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Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control.
Abstract: The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.
Publication Date: 2022-09-04 PubMed ID: 36140757PubMed Central: PMC9498419DOI: 10.3390/genes13091589Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
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.
The research paper discusses the detection and verification of genetic manipulation, also known as gene doping, in thoroughbred racing horses. By employing next-generation sequencing (NGS) techniques, the researchers developed a test capable of detecting instances of gene editing in tested horses.
Methodology
- The researchers designed 1012 amplicons. This process is essentially a detailed study or inspection of multiple sections of specific genes in a DNA sequence. These amplicons targeted 52 individual genes (481 exons), which contributed to 147 single-nucleotide variants (SNVs).
- All the targeted exons underwent next-generation sequencing (NGS) analyses which disclosed that 97.7% of the targets were sequenced to sufficient coverage, ensuring the excellent quality and reliability of the sequence retrieval data.
- The items chosen as subjects of artificial gene editing were defined in the study as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations.
- The researchers constructed four models of gene editing to further the study: three homoALT with 1-bp insertions and one REF/ALT with a 77-bp deletion. This amendment was implemented on the myostatin gene in horse fibroblasts using CRISPR/Cas9 gene-editing technology.
Results
- A developed test was used to screen edited cells alongside 101 thoroughbred horse samples, successfully identifying the three homoALT cells that contained the 1-bp insertions.
- The study further explored 147 SNVs to investigate their usefulness in confirming biological parentage of the horses tested. Out of these, 120 SNVs proved to be accurate and consistent in genotyping.
- Surrogate (non-biological) dams could be differentiated and excluded on average by 9.8 SNVs, which indicated that the 120 SNV could detect foals produced via somatic cloning or embryo transfer. Both practices are sterly prohibited in thoroughbred racing and breeding.
Conclusion
- In conclusion, the study provides evidence that gene-editing tests, including variant calling and SNV genotyping, can prove useful to identify genetically modified racehorses and illegal breeding practices. The new testing protocol could potentially improve the credibility and fairness of horse racing.
Cite This Article
APA
Tozaki T, Ohnuma A, Nakamura K, Hano K, Takasu M, Takahashi Y, Tamura N, Sato F, Shimizu K, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA, Nagata SI.
(2022).
Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control.
Genes (Basel), 13(9), 1589.
https://doi.org/10.3390/genes13091589 Publication
Researcher Affiliations
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, Japan.
- Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu 501-1193, Japan.
- Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu 501-1193, Japan.
- Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1, Yanagido, Gifu 501-1193, Japan.
- Equine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke 329-0412, Japan.
- Equine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke 329-0412, Japan.
- Equine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke 329-0412, Japan.
- Registration Department, Japan Association for International Racing and Stud Book, 4-5-4, Shimbashi, Minato, Tokyo 105-0004, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, Japan.
- Equine Genetics Research Centre, Racing Australia, 2 Randwick Way, Scone, NSW 2337, Australia.
- Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2, Tsurutamachi, Utsunomiya 320-0851, Japan.
MeSH Terms
- Animals
- Gene Editing
- High-Throughput Nucleotide Sequencing
- Horses / genetics
- Myostatin / genetics
- Nucleotides
- Sequence Analysis, DNA
Conflict of Interest Statement
There are no competing interests, including patents, products in development, or marketed products, to declare in relation to this work.
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Citations
This article has been cited 1 times.- Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI. Short Insertion and Deletion Discoveries via Whole-Genome Sequencing of 101 Thoroughbred Racehorses.. Genes (Basel) 2023 Mar 3;14(3).
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