Microsatellite loci in Columbian ground squirrels Spermophilus columbianus

paternity analysis of nine microsatellites in the Columbian ground squirrel (Spermophilus columbianus, family Sciuridae, tribe Marmotini, subtribe Spermophilina) and their amplification in three other species within the same subtribe (S. richardsonii, S. parryii, and Cynomys ludovicianus), one species from the subtribe Marmotini (Marmota monax) and two more distantly related sciurids (Tamias minimus and Tamiasciurus hudsonicus). Queller et al. (1993) were among the first to review the potential utility of microsatellite DNA for studies of parentage and kinship. Although studies of intrasexual competition have traditionally focused on the behavioural mechanisms used to achieve mating success, such competition is not limited to behavioural interactions. Where females mate with more than one male during a single period of sexual receptivity, the gametes of different males compete for access to the gametes of a single female (Parker 1970) and thus mating success may not accurately reflect reproductive success. Therefore, a method of determining the reproductive outcome of mating behaviour is essential to an understanding of mating systems. Isolation of microsatellites follow the procedure outlined in Paetkau & Strobeck (1994). Approximately 20 000 recombinant clones were screened for microsatellites using a synthetic biotinylated (dT-dG)12 oligonucleotide probe and a biotin detection kit (BRL). A total of 68 positive clones were collected of which 35 were sequenced. Sequence template was obtained by using PCR amplification of phage suspension with universal forward and reverse sequencing primers (4 min at 94 °C followed by 30 cycles of 15 s at 94 °C, 15 s at 56 °C, 75 s at 72 °C, followed by 8 min at 72 °C on a Perkin-Elmer-Cetus 9600 thermal cycler). The PCR products were electroeluted and sequenced on a Applied Biosystems Inc. (ABI) 373A automated DNA sequencer using a Taq DyeDeoxyTM Terminator Cycle Sequencing Kit. Primers for PCR amplification of microsatellites were constructed for 14 clones containing at least 10 uninterrupted tandem repeats using O L I G O software (National Biosciences Inc., Version 4.0) and synthesized on a DNA Synthesizer (ABI model 391). Of these 14 primer sets three were labelled with the fluorescent dye HEX (GS3, GS14, GS17), four with FAM (GS12, GS20, GS 22 and GS26) and two with TET (GS25, GS34). The remaining five primers either yielded no product or resulted in multiple banding patterns and were discarded from the analysis. We examined variability of these loci using DNA isolated from blood of 42 adult male Columbian ground squirrels from two meadows (meadow A and meadow B) that are ≈ 150 m apart (for description of study site see Wiggett & Boag 1989). Amplification of the loci was carried out in a 25-μL volume containing ≈ 100 ng of genomic DNA, 120 μM dNTP, 0.16 μM concentrations of each primer, 1 × Taq buffer, and 0.5 units of Taq DNA polymerase. The PCR cycling conditions were: 1 min at 94 °C followed by two cycles of 30 s at 94 °C, 20 s at 58 °C, 5 s at 72 °C and then 33 cycles of 15 s at 94 °C, 20 s at 54 °C, 5 s at 72 °C followed by 30 s at 72 °C. Onemicrolitre of straight PCR product was used for HEX primers and TET and FAM PCR products were diluted 4× before being resolved by electrophoresis using polyacrylamide sequencing gel run on the DNA Sequencer. Microsatellites were sized and visualized using GENESCAN 672 (ABI) and GENOTYPERTM software. The number of alleles in each locus, their size range, and expected heterozygosity, probability of identity, and probability of paternal exclusion based on genotype analysis of the 42 adult male Columbian ground squirrels are shown in Table 1. Calculations assume that loci are unlinked. The probability that two unrelated random individuals will be identical at all loci was calculated as 1 in 13 315 950 (for equation see Paetkau & Strobeck 1994) and the probability that an unrelated male will be excluded as a potential father given prior knowledge of the mother’s genotype is 99.4% (for equation see Chakravarti & Li 1983). These results indicate that analysis of paternity in S. columbianus with these primers is feasible and, indeed, P R I M E R N O T E