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Definitions

• the Thoroughbred horse industry is a multi-billion euro international industry involved in the breeding, training and racing of Thoroughbred horses. Often multi-million euro decisions are made on the purchase of individual animals with perceived racing potential.
• the integration of genomics information into the Thoroughbred racing and breeding industries has huge potential for early 'talent identification'. Thoroughbreds are traditionally selected for racing and breeding based on pedigree information as well as numerous phenotypic characteristics. Early identification of genetic potential, by traditional or new means, is paramount to success. Within the industry the quest to find an 'edge' pushes those involved to constantly consider new methods and techniques. Therefore, genomics information has the potential to directly assist breeders and trainers to fine-tune often multi-million dollar decisions by providing previously inaccessible information.
• Oxygen is an essential regulator of muscle function, influencing energy production, muscle contraction and removal of by-products. During exercise the requirement for energy is greatly limited by the availability of oxygen. Mammalian cells have evolved elaborate adaptive mechanisms to respond to low cellular oxygen environments (Taylor & Colgan 1999). In studies of human exercise, adaptation to such a hypoxic environment in trained skeletal muscle causes a shift in substrate selection to increased oxidation of carbohydrates and stimulates cells to improve conditions for oxygen transport and utilisation (Hoppeler & Vogt 2001).
• “Athletic performance” as used herein includes racing such as competitive racing and equestrian sports such as racing, showjumping, eventing, dressage, endurance events, riding, hunting and the like.
• the equestrian sports may be competitive sports.
• the invention provides single nucleotide polymorphisms (SNPs) that are associated with elite athletic performance.
• the invention provides a method of predicting the athletic performance of a subject comprising the step of assaying a biological sample from the subject for the presence of a single nucleotide polymorphism (SNP) in one or more of the genes listed in the appendices wherein the SNP has a significant association with athletic performance.
• SNP single nucleotide polymorphism
• the biological sample of the subject may be selected from the group comprising: blood, saliva, skeletal muscle, skin, semen, biopsy, bone marrow, soft tissue, internal organs and hair.
• the SNP may be CKM l 5884567 (G/A).
• the presence of an A allele may be indicative of elite athletic performance.
• the presence of a homozygous AA genotype may be indicative of elite athletic performance.
• the DNA may be genomic DNA.
• an assay for use in determining athletic performance of a subject comprising means for detecting the presence or absence of a single nucleotide polymorphism (SNP) in the COX4I2 gene.
• SNP single nucleotide polymorphism
• the SNP may be EquCab 2.0 COX4I2-22676361 - C/T.
• the presence of a homozygous TT genotype may be indicative of elite athletic performance.
• the biological sample of the subject may be selected from the group comprising: blood, saliva, skeletal muscle, semen, biopsy, internal organ, skin, bone marrow (or any other biological tissue) and hair.
• the subject may be from a competitive racing species.
• the subject may be an equine.
• the subject may be a Thoroughbred race horse.
• Fig 9 is a graph showing the genotype frequency for the MSTN 66493737 (T/C) SNP for stallions with a Stamina Index 6 - 8f, 8- 10 f, 10- 12f; and
• the COX4I2 gene contains a glucocorticoid receptor element (TGTT) which may be targeted to increase COX4-2 expression and therefore increase mitochondrial volume. Also, the COX4I2 gene contains a p53 tumor suppressor binding site (CATG). Recent studies have suggested that p53 may play a role in regulation of mitochondrial biogenesis and aerobic metabolism via COX (Matoba et al. 2006; Saleem et al. 2009).
• the PDK4 gene promoter contains a binding site for the FOXOlA transcription factor, a key regulator of insulin signalling in liver and adipose tissue.
• FOXOlA Single nucleotide polymorphisms in FOXOlA have been found to have a protective effect on T2DM development and related phenotypes in humans.
• FOXOlA has also been found among positively selected genomic regions in Thoroughbred and its PDK4 promoter binding site sequence is conserved in horse.
• the transcription factors FOXOl and SMAD have also been shown to be responsible for myostatin (MSTN) gene regulation and therefore play key roles in the regulation of muscle growth.
• HIF- l ⁇ activates the transcription of genes encoding PDKl, LDHA, COX4-2 and LON and controls the switch from COX4-1 to COX4-2.
• Thoroughbred muscle that is deprived of oxygen during intense exercise an enhanced response to reduced oxygen and the ability to generate ATP in the most efficient manner will provide a significant advantage to that individual.
• Glucorticoid receptor elements (Glucocorticoid responsive and related elements) contain the recognition sequence (TGTT).
• the COX4I2 gene contains one of these elements in Intron 2. Therefore glucocorticoid binding may stimulate increased gene expression leading to increased mitochondrial volume and therefore aerobic energy capacity.
• the horse genome EquCab2 assembly is a Whole Genome Shotgun (WGS) assembly at 6.79x and was released in September 2007.
• WGS Whole Genome Shotgun
• a female Thoroughbred named "Twilight” was selected as the representative horse for genome sequencing.
• the project coordination and genome sequencing and assembly is provided by the Broad Institute.
• the N50 size is the length such that
• KASPar competitive allele specific PCR
• Taqman Applied Biosystems
• the present invention identifies significant associations between SNPs and athletic performance phenotypes in a set of these genes including ACN9, ACSSl, ACTN2, ADHFEl, CKM, COX4I2, GSN, MSTN, PONl, PTGSl and PTPNl (see the appendices).
• ACN9 ACN9, ACSSl, ACTN2, ADHFEl, CKM, COX4I2, GSN, MSTN, PONl, PTGSl and PTPNl
• CKM, COX4I2, PDK4 and MSTN were investigated in detail.
• SNPs in three of those genes are associated with elite (Group race winning) performance and a SNP in the MSTN gene is associated with elite sprint race performance.
• the genotypic and dominant/recessive tests will only be conducted if there is a minimum number of observations per cell in the 2-by-3 table: by default, if at least one of the cells has a frequency less than 5, then the alternate tests are skipped (NA is written in the results file).
• the Cochran-Armitage and allelic tests are performed in all cases. This threshold can be altered with the —cell option: plink —file mydata —model —cell 20
• Table 7 Genotype frequencies in elite and non-elite Thoroughbred sub-populations for SNPs: PDK4 (PDK4 38973231); COX4I2 (COX4I2_22684390); CKM (CKM_15884567) and in elite sprinters and elite endurance Thoroughbreds for SNP: MSTN (MSTN_66493737).
• the horse genome EquCab2 assembly is a Whole Genome Shotgun (WGS) assembly at 6.79x and was released in September 2007.
• WGS Whole Genome Shotgun
• a female Thoroughbred named "Twilight” was selected as the representative horse for genome sequencing.
• the project coordination and genome sequencing and assembly is provided by the Broad Institute.
• the N50 size is the length such that 50% of the assembled genome lies in blocks of the N50 size or longer.
• the N50 size of the contigs is 112.38 kb, and the total length of all contigs is 2.43 Gb. When the gaps between contigs in scaffolds are included, the total span of the assembly is 2.68 Gb.
• the horse EquCab2 was annotated using a standard Ensembl mammalian pipeline.
• the invention provides a simple DNA based method (genotype test) for predicting the elite sprint race performance of a thoroughbred race horse based on the presence or absence of a SNP in one or more exercise response gene.
• the exercise response gene may be one or more of the genes listed in the appendices.
• the genotype test may be based on a SNP in one or more of the MSTN, ACN9, PTPNl, PONl, ADHFEl, or GSN genes. Details of some of the SNPs that may be used to predict the elite sprint race performance of a thoroughbred race horse are given in the appendices. It will be appreciated that the genotypic test may be based on a combination of any one or more of these SNPs.
• Genomic DNA was extracted from either fresh whole blood or hair samples using a modified version of a standard phenol/chloroform method (Sambrook & Russell 2001). Thirteen pairs of overlapping PCR primers were designed to cover the entire MSTN genomic sequence using the PCR Suite extension to the Primer3 web-based primer design tool (Rozen & Skaletsky 2000; van Baren & Heutink 2004) (Table 13). Twenty-four unrelated Thoroughbred DNA samples were included in a re-sequencing panel to identify Thoroughbred-specific sequence variants. As such this study was powered to detect 95% of SNPs with MAF > 0.05 in the Thoroughbred population (Kruglyak & Nickerson 2001).
• Quality control analyses included computation of sample allele frequency, percent missing genotypes and deviation from Hardy- Weinberg equilibrium.
• the series of case- control association tests were performed for two loci (g.66493737C>T and g.66494218A>C).
• Statistical significance was assessed using the Cochran-Armitage test for trend and an unconditioned genotypic model. Odds ratios and 95% CIs were calculated for the two most significant associations.
• the linear regression model was used to evaluate quantitative trait association at locus g.66493737C>T using the phenotypes: best race distance and kg / cm ratio.
• n 148 Thoroughbred horses.
• MSTN sequence variants 148 Thoroughbred horses.
• MSTN sequence polymorphisms displayed MAF ⁇ 0.05 in Thoroughbreds (Table 15) and were excluded from the association analyses.
• Genotype trend effects were modeled by estimating the risk associated with a linear trend in magnitude of effect relative to the common homozygote, heterozygote, and rare homozygote genotype using the Cochran-Armitage test for the trend model.
• Table 13 PCR primer details for SNP discovery in the equine myostatin (MSTN) gene
• Table 16 Population summary including details of retrospective racecourse success for each cohort.
• Table 17 a. Case-control association test results for a series of cohort comparisons for g.66493737C>T.
• TBE elite Group race winning Thoroughbreds
• TBO other non-winning Thoroughbreds
• TBE > 8 f, TBE ⁇ 8 f and TBE ⁇ 7 f elite Group race winning Thoroughbreds that won their best (most valuable or highest grade) races over distances > 8 f, ⁇ 8 f and ⁇ 7 f.
• Odds ratios were calculated for the two most significant results
• b Best-fit model results for g.66493737C>T association with elite Group race winning performance over distances ⁇ 7 f .
• MSTNSNP MSTN 66493737 O 1 ZC
• the horse genome EquCab2 assembly is a Whole Genome Shotgun (WGS) assembly at 6.79x and was released in September 2007.
• WGS Whole Genome Shotgun
• a female Thoroughbred named "Twilight” was selected as 0 the representative horse for genome sequencing.
• the project coordination and genome sequencing and assembly is provided by the Broad Institute.
• the N50 size is the length such that 50% of the assembled genome lies in blocks of the N50 size or longer.
• the N50 size of the contigs is 112.38 kb, and the total length of all contigs is 2.43 Gb. When the gaps between contigs in scaffolds are included, the total span of the assembly is 2.68 Gb.
• the horse EquCab2 was annotated using a standard Ensembl mammalian pipeline.
• P 0.0043
• Table 25 below shows the mean best race distance for each genotype for four of the SNPs from Table 24 above.
• the TT genotype has a mean best race distance of 10.54 furlongs (f)
• the TC genotype has a mean best race distance of 9.087 f
• the CC genotype has a mean best race distance of 6.167 f.
• Overall the mean best race distance was 8.55 f.
• a stallions potential can be promoted by:
• the iPLEX® Gold assay based on multiplex PCR followed by a single base primer extension reaction. After the PCR, remaining nucleotides are deactivated by SAP treatment. The single base primer extension step is performed, and the primer extension products analyzed using MALDI TOF MS.

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