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Frontiers in veterinary science2022; 9; 861623; doi: 10.3389/fvets.2022.861623

Development of a 17-Plex of Penta- and Tetra-Nucleotide Microsatellites for DNA Profiling and Paternity Testing in Horses.

Abstract: Tetranucleotide and pentanucleotide short tandem repeat (hereafter termed tetraSTR and pentaSTR) polymorphisms have properties that make them desirable for DNA profiling and paternity testing. However, certain species, such as the horse, have far fewer tetraSTRs than other species and for this reason dinucleotide STRs (diSTRs) have become the standard for DNA profiling in horses, despite being less desirable for technical reasons. During our testing of a series of candidate genes as potentially underlying a heritable condition characterized by megaesophagus in the Friesian horse breed, we found that good tetraSTRs do exist in horses but, as expected, at a much lower frequency than in other species, e.g., dogs and humans. Using a series of efficient methods developed in our laboratory for the production of multiplexed tetraSTRs in other species, we identified a set of tetra- and pentaSTRs that we developed into a 17-plex panel for the horse, plus a sex-identifying marker near the amelogenin gene. These markers were tested in 128 horses representing 16 breeds as well as crossbred horses, and we found that these markers have useful genetic variability. Average observed heterozygosities (Ho) ranged from 0.53 to 0.89 for the individual markers (0.66 average Ho for all markers), and 0.62-0.82 for expected heterozygosity (He) within breeds (0.72 average He for all markers). The probability of identity (PI) within breeds for which 10 or more samples were available was at least 1.1 x 10-11, and the PI among siblings (PIsib) was 1.5 x 10-5. Stutter was ≤ 11% (average stutter for all markers combined was 6.9%) compared to the more than 30% typically seen with diSTRs. We predict that it will be possible to develop accurate allelic ladders for this multiplex panel that will make cross-laboratory comparisons easier and will also improve DNA profiling accuracy. Although we were only able to exclude candidate genes for Friesian horse megaesophagus with no unexcluded genes that are possibly causative at this point in time, the study helped us to refine the methods used to develop better tetraSTR multiplexed panels for species such as the horse that have a low frequency of tetraSTRs.
Copyright © 2022 Luttman, Komine, Thaiwong, Carpenter, Ewart, Kiupel, Langohr and Venta.
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Publication Date: 2022-04-07 PubMed ID: 35464354PubMed Central: PMC9021955DOI: 10.3389/fvets.2022.861623Google 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.

The research article describes a newly developed 17-plex panel of tetra- and penta-nucleotide microsatellites for DNA profiling and paternity testing in horses, despite the usual challenges surrounding the low occurrence of such nucleotides in this species. The researchers were able to test the markers for their genetic variability on horses of various breeds. Although the primary aim of finding possible genetic causes for a hereditary condition in the Friesian horse breed was unsuccessful, the process led to the improvement of methods for creating similar DNA tools.

Overview of the study

  • The researchers, while searching for the genetic basis of a hereditary disease in Friesian horses, found that desirable tetraSTRs and pentaSTRs do exist in horses, albeit at a much lower frequency than in other species. These nucleotide sequences are preferred for DNA profiling and paternity testing due to their properties.
  • The team developed a 17-plex panel of these tetra- and penta-nucleotide short tandem repeats (STRs) for horses. They faced certain challenges in this task due to the rarity of these type of STRs in horses.
  • An added feature in the panel was a marker for identifying the sex of the horse, placed near the amelogenin gene.

Testing and results

  • The panel was tested on 128 horses from 16 different breeds including crossbreeds. The aim was to determine the efficacy of these markers and their genetic variability.
  • Average observed heterozygosities (Ho) ranged between 0.53 to 0.89 for the individual markers and noted an average of 0.66 Ho for all markers. Within the breeds, expected heterozygosity (He) ranged from 0.62-0.82 with an average He of 0.72 for all markers.
  • The probability of identity (PI) or the chance of two individuals having the same genetic profile, within breeds for which 10 or more samples were available, was at least 1.1 x 10^-30. The PI among siblings (PIsib) was noted to be 1.5 x 10^-30.
  • The “stutter” or extraneous DNA fragments, a common issue in STR testing, was found to be very low, ≤ 11% (average stutter for all markers combined was 6.9%) compared to the more than 30% typically seen with diSTRs.

Conclusions and future implications

  • Even though the study was unable to pinpoint any genetic cause for the heritable condition in Friesian horses, it led to the development of improved methods for creating multiplexed tetraSTR panels in species with low frequencies of tetraSTRs.
  • The authors anticipate that it will be possible to develop accurate allelic ladders (reference standards) for this multiplex panel, enabling easier cross-laboratory comparisons and improved DNA profiling accuracy.

Cite This Article

APA
Luttman AM, Komine M, Thaiwong T, Carpenter T, Ewart SL, Kiupel M, Langohr IM, Venta PJ. (2022). Development of a 17-Plex of Penta- and Tetra-Nucleotide Microsatellites for DNA Profiling and Paternity Testing in Horses. Front Vet Sci, 9, 861623. https://doi.org/10.3389/fvets.2022.861623

Publication

Frontiers in veterinary science
ISSN: 2297-1769
NlmUniqueID: 101666658
Country: Switzerland
Language: English
Volume: 9
Pages: 861623

Researcher Affiliations

Luttman, Andrea M
  • Microbiology and Molecular Genetics, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.
  • Genetics and Genomic Sciences, Michigan State University, East Lansing, MI, United States.
Komine, Misa
  • Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.
Thaiwong, Tuddow
  • Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.
Carpenter, Tyler
  • Microbiology and Molecular Genetics, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.
  • Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University College of Human Medicine, Grand Rapids, MI, United States.
Ewart, Susan L
  • Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.
Kiupel, Matti
  • Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.
  • Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.
Langohr, Ingeborg M
  • Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.
  • Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, United States.
Venta, Patrick J
  • Microbiology and Molecular Genetics, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.
  • Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI, United States.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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