Emmeline Hill

University College Dublin | UCD · School of Agriculture & Food Science

Bos taurus (taurine) and Bos indicus (indicine) cattle diverged at least 150,000 years ago and, since that time, substantial genomic differences have evolved between the two lineages. During the last two millennia, genetic exchange in Africa has resulted in a complex tapestry of taurine-indicine ancestry, with most cattle populations exhibiting var...

Thoroughbred horses are bred for competitive racing and undergo intense training regimes. The maintenance of physical soundness and desirable behavioural characteristics are critical to the longevity of a racing career. Horses intended for Flat racing generally enter training as yearlings and undergo introductory training prior to exercise conditio...

Behavioural plasticity enables horses entering an exercise training programme to adapt with reduced stress. We characterised SNPs associated with behaviour in yearling Thoroughbred horses using genomics analyses for two phenotypes: (1) handler‐assessed coping with early training events [coping] (n = 96); and (2) variation in salivary cortisol conce...

Background Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we establ...

The proportion of the genome containing runs of homozygosity (ROH) affects production traits in livestock populations. In European and Australasian Thoroughbreds inbreeding, quantified using ROH (FROH), is associated with the probability of ever racing. Here, we measured FROH using 333 K SNP genotypes from 768 Thoroughbred horses born in North Amer...

Background Thousands of years of natural and artificial selection since the domestication of the horse has shaped the distinctive genomes of Chinese Mongolian horse populations. Consequently, genomic signatures of selection can provide insights into the human-mediated selection history of specific traits and evolutionary adaptation to diverse envir...

Selection for system-wide morphological, physiological, and metabolic adaptations has led to extreme athletic phenotypes among geographically diverse horse breeds. Here, we identify genes contributing to exercise adaptation in racehorses by applying genomics approaches for racing performance, an end-point athletic phenotype. Using an integrative ge...

Background: Thousands of years of natural and artificial selection since the domestication of the horse has shaped the distinctive genomes of Chinese Mongolian horse populations. Consequently, genomic signatures of selection can provide insights into the human-mediated selection history of specific traits and evolutionary adaptation to diverse envi...

Small effective population sizes and active inbreeding can lead to inbreeding depression due to deleterious recessive mutations exposed in the homozygous state. The Thoroughbred racehorse has low levels of population genetic diversity, but the effects of genomic inbreeding in the population are unknown. Here, we quantified inbreeding based on runs...

Although inspiratory muscle training (IMT) is reported to improve inspiratory muscle strength in humans little has been reported for horses. We tested the hypothesis that IMT would maintain and/or improve inspiratory muscle strength variables measured in Thoroughbreds during detraining. Thoroughbreds from one training yard were placed into a contro...

Thoroughbred horse racing is a global sport with major hubs in Europe, North America, Australasia and Japan. Regional preferences for certain traits have resulted in phenotypic variation that may result from adaptation to the local racing ecosystem. Here, we test the hypothesis that genes selected for regional phenotypic variation may be identified...

The Thoroughbred horse is a highly valued domestic animal population under strong selection for athletic phenotypes. Here we present a high resolution genomics-based analysis of inbreeding in the population that may form the basis for evidence-based discussion amid concerns in the breeding industry over the increasing use of small numbers of popula...

While over ten thousand genetic loci have been associated with phenotypic traits and inherited diseases in genome-wide association studies, in most cases only a relatively small proportion of the trait heritability is explained and biological mechanisms underpinning these traits have not been clearly identified. Expression quantitative trait loci (...

Although inspiratory muscle training (IMT) is reported to improve inspiratory muscle strength in humans little has been reported for horses. We tested the hypothesis that IMT would maintain and/or improve inspiratory muscle strength variables measured in Thoroughbreds during detraining. Thoroughbreds from one training yard were placed into a contro...

Coenzyme Q10 (CoQ10) is an essential component of the mitochondrial electron transport chain (ETC). Decreased skeletal muscle CoQ10 content may result in decreased ETC activity and energy production. This study aimed to test the hypothesis that prolonged supplementation with oral CoQ10 will increase plasma CoQ10 concentrations and skeletal muscle C...

The Mongolian horse is one of the oldest extant horse populations and although domesticated, most animals are free-ranging and experience minimal human intervention. As an ancient population originating in one of the key domestication centers, the Mongolian horse may play a key role in understanding the origins and recent evolutionary history of ho...

While over ten thousand genetic loci have been associated with phenotypic traits and inherited diseases in genome-wide association studies, in most cases only a relatively small proportion of the trait heritability is explained and biological mechanisms underpinning these traits have not been clearly identified. Expression quantitative trait loci (...

Thoroughbred horses are finely-tuned athletes with a high aerobic capacity relative to skeletal muscle mass, attributable to centuries of genetic selection for speed and stamina. Polymorphisms in the myostatin gene (MSTN), a pronounced inhibitor of skeletal muscle growth, have been shown to almost singularly account for gene-based race distance apt...

Isolation of MSTN DNA and cloning into pBluescript plasmid. DNA was isolated form skeletal muscle tissue as described in materials and methods section. The MSTN gene along with extensive promoter region was amplified from this DNA. (A) Shows the PCR amplified MSTN DNA (8359 bp) along with restriction enzyme digestions of the same amplified DNA; M1:...

Verification of site-directed mutagenesis modification of Gaussia luciferase plasmid and cloning of MSTN fragment into Gaussia luciferase plasmid. (A) Site-directed mutagenesis (SDM) was employed to alter the Gaussia luciferase to add a KpnI site. After SDM the altered plasmid was transformed into competent cells and was selectively grown in cultur...

Verification of site-directed mutagenesis modification of MSTN-luciferase plasmid to alter SNP g.66493737. A single base change was made by site-directed mutagenesis (SDM) to change the base at SNP g.66493737 from G to A (C to T on negative strand). This alteration was made to two plasmids, the one containing the SINE insertion and the one with the...

The SINE insertion significantly affects myostatin gene expression and protein levels in differentiated C2C12 cells. Differentiated C2C12 cells were transfected with MSTN-luciferase plasmids, 24 hours later the media was refreshed to remove the plasmid and lipofectamine mixture. Samples of cultured media were removed at various time points and luci...

Removal of the MSTN SINE insertion 227 bp polymorphism sequence. (A) Shows sequence of MSTN SINE insertion and surrounding nucleotides, sequence data obtained from GenScript sequencing of MSTN-luciferase plasmid. (B) GenScript removed the MSTN SINE Insertion 227 bp polymorphism sequence by firstly removing the region along with some surrounding nuc...

Background We hypothesised that grade/appearance for upper respiratory tract (URT) disorders identified at the first overground endoscopy (OGE) examination would vary at subsequent examinations. Objectives To compare OGE examinations from horses evaluated on at least two occasions under similar exercise conditions without treatment intervention....

Variation in the myostatin (MSTN) gene has been reported to be associated with race distance, body composition and skeletal muscle fibre composition in the horse. The aim of the present study was to test the hypothesis that MSTN variation influences mitochondrial phenotypes in equine skeletal muscle. Mitochondrial abundance and skeletal muscle fibr...

Expression of CoQ biosynthesis genes in MSTN genotype (CC/II, CT/IN, TT/NN) horses. qPCR was used to measure PDSS1 (A), PDSS2 (B), COQ2 (C), COQ3 (D), COQ5 (E), COQ6 (F), COQ7 (G), COQ9 (H) and ADCK4 (I) gene expression levels. RNA was isolated from gluteus medius skeletal muscle from untrained Thoroughbred horses (21±3 months), reverse transcribed...

CoQ biosynthesis enzymes protein expression in MSTN genotype (CC/II, CT/IN, TT/NN) horses. COQ4, ADCK3 and COQ3 protein levels were measured by western blot analysis in untrained Thoroughbred skeletal muscle protein lysates of three MSTN genotypes. A representative experiment out of six performed is shown for each protein. Densitometry was performe...

Body weight (kg) and body weight/withers height (kg/cm) of subset of Thoroughbred horses biopsied near the time of untrained biopsy compared on the basis of three MSTN g.66493737C>T SNP/SINE insertion 227 bp genotypes (CC/II, CT/IN, TT/NN). Body weight (to the nearest kg) and withers height (to the nearest cm) was measured for each Thoroughbred hor...

Submaximal training pre-biopsy of untrained Thoroughbred horses of three MSTN g.66493737C>T SNP/SINE insertion 227 bp genotypes (CC/II, CT/IN, TT/NN). Information in relation to the amount of submaximal exercise the horses within the study did prior to the gluteus medius biopsy was gathered. (A) Shows the number of days between breaking (teaching t...

Primer sequences for real-time qPCR. (TIF)

Citrate synthase and electron transport chain complex activity values for Thoroughbred horse skeletal muscle tissue. Table of mean ± standard error of the mean (n) values for all spectrophotometric assays. Spectrophotometric results are based on experiments performed in at least duplicate for each sample. (TIF)

Background A single bout of exercise induces changes in gene expression in skeletal muscle. Regular exercise results in an adaptive response involving changes in muscle architecture and biochemistry, and is an effective way to manage and prevent common human diseases such as obesity, cardiovascular disorders and type II diabetes. However, the biomo...

This study examined the effect of submaximal and high-intensity (sprint) training on venous hemoglobin concentration ([Hb]) in Thoroughbred (Tb) racehorses before and after high-intensity exercise on the gallops. Measurements for n=14 Tb yearlings took place during high-intensity exercise on an all-weather gallop during their breaking period before...

Accuracy and precision of a prototype point-of-care (POC) hemoglobin (Hb) and oxygen saturation (SO2) analyzer were compared to a benchtop analyzer, as well as the use of the prototype in a field setting. Arterial and venous Hb concentrations ([Hb]) and SO2 were determined from 180 whole-blood samples from dogs, cats and horses. [Hb] and SO2 values...

Skeletal muscles in horses are characterised by specific adaptations, which are the result of the natural evolution of the horse as a grazing animal, centuries of selective breeding and the adaptability of this tissue in response to training. These adaptations include an increased muscle mass relative to body weight, a great locomotor efficiency ba...

Recurrent laryngeal neuropathy (RLN) is considered more prevalent in taller thoroughbreds. A single nucleotide polymorphism (SNP) (BIEC2‐808543) proximal to the LCORL/NCAPG gene significantly influences height variation in horses. It has been suggested that the C‐allele of this SNP is associated with RLN risk. Our objective was to investigate the a...

Introduction Heritability of Athletic Performance Athletic Performance Genes Encoded by the Nuclear Genome Mitochondrial DNA and Athletic Performance in Thoroughbreds Detection of Genomic Regions under Selection in the Thoroughbred Genome DNA Sequence Variation and Athletic Performance Traits in the Thoroughbred Identification of the Myostatin Gene...

Horses were domesticated from the Eurasian steppes 5,000-6,000 years ago. Since then, the use of horses for transportation, warfare, and agriculture, as well as selection for desired traits and fitness, has resulted in diverse populations distributed across the world, many of which have become or are in the process of becoming formally organized in...

Parsimony relationship among Lusitano and Andalusian individuals. Portion of the parsimony clade shown in Figure 1 consisting of the Lusitano (dark blue) and US Andalusian (light blue) individuals. Bootstrap values greater than 50% are shown. Asterisks indicate horses sampled in the US which were noted to be of Portuguese ancestry. (TIFF)

Example of LD decay over 2 Mb in 9 breeds of horse. Decay of linkage disequilibrium over 2Mb for 9 of the 36 breeds. Landrace populations such as the Mongolian, and large and/or diverse breeds such as the Finnhorse and Quarter Horse, show more rapid decay than those with small population sizes and less diversity (e.g. Clydesdale, Tennessee Walking...

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an F(ST)-based statistic calculated in...

Primers used for sequencing of MSTN. (PDF)

Annotated genes (or other features) within high-frequency, extended haplotypes of interest. (PDF)

Variation at the myostatin (MSTN) gene locus has been shown to influence racing phenotypes in Thoroughbred horses, and in particular, early skeletal muscle development and the aptitude for racing at short distances. Specifically, a single nucleotide polymorphism (SNP) in the first intron of MSTN (g.66493737C/T) is highly predictive of best race dis...

Myostatin, encoded by the MSTN gene, is a member of the TGF-β superfamily that regulates skeletal muscle development. A MSTN SNP significantly associated with Thoroughbred horse racing phenotypes has recently been identified as well as significant reductions in Thoroughbred skeletal muscle gene expression for three transcripts 400-1500 base pairs d...

Selective breeding for speed in the racehorse has resulted in an unusually high frequency of the C-variant (g.66493737C/T) at the myostatin gene (MSTN) in cohorts of the Thoroughbred horse population that are best suited to sprint racing. Here we show using a combination of molecular- and pedigree-based approaches in 593 horses from 22 Eurasian and...

An equine SNP genotyping array was developed and evaluated on a panel of samples representing 14 domestic horse breeds and 18 evolutionarily related species. More than 54,000 polymorphic SNPs provided an average inter-SNP spacing of ∼43 kb. The mean minor allele frequency across domestic horse breeds was 0.23, and the number of polymorphic SNPs wit...

Multidimensional scaling with 14 domestic horse breeds. Metric multidimensional scaling analysis of pair-wise genetic distance was used as described in Materials and Methods to identify relationships between the 14 domestic horse breeds. In these plots dimensions 3–6 (y axes) are always plotted against dimension 1 (x axis). (TIF)

Validation rates for the assayed SNPs. The number of selected SNPs from the seven discovery horses and Twilight that provided genotypes (converted), the number of validated SNPs in the equine sample set, and the validation fraction are indicated. SNPs with the highest validation rate were those originally ascertained in two breeds, especially if on...

Inter-SNP spacing by chromosome. Polymorphic SNPs were defined as having at least one heterozygous individual (i.e., MAF>0) across all 14 breeds. (DOCX)

Proportion of validated SNPs from each discovery breed across all breeds genotyped. The impact of discovery breed on SNP validation rate across the genotyped sample set was determined as described in Materials and Methods. (DOCX)

Inbreeding coefficients in each breed. Individual inbreeding coefficients (F) were estimated with 17,947 SNPs that were pruned for linkage equilibrium as described in Materials and Methods. (DOCX)

Chi-square tests of association for coat color loci within breeds. Chestnut, black and gray phenotypes were either inferred from the genotypes at 8 known coat color loci, or from the genotype of a single locus (designated as MC1R and ASIP) only, using known inheritance models as described in Materials and Methods. Case-control association analyses...

Distance between informative SNPs across all 31 autosomes. Informative SNPs were defined as having MAF>0.05 across all 14 breeds. (TIF)

Decline in genome-wide linkage disequilibrium across and within breeds. Genome-wide linkage disequilibrium (LD) was estimated both within a given breed, and across all breeds, by calculating r2 values between all pairs of SNPs with inter-SNP distances of less than 600 kb as described in Materials and Methods. (TIF)

Multidimensional scaling of Przewalski's Horse and domestic horse breeds. Metric multidimensional scaling analysis of pair-wise genetic distance was used as described in Materials and Methods to identify relationships between the 14 domestic breeds and the Przewalski's Horse. In these plots dimensions 3–6 (y axes) are plotted against dimension 1 (x...

Mapping of the chestnut coat color locus across breeds based on phenotype inferred from all nine genotyped coat color alleles with known interactions (a), or based solely on MC1R genotype and chestnut as a simple recessive trait (b). Phenotypes were inferred as described in Materials and Methods. Unstructured case-control association analyses using...

Tests of Mendelian inheritance in trios. Mendelian errors in each of the 18 nuclear trios were calculated and results are reported as Mendelian agreement as described in the Materials and Methods. (DOCX)

Allele frequencies and genetic diversity in the domestic horse. The mean and median MAF (including all SNPs), the number of polymorphic (MAF≥0.01) and informative SNPs (MAF>0.05), and genetic diversity as determined by heterozygosity (HE), are indicated and calculated as described in Materials and Methods. (DOCX)

Mean pair-wise distances between Przewalski's Horse and domestic horse populations. Genetic distance (D) between pair-wise combinations of individuals was calculated as described in Materials and Methods. (DOCX)

Chi square analysis for mapping of known coat color loci across the 14 breeds. Chestnut, black and gray phenotypes were either inferred from the genotypes at 9 known coat color loci, or from the genotype of a single locus (designated as MC1R and ASIP) only, using known inheritance models as described in Materials and Methods. Case control associati...

CMH analysis for mapping of known coat color loci across the 14 breeds. Phenotypes were inferred from multi or single locus genotypes as described in Table S1 and the Materials and Methods. Stratified genome-wide association analysis was performed using the Cochran-Mantel-Haenszel (CMH) test. Horses were clustered for this test on the basis of the...

Multidimensional scaling of Przewalski's Horse and domestic horse breeds. Metric multidimensional scaling analysis of pair-wise genetic distance was used as described in Materials and Methods to identify relationships between the 14 domestic breeds and the Przewalski's Horse. (TIF)

Mapping of the gray coat color locus across breeds based on gray color as a simple dominant trait. Gray phenotype was inferred as described in Materials and Methods. Corrected p-values after 10,000 label-swapping permutations are indicated. SNPs on each chromosome are labeled with a different color on the X axis as indicated. Also indicated on the...

Species, genotyping rates, sample and locus GC scores for unfiltered data in extant Perissodactyla. Genotyping rates and GC scores reported using Illumina's default calling parameters as described in Materials and Methods. (DOCX)

Mean pair-wise genetic distances in domestic horse populations. Genetic distance (D) between pair-wise combinations of individuals was calculated as described in Materials and Methods. (DOCX)

Mapping of the recessive black coat color locus across breeds based on phenotype inferred from all nine genotyped coat color alleles with known interactions (a), or based solely on ASIP genotype and black color as a simple trait (b). Phenotypes were inferred as described in Materials and Methods. Unstructured case-control association analyses using...

Conserved haplotype at the MC1R locus on ECA3. For all breeds with the exception of the Standardbred, the length of minimal homozygosity for the chestnut allele is depicted. In the Standardbred, where no homozygotes were observed, the shortest length of the haplotype containing the chestnut allele is depicted. The number of chromosomes in each bree...

Breeds included in the study. Breed, number of individuals, and geographic origin of horses genotyped in this study. *The control horses consisted of the 7 SNP discovery horses listed in Table S1 and the Thoroughbred mare Twilight used to generate the equine genome assembly. (DOCX)

Quality scores, SNP conversion and SNP validation rates and mean heterozygosity for extant Perissodactyla after filtering for intensity and genotyping rate. Genotyping rates, conversion and validation rates and observed hetrozygosities after filtering the data as described in Materials and Methods. Conversion and validation rates and heterozygositi...

Summary of MC1R Haplotype Analysis on ECA3. Genotype data was phased as described in Materials and Methods and summary statistics on the chromosomes containing the associated haplotype are presented by breed. (DOCX)

Supporting text includes additional information on: filtering of genotypes in Perissodactyla, the relationships between domestic horse breeds and between the domestic horse and the Przewalski's Horse, and genome wide association analysis. (DOCX)

Sequence variation at the equine myostatin gene (MSTN) locus has previously been shown to have a singular genomic influence on optimum race distance in Thoroughbred racehorses. Myostatin, encoded by the MSTN gene, is a member of the TGF-β superfamily that regulates skeletal muscle development in a range of mammalian species including the horse. In...

Myostatin is a member of the transforming growth factor-β family with a key role in inhibition of muscle growth by negative regulation of both myoblast proliferation and differentiation. Recently, a genomic region on ECA18, which includes the MSTN gene, was identified as a candidate region influencing racing performance in Thoroughbreds. In this st...

Circadian rhythms are innate 24-h cycles in behavioral and biochemical processes that permit physiological anticipation of daily environmental changes. Elucidating the relationship between activity rhythms and circadian patterns of gene expression may contribute to improved human and equine athletic performance. Six healthy, untrained mares were st...

PCR and sequencing primers for re-sequencing MSTN flanking regions. Primers were designed to cover ~2 kb of the 3' UTR and ~2 kb of the 5' UTR of the MSTN gene.

Sequence and structural variation in the coding, intronic and flanking sequences of the equine MSTN gene. Sequences are provided for the + strand.

Quantitative association test results for best race distance. Unadjusted and FDR corrected P-values are given.

Thoroughbred horses have been selected for traits contributing to speed and stamina for centuries. It is widely recognized that inherited variation in physical and physiological characteristics is responsible for variation in individual aptitude for race distance, and that muscle phenotypes in particular are important. A genome-wide SNP-association...

Digital gene expression profiling was used to characterize the assembly of genes expressed in equine skeletal muscle and to identify the subset of genes that were differentially expressed following a ten-month period of exercise training. The study cohort comprised seven Thoroughbred racehorses from a single training yard. Skeletal muscle biopsies...

Variants of the MSTN gene encoding myostatin are associated with muscle hypertrophy phenotypes in a range of mammalian species, most notably cattle, dogs, mice, and humans. Using a sample of registered Thoroughbred horses (n = 148), we have identified a novel MSTN sequence polymorphism that is strongly associated (g.66493737C>T, P = 4.85x10(-8)) wi...

Full list of gene ontologies overrepresented in selected regions (1). The on-line functional annotation bioinformatics tool DAVID (david.abcc.ncifcrf.gov) was used to search for gene ontology (GO) terms that were overrepresented among genes in selected regions. Selected regions were defined by deviation from expected heterozygosity (Ewen-Watterson...

Hardy-Weinberg equilibrium test results for locus g.66493737C>T. (0.03 MB DOC)

Overlapping primer pairs and identified SNPs. (0.04 MB DOC)

Full list of gene ontologies overrepresented in selected regions (2). The on-line functional annotation bioinformatics tool DAVID (david.abcc.ncifcrf.gov) was used to search for gene ontology (GO) terms that were overrepresented among genes in selected regions. Selected regions were defined by the nine high FST regions that had significant (P<0.05)...

Quantitative association test results for g.66493737C>T with kg/cm ratio as phenotype. (0.04 MB DOC)

Population summary including details of retrospective racecourse success for each cohort. RPR = racing post handicap rating. Gr = group race. (0.03 MB DOC)

Abstract Background Digital gene expression profiling was used to characterize the assembly of genes expressed in equine skeletal muscle and to identify the subset of genes that were differentially expressed following a ten-month period of exercise training. The study cohort comprised seven Thoroughbred racehorses from a single training yard. Ske...

Selection for exercise-adapted phenotypes in the Thoroughbred racehorse has provided a valuable model system to understand molecular responses to exercise in skeletal muscle. Exercise stimulates immediate early molecular responses as well as delayed responses during recovery, resulting in a return to homeostasis and enabling long term adaptation. G...

The relative distributions of gene ontology (GO) categories (Level 1) on the equine cDNA microarray. A list of all available human orthologues to equine genes was compared to a list of human orthologues of probes on the microarray using the Database for Annotation, Visualization and Integrated Discovery (DAVID) [28,29] for functional clustering and...