javascript:void(0)DNA quality and sequencing
A section of the mitochondrial cytochrome b (cytB ) gene was amplified for DNA sequencing using PCR and universal primers L14724 (5′–CGAAGCTTGATATGAAAAACCATCGTTG–3′) and H15149 (5′–AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA–3′) (Kocher and White 1989; Irwin et al. 1991). PCRs were carried out using 10 μM of each primer, 0.1 μg/μL of bovine serum albumin (BSA) and 1 X MyTaq Red Mix (Bioline) in a total volume of 20 μL. Thermocycling conditions began with an initial denaturation cycle of 3 mins at 94°C, followed by 40 cycles of denaturation (93°C, 1 min), annealing (50°C, 1 min) and extension (72°C, 1 min) and finished with a final extension cycle of 5 mins at 72°C. Amplicons were separated on a 2% agarose gel stained with SYBR safe (Invitrogen) and visualised under UV light.
A subset of samples from those that successfully amplified thecytB gene were selected for Sanger sequencing (n =37, bloodn =5, scat material n =12 and scat swab n =20). PCR products were purified using ExoSAP-IT express reagent (Applied Biosystems) following the manufacturer’s instructions. DNA sequencing of PCR products was conducted in both directions and sequences aligned, trimmed and compared to reference sequences (NCBI BLAST) using Geneious Prime (Geneious 2023.2.1). A minimum spanning haplotype network was produced using the PopART program (Leigh and Bryant 2015) alongside reference haplotypes for cytB sequence from the Chinese (MT335859), Indian (MG196306) and Malaysian (MG196309) pangolin (Hariet al. 2016; Gaubert et al. 2018). A phylogenetic tree was also produced to visualise relationships between Chinese pangolin from Nepal and those sampled in other studies/regions. The phylogenetic tree included an additional 14 reference sequences that were retrieved from GenBank to include representatives from all eight species of pangolin and Asian pangolin samples originating from various geographical locations (Supplementary material, Table S1). The 17 reference sequences were aligned with the haplotypes identified in this study, along withcytB sequence from the gray wolf (HG998573) as an outgroup, trimmed and used to produce a neighbour joining tree with 1000 bootstrap replicates in Geneious Prime.