DNA extraction, amplification, and sequencing
About 30 μL Genomic DNA was extracted by phenol-chloroform (Sambrock & Russel, 2001 ) from tissue muscle (0.1-0.15g) and verified using 1.0% agarose gel electrophoresis.
The amplification of COI gene was carried out by polymerase chain reaction (PCR). A 658bp fragment was amplified using the forward primer (LCO1490: 5’-GGTCAACAAATCA TAAAGATATTGG-3’) and reverse primer (HCO2198: 5’-TAAACTTCAGGGTGACCAAAAAA TCA-3’) (Folmer, Black, Wr, & Vrijenhoek, 1994 ). PCR reaction was performed in a total volume of 50 μl containing 50-100 ng DNA template, 5 µL of 10× PCR buffer, 1.5 mmol·L–1of MgCl2, 0.2 mmol·L–1of each dNTP , 2 unit(U) of Taq polymerase, 0.2 µmol·L–1of each primer. Thermal cycling began with one cycle pre-denaturation at 94 ºC for 5 min, 35 cycles of denaturation at 94˚C for 30 s, annealing at 50˚C for 45 s, and extension at 72˚C for 45 s, and a final extension holding at 72˚C for 7 min, respectively (Feng, Sun, Cheng, & Li, 2008 ). The PCR product was separated by electrophoresis on 1.0% agarose gels.
Primer synthesis and DNA sequencings were conducted in commercial companies. Total 222 specimens were sequenced in one direction (63.51%) and others were in both directions. Except for the sequences obtained of the genomic DNA in this study, the other COI sequences were obtained from GeneBank to comparative and analyses.