuer and HKY-Tai, by high resolution melting (HRM) analysis conjugated with DNA barcodes (Bar-HRM) validated by comparing melting profiles of verified specimens obtained from a herbarium with suspected specimens to aid in species identification based on the similarity of melting shapes. A Bar-HRM for differentiation among five possible HKY species was developed to authenticate the HKY rhizome. Samples of five HKY species including known species from an herbarium and unknown species from local stores, were subjected to DNA extraction and amplification through real-time PCR. Primer sets were designed from chloroplast DNA, targeting the ndhF, rbcL , and trnK loci for HRM analysis. The shapes of the normalized melting curves were analyzed to distinguish the different plant species. Bar-HRM using HKYndhF(1) and HKYrbcL(1) had the highest specificity and discriminatory power, with a minimum DNA concentration requirement of 0.001 ng per reaction. Bar-HRM using both primers was applied for species authentication of HKY-Nuer and HKY-Tai from local vendors. Results revealed that the melting curves of HKY-Nuer and HKY-Tai closely resembled Smilax sp. ( S. corbularia or S. glabra ) and P. herbacea , respectively. These findings displayed the potential of DNA barcoding coupled with HRM as an effective tool for the precise identification of Hua-Khao-Yen species.