Kathryn Tiplady

Functionally reciprocal mutations of the prolactin signalling pathway define hairy and slick cattle

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.

Sequence-based Association Analysis Reveals an MGST1 eQTL with Pleiotropic Effects on Bovine Milk Composition.

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.

Expression Variants of the Lipogenic AGPAT6 Gene Affect Diverse Milk Composition Phenotypes in Bos taurus

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.

Functional confirmation of PLAG1 as the candidate causative gene underlying major pleiotropic effects on body weight and milk characteristics

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.

Detection and assessment of copy number variation using PacBio long read and Illumina sequencing in New Zealand dairy cattle

Single nucleotide polymorphisms have been the DNA variant of choice for genomic prediction, largely because of the ease of single nucleotide polymorphism genotype collection. In contrast, structural variants (SV), which include copy number variants (CNV), translocations, insertions, and inversions, have eluded easy detection and characterization, particularly in nonhuman species. However, evidence increasingly shows that SV not only contribute a substantial proportion of genetic variation but also have significant influence on phenotypes. Here we present the discovery of CNV in a prominent New Zealand dairy bull using long-read PacBio (Pacific Biosciences, Menlo Park, CA) sequencing technology and the Sniffles SV discovery tool (version 0.0.1; https://github.com/fritzsedlazeck/Sniffles). The CNV identified from long reads were compared with CNV discovered in the same bull from Illumina sequencing using CNVnator (read depth-based tool; Illumina Inc., San Diego, CA) as a means of validation. Subsequently, further validation was undertaken using whole-genome Illumina sequencing of 556 cattle representing the wider New Zealand dairy cattle population. Very limited overlap was observed in CNV discovered from the 2 sequencing platforms, in part because of the differences in size of CNV detected. Only a few CNV were therefore able to be validated using this approach. However, the ability to use CNVnator to genotype the 557 cattle for copy number across all regions identified as putative CNV allowed a genome-wide assessment of transmission level of copy number based on pedigree. The more highly transmissible a putative CNV region was observed to be, the more likely the distribution of copy number was multimodal across the 557 sequenced animals. Furthermore, visual assessment of highly transmissible CNV regions provided evidence supporting the presence of CNV across the sequenced animals. This transmission-based approach was able to confirm a subset of CNV that segregates in the New Zealand dairy cattle population. Genome-wide identification and validation of CNV is an important step toward their inclusion in genomic selection strategies.

DNA and RNA-sequence based GWAS highlights membrane-transport genes as key modulators of milk lactose content

BackgroundLactose 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.ResultsUsing 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.ConclusionsOverall, 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.

Widespread cis-regulation of RNA-editing in a large mammal

Post-transcriptional RNA editing may regulate transcript expression and diversity in cells, with potential impacts on various aspects of physiology and environmental adaptation. A small number of recent genome-wide studies in Drosophila, mouse, and human have shown that RNA editing can be genetically modulated, highlighting loci that quantitatively impact editing of transcripts. The potential gene expression and physiological consequences of these RNA editing quantitative trait loci (edQTL), however, are almost entirely unknown. Here, we present analyses of RNA editing in a large domestic mammal (Bos taurus), where we use whole genome and high depth RNA sequencing to discover, characterise, and conduct genetic mapping studies of novel transcript edits. Using a discovery population of nine deeply-sequenced cows, we identify 2,001 edit sites in the mammary transcriptome, the majority of which are adenosine to inosine edits (97.4%). Most sites are predicted to reside in double-stranded secondary structures (85.7%), and quantification of the rates of editing in an additional 355 cows reveals editing is negatively correlated with gene expression in the majority of cases. Genetic analyses of RNA editing and gene expression highlights 67 cis-regulated edQTL, of which seven appear to co-segregate with expression QTL effects. Trait association analyses in a separate population of 9,988 lactating cows also shows nine of the cis-edQTL coincide with at least one co-segregating lactation QTL. Together, these results enhance our understanding of RNA editing dynamics in mammals, and suggest mechanistic links by which loci may impact phenotype through RNA-editing mediated processes.

Multiple QTL underlie milk phenotypes at the CSF2RB locus

Background Bovine milk provides an important source of nutrition in much of the Western world, forming components of many food products. Over many years, artificial selection has substantially improved milk production by cows. However, the genes underlying milk production quantitative trait loci (QTL) remain relatively poorly characterised. Here, we investigate a previously-reported QTL located at the CSF2RB locus, for several milk production phenotypes, to better understand its underlying genetic and molecular causes. Results Using a population of 29,350 taurine dairy cattle, we conducted association analyses for milk yield and composition traits, and identified highly significant QTL for milk yield, milk fat concentration, and milk protein concentration. Strikingly, protein concentration and milk yield appear to show co-located yet genetically distinct QTL. To attempt to understand the molecular mechanisms that might be mediating these effects, gene expression data were used to investigate eQTL for eleven genes in the broader interval. This analysis highlighted genetic impacts on CSF2RB and NCF4 expression that share similar association signatures to those observed for lactation QTL, strongly implicating one or both of these genes as the cause of these effects. Using the same gene expression dataset representing 357 lactating cows, we also identified 38 novel RNA editing sites in the 3′ UTR of CSF2RB transcripts. The extent to which two of these sites were edited also appears to be genetically co-regulated with lactation QTL, highlighting a further layer of regulatory complexity implicating the CSF2RB gene. Conclusions This chromosome 5 locus presents a diversity of molecular and lactation QTL, likely representing multiple overlapping effects that, at a minimum, highlight the CSF2RB gene as having a causal role in these processes.

Mating strategies to maximize genetic merit in dairy cattle herds

The genetic merit of a herd is a key determinant in productivity for dairy farmers. However, making breeding decisions to maximize the rate of genetic gain can be complex because there is no certainty about which cows will become pregnant with a heifer calf. In this study, breeding worth (BrW) was used as a measure of genetic merit, and several mating strategies were evaluated. These strategies included randomly mating whole herds to the entire bull team, excluding low-ranked cows from producing replacement heifers, and nominating high-ranked cows to the most highly ranked bulls. Simulations were undertaken using 4 bull teams generated from bulls currently marketed in New Zealand and a selection of New Zealand dairy herds. Average replacement heifer BrW was calculated for 1,000 iterations of each combination of mating strategy, herd, and bull team (scenario). Variation in resulting average replacement heifer BrW within scenarios was due to random sampling of which cows became pregnant with a heifer calf. Relative to mating the whole herd to an entire bull team, excluding the lowest ranked cows from producing replacements resulted in the greatest increase in average replacement heifer BrW across all herds and bull teams, with a gain of approximately 0.4 BrW point for each 1% of cows excluded. Nominating top-ranking cows to the highest ranking bulls in the team had little effect (0.06-0.13 BrW increase for each 1% of top cows nominated) in improving BrW of replacement heifers. The number of top bulls nominated had a variable effect depending on the BrW spread of the entire bull team. Although excluding cows with the lowest BrW from producing replacement heifers is most effective for improving BrW, it is important to ensure that the number of heifers born is sufficient to replace cows leaving the herd. It is likely that optimal strategies for improving BrW will vary from farm to farm depending not only on the BrW structure of the herd, the bull team available, and the reproduction success on farm but also on farm management practices. This simulation study provides expected outcomes from a variety of mating strategies to allow informed decision making on farm.

Bovine mammary gland X chromosome inactivation

X chromosome inactivation (XCI) is a process by which 1 of the 2 copies of the X chromosomes present in female mammals is inactivated. The transcriptional silencing of one X chromosome achieves dosage compensation between XX females and XY males and ensures equal expression of X-linked genes in both sexes. Although all mammals use this form of dosage compensation, the complex mechanisms that regulate XCI vary between species, tissues, and development. These mechanisms include not only varying levels of inactivation, but also the nature of inactivation, which can range from being random in nature to driven by parent of origin. To date, no data describing XCI in calves or adult cattle have been reported and we are reliant on data from mice to infer potential mechanisms and timings for this process. In the context of dairy cattle breeding and genomic prediction, the implications of X chromosome inheritance and XCI in the mammary gland are particularly important where a relatively small number of bulls pass their single X chromosome on to all of their daughters. We describe here the use of RNA-seq, whole genome sequencing and Illumina BovineHD BeadChip (Illumina, San Diego, CA) genotypes to assess XCI in lactating mammary glands of dairy cattle. At a population level, maternally and paternally inherited copies of the X chromosome are expressed equally in the lactating mammary gland consistent with random inactivation of the X chromosome. However, average expression of the paternal chromosome ranged from 10 to 90% depending on the individual animal. These results suggest that either the mammary gland arises from 1 or 2 stem cells, or a nongenetic mechanism that skews XCI exists. Although a considerable amount of future work is required to fully understand XCI in cattle, the data reported here represent an initial step in ensuring that X chromosome variation is captured and used in an appropriate manner for future genomic selection.