COI Gene Amplification and Sequencing Protocol

Universal primer sequences for the COI gene were taken from the literature (Table 2) [13 (link), 14 (link), 24 (link)–26 (link)], and PCRs were performed using a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The PCR reaction conditions consisted of an initial denaturation step at 95°C for 11 min, followed by 35 cycles at 95°C for 30 s, 50°C for 1 min, and 72°C for 1 min, and then a final elongation step at 72°C for 15 min. Each reaction mixture was prepared using 50 ng of template DNA, 2.5 μL 10× Amplitaq Gold Buffer, 0.5 U AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA, USA), 10 pmol (each) upstream and downstream primers, 62.5 nmol MgCl₂, 5 nmol (each) dNTPs, and sterile distilled water to a final volume of 25 μL. After purification of the PCR products, cycle sequencing reactions were performed according to the manufacturer's instructions using a BigDye v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequencing products were analyzed using an ABI 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Assembled sequences were deposited into the NCBI GenBank database (JX861406–JX861482).