The development of microsatellite markers helps to identify and develop appropriate management actions to aid in population recovery (Abdul-Muneer 2014), such as in woodrat recovery programs. Musser and Carleton (2005) reclassified the woodrat genus, Neotoma, into 22 distinct species. At least four of these species were endangered or possibly extinct, with many more declining throughout their range (Feldhamer and Poole 2008). Illinois’ subspecies of the eastern woodrat (Neotoma floridana illinoensis) was placed on the Illinois Endangered Species list during 1977–2020 due to restricted habitats and small populations (Mankowski 2012). A historical metapopulation stretching across five counties and the entire east–west extent of southern Illinois collapsed into several isolated populations due to habitat fragmentation (Monty et al. 2003). By the 1960s, Illinois’ subspecies of the eastern woodrat was restricted to three populations located in Union (Pine Hills) and Jackson (Fountain Bluff and Horseshoe Bluff) Counties in southwest Illinois (Crim 1961). By 1974 the Fountain Bluff population was extirpated and the population at Horseshoe Bluff had severely declined (Nawrot and Klimstra 1976). Although woodrat numbers were very low in Jackson County, genetic analyses by Monty et al. (2003) showed significant genetic differences between these geographically-proximate populations. The Illinois Department of Natural Resources implemented genetic augmentations and reintroductions across the SNF during 2003–2014. Microsatellite markers provide a means to assess the genetic structure of these populations.

Tissue samples of eastern woodrats were collected from reintroduced populations in the eastern SNF at Garden of the Gods. Capture and handling activities were conducted in accordance with Institutional Animal Care and Use protocol # 19-003 at Southern Illinois University. Genomic libraries were constructed and sequenced by University of Missouri DNA Core Facility (Columbia, MO) using Illumina sequencing. PCR primers were designed using QDD (Meglecz et al. 2010). We tested 24 primer sets on the 32 individuals from Garden of the Gods (Table 1). DNA was extracted using a section of a 3 mm ear biopsy and the Qiagen DNeasy DNA Micro kit (Qiagen Inc., Valencia, California) and concentrations were quantified using Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, USA). Microsatellites were amplified using PCR in 10 µl reactions using 8–40 ng genomic DNA, 5 µl DreamTaq PCR Master Mix (2X) (ThermoFisher Scientific, USA), 0.5–0.9 µM each of a fluorescently-tagged forward primer and an untagged reverse primer. Microsatellite PCR parameters were as follows: initial denature at 94 °C for 4 min, 32 cycles at 94 °C for 45 s, annealing temperature(s) for 30 s, a 4-min extension at 72 °C, and then a 20-min extension at 70 °C (Castleberry et al. 2002; Matocq 2002; Sousa et al. 2007; Kanine 2013). Products were denatured with HiDi formamide. Reactions were resolved on an ABI 3130XL Gene Analyzer (Applied Biosystems Inc., Warrington, UK) against a 70–400 bp standard (Gel Company, San Francisco) and genotyped using GeneMapper® ID-X software version 4.0 (ThermoFisher Scientific, USA). GENEPOP 4.7.5 (Raymond and Rousset 1995) was utilized to calculate Expected (HE) and observed (HO) heterozygosity and estimate deviations from Hardy–Weinberg equilibrium (Table 1).

Table 1 Primer sequences and properties of 24 loci developed for Neotoma floridana
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