Abstract
Microsatellites are powerful markers for tracking genetic variation in wildlife populations due to their high polymorphism and genome-wide abundance. While PCR-based fragment size analysis has been the standard for genotyping microsatellites, high-throughput sequencing offers greater resolution and the opportunity to sync historical datasets with modern analyses. We evaluated how genotypes from whole-genome sequencing align with PCR data for 15 microsatellite loci in 11 North American brown bears (Ursus arctos). Brown bear populations in the lower 48 United States have declined from approximately 50,000 to fewer than 2,000 over the past decades. Their endangered status has prompted extensive research and genetic monitoring, yielding large, multi-year microsatellite datasets upon which future conservation efforts can build. We achieved a microsatellite genotype concordance rate of 94.5% with PCR results. All discrepancies occurred at complex loci containing multiple insertions and/or deletions (indels). Physically linked indels or single nucleotide polymorphisms (SNPs) occurring within the loci were misinterpreted as independent insertions, underscoring the need for genotyping tools that incorporate phasing when genotyping. To evaluate coverage effects, we downsampled from 30x to 2x. Concordance remained high at 20-30x but dropped sharply at 10x, with 5x and 2x having discordant genotypes or insufficient coverage for genotyping. Accurate genotyping required both sufficient depth and number of reads spanning the entire repeat regions. Our results show that short-read whole-genome sequencing can recover microsatellite genotypes with high accuracy when paired with careful variant interpretation. By aligning historical PCR datasets with modern sequencing data, we can preserve decades of genetic insight and strengthen long-term monitoring of at-risk populations.