Microsatellite loci for the Siberian flying squirrel, Pteromys volans

The Siberian flying squirrel is a nocturnal, arboreal rodent resident in spruce-dominated boreal forests in Eurasia. We have been studying home ranges, space use, movements, natal dispersal and mating behaviour of the flying squirrel in Finland using radio telemetry from 1996 to 2002 (Hanski 1998; Hanski et al. 2000b; Selonen 2002). Male flying squirrels have home ranges five to 10 times larger than those of females, which usually overlap the ranges of several females. The females seem to live in separate home ranges, whereas the ranges of males overlap with each other (Hanski et al. 2000a and unpublished). Due to their nocturnal habits many aspects of flying squirrel behaviour, such as mating and copulations, are difficult to observe. We developed microsatellite loci for the Siberian flying squirrel in order to investigate questions of paternity and the mating system and also the distribution of genetic variation amongst flying squirrel populations.

DNA for the construction of genomic libraries was extracted from muscle tissue using a standard phenol–chloroform extraction procedure. Two different methods were used in the construction of the genomic libraries used to screen for microsatellite repeat motifs. The first method approximately followed the protocol of Painter et al. (1997). Briefly, 2 µg of DNA were digested with two restriction enzymes, Sau3AI and RsaI (Amersham). The digested DNA was ligated into pUC19 vector (Amersham) and then transformed into ELECTROMAX™ DH10B™ cells (Gibco BRL). Approximately 10 000 colonies were screened for microsatellite repeat motifs by hybridization at 55 °C overnight using the following γP³³ end-labelled probes: (AC)₁₅, (AG)₁₅, (AAT)₁₅, (CCT)₁₅, (AAAG)₈ and (ATAG)₈. Thirty positive clones were polymerase chain reaction (PCR) amplified and sequenced with both the pUCF23 and pUCR24 primers, using BIGDYE terminators (ABI PRISM). Most inserts contained AC repeats. Primers were designed for 16 loci that contained repeat motifs longer than 10 repeat units in length and checked for self and pair complementarity using primer3.0 (Rozen & Skaletsky 2000). Five of the loci were polymorphic (Table 1).

The second method was a modification of the enrichment protocol of (Fleischer & Loew 1995; Hamilton & Fleischer 2001; Hamilton et al. 1999). Briefly, 2 µg of genomic DNA were digested with Sau3AI, HaeIII and NheI. Single-stranded overhangs were removed using S1 nuclease (MBI Fermentas) and, following column purification, the 5′ phosphates were removed using calf intestinal alkaline phosphastase (MBI Fermentas). This DNA was then ligated to the SNX linkers and the ‘library’ enriched by hybridizing a biotinylated (GA)₁₀ oligonucleotide to the insert DNA at 55 °C overnight. The oligonucleotide–DNA complexes were isolated using streptavidin MagneSphere paramagnetic particles (Promega) according to the manufacturer's instructions. After PCR amplification and the subsequent removal of the linkers by digestion with NheI, the insert DNA was ligated into pUC18 (Amersham) previously digested with XbaI and dephosphorylated. The ligated DNA was then transformed as above. Only 116 white colonies were obtained and, of these, 21 contained a microsatellite motif of more than 10 repeat units. Primers were designed for 13 loci as there was too little or no flanking sequence on one side of the microsatellite in the remaining inserts. Again, five of the loci were polymorphic (Table 1).

DNA for the amplification of microsatellite loci was extracted from flying squirrel hair follicles by incubating 1 cm pieces (including the follicle) of approximately 20 hairs in 5% Chelex 100 (Bio-Rad) with 20 µg proteinase K at 56 °C for 1 h, followed by 10 min at 95 °C. The extractions were subsequently stored at 4 °C. For each primer pair the forward primer was 5′ end-labelled with either HEX or FAM fluorescent dyes (DNA Technology). The ratios of the two primers in the PCR mix were dependent on the dye, 1 : 2 labelled : unlabelled for HEX and 1 : 10 labelled : unlabelled for FAM. Each 10 µL PCR mix contained 1× PCR buffer plus NHSO₄ (MBI Fermentas), 1.5 µm MgCl₂, 100 µm each dNTP, 1 pmol (FAM) or 5 pmol (HEX) of labelled primer, 10 pmol of unlabelled primer, 10 µg bovine serum albumin, 0.5 U Taq and 1 µL of DNA. The PCR profile consisted of denaturation for 2 min at 92 °C, 30 s annealing (see Table 1 for temperatures) and extension at 72 °C for 30 s, followed by 32 cycles of 92 °C for 30 s, annealing for 30 s and 72 °C for 30 s. During the final cycle the extension time was extended to 10 min. The Pvol74 products were then incubated at 60 °C for 1 h to allow additional time for the Taq enzyme to add an A to the fragments.

Ten of the 29 microsatellite loci for which primers were designed were polymorphic (Table 1). However, we have used only seven of these markers for the paternity analyses as for two loci (PvolI1 and PvolI3) almost all individuals were homozygous for one common allele, whilst a third locus (PvolE25) was often difficult to interpret due to stuttering and requires further optimization. The seven most useful loci had between six and 11 alleles and expected heterozygosities ranging from 0.477 to 0.866.

We tested the utility of the seven most useful primers for cross-species amplification in three additional Sciuridae species, the common red squirrel Scurius vulgaris and two species of northern flying squirrels, Glaucomys sabrinus and G. volans. The PCR conditions were as for Siberian flying squirrels except that a touchdown amplification protocol (55–45 °C) was employed. Siberian flying squirrels are relatively distantly related to other squirrel species. In limited trials we were unable to successfully amplify microsatellite loci using a subset of the primers that have been designed for other squirrel species: from Columbian ground squirrels (Spermophilus columbians) GS14, GS17, GS22, GS25 and GS26 (Stevens et al. 1997); from northern Idaho ground squirrels (S. brunneus brunneus) IGS-1 and IGS-6 (May et al. 1997) and from northern flying squirrels (G. sabrinus) GS-2, GS-8, GS-10, GS-13 and GS-16 (Zittlau et al. 2000). In our cross-species trials a resolvable product was obtained for five of the Siberian flying squirrel loci in all three species (Table 2). Three of the loci were polymorphic in at least one additional species, with allele sizes within the ranges seen in Siberian flying squirrels. Hence, the microsatellite loci that we designed for the Siberian flying squirrel may have limited utility for use in other squirrel species.