Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports.
Abstract: Gene doping is prohibited in horseracing and equestrian sports. In previous studies, we developed non-targeted transgene and genome editing detection methods based on whole genome resequencing (WGR) using genomic DNA extracted from whole blood. In this study, we aimed to develop a WGR method using DNA extracts from hair roots. Hair roots are a preferred substrate because their collection is less invasive than blood collection. Hair is also easier to store for long periods of time. Although almost all genomic DNA extracted from hair root samples stored for years at room temperature was degraded, the quality of genomic DNA from samples stored for years at refrigerated temperatures (4-8°C) was maintained. High-molecular-weight genomic DNA was isolated from hair roots using a magnetic silica beads method of extraction, enabling WGR from horsehair root extracts. Nucleotide sequencing results and numbers of single-nucleotide polymorphisms and insertions/deletions concurred with those previously reported for WGR of DNA extracted from whole blood. Therefore, we consider that storing hair samples at refrigerated temperatures prevents degradation of DNA, allowing the detection of gene doping in these samples based on WGR. It is likely this finding will also have a deterrent effect, as it is now possible to test horses with archived samples even if they or their parents are deceased. To our knowledge, this is the first report employing WGR on horsehair roots stored for a long term.
©2020 The Japanese Society of Equine Science.
Publication Date: 2020-12-18 PubMed ID: 33376443PubMed Central: PMC7750640DOI: 10.1294/jes.31.75Google 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
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 study presents a new method for detection of gene doping in horses using DNA extracted from horsehair roots. The method employs whole genome resequencing and is particularly useful because hair roots are less invasive to collect and easier to store than blood.
Objective of the Research
- The primary aim of this research was to develop a whole genome resequencing (WGR) method using DNA extracted from horsehair roots as an innovative approach in controlling gene doping in horse sports.
- The research intends to provide a less invasive method of sample collection and easy storage condition compared to the conventional blood sample extraction method.
Methodology
- The extracted genomic DNA for this research was sourced from horsehair roots through a magnetic silica beads method of extraction.
- These hair roots were stored under refrigerated conditions to prevent degradation of their genomic DNA.
- From the extracted hair root genomic DNA, Whole Genome Resequencing (WGR) was conducted.
Findings
- The study found that most of the genomic DNA from hair roots stored at room temperature over the years was degraded. However, samples stored at refrigerated temperatures (4-8°C) were able to maintain their DNA quality.
- Results from nucleotide sequencing and the numbers of single-nucleotide polymorphisms and insertions/deletions concurred with previous reports for WGR of DNA extracted from whole blood.
- A significant finding of the study is that storing hair samples at refrigerated temperatures prevents degradation of DNA; this allows for the detection of gene doping in these samples based on WGR.
- The research successfully demonstrated the potential of WGR using horsehair roots for gene doping control.
Implications of the Research
- The findings of this study could have a deterrent effect as it is now possible to test horses for gene doping with archived samples even if they or their parents are deceased.
- This is the first report to use WGR on horse hair roots stored for a long term, highlighting the potential for more comprehensive and retrospective doping control in horse sports.
- The detection method can be especially useful in equestrian sports, where it is required to prohibit gene doping.
Cite This Article
APA
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA, Kusano K, Nagata SI.
(2020).
Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports.
J Equine Sci, 31(4), 75-83.
https://doi.org/10.1294/jes.31.75 Publication
Researcher Affiliations
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
- Equine Genetics Research Centre, Racing Australia, NSW 2337, Australia.
- Equine Department, Japan Racing Association, Tokyo 106-8401, Japan.
- Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
References
This article includes 24 references
- Ablondi M, Viklund Å, Lindgren G, Eriksson S, Mikko S. Signatures of selection in the genome of Swedish warmblood horses selected for sport performance.. BMC Genomics 2019 Sep 18;20(1):717.
- Anti-Doping Rules,nFédération Equestre Internationale (FEI)nhttps://inside.fei.org/content/anti-doping-rules [accessed on October 1,n2020].
- Bower MA, McGivney BA, Campana MG, Gu J, Andersson LS, Barrett E, Davis CR, Mikko S, Stock F, Voronkova V, Bradley DG, Fahey AG, Lindgren G, MacHugh DE, Sulimova G, Hill EW. The genetic origin and history of speed in the Thoroughbred racehorse.. Nat Commun 2012 Jan 24;3:643.
- Brzeziańska E, Domańska D, Jegier A. Gene doping in sport - perspectives and risks.. Biol Sport 2014 Dec;31(4):251-9.
- Chu Y, Bartus RT, Manfredsson FP, Olanow CW, Kordower JH. Long-term post-mortem studies following neurturin gene therapy in patients with advanced Parkinson's disease.. Brain 2020 Mar 1;143(3):960-975.
- Ducro BJ, Gorissen B, van Eldik P, Back W. Influence of foot conformation on duration of competitive life in a Dutch Warmblood horse population.. Equine Vet J 2009 Feb;41(2):144-8.
- Dupont JB, Guo J, Renaud-Gabardos E, Poulard K, Latournerie V, Lawlor MW, Grange RW, Gray JT, Buj-Bello A, Childers MK, Mack DL. AAV-Mediated Gene Transfer Restores a Normal Muscle Transcriptome in a Canine Model of X-Linked Myotubular Myopathy.. Mol Ther 2020 Feb 5;28(2):382-393.
- George LA, Ragni MV, Rasko JEJ, Raffini LJ, Samelson-Jones BJ, Ozelo M, Hazbon M, Runowski AR, Wellman JA, Wachtel K, Chen Y, Anguela XM, Kuranda K, Mingozzi F, High KA. Long-Term Follow-Up of the First in Human Intravascular Delivery of AAV for Gene Transfer: AAV2-hFIX16 for Severe Hemophilia B.. Mol Ther 2020 Sep 2;28(9):2073-2082.
- Haase B, Rieder S, Tozaki T, Hasegawa T, Penedo MC, Jude R, Leeb T. Five novel KIT mutations in horses with white coat colour phenotypes.. Anim Genet 2011 Jun;42(3):337-9.
- Haughan J, Jiang Z, Stefanovski D, Moss KL, Ortved KF, Robinson MA. Detection of intra-articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma.. Drug Test Anal 2020 Jun;12(6):743-751.
- InternationalnAgreement on Breeding Racing and Wagering (IABRW). InternationalnFederation of Horseracing Authorities (IFHA). https://www.ifhaonline.org/Default.asp?section=IABRW&area=15 [accessednon October 1, 2020].
- Kakoi H, Nagata S, Kurosawa M. DNA typing with 17 microsatellites for parentage verification of racehorses in Japan. Anim. Sci. J. 72: 453–460.
- Kovac M, Litvin YA, Aliev RO, Zakirova EY, Rutland CS, Kiyasov AP, Rizvanov AA. Gene Therapy Using Plasmid DNA Encoding VEGF164 and FGF2 Genes: A Novel Treatment of Naturally Occurring Tendinitis and Desmitis in Horses.. Front Pharmacol 2018;9:978.
- Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.. Bioinformatics 2009 Jul 15;25(14):1754-60.
- Martin NP, Myers P, Goulding E, Chen SH, Walker M, Porter TM, Van Gorder L, Mathew A, Gruzdev A, Scappini E, Romeo C. Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs.. J Vis Exp 2018 Nov 1;(141).
- McLaren W, Gil L, Hunt SE, Riat HS, Ritchie GR, Thormann A, Flicek P, Cunningham F. The Ensembl Variant Effect Predictor.. Genome Biol 2016 Jun 6;17(1):122.
- Moss KL, Jiang Z, Dodson ME, Linardi RL, Haughan J, Gale AL, Grzybowski C, Engiles JE, Stefanovski D, Robinson MA, Ortved KF. Sustained Interleukin-10 Transgene Expression Following Intra-Articular AAV5-IL-10 Administration to Horses.. Hum Gene Ther 2020 Jan;31(1-2):110-118.
- Neuberger EWI, Simon P. Gene and Cell Doping: The New Frontier - Beyond Myth or Reality.. Med Sport Sci 2017;62:91-106.
- Rosengren Pielberg G, Golovko A, Sundström E, Curik I, Lennartsson J, Seltenhammer MH, Druml T, Binns M, Fitzsimmons C, Lindgren G, Sandberg K, Baumung R, Vetterlein M, Strömberg S, Grabherr M, Wade C, Lindblad-Toh K, Pontén F, Heldin CH, Sölkner J, Andersson L. A cis-acting regulatory mutation causes premature hair graying and susceptibility to melanoma in the horse.. Nat Genet 2008 Aug;40(8):1004-9.
- Tozaki T, Kakoi H, Mashima S, Hirota K, Hasegawa T, Ishida N, Miura N, Choi-Miura NH, Tomita M. Population study and validation of paternity testing for Thoroughbred horses by 15 microsatellite loci.. J Vet Med Sci 2001 Nov;63(11):1191-7.
- Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control.. Genes (Basel) 2020 Apr 23;11(4).
- Tozaki T, Ohnuma A, Takasu M, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI. Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control.. Genes (Basel) 2019 Mar 21;10(3).
- Tozaki T, Ohnuma A, Takasu M, Nakamura K, Kikuchi M, Ishige T, Kakoi H, Hirora KI, Tamura N, Kusano K, Nagata SI. Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control.. Gene Ther 2021 Apr;28(3-4):199-205.
- Zhang J, Liu J, Yang W, Cui M, Dai B, Dong Y, Yang J, Zhang X, Liu D, Liang H, Cang M. Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats.. Theriogenology 2019 Jul 1;132:1-11.
Citations
This article has been cited 7 times.- 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. Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control. Genes (Basel) 2022 Sep 4;13(9).
- O'Hara V, Cowan A, Riddell D, Massey C, Martin J, Piercy RJ. A highly prevalent SINE mutation in the myostatin (MSTN) gene promoter is associated with low circulating myostatin concentration in Thoroughbred racehorses. Sci Rep 2021 Apr 12;11(1):7916.
- 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.
- Maehara K, Hirokawa A, Watanabe H, Otani N, Wan J, Shirai T, Takemasa T, Watanabe K, Nishi T, Sato K, Shimmura S, Nguyen KDM, Takahashi Y, Sugasawa T. Development of Detection Method Using Dried Blood Spot with Next-Generation Sequencing and LabDroid for Gene Doping Control. Int J Mol Sci 2025 Jun 26;26(13).
- Puchalska M, Witkowska-Piłaszewicz O. Gene doping in horse racing and equine sports: Current landscape and future perspectives. Equine Vet J 2025 Mar;57(2):312-324.
- Rubin CJ, Hodge M, Naboulsi R, Beckman M, Bellone RR, Kallenberg A, J'Usrey S, Ohmura H, Seki K, Furukawa R, Ohnuma A, Davis BW, Tozaki T, Lindgren G, Andersson L. An intronic copy number variation in Syntaxin 17 determines speed of greying and melanoma incidence in Grey horses. Nat Commun 2024 Aug 29;15(1):7510.
- Muko R, Ojima Y, Matsuda H, Toishi Y, Oikawa MA, Shin T, Sato H, Tanaka A. Comparison of DNA extraction methods for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms using oral mucosa swabs and feces. Vet Anim Sci 2024 Sep;25:100361.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
