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.