Species Identification of Sea Turtles in Thailand Using DNA Barcoding Coupled With High-Resolution Melting (Bar-HRM) Analysis.

Abstract

The identification of sea turtle species relies on morphological characteristics, presenting challenges in incomplete or degraded specimens, with DNA barcoding now increasingly recognized for its potential in expediting species identification across various organisms. This study proposed a combination of DNA barcoding with high-resolution melting (Bar-HRM) analysis for accurate identification of sea turtles at the species level. Mitochondrial DNA (D-loop, cytb, and cox1) and nuclear DNA (Ahr, Bdnf, Bmp2, C-mos, Hmgb2, Ngf, and Pax1p1) were employed for Bar-HRM to differentiate the five sea turtle species (green turtle, Chelonia mydas; hawksbill, Eretmochelys imbricata; leatherback, Dermochelys coriacea; loggerhead, Caretta caretta; and olive ridley, Lepidochelys olivacea) commonly found in Thai waters and some tortoise species used as outgroup. Results demonstrated that Bar-HRM using TurtleDloop2 and TurtleCytb2(R1) primers produced distinct normalized melting curves for all five species (100%) that their Tm value ranged 77°C-81°C and 81°C-83°C, respectively. The performance of the established approach was evaluated by a blind test of 30 samples, randomly selected for species identification by Bar-HRM using both primers, with accuracy rates of 100%. The minimum DNA concentrations required to discriminate sea turtle and tortoise species using this method were 0.001 and 0.01 ng per reaction for TurtleDloop2 and TurtleCytb2(R1), respectively. Our findings suggested that Bar-HRM using TurtleDloop2 and TurtleCytb2(R1) was a promising tool for the identification of sea turtle species, contributing to effective conservation endeavors and precise evaluations of their endangered status, thereby enhancing the efficiency of conservation planning initiatives.