DNA Amplification and Genotyping
A suite of 8 dimorphic microsatellite primers originally designed for domestic cats (Menotti-Raymond et al. 1999, 2005) and previously amplified with bobcats (Morin et al. 2018) were optimized using bobcat tissue DNA (Table 1). We used a multitube process to test the quality of scat DNA and extraction success and eliminate samples of low quality. All bobcat identified or potential samples were first amplified with a primer multiplex containing 4 loci (FCA096, FCA275, FCA391, and FCA126; i.e. Multiplex 1). Samples that amplified at ≤1 locus were re-extracted and samples that amplified at 2 loci (including re-extractions) were re-amplified. Samples that amplified at 3-4 loci then moved onto a subsequent multiplex (i.e., Multiplex 2) containing 4 primers (FCA090, FCA043, FCA124, and F124) plus a sex identification primer (AmelX/Y ; Pilgrim et al. 2005). DNA was amplified in 15µl polymerase chain reactions (PCR) containing 4µl DNA plus 7.5µl 2X Multiplex Master Mix (Qiagen), 1.5µl Q Solution (Qiagen), 0.375µl Bovine Serum Albumin, primer mix at 0.2µM concentration of each forward and reverse primer, and 0.425µl of diH2O. Reactions were initiated at 95°C for 15 minutes to activate hot-start TAQ, then denatured at 94°C for 30 seconds, annealed at 55°C for 3 minutes, and extended at 72°C for 1 minute for 40 cycles; then finished with a final extension at 60°C for 30 minutes. Amplified product was plated with 9.5µl formamide and 0.5µl GeneScan 500 LIZ dye Size Standard (Applied Biosystems) and genotyped on an ABI 3730xl at the Genomics Shared Resource in The James Comprehensive Cancer Center at The Ohio State University (NCI Cancer Center Support Grant P30CA016058). Genotypes were scored using Geneious Prime software (version 2021.0, Dotmatics, Boston MA). Samples were amplified 2-9 times at each locus and included in the dataset used for individual analyses if they had at least 2 consensus genotypes for homozygotes and at least 3 consensus amplifications of each allele for heterozygotes at 5 or more loci.