Microsatellites in the bottlenose dolphin Tursiops truncatus

common marine mammal species, found world-wide in temperate and tropical waters (Caldwell & Caldwell 1972). Over the last two decades this species has been the focus of extensive research, under captive and natural conditions, that has increased our knowledge of both its behaviour and social structure (e.g. Wells et al. 1987; Smoker et al. 1992). However, the lack of reports describing the genetic structure of the species makes it difficult to interpret behavioural and social findings from an evolutionary perspective. In this study, we describe five primer sets, designed from the sequences flanking the (GT)n dinucleotide repeat microsatellite regions, isolated from a male bottlenose dolphin genomic library. Our aim was to obtain sufficient variable genetic markers to permit the application of microsatellite technology to behavioural and social studies of wild populations of bottlenose dolphins. We also test the utility of the primer sets for the amplification of microsatellite regions in four other cetacean species. Nuclear DNA was isolated from a male bottlenose dolphin captured by the traditional driving fishery method, off the Pacific coast of Taiji, Japan. The procedures for the construction of the genomic library, detection of colonies containing (GT)n regions, plasmid isolation and sequencing were essentially the same as those described in Takenaka et al. (1993). Five simple sequence repeat loci were selected for long and perfect repeats and PCR primers were designed from the sequences flanking their repetitive regions. Muscle or blood samples were derived from 48 bottlenose dolphins, 14 Risso’s dolphins Grampus griseus, one spotted dolphin Stenella attenuata and 36 shortfinned pilot whales Globicephala macrorhynchus. All these animals were captured off the Pacific Coast of Taiji, Japan. Sloughed skin samples were collected from two humpback whales Megaptera novaeangliae found around Ogasawara waters. Template DNA was extracted from these samples by standard phenol–chloroform procedures. We performed PCR in the presence of 100 ng of template DNA for a final volume of 25 μL. The reaction mixture contained 12.5 pmol of each primer, 10% DMSO, 2 mM MgCl2, and 400 μM dNTP. An initial denaturation at 95 °C for 5 min was performed before the addition of 0.6 U of Taq DNA polymerase (Promega & Perkin Elmer Cetus). The reaction was carried out for 28 or 30 cycles including 1 min at 94 °C, 30 s at the annealing temperature, and 30 s at 72 °C. PCR products were run in 7.5% polyacrylamide gels and detected by silver staining following the protocol described by Tegelström (1986). Several gels were run, exchanging the order of the samples, to determine precisely the number and order of alleles found for each primer set and species. Table 1 shows the sequences of the five microsatellite regions selected, their corresponding primers, and the length of the fragment to be amplified for the dolphin used in the library construction. Five primer sets successfully amplified the target sequence and showed high levels of polymorphism in the bottlenose dolphin. Successful amplification was attained at the annealing temperature of 56 °C for all primers except for D22 which performed better at 54 °C. The latter primer was run in the PCR machine for 28 cycles, whereas the other four were run for 30 cycles. Table 1 shows the number of alleles and expected heterozygosities obtained for the bottlenose dolphins. Expected heterozygosity was calculated as (1–ΣPi2), where Pi is the frequency of the ith allele. These primer sets also amplified the target region in the samples from other four cetaceans. Table 2 shows the number of alleles and expected heterozygosities obtained for each primer set for each species. The cross-species amplification in other Odontoceti species and even in the humpback whale, a Mysticeti, confirms the findings of Schlötterer et al. (1991) that the sequences flanking microsatellite loci in cetaceans show an unusually high degree of conservation. The heterologous capability of the primers described in this and previous studies of cetaceans (Schlötterer et al. 1991; Amos et al. 1993; Buchanan et al. 1996) will permit the acquisition of genetic information essential to interpret numerous behavioural and ecological characteristics of the bottlenose dolphin and other cetacean species. P R I M E R N O T E