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).