Kangaroo mice, Microdipodops megacephalus and M. pallidus, are sand-obligate rodents endemic to the Great Basin Desert of western North America. These two species diverged roughly 8 million years ago (Hafner et al. 2007) and subsequently encountered large-scale climatic fluctuations during the late Pleistocene and Holocene. As a result of these climatic events in conjunction with restricted habitat preferences, populations of both M. megacephalus and M. pallidus are often isolated. In fact, several recent studies have revealed that some of these isolated populations may represent unique genetic clusters (Hafner et al. 2006, 2008) resulting in conservation concerns for both M. megacephalus and M. pallidus. Microsatellite markers will be used to help elucidate how Microdipodops populations responded to past climatic changes as well as gain a better understanding of the interactions among extant populations of M. megacephalus and M. pallidus.

We extracted total DNA from one individual of M. megacephalus, using the DNeasy tissue kit protocol (Qiagen, Valencia, CA) following manufacturers protocols. We followed the enrichment procedure of Glenn and Schable (2005) with some exceptions. Briefly, DNA was digested with restriction enzyme RsaI (New England Biolabs), ligated to double-stranded linkers, denatured and hybridized to biotinylated microsatellite oligonucleotide mixes (mix 2 = (AG)12, (TG)12, (AAC)6, (AAG)8, (AAT)12, (ACT)12, (ATC)8; mix 3 = (AAAC)6, (AAAG)6, (AATC)6, (AATG)6, (ACAG)6, (ACCT)6, (ACTC)6, (ACTG)6; mix 4 = (AAAT)8, (AACT)8, (AAGT)8, (ACAT)8, (AGAT)8), then captured on magnetic streptavidin beads (Dynal). Unhybridized DNA was washed away and remaining DNA was eluted from the beads, amplified in polymerase chain reactions (PCR) using the forward SimpleX4 as a primer. There were two primary changes to the Glenn and Schable (2005) protocol. First, a new linker was used (SimpleX-4 Forward 5′-AAAAGCAGCAGCGGAATC and SimpleX-4 Reverse 5′- pGATTCCGCTGCTGC). Second, the enriched libraries were sequenced on a 454 using titanium chemistry following standard Roche 454 library protocols (454 Life Sciences, a Roche company, Branford CT). Sequences were subjected to a 3′ quality trim where only one base in the last 25 bases of the sequence contains a quality score less than 20 or alternatively contains one ambiguous base. CAP3 [33] was then used to assemble sequences at 98% sequence identity using a minimal overlap of 75 bp. Along with singlets, contigs of two or three sequences were searched for the presence of microsatellite DNA loci using the program MSATCOMMANDER version 0.8.1 (Faircloth 2008) and primers designed with Primer3. One primer from each pair was modified on the 5′ end with an engineered sequence (CAG tag 5′-CAGTCGGGCGTCATCA-3′) to enable use of a third primer in the PCR (identical to the CAG tag) that was fluorescently labeled for detection.

Forty-eight primer pairs were tested for amplification and polymorphism using DNA obtained from eight individuals of M. megacephalus. PCR amplifications were performed in a 12.5 μL volume (10 mM Tris pH 8.4, 50 mM KCl, 25.0 μg/ml BSA, 0.4 μM unlabeled primer, 0.04 μM tag labeled primer, 0.36 μM universal dye-labeled primer, 3.0 mM MgCl2, 0.8 mM dNTPs, 0.5 units JumpStart Taq DNA Polymerase (Sigma), and ~20 ng DNA template) using an Applied Biosystems GeneAmp 9700. Touchdown thermal cycling programs (Don et al. 1991) encompassing a 10°C span of annealing temperatures ranging between 65–55°C were used for all loci. Touchdown cycling parameters consisted of 20 cycles of 96°C for 30 s, highest annealing temperature of 65°C (decreased 0.5°C per cycle) for 30 s, and 72°C for 30 s; and 20 cycles of 96°C for 30 s, 55°C for 30 s, and 72°C for 30 s. PCR products were run on an ABI-3130xl sequencer and sized with Naurox size standard prepared as described in DeWoody et al. (2004), except that unlabeled primers started with GTTT. Results were analyzed using GeneMapper version 3.7 (Applied Biosystems). Seventeen of the tested primer pairs amplified high quality PCR product that exhibited polymorphism.

We assessed the variability of 17 polymorphic loci in 24 specimens (15 M. megacephalus and 9 M. pallidus) that came from a total of 21 general localities across the distributional range of Microdipodops in the Great Basin Desert. Specimen voucher information (museum numbers and collection localities) is available from J.C.H. Conditions and characteristics of the 17 loci are given in Table 1. We estimated number of alleles per locus (k), observed and expected heterozygosity (H o and H e), probability of identity (PI) using GenAlEx v6.0 (Peakall and Smouse 2006). Tests for deviations from Hardy–Weinberg equilibrium (HWE) and for linkage disequilibrium were conducted using GENEPOP v4.0 (Rousset 2008). For M. megacephalus the number of alleles per locus ranged from 5 to 16, observed heterozygosity ranged from 0.357 to 0.933, and the probability of identity values ranged from 0.013 to 0.107. For M. pallidus, 16 of 17 microsatellite loci amplified although one locus was monomorphic. Of the remaining 15 loci, the number of alleles per locus ranged from 4 to 12, observed heterozygosity ranged from 0.333 to 1.0, and the probability of identity values ranged from 0.022 to 1.0. After Bonferroni correction for multiple comparisons only one locus showed significant deviations from expectations under HWE and no linkage disequilibrium was detected for any of the 136 paired loci comparisons. These microsatellite loci may be useful in future analyses to determine how past climatic changes may have affected populations of M. megacephalus and M. pallidus and assess population dynamics within these two rare species.

Table 1 Details for 17 polymorphic microsatellite loci developed for Microdipodops megacephalus and screened for both M. megacephalus and its sister species, M. pallidus
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