Isolation and characterization of simple sequence repeats in the genome of the common shrew

the most remarkable degrees of chromosomal variability in mammals (Zima et al. in press). The geographical distribution of these races, their complex pattern of genetic and karyotypic differentiation and the existence of several hybrid zones has attracted much attention over the past 10 years (review: Searle 1993). Consequently, the common shrew has become a model species to study the role of chromosomes in the process of population differentiation and speciation. More recently it has become the subject of gene mapping projects (Pack et al. 1995; Matyakhina 1996). At present, using a set of shrew–rodent somatic cell hybrid clones, 17 genes have been mapped (Pack et al. 1995; Matyakhina 1996). However, a major obstacle of using the common shrew in genetic studies is the current lack of sufficient polymorphic markers in the nuclear genome. An attractive source of markers which show abundant polymorphism is provided by simple sequence repeats (SSRs), also called microsatellites. SSRs are tandemly repeated DNA sequences which have proved to be both abundant and highly polymorphic in mammalian genomes (Hamada et al. 1982; Weber 1990; Stallings et al. 1991). Besides their importance in human genetic diseases, such as neurological disorders and cancer (Hamada et al. 1982; Weber 1990; Stallings et al. 1991; Wooster et al. 1994; Sutherland & Richards 1995), and their usefulness in genome mapping (Hamada et al. 1982; Weber 1990; Stallings et al. 1991; Weissenbach et al. 1992; Wooster et al. 1994), there is a great potential for the use of SSRs in population genetic studies (Bruford & Wayne 1993). In this paper we show that the genome of the common shrew contains abundant SSRs. We characterize eight polymorphic loci for use in population genetic studies in Sorex and investigate the level of conservation and polymorphism of these loci in other members of the order Insectivora. A partial genomic library was constructed with genomic DNA purified from common shrew spleen. The protocol as described by Rassmann et al. (1991) was followed with the following modifications: DNA fragments between 300 and 600 bp were cloned in a pBluescript II KS+ vector (Stratagene). The library contained 2000 recombinant clones. Seventy (3.5%) gave a positive hybridization signal after screening with a mixture of (TC)10–(TG)10 oligonucleotide probe labelled with the DIG system (Boehringer) and in subsequent Southern blotting assays. Inserts of 25 clones were sequenced from which 20 clones contained a perfect (AC)n repeat (two compound with a trinucleotide), two clones an imperfect (AC)n repeat and three clones a perfect (AG)n repeat following the nomenclature of Weber (1990). The same procedure was applied to screen 1500 clones with a (CCT)8oligonucleotide probe. Eight positive clones were found, from which three were sequenced and confirmed to be (GGA)n repeats. Primers flanking each repeat were designed for 15 loci using the computer program PRIMER 1.0 (Lincoln et al. 1993). Seven SSR-loci were monomorphic or showed unclear banding patterns, whereas eight SSR-loci gave satisfactory amplification results and, for the most part, showed extensive polymorphism (3–37 different alleles) (Table 1). These data are based on samples from four chromosomal races of Switzerland and France. Within chromosomal races, loci exhibited between three and 15 alleles and only locus 9 had 28 alleles (data not shown). When pooling all data, the allele frequency distributions at the eight loci were irregular showing bimodal or multimodal distributions. In order to test whether the loci were in Hardy– Weinberg equilibrium and if they were independent, we performed exact tests using the G E N E P O P package version 1.2 (Raymond & Rousset 1995). The samples used for this analysis were collected in 1994 in one valley (5 km stretch) of the French Alps and were partitioned into 15 subsets (n = 5–15). There was no departure from panmixia locally, but a classical F-statistic analysis using F S T A T 1.2 (Goudet 1995) indicated some degree of population subdivision (FST = 0.02, P < 0.001) and thus overall a slight departure from Hardy–Weinberg equilibrium. Exact tests for P R I M E R N O T E