2.3 | Amplification part of mtDNA control regions (D-Loop) and sequencing
The proximal part of the D-loop region (418 bp, between 15 419 bp to 15 836 bp, GenBank accession number X97337(Xu et al., 1996)) between thetrnP gene and the central conserved sequence block (Ishida et al., 1994) , was amplified by PCR using appropriate primers (F: 5’-ACCACTCGCAAGCACCA-3’; R: 5’-CACAGCATCCCCAAATA-3’) as previously described (Ivankovic et al., 2002). The primers were designed by Primer3 (Untergasser et al., 2012) and synthesized and purified by TSINGKE Biological Technology (Nanjing, China). PCR amplification was performed in a 50 μL system containing 300-500 ng of template DNA, 25 μL 2 × Taq Master Mix, 2 μL of each primer, and 19 μL of ddH2O. Amplification conditions were as follows: 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension step at 72 °C for 10 min. The complete mtDNA of one HGD was obtained by whole-genome shotgun sequencing, using an Illumina NovaSeq 6000 platform, with paired-end read lengths of 150 bp.