The first step in developing our method was to make a reference collection of leaf samples from tree species that koalas might feed from. We then extracted and sequenced DNA from these samples and looked for differences in the DNA sequence that were unique to, or characteristic of, each tree species. Some of these DNA differences were eventually suitable for use as markers, meaning we could look for these sequences in DNA extracted from koala pellets to reveal whether the koala in question had recently fed from a tree of that species. To ensure that the potential markers were common to all or most individuals of a tree species, and that they did not commonly occur in other tree species as well, we collected leaves from multiple individuals of each tree species, across multiple sites. To build a panel of markers for analysing koala diet composition in Queensland, we used DNA from samples collected specifically for this project and for other previous projects, where appropriate. The locations of trees that we used to build our panel are shown on the map below. In total we included DNA from 730 trees. These represented 99 tree species, including some non-eucalypts such as brush box, turpentine and Acacia.

A complete list of marker allele sequences for potential koala food tree species can be downloaded here: accepted_set_of_qld_panel_markers.xlsx