Paternity exclusion in koalas using hypervariable microsatellites

Koala microsatellite loci containing the dinucleotide motif (CA)n were isolated from a size-fractionated (250-500 bp) koala genomic library and sequenced. Six locus-specific primer pairs were designed and synthesized for DNA amplification using the polymerase chain reaction (PCR). Microsatellite genotyping of 12 individuals generated unique "fingerprints" for each koala. All six microsatellite loci were polymorphic, with a mean of 6.5 +/- 0.6 alleles/locus. This level of allelic diversity is capable of generating > 4 x 10(9) DNA profiles, making it the most powerful technology for fingerprinting koalas currently available. Observed heterozygosities (H(o)) in the eight unrelated individuals surveyed ranged from 0.25 to 0.75, with a mean of 0.54 +/- 0.06. Mendelian inheritance of the observed polymorphism was confirmed by family studies. We demonstrate that microsatellite loci are ideal genetic markers for paternity exclusion and pedigree analysis of koalas, which have shown little genetic variation using most other methods.