DNA extraction, PCR amplification and sequencing
Total genomic DNA was extracted using the salt-extraction protocol
(Aljanabi & Martinez, 1997). The cytochrome b gene (Cyt b ) was
amplified with the primers L14724 and H15915 (Tang et al., 2012). Each
30 μL reaction volume contained 3 μL 10x Easytaq Buffer, 1.5 μL dNTPs
(each 2.5 mM), 0.25 μL Taq DNA polymerase, 1μL each primer, 2 μL
template DNA, and 21.25 μL sterile double distilled water. Thermocycling
program for the polymerase chain reaction (PCR) included: an initial
denaturation at 94°C for 5 min, 35 denaturation cycles at 94°C for 30 s,
annealing at 54°C for 45 s, extension at 72°C for 1 min, and a final
extension at 72°C for 10 min. PCR products were sent to commercial
sequencing companies for purification and sequencing.