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.