Functional and Genetic Characterisation of Major Effect Milk Composition Variants in Dairy Cattle

Abstract

Bovine milk production and composition traits are complex quantitative phenotypes, reflecting the interplay of many environmental and genetic factors influencing mammary and lactation biology. Despite a largely quantitative architecture, genetic mapping studies have identified a handful of major effect loci in dairy cattle, each containing a large number of trait-associated sequence variants. Identifying the specific variant causally responsible at these genetic signals requires extensive experimental follow-up, where many of the genomic regions implicate present no obvious candidate mutations. The association signal is instead represented by many non-coding variants that are difficult to functionally prioritise. The aim of the research described in this thesis was to investigate four such loci segregating in NZ dairy cattle, aiming to mechanistically link causative genes and variants to the milk production and composition effects. The results of this thesis describe the identification of expression quantitative trait loci at each of the four major associated regions, providing evidence that the variants responsible exert their phenotypic influence through the modulation of mammary gene expression. Further genetic and functional experiments were conducted to elucidate the mechanism by which the implicated variants impact gene expression. Highlights of this work include the demonstration of a new mechanism of effect for the largest and most highly cited causal mutation in the bovine literature. This work also reports the discovery of major milk production effects for a mutation previously only implicated in bovine growth and stature. This thesis also reports the development and optimisation of CRISPR-Cas9 mediated genome editing as a method for testing the function of regulatory variants directly. By presenting an efficient in vitro engineering approach in mammary cells, the work creates a framework to genetically isolate candidate variants, creating opportunities to study the major effect variants reported in this thesis, and investigate other as yet undefined milk composition loci. Taken together, the work presented in this thesis provides enhanced understanding of the mechanisms linking major effect genetic variants to bovine milk production and composition, and raises the profile of these and other variants as markers to incorporate in animal selection and breeding strategies.