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Home / Equine Research Bank / Front Vet Sci / Development of a 17-Plex of Penta- and Tetra-Nucleotide Micr...
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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  • 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.

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.

References

This article includes 47 references
  1. Weber JL, Broman KW. Genotyping for human whole-genome scans: past, present, and future.. Adv Genet (2001) 42:77–96.
    doi: 10.1016/S0065-2660(01)42016-5pubmed: 11037315google scholar: lookup
  2. Wictum E, Kun T, Lindquist C, Malvick J, Vankan D, Sacks B. Developmental validation of DogFiler, a novel multiplex for canine DNA profiling in forensic casework.. Forensic Sci Int Genet (2013) 7:82–91.
    doi: 10.1016/j.fsigen.2012.07.001pubmed: 22832398google scholar: lookup
  3. Komine M, Langohr IM, Kiupel M. Megaesophagus in Friesian horses associated with muscular hypertrophy of the caudal esophagus.. Vet Pathol (2014) 51:979–85.
    doi: 10.1177/0300985813511126pubmed: 24227010google scholar: lookup
  4. Ploeg M, Gröne A, Saey V, de Bruijn CM, Back W, van Weeren PR. Esophageal dysfunction in Friesian horses: morphological features.. Vet Pathol (2015) 52:1142–7.
    doi: 10.1177/0300985814556780pubmed: 25367366google scholar: lookup
  5. Winkler PA, Bartoe JT, Quinones CR, Venta PJ, Petersen-Jones SM. Exclusion of eleven candidate genes for ocular melanosis in Cairn terriers.. J Negat Results Biomed (2013) 12:6.
    doi: 10.1186/1477-5751-12-6pmc: PMC3599239pubmed: 23448350google scholar: lookup
  6. Winkler PA, Gornik KR, Ramsey DT, Dubielzig RR, Venta PJ, Petersen-Jones SM. A partial gene deletion of SLC45A2 causes oculocutaneous albinism in Doberman pinscher dogs.. PLoS ONE (2014) 9:e92127.
    doi: 10.1371/journal.pone.0092127pmc: PMC3960214pubmed: 24647637google scholar: lookup
  7. Binns M, Swinburne JE, Breen M. Molecular genetics of the horse.. In: Bowling AT. and Ruvinsky A, editors. The Genetics of the Horse. Cambridge: CABI Pub; (2000). p. 109–22.
  8. Clark LA, Tsai KL, Steiner JM, Williams DA, Guerra T, Ostrander EA. Chromosome-specific microsatellite multiplex sets for linkage studies in the domestic dog.. Genomics (2004) 84:550–4.
    doi: 10.1016/j.ygeno.2004.06.006pubmed: 15498461google scholar: lookup
  9. Dorji J, Tamang S, Tshewang T, Dorji T, Dorji TY. Genetic diversity and population structure of three traditional horse breeds of Bhutan based on 29 DNA microsatellite markers.. PLoS ONE (2018) 13:e0199376.
    doi: 10.1371/journal.pone.0199376pmc: PMC6021118pubmed: 29949614google scholar: lookup
  10. van de Goor LH, Panneman H, van Haeringen WA. A proposal for standardization in forensic equine DNA typing: allele nomenclature for 17 equine-specific STR loci.. Anim Genet (2010) 41:122–7.
    doi: 10.1111/j.1365-2052.2009.01975.xpubmed: 19821810google scholar: lookup
  11. Conant EK, Juras R, Cothran EG. A microsatellite analysis of five Colonial Spanish horse populations of the southeastern United States.. Anim Genet (2012) 43:53–62.
    doi: 10.1111/j.1365-2052.2011.02210.xpubmed: 22221025google scholar: lookup
  12. Srivastava S, Avvaru AK, Sowpati DT, Mishra RK. Patterns of microsatellite distribution across eukaryotic genomes.. BMC Genomics (2019) 20:153.
    doi: 10.1186/s12864-019-5516-5pmc: PMC6387519pubmed: 30795733google scholar: lookup
  13. De Barba M, Miquel C, Lobréaux S, Quenette PY, Swenson JE, Taberlet P. High-throughput microsatellite genotyping in ecology: improved accuracy, efficiency, standardization and success with low-quantity and degraded DNA.. Mol Ecol Resour (2017) 17:492–507.
    doi: 10.1111/1755-0998.12594pubmed: 27505280google scholar: lookup
  14. Sun JX, Helgason A, Masson G, Ebenesersdóttir SS, Li H, Mallick S. A direct characterization of human mutation based on microsatellites.. Nat Genet (2012) 44:1161–5.
    doi: 10.1038/ng.2398pmc: PMC3459271pubmed: 22922873google scholar: lookup
  15. Baric S, Monschein S, Hofer M, Grill D, Dalla Via J. Comparability of genotyping data obtained by different procedures – an inter-laboratory survey.. J Horticult Sci Biotechnol (2008) 83:183–90.
    doi: 10.1080/14620316.2008.11512368google scholar: lookup
  16. Hirota K, Kakoi H, Gawahara H, Hasegawa T, Tozaki T. Construction and validation of parentage testing for thoroughbred horses by 53 single nucleotide polymorphisms.. J Vet Med Sci (2010) 72:719–26.
    doi: 10.1292/jvms.09-0486pubmed: 20124759google scholar: lookup
  17. Holl HM, Vanhnasy J, Everts RE, Hoefs-Martin K, Cook D, Brooks SA. Single nucleotide polymorphisms for DNA typing in the domestic horse.. Anim Genet (2017) 48:669–76.
    doi: 10.1111/age.12608pubmed: 28901559google scholar: lookup
  18. Shubitowski DM, Venta PJ, Douglass CL, Zhou RX, Ewart SL. Polymorphism identification within 50 equine gene-specific sequence tagged sites.. Anim Genet (2001) 32:78–88. Erratum in: Anim Genet (2001) 32: 332.
    doi: 10.1046/j.1365-2052.2001.00738.xpubmed: 11421942google scholar: lookup
  19. Andersson LS, Wilbe M, Viluma A, Cothran G, Ekesten B, Ewart S. Equine multiple congenital ocular anomalies and silver coat colour result from the pleiotropic effects of mutant PMEL.. PLoS ONE (2013) 8:e75639.
    doi: 10.1371/journal.pone.0075639pmc: PMC3781063pubmed: 24086599google scholar: lookup
  20. Brownstein MJ, Carpten JD, Smith JR. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping.. Biotechniques (1996) 20:1004-6, 1008–10.
    doi: 10.2144/96206st01pubmed: 8780871google scholar: lookup
  21. Gouy M, Guindon S, Gascuel O. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building.. Mol Biol Evol (2010) 27:221–4.
    doi: 10.1093/molbev/msp259pubmed: 19854763google scholar: lookup
  22. Blacket MJ, Robin C, Good RT, Lee SF, Miller AD. Universal primers for fluorescent labelling of PCR fragments–an efficient and cost-effective approach to genotyping by fluorescence.. Mol Ecol Resour (2012) 12:456–63.
    doi: 10.1111/j.1755-0998.2011.03104.xpubmed: 22268566google scholar: lookup
  23. Hill CR, Butler JM, Vallone PM. A 26plex autosomal STR assay to aid human identity testing*.. J Forensic Sci (2009) 54:1008–15.
    doi: 10.1111/j.1556-4029.2009.01110.xpubmed: 19627417google scholar: lookup
  24. Vallone PM, Butler JM. AutoDimer: a screening tool for primer-dimer and hairpin structures.. Biotechniques (2004) 37:226–31.
    doi: 10.2144/04372ST03pubmed: 15335214google scholar: lookup
  25. Brookes C, Bright JA, Harbison S, Buckleton J. Characterising stutter in forensic STR multiplexes.. Forensic Sci Int Genet (2012) 6:58–63.
    doi: 10.1016/j.fsigen.2011.02.001pubmed: 21388903google scholar: lookup
  26. Corner S, Yuzbasiyan-Gurkan V, Agnew D, Venta PJ. Development of a 12-plex of new microsatellite markers using a novel universal primer method to evaluate the genetic diversity of jaguars (Panthera onca) from North American zoological institutions.. Conserv Genet Resour (2019) 11:487–97.
    doi: 10.1007/s12686-018-1070-8google scholar: lookup
  27. Benson G. Tandem repeats finder: a program to analyze DNA sequences.. Nucleic Acids Res (1999) 27:573–80.
    doi: 10.1093/nar/27.2.573pmc: PMC148217pubmed: 9862982google scholar: lookup
  28. Bär W, Brinkmann B, Budowle B, Carracedo A, Gill P, Lincoln P. DNA recommendations. Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems. International Society for Forensic Haemogenetics.. Int J Legal Med (1997)110:175–6.
    pubmed: 9274938
  29. Parson W, Ballard D, Budowle B, Butler JM, Gettings KB, Gill P. Massively parallel sequencing of forensic STRs: Considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) on minimal nomenclature requirements.. Forensic Sci Int Genet (2016) 22:54–63.
    doi: 10.1016/j.fsigen.2016.01.009pubmed: 26844919google scholar: lookup
  30. Peakall R, Smouse PE. Genalex 6: genetic analysis in Excel. Population genetic software for teaching and research.. Mol Ecol Notes (2006)6:288–95.
    doi: 10.1111/j.1471-8286.2005.01155.xpmc: PMC3463245pubmed: 22820204google scholar: lookup
  31. Peakall R, Smouse PE. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research–an update.. Bioinformatics (2012) 28:2537–9.
    doi: 10.1093/bioinformatics/bts460pmc: PMC3463245pubmed: 22820204google scholar: lookup
  32. Marshall TC, Slate J, Kruuk LE, Pemberton JM. Statistical confidence for likelihood-based paternity inference in natural populations.. Mol Ecol (1998) 7:639–55.
    doi: 10.1046/j.1365-294x.1998.00374.xpubmed: 9633105google scholar: lookup
  33. Kun T, Lyons LA, Sacks BN, Ballard RE, Lindquist C, Wictum EJ. Developmental validation of Mini-DogFiler for degraded canine DNA.. Forensic Sci Int Genet (2013) 7:151–8.
    doi: 10.1016/j.fsigen.2012.09.002pubmed: 23040244google scholar: lookup
  34. Keven JB, Walker ED, Venta PJ. A Microsatellite multiplex assay for profiling pig DNA in mosquito bloodmeals.. J Med Entomol (2019) 56:907–14.
    doi: 10.1093/jme/tjz013pmc: PMC6595529pubmed: 30768665google scholar: lookup
  35. Innis M, Gelfand D. Optimization of PCR: conversations between Michael and David.. In Innis MA, Gelfand DH, Sninsky JJ, editors. PCR Applications: Protocols for Functional Genomics. San Diego: Academic Press; (1999). p. 3–22.
    doi: 10.1016/B978-012372185-3/50002-Xgoogle scholar: lookup
  36. Te Meerman GJ, Van der Meulen MA, Sandkuijl LA. Perspectives of identity by descent (IBD) mapping in founder populations.. Clin Exp Allergy (1995) 25(Suppl 2):97–102.
    doi: 10.1111/j.1365-2222.1995.tb00433.xpubmed: 8590355google scholar: lookup
  37. Sutton JT, Robertson BC, Jamieson IG. Dye shift: a neglected source of genotyping error in molecular ecology.. Mol Ecol Resour (2011) 11:514–20.
    doi: 10.1111/j.1755-0998.2011.02981.xpubmed: 21481209google scholar: lookup
  38. Gettings KB, Aponte RA, Vallone PM, Butler JM. STR allele sequence variation: current knowledge and future issues.. Forensic Sci Int Genet (2015) 18:118–30.
    doi: 10.1016/j.fsigen.2015.06.005pubmed: 26197946google scholar: lookup
  39. Guichoux E, Lagache L, Wagner S, Chaumeil P, Léger P, Lepais O. Current trends in microsatellite genotyping.. Mol Ecol Resour (2011) 11:591–611.
    doi: 10.1111/j.1755-0998.2011.03014.xpubmed: 21565126google scholar: lookup
  40. Budowle B, Garofano P, Hellman A, Ketchum M, Kanthaswamy S, Parson W. Recommendations for animal DNA forensic and identity testing.. Int J Legal Med (2005) 119:295–302.
    doi: 10.1007/s00414-005-0545-9pubmed: 15834735google scholar: lookup
  41. Chen JM, Férec C, Cooper DN. Complex multiple-nucleotide substitution mutations causing human inherited disease reveal novel insights into the action of translesion synthesis DNA polymerases.. Hum Mutat (2015) 36:1034–8.
    doi: 10.1002/humu.22831pubmed: 26172832google scholar: lookup
  42. Brinkmann B, Klintshar M, Neuhuber F, Hühne F, Rolf B. Mutation rate in human microsatellites: influence of the structure and length of the tandem repeat.. Am J Hum Genet (1998) 62:1408–15.
    doi: 10.1086/301869pmc: PMC1377148pubmed: 9585597google scholar: lookup
  43. Zangenberg G, Saiki RK, Reynolds K. Multiplex PCR: optimization guidelines.. In: Innis MA, Gelfand DH, Sninsky JJ, editors. PCR Applications: Protocols for Functional Genomics. San Diego: Academic Press; (1999). p. 73–94.
    doi: 10.1016/B978-012372185-3/50007-9google scholar: lookup
  44. Nagy K, Sung HK, Zhang P, Laflamme S, Vincent P, Agha-Mohammadi S. Induced pluripotent stem cell lines derived from equine fibroblasts.. Stem Cell Rev Rep (2011) 7:693–702.
    doi: 10.1007/s12015-011-9239-5pmc: PMC3137777pubmed: 21347602google scholar: lookup
  45. Horbach SPJM, Halffman W. The ghosts of HeLa: How cell line misidentification contaminates the scientific literature.. PLoS ONE (2017) 12:e0186281.
    doi: 10.1371/journal.pone.0186281pmc: PMC5638414pubmed: 29023500google scholar: lookup
  46. Venta PJ, Nguyen AK, Senut M-C, Poulos WG, Prukudom S, Cibelli JB. A 13-plex of tetra- and penta-STRs to identify zebrafish.. Sci Rep (2020) 10:3851–8.
    doi: 10.1038/s41598-020-60842-5pmc: PMC7052278pubmed: 32123258google scholar: lookup
  47. Durco BJ, Bovenhuis H, Neuteboom M, Hellinga I. Genetic diversity in the Dutch Friesian Horse.. In: Proceeding of the 8th World Congress on Genetics Applied to Livestock Production (Belo Horizonte: ). (2006). p. 08–03.
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