DNA extraction, PCR amplification, and genotyping

DNA was extracted by the phenol-chloroform method [25]. The high altitude DNA pool was constructed from 50 samples, each containing 100 ng DNA from randomly selected TC. The LC pool was constructed in the same way. These two gene pools were used to detect SNPs.

After blasting chicken EPAS1 mRNA sequence (Ensembl accession ENSGALT00000016253) in the chicken genome, we designed 15 primer pairs (Table 2) to amplify the 15 exons using Primer V6.0 software [26]. PCR was performed in 25 μL of reaction volume. The ingredient contain 50 ng DNA template, 1×buffer (including 1500 μmol L^-1 Mg₂Cl₂, 200 μmol L^-1 dNTPs, and 1.5 U of Taq DNA polymerase) and 1 μmol L^-1 of each primer. Besides, the PCR procedure was as following: initial denaturation for 5 min at 9500, followed by 35 cycles of 95°C for 30 s; annealing at prescribed annealing temperature for 30 s; and primer extension at 72°C for 45 s. The final extension was performed at 72°C for 7 min. PCR products were sequenced on an ABI 3730 DNA sequencer. Then, the SNPs were determined and pairs of 4 primers (P1, P7, P12, and P14) were used for individual genotyping. PCR procedures and systems were the same as above.

^1.2 F and R are the abbreviation of forward and reverse primer, respectively.