DNA Amplification and Sequencing
To DNA extraction, high-salt method was performed (Sambrook et al., 1989). A partial sequence of cytochrome b (cytb) gene was selected for molecular analysis. The desired gene was amplified with forward F08_F (5′ GACTTGAAAAACCACCGTTG-3′) and reverse E08_R (5′ CTCCGATCTCCGGATTACAAGAC -3′) primers (Wang et al., 2021). Polymerase Chain Reactions (PCRs) were performed with 1 μl template DNA (50–100 ng), 0.5 μl of each primer, 12.5 μl Master Mix Red (Ampliqon) and 10.5 μl of ddH2O up to 25 μl of reaction mix. The amplification of the PCR, carried out on an MJ Mini™ thermocycler (Bio-Rad), started with an initial denaturation at 95°C for 2 min, followed by 35 cycles consisting of second denaturation at 95°C for 1 min, annealing at 56°C for 30 s and elongation at 72°C for 30 s; the last step was final elongation at 72°C for 10 min. The quality of PCR products were examined with agarose gel 1% stained with Safe-Red ™. The suitable amplicons were sent to Pishgam Inc, for purification and sequencing.
In this study, apart from the newly determined sequences, additional sequences for 84 species were downloaded from GenBank (Table. S1).