Computational protocol: A Sequence Polymorphism in MSTN Predicts Sprinting Ability and Racing Stamina in Thoroughbred Horses

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Protocol publication

[…] The highest standard and most valuable elite Flat races are known as Group (Europe and Australasia) or Stakes races (North America). The most prestigious of these races include The Breeders' Cup races (United States), The Kentucky Derby (United States), The Epsom Derby (Great Britain) etcetera. In the United Kingdom and Ireland 196 Group races are competed annually (43 Group 1, 50 Group 2 and 103 Group 3). After Group races, Listed races are the next highest grade of race. To minimize confounding effects of racing over obstacles only horses with performance records in Flat races were considered for inclusion in the principal study cohorts. Horses were categorized based on retrospective racecourse performance records as “Thoroughbred-elite” (TBE) or “Thoroughbred-other” (TBO). Elite Thoroughbreds were Flat racehorses that had won at least one Group race. Other Thoroughbreds were those that had never won a Flat race or had a handicap rating (Racing Post Rating, RPR) <89.Association sample: The International Federation of Horseracing Authorities recognizes five distance categories: Sprint (5–6.5 f, ≤1,300 m), Mile (6.51–9.49 f, 1,301–1,900 m), Intermediate (9.5–10.5 f, 1,901–2,112 m), Long (10.51–13.5 f, 2,114–2,716 m) and Extended (>13.51 f, >2,717 m) races (International Federation of Horseracing Authorities Classifications, [Note: 1 furlong = 1/8 mile = 201.2 meters]. However, for the case-control investigations we compared two cohorts: samples were subdivided into short (≤8 f and ≤7 f) and long (>8 f) distance racing cohorts. To avoid animals with excessive consanguinity (within two generations) and over-representation of popular sires within the pedigrees, a set of Thoroughbred DNA samples (n = 148) was selected from a large DNA sample repository (n>1,000) collected with informed owners' consent from Thoroughbred training, breeding and sales establishments in Ireland and New Zealand during 1998–2009.Replication samples: To validate the findings, a replication sample of n = 62 unrelated elite (Group and Listed race winners) Thoroughbreds was re-sampled from the original repository and supplemented with additional samples collected following the original analyses and genotyped for the g.66493737C>T SNP (Replication sample I).To minimize non-genetic influences on performance we further validated the findings by genotyping elite (Group and Listed race winning) racehorse samples (n = 39) selected from a repository of DNA samples (n = 419) from horses trained by the same trainer in Ireland during 2004–2008. This sample had some sharing of relatives, accounted for in the analyses (Replication sample II).A subset (n = 142) of this repository was evaluated for genotypic trends with parameters of racecourse success in two-year-old racehorses. Race records were derived from three sources: European race records, The Racing Post on-line database (; Australasian and South East Asian race records, Arion Pedigrees (; and North American race records, Pedigree Online Thoroughbred database ( [...] Genomic DNA was extracted from either fresh whole blood or hair samples using a modified version of a standard phenol/chloroform method . 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 , []. 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 . Bidirectional DNA sequencing of PCR products was outsourced to Macrogen Inc. (Seoul, Korea) and carried out using AB 3730xl sequencers (Applied Biosystems, Foster City, CA). Sequence variants were detected by visual examination of sequences following alignment using Consed version 19.0 (090206) . Genotyping was carried out using Sequenom (San Diego, USA) iPlex technology at Sequenom facilities in San Diego, USA (Association samples) and Hamburg, Germany (Replication samples). […]

Pipeline specifications

Software tools PedigreeQuery, Primer3, Consed
Applications Population genetic analysis, qPCR
Organisms Equus caballus, Homo sapiens, Bos taurus, Canis lupus familiaris, Mus musculus
Diseases Athletic Injuries