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Home / Equine Research Bank / Anim Genet / Long-read sequencing assays designed to detect potential gen...
Animal genetics2023; 54(4); 470-482; doi: 10.1111/age.13332

Long-read sequencing assays designed to detect potential gene editing events in the myostatin gene revealed distinct haplotype signatures in the Thoroughbred horse population.

Abstract: We present here the use of targeted, long-read sequencing of the myostatin (MSTN) gene as a model to detect potential gene editing events in Thoroughbred horses. MSTN is a negative regulator of muscle development, making the gene a prime candidate target for gene doping. By sequencing the complete gene in one PCR product, we can catalogue all mutations without the need to produce short-fragment libraries. A panel of reference material fragments with defined mutations was constructed and successfully sequenced by both Oxford Nanopore and Illumina-based methods, showing that gene doping editing events can be detected using this technology. To ascertain the normal variation within the population, we sequenced the MSTN gene in 119 UK Thoroughbred horses. Variants from the reference genome were assigned to haplotypes and eight distinct patterns, designated Hap1 (reference genome) to Hap8, were determined with haplotypes Hap2 and Hap3 (which includes the 'speed gene' variant) being far the most prevalent. Hap3 was most abundant in flat-racing horses, whereas Hap2 was most abundant in jump-racing. Within this data set, results for 105 racehorses from out-of-competition sampling were compared between matrices of extracted DNA and direct PCR of whole blood from lithium heparin gel tubes, and strong agreement was found between the two methods. The direct-blood PCR was achieved without compromising the sample prior to plasma separation for analytical chemistry, and could thus be used as part of a routine screening workflow for gene editing detection.
© 2023 Stichting International Foundation for Animal Genetics.
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Publication Date: 2023-06-08 PubMed ID: 37288798DOI: 10.1111/age.13332Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research paper uncovers how long-read sequencing of the myostatin (MSTN) gene can be utilized to detect potential gene editing events in Thoroughbred horses and portrays the variation within the gene in the Thoroughbred population through eight distinct haplotype patterns.

Methodology

  • The researchers employed targeted, long-read sequencing of the MSTN gene to discover potential gene “doping”. MSTN is known to negatively regulate muscle growth, therefore making it a suitable target for gene doping.
  • Complete gene sequencing allowed the researchers to record all mutations without having to create short-fragment libraries.
  • A panel of defined, referential mutation fragments was constructed and sequenced through the use of both Oxford Nanopore and Illumina methodologies.

Results

  • The researchers sequenced the MSTN gene in 119 UK Thoroughbred horses in order to understand the standard variation within the breed.
  • Divergent from the reference genome, these variants were assigned to haplotypes. Eight unique patterns, named Hap1 (reference genome) to Hap8, were identified.
  • The two most commonplace haplotypes were Hap2 and Hap3, which contains the ‘speed gene’ variant. Hap2 was most abundant in jump-racing horses while Hap3 was most common in flat-racing horses.
  • Within this data set, the researchers compared the results for 105 racehorses collected from out-of-competition sampling between matrices of extracted DNA and direct PCR of whole blood from lithium heparin gel tubes, finding a strong agreement between the two methods.

Implications

  • The direct blood PCR was executed without compromising the sample prior to plasma separation for chemical analysis.
  • Therefore, this sampling method could be exploited as a part of a regular screening workflow to detect gene editing.

The findings of this study indicate that long-read sequencing techniques can be effectively used to detect potential gene editing events in Thoroughbred horses, aiding in the fight against gene doping in horse racing.

Cite This Article

APA
Maniego J, Giles O, Hincks P, Stewart G, Proudman C, Ryder E. (2023). Long-read sequencing assays designed to detect potential gene editing events in the myostatin gene revealed distinct haplotype signatures in the Thoroughbred horse population. Anim Genet, 54(4), 470-482. https://doi.org/10.1111/age.13332

Publication

Animal genetics
ISSN: 1365-2052
NlmUniqueID: 8605704
Country: England
Language: English
Volume: 54
Issue: 4
Pages: 470-482

Researcher Affiliations

Maniego, Jillian
  • Sport and Specialised Analytical Services, LGC Assure, Cambridgeshire, UK.
Giles, Orla
  • Sport and Specialised Analytical Services, LGC Assure, Cambridgeshire, UK.
Hincks, Pamela
  • Sport and Specialised Analytical Services, LGC Assure, Cambridgeshire, UK.
Stewart, Graham
  • School of Biosciences and Medicine, University of Surrey, Guildford, UK.
Proudman, Christopher
  • School of Veterinary Medicine, University of Surrey, Guildford, UK.
Ryder, Edward
  • Sport and Specialised Analytical Services, LGC Assure, Cambridgeshire, UK.

MeSH Terms

  • Horses / genetics
  • Animals
  • Haplotypes
  • Myostatin / genetics
  • Gene Editing
  • DNA
  • Base Sequence

Grant Funding

  • British Horseracing Authority

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Citations

This article has been cited 2 times.
  1. Jafari H, Abebe BK, Cong L, Ahmed Z, Zhaofei W, Sun M, Muhatai G, Chuzhao L, Dang R. Review: Genomic insights into the adaptive traits and stress resistance in modern horses. Stress Biol 2026 Jan 12;6(1):5.
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  2. Wu D, Ding S, Liu N, Shi Y, Su P, Shi H, Shi Y, Han B, Cheng S, Ren X, Tian F, Chen P, Wu J, Su X, Li R. Codon changes challenge PCR-based gene doping detection. Gene Ther 2025 Dec;32(6):632-640.
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