Lactation, hair development and homeothermy are characteristic evolutionary features that define mammals from other vertebrate species. Here we describe the discovery of two autosomal dominant mutations with antagonistic, pleiotropic effects on all three of these biological processes, mediated through the prolactin signalling pathway. Most conspicuously, mutations in prolactin (PRL) and its receptor (PRLR) have an impact on thermoregulation and hair morphology phenotypes, giving prominence to this pathway outside of its classical roles in lactation.

The mammary gland is a prolific lipogenic organ, synthesising copious amounts of triglycerides for secretion into milk. The fat content of milk varies widely both between and within species and recent independent genome-wide association studies have highlighted a milk fat percentage quantitative trait locus (QTL) of large effect on bovine chromosome 5. Although both EPS8 and MGST1 have been proposed to underlie these signals, the causative status of these genes has not been functionally confirmed. To investigate this QTL in detail, we report genome sequence-based imputation and association mapping in a population of 64,244 taurine cattle. This analysis reveals a cluster of 17 non-coding variants spanning MGST1 that are highly associated with milk fat percentage and a range of other milk composition traits. Further, we exploit a high-depth mammary RNA sequence dataset to conduct expression QTL (eQTL) mapping in 375 lactating cows, revealing a strong MGST1 eQTL underpinning these effects. These data demonstrate the utility of DNA and RNA sequence-based association mapping and implicate MGST1, a gene with no obvious mechanistic relationship to milk composition regulation, as causally involved in these processes.

Lactose provides an easily-digested energy source for neonates, and is the primary carbohydrate in milk in most species. Bovine lactose is also a key component of many human food products. However, compared to analyses of other milk components, the genetic control of lactose has been little studied. Here we present the first GWAS focussed on analysis of milk lactose traits. Using a discovery population of 12,000 taurine dairy cattle, we detail 27 QTL for lactose concentration and yield, and subsequently validate the effects of 26 of these loci in a distinct population of 18,000 cows. We next present data implicating causative genes and variants for these QTL. Fine mapping of these regions using imputed, whole genome sequence-resolution genotypes reveals protein-coding candidate causative variants affecting the ABCG2, DGAT1, STAT5B, KCNH4, NPFFR2 and RNF214 genes. Eleven of the remaining QTL appear to be driven by regulatory effects, suggested by the presence of co-locating, co-segregating eQTL discovered using mammary RNA sequence data from a population of 357 lactating cows. Pathway analysis of genes representing all lactose-associated loci shows significant enrichment of genes located in the endoplasmic reticulum, with functions related to ion channel activity mediated through the LRRC8C, P2RX4, KCNJ2 and ANKH genes. A number of the validated QTL are also found to be associated with additional milk volume, fat and protein phenotypes. Overall, these findings highlight novel candidate genes and variants involved in milk lactose regulation, whose impacts on membrane transport mechanisms reinforce the key osmo-regulatory roles of lactose in milk.

Milk is composed of a complex mixture of lipids, proteins, carbohydrates and various vitamins and minerals as a source of nutrition for young mammals. The composition of milk varies between individuals, with lipid composition in particular being highly heritable. Recent reports have highlighted a region of bovine chromosome 27 harbouring variants affecting milk fat percentage and fatty acid content. We aimed to further investigate this locus in two independent cattle populations, consisting of a Holstein-Friesian x Jersey crossbreed pedigree of 711 F2 cows, and a collection of 32,530 mixed ancestry Bos taurus cows. Bayesian genome-wide association mapping using markers imputed from the Illumina BovineHD chip revealed a large quantitative trait locus (QTL) for milk fat percentage on chromosome 27, present in both populations. We also investigated a range of other milk composition phenotypes, and report additional associations at this locus for fat yield, protein percentage and yield, lactose percentage and yield, milk volume, and the proportions of numerous milk fatty acids. We then used mammary RNA sequence data from 212 lactating cows to assess the transcript abundance of genes located in the milk fat percentage QTL interval. This analysis revealed a strong eQTL for AGPAT6, demonstrating that high milk fat percentage genotype is also additively associated with increased expression of the AGPAT6 gene. Finally, we used whole genome sequence data from six F1 sires to target a panel of novel AGPAT6 locus variants for genotyping in the F2 crossbreed population. Association analysis of 58 of these variants revealed highly significant association for polymorphisms mapping to the 5′UTR exons and intron 1 of AGPAT6. Taken together, these data suggest that variants affecting the expression of AGPAT6 are causally involved in differential milk fat synthesis, with pleiotropic consequences for a diverse range of other milk components.

A major pleiotropic quantitative trait locus (QTL) located at ~25 Mbp on bovine chromosome 14 affects a myriad of growth and developmental traits in Bos taurus and indicus breeds. These QTL have been attributed to two functional variants in the bidirectional promoter of PLAG1 and CHCHD7. Although PLAG1 is a good candidate for mediating these effects, its role remains uncertain given that these variants are also associated with expression of five additional genes at the broader locus. In the current study, we conducted expression QTL (eQTL) mapping of this region using a large, high depth mammary RNAseq dataset representing 375 lactating cows. Here we show that of the seven previously implicated genes, only PLAG1 and LYN are differentially expressed by QTL genotype, and only PLAG1 bears the same association signature of the growth and body weight QTLs. For the first time, we also report significant association of PLAG1 genotype with milk production traits, including milk fat, volume, and protein yield. Collectively, these data strongly suggest PLAG1 as the causative gene underlying this diverse range of traits, and demonstrate new effects for the locus on lactation phenotypes.

Polymorphisms underlying complex traits often explain a small part (less than 1 %) of the phenotypic variance (σ2 P). This makes identification of mutations underling complex traits difficult and usually only a subset of large-effect loci are identified. One approach to identify more loci is to increase sample size of experiments but here we propose an alternative. The aim of this paper is to use secondary phenotypes for genetically simple traits during the QTL discovery phase for complex traits. We demonstrate this approach in a dairy cattle data set where the complex traits were milk production phenotypes (fat, milk and protein yield; fat and protein percentage in milk) measured on thousands of individuals while secondary (potentially genetically simpler) traits are detailed milk composition traits (measurements of individual protein abundance, mineral and sugar concentrations; and gene expression). Quantitative trait loci (QTL) were identified using 11,527 Holstein cattle with milk production records and up to 444 cows with milk composition traits. There were eight regions that contained QTL for both milk production and a composition trait, including four novel regions. One region on BTAU1 affected both milk yield and phosphorous concentration in milk. The QTL interval included the gene SLC37A1, a phosphorous antiporter. The most significant imputed sequence variants in this region explained 0.001 σ2 P for milk yield, and 0.11 σ2 P for phosphorus concentration. Since the polymorphisms were non-coding, association mapping for SLC37A1 gene expression was performed using high depth mammary RNAseq data from a separate group of 371 lactating cows. This confirmed a strong eQTL for SLC37A1, with peak association at the same imputed sequence variants that were most significant for phosphorus concentration. Fitting any of these variants as covariables in the association analysis removed the QTL signal for milk production traits. Plausible causative mutations in the casein complex region were also identified using a similar strategy. Milk production traits in dairy cows are typical complex traits where polymorphisms explain only a small portion of the phenotypic variance. However, here we show that these mutations can have larger effects on secondary traits, such as concentrations of minerals, proteins and sugars in the milk, and expression levels of genes in mammary tissue. These larger effects were used to successfully map variants for milk production traits. Genetically simple traits also provide a direct biological link between possible causal mutations and the effect of these mutations on milk production.