Dhole Scat Identification via Microsatellite Genotyping

For individual identification from the confirmed dhole scats, we used the earlier validated 12 microsatellite loci panel described in Modi et al.^{26 (link)} (Supplementary Table 1). We performed PCR reactions in 10 μl reaction volumes containing 4 μl of Multiplex master mix (QIAGEN Inc., Hilden, Germany), 4 μM (2.5 μl) BSA, 0.5 μM of primer mix and 3 μl of DNA extract with PCR conditions including initial denaturation (95 °C for 15 min); 50 cycles of denaturation (94 °C for 30 s), annealing (50 °C for 30 s) and extension (72 °C for 35 s); followed by a final extension (72 °C for 10 min)^{26 (link)}. Negative and extraction controls were included to monitor contaminations. Amplified products were mixed with HiDi formamide and LIZ 500 size standard (Applied Biosystems, California, United States) and genotyped in an ABI genetic analyzer (Applied Biosystems, California, United States). We scored the fragment lengths manually using the same reference sample and following stringent criteria described in Modi et al.^{26 (link)}. All samples were genotyped three independent times to ensure good data quality for subsequent analyses. We have also included 101 individual genotypes from our previous study^{26 (link)} collected from five protected areas (MTR, TATR, PTR, NNTR, UKWLS) along with the newly generated data for further analysis.