2.2 PCR amplification and Sequencing
According to the previously reported methods (Yang et al., 2009), the complete Cytb sequences were amplified using a pair of primers: PF 5’-gttccgtagccatagccg-3’; PR 5’- ttgagtcttagggaggtt-3’. PCR amplification was conducted in a 25-μL reaction mixture containing 12.5 μL of 2×PreMix, 1 μL of each primer (10 pmol/μL), 1 μL of genomic DNA (10-20ng/μL), and 9.5 μL of ddH2O. The PCR was carried out using a standard program with 4 min predenaturation , then consisted of 35 thermal cycles with a denaturation step (45 sec at 95℃), a hybridization step (50 sec at 53.2℃) and an elongation step (120 sec at 72℃) for each cycle, and final extension for 5 min at 72℃, followed by cooling to 4 ℃. PCR products with appropriate band size were then sent to Beijing Tsingke Biotechnology Co., Ltd (Beijing, China) for sequencing.
2.3. Sequences download and Data Analysis
161 previously reported Cytb sequences from wild yak and four Qinghai domestic yak breeds/populations were downloaded in Genbank (Table S1). Combined with five new Cytb sequences from two wild yak and three Qinghai-Gaoyuan yak, 166 mtDNA Cytb sequences were analyzed totally in this study (Table S1). Raw Cytb sequences were checked and aligned with the Clustal W multiple alignment algorithm of BioEdit v7.2.5 software (Hall, 1999). The number of variable sites, haplotype diversity (Hd) and nucleotide diversity (Pi) were estimated using Dnasp 5.10 (Librado& Rozas, 2009) and Arlequin 3.11 (Excoffier et al., 2007). Fixation index (F ST) was also calculated using the Arlequin 3.11 software. TheR ST values were calculated by the linearizedFST value, namely, R ST =F ST/(1-F ST). Multidimensional scaling (MDS) analysis was performed based onR ST values using SPSS 18.0 software. Taking the American bison (Bison bison ) counterpart as an out-group (Genbank accession No.: EU177871), the phylogenetic tree was constructed by using the Neighbor-joining (NJ) method in Mega 6.0 (Tamura et al., 2013). A median-joining (MJ) network was generated using the Network 10.2.0 software (Bandelt et al., 1999).