Characterization of microsatellite loci in the Northern Idaho ground squirrel Spermophilus brunneus brunneus

rarest sciurid in North America. A northern (S. b. brunneus) and a southern subspecies (S. b. endemicus) have recently been described (Yensen 1991). The former is currently found in only 18 populations in Adams and Valley counties (west–central Idaho); the largest population is < 300 animals and the subspecies consists of < 1000 animals (Yensen & Sherman, in press). Reductions in S. b. brunneus’s range and population sizes are apparently due to major alterations of the habitat in the last 100 years. Many of the resultant small disjunct populations have recently become extinct, and others are in imminent danger of extinction. Previously we used allozymes to study mating behaviour (Sherman 1989) and population structure (T. A. Gavin et al. unpublished data). Recently we developed microsatellite markers in S. b. brunneus to enhance the resolving power of our data for understanding the extant and historical population structuring of S. b. brunneus, prior to restorative efforts. Samples of S. b. brunneus consisted of (i) blood taken from the suborbital sinus or (ii) buccal scrapings taken with a toothpick and placed in 1.5-mL cryovials and either air-dried or immersed in 0.5 mL of lysis buffer (White & Densmore 1992). DNA was extracted from 150 to 200 μL blood or buccal scrapings using QIAGEN Blood Kits. Genomic libraries were constructed from the DNA from a single individual by digesting with Sau3AI, ligating into pUC 18, and transforming DH5α competent cells (see May et al. in press for more detail). One library of 196 colonies (Library I) was dot-blotted and screened with total genomic DNA. A second library of 53 000 colonies (Library II) was lifted and screened with trimer and tetramer oligonucleotides from BIOS Laboratories (AAAT, AAAG, AAAC, CCG, AAG, AAT, AAC, and CAG). Total genomic DNA and oligos were end labeled (tailed) with either the Oligonucleotide 3′-End Labeling Kit or the Oligonucleotide Tailing Kit from Boehringer Mannheim. Positives were detected with the Lumi-Phos Chemiluminescent Kit or the colorimetric (chromogenic) method using the NBT/BCIP Kit (Boehinger Mannheim). Initially positive colonies were screened two more times for confirmation. Tertiary positives with inserts of 300–1500 bp were sequenced on an ABI 373A automated sequencer. Microsatellite sequences were located in 8/8 positives from Library I and 17/26 positives from Library II, and primers were designed for five and 15 of the sequences, respectively. Polymerase chain reaction amplifications were carried out in an MJR PTC-100 thermocyler in 50-μL reactions containing 3–10 ng DNA, 1.5 mM MgCl2, 0.4 μM each primer, 175 μM dNTPs, and 1 unit of GIBCO Taq. Amplification conditions were 94 °C for 3 min; 34 cycles of 94 °C for 1 min, 52° (57 °C for IGS-BM1) for 30 s, and 72 °C for 30 s; and a final extension of 5 min at 52 °C (or 57 °C). Amplified samples were concentrated from 25 μL down to 10 μL in a vacuum centrifuge and run on 4% 1 × TBE MetaPhor (FMC) agarose gels (Fig. 1) at 460 V (17 V/cm), with the circulating buffer (0.5 × TBE) at 12 °C for the first 5 min and the remainder of the run at 20 °C. Gels were run for 45 min to 1.5 h depending on size of PCR product (about 15 min per 25 base pairs). Gels were stained with ethidium bromide. The amplification effectiveness of the 20 primer pairs was tested against the clone and three individual squirrel DNA extracts. Thirteen pairs produced a resolvable amplicon in all four samples. The other seven amplified only in the clone or did not produce sufficient product to be useful. Each of the useable 13 primer pairs was subsequently tested against 10 individuals from across the range of S. b. brunneus. Primer sequences, GenBank accession numbers, repeat motif, clone amplicon size, and number of alleles detected for these 13 loci are presented in Table 1. The first three loci were from Library I and the latter 10 loci were from Library II. Usually only the primary amplicons were observed, as well as one or two heteroduplex bands in heterozygous individuals, demonstrating that amplification was PRIMER NOTE