Microsatellite marker development from next-generation sequencing in the New England cottontail ( Sylvilagus transitionalis ) and cross-amplification in the eastern cottontail ( S. floridanus )

Objective The New England cottontail ( Sylvilagus transitionalis ) is a species of high conservation priority in the Northeastern United States, and was a candidate for federal listing under the Endangered Species Act until a recent decision determined that conservation actions were sufficient to preclude listing. The aim of this study was to develop a suite of microsatellite loci to guide future research efforts such as the analysis of population genetic structure, genetic variation, dispersal, and genetic mark-recapture population estimation. Results Thirty-five microsatellite markers containing tri- and tetranucleotide sequences were developed from shotgun genomic sequencing of tissue from S. transitionalis , S. obscurus , and S. floridanus . These loci were screened in n = 33 wild S. transitionalis sampled from a population in eastern Massachusetts, USA. Thirty-two of the 35 loci were polymorphic with 2–6 alleles, and observed heterozygosities of 0.06–0.82. All loci conformed to Hardy–Weinberg Equilibrium proportions and there was no evidence of linkage disequilibrium or null alleles. Primers for 33 of the 35 loci amplified DNA extracted from n = 6 eastern cottontail ( S. floridanus ) samples, of which nine revealed putative species-diagnostic alleles. These loci will provide a useful tool for conservation genetics investigations of S. transitionalis and a potential diagnostic species assay for differentiating sympatric eastern and New England cottontails.