DNA studies
DNA was extracted from blood, epithelial cells, and calamus of feathers by a phenol–chloroform method according to standard protocol (Sambrook et al. 1989, Smith and Schantz 1996). Two fragments of mtDNA were amplified by PCR. The control region (CR) of mtDNA was amplified using the primers L98 and H772 (Wenink et al. 1993). In addition, the primers L13706 and H14562 (L13706, 5′–AATGGCCCCAAACCTCCGAAAAT; H14562, 5′–GGATTGAGCGTAGGATGGCGTAT) were designed to amplify the cytochrome b(Cytb ) gene. The CR and Cyt b segments were both amplified for 35 cycles, using the following temperature profile: denaturation at 94°C for 30 s, annealing at 50.9°C (CR) / 50°C (Cytb ) for 30 s, and a final extension at 72°C for 1.5 min. The PCR amplifications were conducted in a 25-μL reaction system containing 1.0 μL of DNA template (100 ng/μL), 1.0 μL of each primer (10 μM), 3.0 μL of 10 × buffer, 2.0 μL of MgCl2 (25 mmol/l), 2.0 μL of dNTPs (2 mmol/l), 1.0 μL of Taq DNA polymerase (1U; Takara, Japan), and 15 μL of sterilized double-distilled water. After checking the PCR amplifications by electrophoresis, PCR products were sequenced in two directions with each primer on an ABI 3731XL sequencer.
The sex of individuals was identified by a molecular genetic sexing method. After DNA was extracted, the primers P2 and P8 were used in PCR to amplify the sex chromosomes (Griffiths et al. 1998). Then, the sex of individuals was assigned by reading the numbers of bands separated by electrophoresis in a 2% agarose gel.