Optimisation of a microsatellite panel for the individual identification of brushtail possums using low template DNA

The Australian brushtail possum Trichosurus vulpecula is a pervasive marsupial pest of New Zealand. Impacting on the native flora and fauna and the nation’s livestock industry as a vector of bovine tuberculosis, T. vulpecula is a priority for control and eventual eradication. Possum control at present relies on conventional trapping and poisoning methods. Efficient allocation of control depends on accurate quantification of abundance, which could be achieved with the implementation of non-invasive sampling schemes. We evaluated the use of salivary DNA retrieved remotely as a source of DNA for microsatellite amplification. A panel of six loci were optimised using tissue samples from possums from three locations in the Canterbury Region, South Island, New Zealand. Optimised loci were then assembled into a multiplex PCR assay. Microsatellite diversity patterns revealed moderate to high polymorphism and heterozygosity, and a sufficiently low overall probability of identity adjusted for siblings (PI_sib = 3.0 × 10⁻³) to ensure a robust identification of individual possums based on their multi-locus genotype. While dilution of DNA extracted from tissue did not affect the results, the use of DNA from saliva significantly decreased the performance of the microsatellite amplification system. Altogether these results indicate that locus characteristics (i.e. amplicon size) and DNA quality are crucial factors affecting the sensitivity and reliability of this method.