PCR Analysis and Sequence Alignment

All the polymerase chain reactions conducted in the current study, except those performed during plasmid construction, were performed using EasyTaq DNA Polymerase and 25 μl reaction mixtures containing 12.5 μl 2x Master Mix (Tsingke, Beijing, China), 0.5 μl Forward Primer (10 μM), 0.5 μl Reverse Primer (10 μM), and 500–1000 ng template DNA. The PCR itself was conducted using a Bio-gener GT9612 thermocycler (Bio-gener, Hangzhou, China) and the following program: initial denaturing at 94°C for 4 min, followed by 34 cycles of denaturing at 94°C for 30 s, annealing at 55–62°C (depending on the primer) for 30 s and extension at 72°C for 1 min for each 1 kb fragment, with a final extension at 72°C for 10 min. The resulting PCR products were visualized by agarose gel electrophoresis and sequenced by Tsingke (Beijing, China). Multiple sequence alignments were prepared using the DNAMAN 9.0.1.116 software package (Lynnon Biosoft, Quebec, QC, Canada).