Three mitochondrial protein-coding genes (COI, COII, and CytB) were utilized as molecular markers in the present study. Based on the published mitochondrial genome sequence of A. mali (Sun et al., 2020 (link)), we designed specific primers for the amplification of complete COI, COII, and CytB sequences using the software Primer Premier version 5 (Lalitha, 2000 (link)). Details of the primer sequences and product sizes are provided in Supplementary Table S1 .
Polymerase Chain Reaction (PCR) amplification was conducted in a total volume of 25 μl containing 1.5 μl genomic DNA, 1 μl each of forward and reverse primers (10 μmol/L), 12.5 μl 2× Flash PCR MasterMix (CWBIO, China), and 9 μl ddH2O. PCR amplification was performed with an initial denaturation at 98°C for 1 min; 35 cycles of 10 s at 94°C, 15 s at a primer-specific annealing temperature (46°C for COI, 49°C for COII, and 53°C for CytB), and extension at 72°C for 30 s; and a final elongation at 72°C for 1 min. The PCR products were visualized on 1.0% agarose gels under UV light. The sequencing reaction was carried out using an ABI BigDye Terminator version 3.1 cycle sequencing kit on an ABI 3730XL (Tsingke Biotechnology Co., Ltd., Beijing, China) for Sanger sequencing in both directions.
Polymerase Chain Reaction (PCR) amplification was conducted in a total volume of 25 μl containing 1.5 μl genomic DNA, 1 μl each of forward and reverse primers (10 μmol/L), 12.5 μl 2× Flash PCR MasterMix (CWBIO, China), and 9 μl ddH2O. PCR amplification was performed with an initial denaturation at 98°C for 1 min; 35 cycles of 10 s at 94°C, 15 s at a primer-specific annealing temperature (46°C for COI, 49°C for COII, and 53°C for CytB), and extension at 72°C for 30 s; and a final elongation at 72°C for 1 min. The PCR products were visualized on 1.0% agarose gels under UV light. The sequencing reaction was carried out using an ABI BigDye Terminator version 3.1 cycle sequencing kit on an ABI 3730XL (Tsingke Biotechnology Co., Ltd., Beijing, China) for Sanger sequencing in both directions.