Null microsatellite alleles due to nucleotide sequence variation in the grey‐sided vole Clethrionomys rufocanus

ed to occur at microsatellite loci in humans (Callen et al. 1993), deer (Pemberton et al. 1995) and bear (Paetkau & Strobeck 1995). Such null alleles are considered to be due to nucleotide sequence variation (i.e. a deletion or a base substitution) in the priming site of the oligonucleotide primers used for amplification. The presence of null alleles warrants caution for applying microsatellite data to parentage analyses and, particularly, population genetic studies, because the presence of null alleles might decrease the estimated frequency of heterozygotes, resulting in an overestimation of the level of inbreeding (Pemberton et al. 1995). In our previous population study, a significant heterozygote deficiency was observed at one of five microsatellite loci (MSCRB-3) in the grey-sided vole Clethrionomys rufocanus (Ishibashi et al. 1995). We ascribed this deficiency to the occurrence of null allele(s) at that locus or linkage to a selectively non-neutral locus (i.e. genetic hitchhiking). Here, we used redesigned primers to confirm the presence of two null alleles at the locus in individuals previously scored as homozygotes. These null alleles apparently reflect base substitutions in the nucleotide sequence of the priming site. In an attempt to identify null allele(s) at locus MSCRB-3, two primers, prF2, 5′-GCTACTTCTCACCTCTCTAGAT-3′, and prR2, 5′-CTCTAGCATGATGTTACTGT-3′ , were designed on the basis of the previously reported nucleotide sequence (DDBJ/EMBL D37834). These primer sites are 53 and 78 base pairs upstream from the 5′ end of the original primers, prF1, 5′-CATGACCTTCTATTTCTGTCAG-3′, and prR1, 5′-TAAAACCCCAGACAAAGAAGAT-3′, respectively (Ishibashi et al. 1995). Amplifications were performed using each of four combinations of these primers in 20 μl of reaction mixture containing 0.2 mM of dNTP, 0.25 μM of each primer, and 0.5 unit of Taq DNA polymerase (TaKaRa), in which one of the paired primers were 5’-end labelled with P. About 100 ng of genomic DNA extracted from 16 voles ‘homozygous’ at MSCRB-3 was used for each reaction. After denaturation at 93 °C for 2 min, the reaction was carried out for all primer combinations for 30–40 cycles under the following condition; 90 °C for 30 s, 48 °C for 30 s and 72 °C for 20 s. The products were separated by electrophoresis using 6% denatured polyacrylamide gels containing 7 M urea. When a novel band (allele), which had not been amplified previously with prF1 and prR1, was yielded using any of new primer combinations, the product of a vole was cloned and sequenced using the TA CloningTM Kit (Invitrogen Corporation). Direct sequencing from gelpurified DNA templates was also attempted for the other individuals. Of the 16 individuals examined, three showed two novel alleles when amplification was performed with the primer combination of prR2 and prF1 (arrowheads in Fig. 1a) or prF2 (data not shown), although no such alleles were observed in the products amplified with the combination of prR1 and prF1 (Fig. 1b) or prF2. The other 13 individuals, however, remained homozygous with the new primers. The nucleotide sequence of these novel alleles in all three individuals showed the same base substitutions around the priming site of prR1 (Fig. 2b) compared with the sequences described previously (Fig. 2a). No such sequence variation was observed around the priming site of prF1. The observed variation in the size of alleles was due to variation in the different number of CA and GA repeats. These results suggest that the nonamplifying alleles in these three voles can be explained by base substitutions within the priming site of prR1. Taking the present data, corrected heterozygosity for a total of 81 individuals in the examined population (Ishibashi et al. 1995) was 0.840, which, however, was still significantly smaller than the expected value, 0.939 (SE 0.0072; according to Nei 1987) (t = –13.83, P < 0.001). Failure in amplifying a novel allele using redesigned primers in the remaining 13 ‘homozygotes’ implies other sequence variations at the MSCRB-3, e.g. a deletion TECHNICAL NOTE