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Home / Equine Research Bank / Equine Vet J / Screening of Y-chromosomal STR loci and development of a mul...
Equine veterinary journal2026; doi: 10.1002/evj.70153

Screening of Y-chromosomal STR loci and development of a multiplex PCR system for paternal lineage identification in horses.

Abstract: With growing demand for pedigree verification and breed management in horses (Equus caballus), reliable paternal lineage tools are essential. Y-chromosomal STRs (Y-STRs) have advantages over autosomal STRs due to paternal inheritance and lack of recombination. However, few validated loci and no standardised efficient genotyping systems limit their use. Current methods often require multiple reactions, increasing cost and labour. Thus, identifying informative Y-STR loci and developing a multiplex PCR system for cost-effective paternal lineage analysis is urgently needed. Objective: To identify informative horse Y-STR loci and develop a multiplex PCR system for paternal lineage tracing, breed conservation, pedigree verification, and forensic settings. Methods: Experimental study. Methods: Y-STR loci were screened from the horse reference genome based on repeat motif, number, and location. Primers were designed and tested on stallions and mares to confirm Y-specificity. Loci with stable, male-specific amplification were combined with known markers into a multiplex PCR system. Sensitivity, repeatability, reproducibility, and performance were evaluated, and locus polymorphism was assessed in Debao ponies, Mongolian horses, Thoroughbreds, and Arabian horses. Results: Twenty candidate loci were screened, and 15 of them showed clear, reproducible male-specific amplification. Three novel polymorphic loci were identified and combined with 5 known markers into 2-plex and 6-plex panels. Most loci were polymorphic in Debao ponies (PIC: 0.000-0.575) and Mongolian horses (PIC: 0.000-0.375), while less polymorphism was detected in Thoroughbreds (PIC: 0.000-0.099) and Arabian horses (PIC: 0.000-0.099). The optimised multiplex PCR system demonstrated high sensitivity, repeatability, and compatibility. Conclusions: Reproducibility and stability across labs and platforms require further validation. Conclusions: Fifteen new Y-STR loci were identified, and 3 of them were polymorphic. Novel optimised multiplex PCR systems were developed for the identification of horse paternal lines, providing reliable tools for equine genetic study, breeding, and forensic applications. 背景: 随着马(Equus caballus)系谱核查及育种管理需求的不断提升，建立可靠的父系谱系鉴定技术尤为重要。Y染色体短串联重复序列(Y‐chromosomal short tandem repeats，Y‐STR)因其父系遗传且不发生重组的特性，在父系谱系追溯中相较常染色体STR具有显著优势。然而，目前马Y‐STR已验证的位点数量有限，且缺乏标准化高效的基因分型系统，严重制约了其推广应用。现有检测方法多依赖多次扩增，检测成本高、操作复杂，亟需系统筛选具有应用价值的Y‐STR位点并构建低成本且高效的复合PCR父系谱系检测体系。 目的: 筛选有价值的马Y‐STR位点，构建复合PCR检测体系，为马父系谱系追溯、品种保护、系谱验证及法医学应用提供可靠技术手段。 研究设计: 试验性研究。 方法: 从马参考基因组，基于重复特征序列、数目及位置系统筛选Y‐STR候选位点。针对候选位点设计特异性引物，并在公马与母马样本中进行扩增验证，以确认其Y染色体特异性。将扩增稳定、具有雄性特异性的位点与已知的Y‐STR标记相结合，构建复合PCR体系。对所建体系的灵敏度、重复性、再现性及整体检测性能进行系统评估，并在德保矮马、蒙古马、纯血马和阿拉伯马群体中分析各位点的多态性水平。 结果: 本试验共筛选获得20个Y‐STR候选位点，其中15个位点表现出清晰且可重复的雄性特异性扩增。本试验鉴定出3个新的多态性Y‐STR位点，并与5个已知标记组合，成功构建2重和6重复合PCR检测体系。多态性分析结果显示，多数位点在德保矮马(多态信息含量PIC:0.000–0.575)和蒙古马(PIC:0.000–0.375)群体中表现出较高多态性，而在纯血马(PIC:0.000–0.099)和阿拉伯马(PIC:0.000–0.099)中多态性相对较低。所优化的复合PCR体系具有较高灵敏度、良好的重复性及兼容性。 局限性: 该复合PCR检测体系在不同实验室条件及不同检测平台间的稳定性与再现性仍需进一步验证。 结论: 本研究系统筛选并鉴定了15个新的马Y‐STR位点，其中3个位点具有多态性，并成功构建了优化的复合PCR检测体系。该体系可作为马父系谱系鉴定的有效工具，为马遗传学研究、育种实践及法医学应用提供了可靠的技术支撑。.
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Publication Date: 2026-03-10 PubMed ID: 41806849DOI: 10.1002/evj.70153Google Scholar: Lookup
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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.

Overview

  • This study identified new Y-chromosomal short tandem repeat (Y-STR) markers in horses and developed a multiplex PCR system for efficient and cost-effective paternal lineage identification useful in breeding, pedigree verification, and forensic analysis.

Background

  • Pedigree verification and breed management in horses require reliable tools to track paternal lineage.
  • Y-chromosomal STRs are ideal because they are inherited only from the father and do not undergo recombination, making them stable markers for paternal lineage tracing.
  • Existing Y-STR markers in horses are limited and lack standardized and efficient genotyping systems.
  • Current methods often require multiple PCR reactions, raising costs and labor intensity.
  • There is an urgent need to identify informative Y-STR loci and create a multiplex PCR system that is low-cost and efficient.

Objectives

  • Identify informative and polymorphic Y-STR loci in horses.
  • Develop a multiplex PCR detection system for analyzing paternal lineage in various horse breeds.
  • Provide a reliable technical method suitable for breed conservation, pedigree verification, and forensic applications.

Methods

  • Screened candidate Y-STR loci from the horse reference genome based on repeat motif, number of repeats, and genomic location.
  • Designed primers specific for these loci and tested them on male (stallion) and female (mare) samples to confirm Y chromosome specificity.
  • Selected loci showing stable, male-specific amplification for further study.
  • Combined novel polymorphic loci with previously known Y-STR markers to build multiplex PCR panels (2-plex and 6-plex systems).
  • Evaluated the multiplex system for sensitivity (ability to detect low DNA amounts), repeatability (consistency within the same lab), reproducibility (consistency between labs), and overall performance.
  • Assessed polymorphism (genetic variability) of loci in horse populations: Debao ponies, Mongolian horses, Thoroughbreds, and Arabian horses.

Results

  • Twenty candidate Y-STR loci were initially screened from the genome.
  • Fifteen loci showed clear, consistent male-specific amplification, confirming Y-chromosome specificity.
  • Among these, three novel loci were identified as polymorphic (i.e., variable between individuals), enhancing their usefulness as genetic markers.
  • These three loci were combined with five known markers to construct effective multiplex PCR panels (two-marker and six-marker panels).
  • Polymorphism analysis revealed that:
    • Debao ponies showed substantial genetic variation (Polymorphism Information Content, PIC: 0.000 to 0.575).
    • Mongolian horses had moderate variation (PIC: 0.000 to 0.375).
    • Thoroughbreds and Arabian horses showed low polymorphism (PIC: 0.000 to 0.099), indicating less genetic variation at these markers in these breeds.
  • The optimized multiplex PCR system demonstrated:
    • High sensitivity — able to detect low concentrations of DNA.
    • Good repeatability — consistent results in repeated tests.
    • Compatibility — worked well across different loci combined in the panel.

Limitations

  • The stability and reproducibility of the multiplex PCR system across different laboratories and detection platforms still need further validation.

Conclusions and Implications

  • The study contributes significantly by identifying 15 new Y-STR loci on the horse Y chromosome, including three with useful polymorphism for lineage differentiation.
  • The multiplex PCR system developed allows researchers and breeders to efficiently analyze paternal lines in horses with reduced costs and simpler protocols versus previous methods requiring multiple PCR reactions.
  • This tool is valuable for:
    • Tracing paternal lineage for breeding programs and maintaining breed integrity.
    • Confirming pedigrees in studbooks and breeding operations.
    • Forensic investigations involving horses.
  • Further testing across labs will enhance confidence in the system’s broad application.

Cite This Article

APA
Li Y, Fu C, Yun X, Zhang H, Yang T, Feng M, Wang X, Qian S, Xing W, Yang R, Wu J, Liu Y, Zhao C. (2026). Screening of Y-chromosomal STR loci and development of a multiplex PCR system for paternal lineage identification in horses. Equine Vet J. https://doi.org/10.1002/evj.70153

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English

Researcher Affiliations

Li, Yuanyuan
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
Fu, Chunzheng
  • Institute of Sericulture, Chengde Medical University, Chengde, China.
Yun, Xirong
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
Zhang, Hetong
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
Yang, Tao
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
Feng, Mo
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
Wang, Xinyu
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
Qian, Shiyu
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
Xing, Wenhui
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
Yang, Ran
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
Wu, Jiahui
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
Liu, Yu
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
Zhao, Chunjiang
  • College of Animal Science and Technology, China Agricultural University, Beijing, China.
  • State Key Laboratory of Animal Biotech Breeding, Beijing, China.
  • Equine Center, China Agricultural University, Beijing, China.
  • National Engineering Laboratory for Animal Breeding, Beijing, China.
  • Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture, Beijing, China.
  • Beijing Key Laboratory of Animal Genetic Improvement, Beijing, China.

Grant Funding

  • 32302731 / National Natural Science Foundation of China
  • IRT1191 / Program for Changjiang Scholars and Innovative Research Team in University
  • 19221073 / Project on the Third National Survey of the Livestock and Poultry Genetic Resources
  • Z171100002217072 / Beijing Key Laboratory for Genetic Improvement of Livestock and Poultry
  • National Germplasm Center of Domestic Animal Resources

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