Materials and methods

Study area and sample collection
Our study area encompassed Heizhugou, Meigu Dafengding and Mabian Nature Reserves of the Liangshan Mountains (Table 1).
Giant pandas fecal samples were collected by ranger staff during their daily monitoring and patrol work in the reserves. The staff used sterile gloves to collect fresh fecal samples when they detected giant panda activity. Samples were considered fresh based on the color and surface sheen, with dark colored and dull feces being discarded. Each sample was collected in 1-2 copies and stored in a 500 ml sample bottle containing anhydrous ethanol. Spatial coordinates were recorded from the deposition site (e.g., longitude, latitude, elevation) using GPS units and the distribution of samples was mapped as shown in Figure 2, using ArcGIS 10.6 (Price, 2010).
DNA extraction and PCR amplification of mitochondrial D-loop
Fecal DNA was extracted using the kit (Biobase Upure DNA stool kit, Chengdu, China) and nucleic acid purifier (Thermo KlngFisher, USA). Fecal samples collected in the field were soaked in anhydrous ethanol and frozen at −20°C. DNA extraction was undertaken according to the manufacturer’s instructions, except for DNA samples being amplified by PCR using the mitochondrial control region primers of the giant panda (Zhang et al., 2007). The total length of the amplification was about 750 bp. The amplification primers were:
P - tp: 5 ’- CTCCCTAAGACTCAAGGAAG - 3’
BEDH: 5 ’- GGGTGATCTATAGTGTTATGTCC - 3’
PCR amplifications were performed in a 20 μL reaction volume containing 10 μL 2xTaq PCR Pre Mix (+dye), 1μL MgCl2 (25 mmol/L, 0.8 μL BSA (1 mg/ml), 0.8 μL Ptp primer (15 pmol/ L), 0.8 μL BEDH primer (15 pmol/ L), 5 μL template DNA (50 ng/ μL), and 1.6 μL ddH2O. Amplifications were performed using the following PCR procedure: an initial denaturation step for 5 min at 94°C, followed by 40 cycles of 94°C for 50 s, 55°C annealing for 45 s, 72°C elongation for 50 s and a final elongation for 10 min at 72°C. Finally, samples were stored at 4°C. PCR conditions were optimized by changing the concentration of Mg2+, annealing temperature and increasing the amount of template DNA.
Selection and amplification of microsatellite markers
Our laboratory has screened giant panda DNA for standardized microsatellite loci and obtained 15 loci that can be effectively applied to giant panda fecal DNA samples (Huang, 2015). We selected seven of these 15 microsatellite loci for population analysis, which were GPL8, GPL29, GPL60, gpz20, gpz47, gpy5 and gpy20 ( Table2). PCR amplifications were performed in a 20 μL reaction volume comprising about 10 μL 2xTaq PCR Pre Mix (+dye), 1μL MgCl2 (25 mmol/L), 0.8 μL BSA (1 mg/ml), 0.8 μLF-primer (15 pmol/ L), 0.8 μL R-primer (15 pmol/ L), 5 μL template DNA (50 ng/ μL), and 1.6 μL ddH2O. Amplifications were performed using the following PCR procedure: an initial denaturation step for 5 min at 94°C, followed by 40 cycles of 94°C for 50 s, 55°C-63°C annealing for 45 s, 72°C elongation for 30 s, and a final elongation for 10 min at 72°C. Finally, samples was stored at 4°C. After the PCR amplification, 5 μL of PCR products from each sample was applied to agarose gel electrophoresis with a concentration of 1.5% to detect whether each sample was successfully amplified. At the end of electrophoresis, the PCR products were stored at 4°C away from light (using tin foil box) for genotyping.
Microsatellite genotyping
Genetic analysis and detection of all samples were undertaken at Chengdu Qingke Zixi Biotechnology Co., Ltd. During genotyping, the amplification products of each fluorescent primer were separately placed in a single lane for electrophoresis. Genotyping of all samples was conducted using ABI 3730 DNA Analyzer. The number of alleles in each sample was determined by using Gene Mapper v4.0. The allele size was determined relative to the intramolecular GS500LIZ.
Data analysis
The results of genotyping data were estimated with Micro-Checker (Van Oosterhout et al., 2004). Individual identification was analyzed using Microsatellite tools (Park et al., 2001). PID and PID (sib) were calculated using Gimlet (Valière, 2010). The Cervus v3.0 (Marshall et al.,2010) was used to calculate Allele number (A), observe Heterozygosity (Ho), expected Heterozygosity (He) and polymorphic information content (PIC). Deviations from the Hardy–Weinberg equilibrium (HWE) and Linkage Disequilibrium (LD) were analyzed using Genepop 3.4 (Raymond and Rousset, 1995). Mitochondrial sequence alignment was performed using MEGA v5.2 (Tamura et al., 2011) and was manually calibrated. DNA ASP v5.10 (Librado and Rozas, 2009) was used to calculate haplotype diversity (h), nucleotide diversity (π) and other genetic diversity indices. Popgene 32 was used to calculate the inbreeding coefficient of population (Yeh, 2000). Population genetic structure analysis was undertaken using STRUCTURE (Pritchard, Stephens & Donnelly, 2000).