Non-additive QTL mapping of lactation traits in 124,000 cattle reveals novel recessive loci.

BACKGROUND: Deleterious recessive conditions have been primarily studied in the context of Mendelian diseases. Recently, several deleterious recessive mutations with large effects were discovered via non-additive genome-wide association studies (GWAS) of quantitative growth and developmental traits in cattle, which showed that quantitative traits can be used as proxies of genetic disorders when such traits are indicative of whole-animal health status. We reasoned that lactation traits in cattle might also reflect genetic disorders, given the increased energy demands of lactation and the substantial stresses imposed on the animal. In this study, we screened more than 124,000 cows for recessive effects based on lactation traits. RESULTS: We discovered five novel quantitative trait loci (QTL) that are associated with large recessive impacts on three milk yield traits, with these loci presenting missense variants in the DOCK8, IL4R, KIAA0556, and SLC25A4 genes or premature stop variants in the ITGAL, LRCH4, and RBM34 genes, as candidate causal mutations. For two milk composition traits, we identified several previously reported additive QTL that display small dominance effects. By contrasting results from milk yield and milk composition phenotypes, we note differing genetic architectures. Compared to milk composition phenotypes, milk yield phenotypes had lower heritabilities and were associated with fewer additive QTL but had a higher non-additive genetic variance and were associated with a higher proportion of loci exhibiting dominance. CONCLUSIONS: We identified large-effect recessive QTL which are segregating at surprisingly high frequencies in cattle. We speculate that the differences in genetic architecture between milk yield and milk composition phenotypes derive from underlying dissimilarities in the cellular and molecular representation of these traits, with yield phenotypes acting as a better proxy of underlying biological disorders through presentation of a larger number of major recessive impacts.

Non-additive association analysis using proxy phenotypes identifies novel cattle syndromes.

Mammalian species carry ~100 loss-of-function variants per individual1,2, where ~1-5 of these impact essential genes and cause embryonic lethality or severe disease when homozygous3. The functions of the remainder are more difficult to resolve, although the assumption is that these variants impact fitness in less manifest ways. Here we report one of the largest sequence-resolution screens of cattle to date, targeting discovery and validation of non-additive effects in 130,725 animals. We highlight six novel recessive loci with impacts generally exceeding the largest-effect variants identified from additive genome-wide association studies, presenting analogs of human diseases and hitherto-unrecognized disorders. These loci present compelling missense (PLCD4, MTRF1 and DPF2), premature stop (MUS81) and splice-disrupting (GALNT2 and FGD4) mutations, together explaining substantial proportions of inbreeding depression. These results demonstrate that the frequency distribution of deleterious alleles segregating in selected species can afford sufficient power to directly map novel disorders, presenting selection opportunities to minimize the incidence of genetic disease.

Development of Epigenetic Clocks for Key Ruminant Species.

Robust biomarkers of chronological age have been developed in humans and model mammalian species such as rats and mice using DNA methylation data. The concept of these so-called "epigenetic clocks" has emerged from a large body of literature describing the relationship between genome-wide methylation levels and age. Epigenetic clocks exploit this phenomenon and use small panels of differentially methylated cytosine (CpG) sites to make robust predictions of chronological age, independent of tissue type. Here, we present highly accurate livestock epigenetic clocks for which we have used the custom mammalian methylation array "HorvathMammalMethyl40" to construct the first epigenetic clock for domesticated goat (Capra hircus), cattle (Bos taurus), Red (Cervus elaphus) and Wapiti deer (Cervus canadensis) and composite-breed sheep (Ovis aries). Additionally, we have constructed a 'farm animal clock' for all species included in the study, which will allow for robust predictions to be extended to various breeds/strains. The farm animal clock shows similarly high accuracy to the individual species' clocks (r > 0.97), utilizing only 217 CpG sites to estimate age (relative to the maximum lifespan of the species) with a single mathematical model. We hypothesise that the applications of this livestock clock could extend well beyond the scope of chronological age estimates. Many independent studies have demonstrated that a deviation between true age and clock derived molecular age is indicative of past and/or present health (including stress) status. There is, therefore, untapped potential to utilize livestock clocks in breeding programs as a predictor for age-related production traits.

De novo assembly of the cattle reference genome with single-molecule sequencing.

BACKGROUND: Major advances in selection progress for cattle have been made following the introduction of genomic tools over the past 10-12 years. These tools depend upon the Bos taurus reference genome (UMD3.1.1), which was created using now-outdated technologies and is hindered by a variety of deficiencies and inaccuracies. RESULTS: We present the new reference genome for cattle, ARS-UCD1.2, based on the same animal as the original to facilitate transfer and interpretation of results obtained from the earlier version, but applying a combination of modern technologies in a de novo assembly to increase continuity, accuracy, and completeness. The assembly includes 2.7 Gb and is >250× more continuous than the original assembly, with contig N50 >25 Mb and L50 of 32. We also greatly expanded supporting RNA-based data for annotation that identifies 30,396 total genes (21,039 protein coding). The new reference assembly is accessible in annotated form for public use. CONCLUSIONS: We demonstrate that improved continuity of assembled sequence warrants the adoption of ARS-UCD1.2 as the new cattle reference genome and that increased assembly accuracy will benefit future research on this species.

Genome-wide association analysis reveals QTL and candidate mutations involved in white spotting in cattle.

BACKGROUND: White spotting of the coat is a characteristic trait of various domestic species including cattle and other mammals. It is a hallmark of Holstein-Friesian cattle, and several previous studies have detected genetic loci with major effects for white spotting in animals with Holstein-Friesian ancestry. Here, our aim was to better understand the underlying genetic and molecular mechanisms of white spotting, by conducting the largest mapping study for this trait in cattle, to date. RESULTS: Using imputed whole-genome sequence data, we conducted a genome-wide association analysis in 2973 mixed-breed cows and bulls. Highly significant quantitative trait loci (QTL) were found on chromosomes 6 and 22, highlighting the well-established coat color genes KIT and MITF as likely responsible for these effects. These results are in broad agreement with previous studies, although we also report a third significant QTL on chromosome 2 that appears to be novel. This signal maps immediately adjacent to the PAX3 gene, which encodes a known transcription factor that controls MITF expression and is the causal locus for white spotting in horses. More detailed examination of these loci revealed a candidate causal mutation in PAX3 (p.Thr424Met), and another candidate mutation (rs209784468) within a conserved element in intron 2 of MITF transcripts expressed in the skin. These analyses also revealed a mechanistic ambiguity at the chromosome 6 locus, where highly dispersed association signals suggested multiple or multiallelic QTL involving KIT and/or other genes in this region. CONCLUSIONS: Our findings extend those of previous studies that reported KIT as a likely causal gene for white spotting, and report novel associations between candidate causal mutations in both the MITF and PAX3 genes. The sizes of the effects of these QTL are substantial, and could be used to select animals with darker, or conversely whiter, coats depending on the desired characteristics.

Multiple QTL underlie milk phenotypes at the CSF2RB locus.

BACKGROUND: Over many years, artificial selection has substantially improved milk production by cows. However, the genes that underlie milk production quantitative trait loci (QTL) remain relatively poorly characterised. Here, we investigate a previously reported QTL located at the CSF2RB locus on chromosome 5, for several milk production phenotypes, to better understand its underlying genetic and molecular causes. RESULTS: Using a population of 29,350 taurine dairy cows, 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 11 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 responsible for 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 that involves the CSF2RB gene. CONCLUSIONS: This 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.

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, characterize, and conduct genetic mapping studies of novel transcript edits. Using a discovery population of nine deeply sequenced cows, we identify 2413 edit sites in the mammary transcriptome, the majority of which are adenosine to inosine edits (98.6%). Most sites are predicted to reside in double-stranded secondary structures (85.1%), 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 highlight 152 cis-regulated edQTL, of which 15 appear to cosegregate with expression QTL effects. Trait association analyses in a separate population of 9989 lactating cows also shows 12 of the cis-edQTL coincide with at least one cosegregating 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.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

The CD4(+) T cell methylome contributes to a distinct CD4(+) T cell transcriptional signature in Mycobacterium bovis-infected cattle.

We hypothesised that epigenetic regulation of CD4(+) T lymphocytes contributes to a shift toward a dysfunctional T cell phenotype which may impact on their ability to clear mycobacterial infection. Combined RNA-seq transcriptomic profiling and Reduced Representation Bisulfite Sequencing identified 193 significantly differentially expressed genes and 760 differentially methylated regions (DMRs), between CD4(+) T cells from M. bovis infected and healthy cattle. 196 DMRs were located within 10 kb of annotated genes, including GATA3 and RORC, both of which encode transcription factors that promote TH2 and TH17 T helper cell subsets respectively. Gene-specific DNA methylation and gene expression levels for the TNFRSF4 and Interferon-γ genes were significantly negatively correlated suggesting a regulatory relationship. Pathway analysis of DMRs identified enrichment of genes involved in the anti-proliferative TGF-ß signaling pathway and TGFB1 expression was significantly increased in peripheral blood leukocytes from TB-infected cattle. This first analysis of the bovine CD4(+) T cell methylome suggests that DNA methylation directly contributes to a distinct gene expression signature in CD4(+) T cells from cattle infected with M. bovis. Specific methylation changes proximal to key inflammatory gene loci may be critical to the emergence of a non-protective CD4(+) T cell response during mycobacterial infection in cattle.

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.

Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project.

We describe the organization of a nascent international effort, the Functional Annotation of Animal Genomes (FAANG) project, whose aim is to produce comprehensive maps of functional elements in the genomes of domesticated animal species.

Identification of animals produced by somatic cell nuclear transfer using DNA methylation in the retrotransposon-like 1 promoter.

Public perception of somatic cell nuclear transfer (SCNT) in the production of agricultural animals is surrounded by fear, which is exacerbated by the inability to differentiate animals generated by SCNT from those generated by natural mating or artificial insemination (AI). Unfortunately, the DNA sequence of animals produced by SCNT is indistinguishable from those generated by fertilization. With the current banning of all SCNT animal products from entering the food supply in some countries, the lack of a diagnostic test to identify SCNT animals may jeopardize market access for producers. The aim of this research was to exploit differences in epigenetic reprogramming that occur during SCNT and fertilization in the early embryo. The resulting differences in epigenetic signatures that persist to adulthood are proposed as the basis for a diagnostic test to identify animals generated by SCNT. Here we describe differences in DNA methylation at eight CpG sites in the retrotransposon-like 1 (Rtl1) promoter region in cattle blood and test whether these differences could be used as a diagnostic tool. For a definitive diagnosis, it is critical that no overlap in DNA methylation levels is observed between individuals produced by SCNT and fertilization. This was the case for the cohort of SCNT animals studied, their female half-siblings generated by AI, and a collection of unrelated cows also generated by AI. Further rigorous testing is required to determine what effects donor cell type, age, sex, genetic background, SCNT methods, and the environment have on the DNA methylation across this region, but the Rtl1 promoter is currently a promising candidate for the identification of SCNT generated cattle.

Assessing DNA methylation levels in animals: choosing the right tool for the job.

Selection of agricultural animals for improved performance based on genetics has seen significant progress made over the past few decades. Further improvements are likely by combining genetic selection with epigenetic selection or manipulation. However, before this can be undertaken, an understanding of epigenetic mechanisms is required, and this can be obtained only by precise and accurate analysis of epigenetic patterns. Even when one only considers a single epigenetic modification such as DNA methylation, the last 10 years have seen a wide array of technologies developed. For scientists whose primary training is in a field other than epigenetics, the choices can be confusing, and it can be challenging to determine which technology is best for the task at hand. There are many factors to take into consideration before beginning analysis of DNA methylation in animals. It is crucial that the most appropriate tools are selected to ensure that the best possible results are achieved. This review provides an overview of the most common methods of analysing DNA methylation in animals, when they are appropriate, what resolution of information they can provide and what their limitations are.

Genome-wide DNA methylation patterns and transcription analysis in sheep muscle.

DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS). While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep Longissimus dorsi muscles. RRBS analysis of ∼1% of the genome from Longissimus dorsi muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep Longissimus dorsi muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species.

Exploring genome wide bisulfite sequencing for DNA methylation analysis in livestock: a technical assessment.

Recent advances made in "omics" technologies are contributing to a revolution in livestock selection and breeding practices. Epigenetic mechanisms, including DNA methylation are important determinants for the control of gene expression in mammals. DNA methylation research will help our understanding of how environmental factors contribute to phenotypic variation of complex production and health traits. High-throughput sequencing is a vital tool for the comprehensive analysis of DNA methylation, and bisulfite-based strategies coupled with DNA sequencing allows for quantitative, site-specific methylation analysis at the genome level or genome wide. Reduced representation bisulfite sequencing (RRBS) and more recently whole genome bisulfite sequencing (WGBS) have proven to be effective techniques for studying DNA methylation in both humans and mice. Here we report the development of RRBS and WGBS for use in sheep, the first application of this technology in livestock species. Important technical issues associated with these methodologies including fragment size selection and sequence depth are examined and discussed.

DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low.

Primary transgenic bovine cells and their rejuvenated cloned equivalents show transgene-specific epigenetic differences.

Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, have a limited lifespan, and complex genetic modifications that require sequential transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is frequently used to rejuvenate the cell lines and restore exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment. Our transgene contains a puromycin resistance marker gene and an enhanced green fluorescence protein (EGFP) expression cassette, both driven by a strong mammalian promoter, and flanked by loxP sites and sequences from the bovine ß-casein locus. Several transgenic cell lines were generated by random insertion into primary bovine cell lines. Two of these original cell lines were rederived by SCNT and new primary cells, with the same genetic makeup as the original donors, were established. While the original cell lines were puromycin-resistant and had a characteristic EGFP expression profile, all rejuvenated cell lines were sensitive to puromycin, and displayed varied EGFP expression, indicative of various degrees of silencing. When the methylation states of individual CpG sites within the transgene were analyzed, a striking increase in transgene-specific methylation was observed in all rederived cell lines. The results indicate that original transgenic donor cells and their rejuvenated derivatives may not be equivalent and differ in the functionality of their transgene sequences.

DNA methylation patterns are appropriately established in the sperm of bulls generated by somatic cell nuclear transfer.

The cloning of animals by somatic cell nuclear transfer (SCNT) has the potential to allow rapid dissemination of desirable traits from elite animals. However, concern has been expressed that aberrant epigenetic marks in SCNT-derived animals may be passed onto the next generation, even though the offspring of clones appear to be mainly normal. Here, we compared the DNA methylation patterns at 10 genomic regions in sperm from SCNT bulls with that from normal, naturally conceived bulls and with the nuclear donor somatic cells. Eight of the 10 genomic regions were differentially methylated in sperm compared with the donor cell DNA. All three satellite sequences examined here were less methylated in sperm than in the donor cells, contradicting the belief that the sperm genome is always highly methylated. The DNA methylation patterns at all 10 regions were almost identical between SCNT and control sperm, with only one out of the 175 CpG sites/groups of sites examined showing significant difference. These results provide the first molecular evidence that the donor cell genome is correctly reprogrammed upon passage through the germ line in males, and that any epigenetic aberrations harbored by SCNT bulls are unlikely to be passed onto their offspring.

DNA methylation patterns in tissues from mid-gestation bovine foetuses produced by somatic cell nuclear transfer show subtle abnormalities in nuclear reprogramming.

BACKGROUND: Cloning of cattle by somatic cell nuclear transfer (SCNT) is associated with a high incidence of pregnancy failure characterized by abnormal placental and foetal development. These abnormalities are thought to be due, in part, to incomplete re-setting of the epigenetic state of DNA in the donor somatic cell nucleus to a state that is capable of driving embryonic and foetal development to completion. Here, we tested the hypothesis that DNA methylation patterns were not appropriately established during nuclear reprogramming following SCNT. A panel of imprinted, non-imprinted genes and satellite repeat sequences was examined in tissues collected from viable and failing mid-gestation SCNT foetuses and compared with similar tissues from gestation-matched normal foetuses generated by artificial insemination (AI). RESULTS: Most of the genomic regions examined in tissues from viable and failing SCNT foetuses had DNA methylation patterns similar to those in comparable tissues from AI controls. However, statistically significant differences were found between SCNT and AI at specific CpG sites in some regions of the genome, particularly those associated with SNRPN and KCNQ1OT1, which tended to be hypomethylated in SCNT tissues. There was a high degree of variation between individuals in methylation levels at almost every CpG site in these two regions, even in AI controls. In other genomic regions, methylation levels at specific CpG sites were tightly controlled with little variation between individuals. Only one site (HAND1) showed a tissue-specific pattern of DNA methylation. Overall, DNA methylation patterns in tissues of failing foetuses were similar to apparently viable SCNT foetuses, although there were individuals showing extreme deviant patterns. CONCLUSION: These results show that SCNT foetuses that had developed to mid-gestation had largely undergone nuclear reprogramming and that the epigenetic signature at this stage was not a good predictor of whether the foetus would develop to term or not.