A multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep

Abstract

Sex is an important biological variable as many physiological as well as disease processes differ between females and males. The fundamental biological distinction between females and males starts with chromosomal sex, and the establishment of XX and XY cells and tissues. Polymerase Chain Reaction (PCR) is a simple and effective method to easily determine chromosomal or genetic sex of cells and tissues. The goal of this study was to develop a simple multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep. Primers were designed to amplify a fragment of the autosomal gene myogenin (MYOG) and sex determining region on the Y chromosome (SRY). PCR analysis was performed on a variety of genomic DNA samples isolated from fetal sheep skeletal muscle, brain, liver, and placenta, and revealed a single 259 bp band for MYOG in XX females, and a 259 bp band for MYOG and a 167 bp band for SRY in XY males. Amplicons were clearly distinguishable by gel electrophoresis, and their sequences revealed 100% identity to the known ovine MYOG and SRY sequence. The reported multiplex PCR genotyping assay provides a rapid means to distinguish XX and XY sheep tissues using low volume samples.

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Abbreviations

PCR:

Polymerase chain reaction

MYOG:

Myogenin

SRY:

Sex determining region on the Y chromosome

AMEL:

Amelogenin

References

  1. 1.

    Anthony RV, Barry JS (2008) The pregnant sheep as a model for human pregnancy. Theriogenology 69(1):55–67

    Article  Google Scholar 

  2. 2.

    Pfeiffer I, Brenig B (2005) X- and Y-chromosome specific variants of the amelogenin gene allow sex determination in sheep (Ovis Aries) and European Red Deer (Cervus Elaphus). BMC Genet 6:16

    CAS  Article  Google Scholar 

  3. 3.

    Primer3Web version 4.1.0. http://bioinfo.ut.ee/primer3-0.4.0/. Accessed July, Sept 2019

  4. 4.

    NCBI National Center for Biotechnology Information. https://blast.ncbi.nlm.nih.gov/Blast.cgi. Accessed July, Sept 2019

  5. 5.

    Genewiz. https://www.genewiz.com/. Accessed Sept 2019

  6. 6.

    Tunster SJ (2017) Genetic sex determination of mice by simplex PCR. Biol Sex Differ 8:31

    Article  Google Scholar 

  7. 7.

    Ito H et al (2003) Sex identification by alternative polymerase chain reaction methods in falcorniformes. Zool Sci 20(3):339–344

    Article  Google Scholar 

  8. 8.

    Gokulakrishnan P (2012) Sex determination of cattle meat by polymerase chain reaction amplification of the DEAD box protein (DDX3X/DDX3Y) gene. Asian-Aust J Anim Sci 25(5):733–737

    CAS  Article  Google Scholar 

  9. 9.

    Ennis S, Gallagher TFA, PCR-Based (1994) Sex-determination assay in cattle based on the bovine amelogenin locus. Anim Genet 25(6):425–427

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors like to Asma Omar for her help with the tissue collections and providing the blinded samples. Funding for this work was provided by supported by Agriculture and Food Research Initiative Competitive Grant 2019-67015-29000 from the U.S. Department of Agriculture (USDA) National Institute of Food and Agriculture.

Funding

Funding for this work was provided by supported by Agriculture and Food Research Initiative Competitive Grant 2019-67015-29000 from the U.S. Department of Agriculture (USDA) National Institute of Food and Agriculture.

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Authors

Contributions

RE and HJE carried out the PCR experiments. RE and GJB wrote the manuscript. QAW and AJC contributed tissue samples and reagents. GJB conceived and designed the study.

Corresponding author

Correspondence to Gerrit J. Bouma.

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Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All tissues were collected as part of a separate study, and all animal procedures were approved by Institutional Animal Care and Use Committee at Colorado State University, Fort Collins, Colorado, USA.

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Evans, R., Engelhorn, H.J., Winger, Q.A. et al. A multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep. Mol Biol Rep (2020). https://doi.org/10.1007/s11033-020-05701-2

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Keywords

  • XX
  • XY
  • Sheep
  • PCR
  • Genotyping