The possession of fur or hair is a defining characteristic of mammals and can occur in a variety of colours and patterns. While genetic determinants of coat colour are well described in eutherian ‘placental’ mammals, the other major mammalian infraclass, marsupials, is grossly understudied. The fur of the common brushtail possum (Trichosurus vulpecula), an iconic native mammal found throughout Australia and introduced into Aotearoa New Zealand, possesses two main colour morphs: grey and black. To identify genetic variants associated with coat colour, we performed a genome wide association study (GWAS) with genotype by sequencing (GBS) data. Single nucleotide polymorphisms (SNPs) on chromosome 3, close to the agouti signalling protein (ASIP) gene that controls the temporal and spatial distribution of pigments in eutherian mammals, were identified. Fine-mapping identified a C>T variant at chr3:100,483,705 that results in a p.Arg115Cys substitution of the ASIP protein, and animals homozygous for this variant have black fur. In addition to uncovering the first genetic determinant of coat colour in a natural marsupial population, comparative analysis of ASIP in divergent marsupial species identified the Dasyurids as having accelerated evolution, reflecting their well described diversity of coat colour and pattern.

A dual-indexing, four-primer PCR-based assay was used for amplicon sequencing, as described in Bond et al., 2023. Both the first and second round of PCR amplification were performed using Phusion High-Fidelity DNA Polymerase (M0530L; New England Biolabs) according to the manufacturers’ protocol. For the first round of amplification, approximately 10–25 ng of DNA was used in the following PCR mix: 1x HF Buffer with 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.5 µM of each primer, and 0.02 U/µL units of Phusion, with the following PCR cycling conditions:  98 °C for 3 min; 27 cycles of 98 °C for 10 s, 62 °C for 20 s, and 72 °C for 15 s; 72 °C for 2 mins. The PCR reaction was cleaned up and sized selected using 0.9X SPRI beads diluted in standard PEG buffer (18% w/v polyethylene glycol 8000 (PEG), 1 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 0.05% v/v Tween-20) (Oberacker et al., 2023). The DNA was eluted in 10 µL nuclease-free water and 2 µL was used as a template in a second round of PCR amplification (using the same PCR conditions as described above) with 0.2 µM of each second step primer (indexed Truseq-type oligos for Illumina sequencing). PCR cycling parameters for the second step PCR were: 98 °C for 3 min; 5 cycles of 98 °C for 10 s, 62 °C for 20 s, and 72 °C for 15 s; 72 °C for 2 min. The final libraries were pooled in equimolar amounts, cleaned-up and size-selected using 0.9X SPRI beads diluted in standard PEG buffer and sequenced on the iSeq100 (Illumina) to generate 150 bp paired-end reads. All primers used for amplicon sequencing are given in S2 Table.