Taq polymerase buffer

COI sequences were amplified using the primer pair of BirdF1 (5’-TTCTCCAACCACAAAGACATT GGCAC-3’) and BirdR1 (5’-ACGTGGGAGATAATTCCA AATCCTG-3’) (Hebert et al. 2004 ). PCR amplification was carried out in 50 µL using a reaction mix composed of 10 µL Taq polymerase buffer (1 x), 1 µL of dNTPs (0.2 µM), 1.5 µL of MgCl₂ (1.5 mM), 2 µL of each primer (0.4 µM), 0.5 µL of Taq DNA polymerase (0.05 U/ µL) (Kapa Biosystems), 7.5 µL of template DNA (100 ng/µL), and 25.5 µL of distilled water DNAse, RNAse free (Gibco). The thermal cycling was performed as follows: initial denaturation at 94 °C for 2 min followed by 40 cycles of 94 °C for 45 s, annealing temperature of 58 °C for 45 s, 72 °C for 45 s and a final extension step at 72 °C for 5 min. PCR products were visualized on 2% agarose gels, and prior to sequencing, these were cleaned with QIA quick gel extraction kit (Qiagen). PCR products were bi-directionally sequenced on an Applied Biosystems ABI377 automated sequencer. Sequence records were assembled from forward and reverse reads, and aligned and edited using GENEIOUS 4.0.2 software (Biomatters Ltd.). The sequences are deposited at GenBank (accession numbers KM377628–KM377638).

The control region sequences were amplified using the primer pair of Ctrl Reg-L19 (5’-CCACTAGCTCCCAAAGCTA-3’) and Ctrl Reg-03-R (5’-GTGGGTAACGGGCAATAAGA-3’). PCR amplification was carried out in 45 µl using a reaction mix composed of 9 µl Taq polymerase buffer A (1×), 0.9 µl of dNTPs (0.2 mM), 1.35 µl of MgCl₂ (1.5 mM), 0.9 µl of each primer (0.2 µM), 0.18 µl of Taq DNA polymerase (0.02 U/µl) (Kapa Biosystems), 9.0 µl of template DNA (20 ng/µl), and 22.77 µl of DNAse-free and RNAse-free distilled water (Gibco). The thermal profile was performed as follows: initial denaturation at 94°C for 2 min followed by 25 cycles at 94°C for 45 s, 62°C for 45 s with −0.5°C per cycle and 72°C for 55 s, then 15 cycles at 94°C for 45 s, 54°C for 45 s, 72°C for 55 s and a final extension step at 72°C for 5 min. PCR products were visualized on 2% agarose gels, and prior to sequencing, these products were cleaned with a QIA-quick Gel Extraction Kit (Qiagen). PCR products were bi-directionally sequenced on an Applied Biosystems ABI377 automated sequencer. The sequences from forward and reverse reads were aligned and edited using GENEIOUS 4.0.2 software (Biomatters Ltd.) to obtain consensus sequences for all individuals. The sequences were deposited in GenBank under accession numbers MN166836–MN166903.