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).