Characterization of microsatellite loci in the endangered long‐footed potoroo Potorous longipes

species with low allozyme variation (Hughes & Queller 1993; Paetkau et al. 1995). Such loci are increasingly used in population studies to elucidate mating systems, genetic relatedness, inbreeding, and the history of population size. Microsatellites can be analysed using noninvasive sampling techniques and minuscule samples such as single hairs, and therefore are especially useful for studying endangered species and individuals that are difficult to capture (Morin et al. 1994; Taylor et al. 1994). The long-footed potoroo Potorous longipes is a small (1.6–2.2 kg) macropod in the rat-kangaroo family (Potoriodae). The major subfamily of rat-kangaroos, the Potorinae, comprises four genera with nine species, five of which are rare or endangered, and two of which have recently become extinct (Seebeck et al. 1989). Potorous longipes is known to occur in only two small areas of eastern and north-eastern Victoria, Australia, where logging and fox predation may be severely reducing potoroo population sizes. The status of the wild populations is difficult to ascertain because wild individuals are difficult to capture. The Zoological Society of Victoria maintains a captivebreeding population at the Healesville Sanctuary, but it may be suffering from inbreeding depression. This captive population was founded from only four individuals in 1980, and has recently suffered contagious disease epizootics and reproductive failures. Here, we characterize six microsatellite loci in the long-footed potoroo for use in managing and conserving both wild and captive populations. A partial genomic library was constructed using standard techniques (Estoup et al. 1993; Queller et al. 1993). Six clones containing (AC)n were identified with ‘n’ ranging from 10 to 22. Oligonucleotide primers were then designed for PCR amplification of the (AC)n microsatellites (Table 1). Microsatellite PCR was conducted on DNA isolated from frozen blood and/or hair samples from nine wild and nine captive potoroos. PCR amplifications were conducted in 10-μL volumes containing ≈ 1–100 ng of DNA, 125 μM dNTP, 0.15 μM concentrations of each primer (one of which was end-labelled with 33P-γATP), 1X Taq buffer, and 0.5 units of Taq DNA polymerase (Perkin Elmer). PCR was conducted in a Perkin-Elmer-Cetus 480 Thermocycler under the following conditions: 3 min at 94 °C followed by 30 cycles of 30 s at 94 °C, 30 s at the annealing temperature (Table 1), 30 s at 71 °C, followed by 5 min at 71 °C. PCR products were resolved on urea-polyacrylamide (6%) gels, and allele lengths were determined using a sequencing size ladder. The mean number of alleles per locus was 3.7 in the wild population from Bellbird Creek in East Gippsland, Victoria (Table 1). The number of alleles is encouraging given that only nine individuals were assayed. However, more individuals and loci must be analysed before inferring whether the population is suffering a severe a reduction in size, as is feared. Surveys of the population at Buffalo River in north-eastern Victoria, would improve assessment of the recent history of the species. Newly developed statistical tests for detecting recent historical bottlenecks from allele frequency data would also help determine the status of the wild populations (Cornuet & Luikart 1996). The P. longipes primers were also used to try to amplify microsatellite loci from other members of the sub-family Potorinae. These were: P. tridactylus, Bettongia tropica, B. penicillata, and B. lesueur. Only one or two individuals from each species were assayed. None the less, microsatellite PCR products were similar in pattern and length to those from P. longipes. Such results are important because the three Bettongia species are currently regarded as rare or endangered. The cross-species amplification demonstrated above suggests that primers isolated from one species can be used to ascertain levels of genetic variation in closely related species, as has been found in other microsatellite studies of vertebrates (Taylor et al. 1994; Valsecchi & Amos 1996; Vaiman et al. 1996). Thus, we are optimistic that the markers reported here will assist in studies of other endangered Potoroines, such as the recently rediscovered Gilbert’s potoroo from Western Australia. P R I M E R N O T E