The genetic characterisation of coat colour and patterning traits in cattle : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, AL Rae Centre for Genetics and Animal Breeding, Hamilton, New Zealand. EMBARGOED until until 1 January 2024.
Coat colour and patterning traits have been of interest to cattle breeders for centuries and have undergone intense selection due to their ability to provide easy means of breed identification. The striking coat colour and patterning traits observed in modern-day cattle breeds have reached fixation in many breed populations, and causal mutations for almost all major coat patterning traits in cattle have been resolved, with the exception of the white spotting trait characteristic of Holstein cattle. Despite this, the molecular mechanisms through which these mutations modulate pigmentation, and how they might interact with each other to produce different patterning traits, remain poorly understood. The aims of this thesis were to uncover the causal variants that contribute toward the proportion of white spotting on the coat, identify epistatic interactions between coat colour loci, and explore the implementation of alternative breeding solutions to introgress favourable coat-colour relevant genetic variants with minimal genetic drag. A combination of molecular, quantitative, and bioinformatic tools were utilised to discover a mutation in the protein-coding sequence of the PAX3 gene, a mutation within a highly conserved region of the MITF gene, and a novel structural variant upstream of the KIT gene, that all likely contribute towards the proportion of white spotting on the coat. Using genome-wide association analyses we also described an epistatic interaction between the MITF and KIT loci that causes a splotchy face in cattle with Hereford parentage, and an epistatic interaction between two mutations at the KIT locus that causes pigmentation around the eyes in white-faced Hereford cattle. Finally, unbiased whole-genome sequence analysis and long-molecule sequencing demonstrated that CRISPR-Cas9 gene-editing could be used to introgress a PMEL coat colour dilution mutation into a Holstein-Friesian background with no detectable off-target mutagenesis or genetic drag. This thesis reports novel candidate causal mutations and epistatic interactions previously unreported, and also the first study in cattle to investigate off-target mutations with the application of CRISPR-Cas9 gene-editing technologies. The results presented here provide insight into biological and physiological aspects of pigment biology, and enhance our understanding of these processes with relevance across mammals.