Microsatellites, also known as short tandem repeats (STRs), are repetitive sequences of DNA that consist of short motifs repeated in tandem. In horses, these genetic markers are distributed throughout the genome and exhibit high levels of polymorphism, making them valuable tools in genetic studies. Microsatellites are commonly used in parentage verification, genetic diversity assessments, and population structure analyses in equine research. They provide insights into hereditary traits and can assist in the management of breeding programs. This page compiles peer-reviewed research studies and scholarly articles that explore the application, variation, and genetic implications of microsatellites in horse populations.
Tozaki T, Kakoi H, Mashima S, Hirota K, Hasegawa T, Ishida N, Miura N, Choi-Miura NH, Tomita M.Microsatellite 15 TKY System was characterized for parentage verification of horse registry. The Microsatellite 15 TKY System was constructed by using 15 microsatellites, TKY279, TKY287, TKY294, TKY297, TKY301, TKY312, TKY321, TKY325, TKY333, TKY337, TKY341, TKY343, TKY344, TKY374, and TKY394, to provide stringent PCR-based microsatellite typing specifically optimized for multicolor fluorescence detection. The Microsatellite 15 TKY System showed good resolutions for 250 unrelated Thoroughbred horses, and the probability of exclusion (PE) at each microsatellite ranged from 0.437 to 0.621, resul...
Locke MM, Ruth LS, Millon LV, Penedo MC, Murray JD, Bowling AT.The colour locus historically referred to as C in the horse is linked to microsatellites markers on horse chromosome 21. Preliminary results demonstrated linkage of Ccr, thought to be the cream dilution variant of the C locus, to HTG10. An analysis of horse chromosome 21 using additional families confirmed and established a group of markers linked to Ccr. This work also improved the resolution of previously reported linkage maps for this chromosome. Linkage analysis unambiguously produced the map order: SGCV16-(19.1 cM)-HTG10-(3.8 cM)-LEX60/COR73-(1.3 cM)-COR68-(4.5 cM)- Ccr-(11.9 cM)-LEX31. C...
Cunningham EP, Dooley JJ, Splan RK, Bradley DG.The thoroughbred (TB) horse is one of the oldest breeds of domestic animals, with pedigree records spanning three centuries. Because the population is essentially closed, there is concern about loss of genetic variation. Here we report two parallel analyses. In the first, genetic variation in the current population is measured using data from 13 microsatellite loci in 211 horses with relationships calculated based on allele sharing. In the second analysis, pedigree information is used to calculate genetic relationships between animals based on shared ancestry. These two measures of relationshi...
Aranguren-Méndez J, Jordana J, Gomez M.Genetic diversity at 13 equine microsatellite loci was compared in five endangered Spanish donkey breeds: Andaluza, Catalana, Mallorquina, Encartaciones and Zamorano-Leonesa. All of the equine microsatellites used in this study were amplified and were polymorphic in the domestic donkey breeds with the exception of HMS1, which was monomorphic, and ASB2, which failed to amplify. Allele number, frequency distributions and mean heterozygosities were very similar among the Spanish donkey breeds. The unbiased expected heterozygosity (H(E)) over all the populations varied between 0.637 and 0.684 in t...
Shubitowski DM, Venta PJ, Douglass CL, Zhou RX, Ewart SL.The continued discovery of polymorphisms in the equine genome will be important for future studies using genomic screens and fine mapping for the identification of disease genes. Segments of 50 equine genes were examined for variability in 10 different horse breeds using a pool-and-sequence method. We identified 11 single nucleotide polymorphisms (SNPs) in 9380 bp of sequenced exon, and 25 SNPs, six microsatellites, and one insertion/deletion in 16961 bp of sequenced intron. Of all genes studied 52% contained at least one polymorphism, and polymorphisms were found at an overall rate of 1/613 b...
Terry RR, Bailey E, Bernoco D, Cothran EG.The appaloosa coat colour pattern of the horse is similar to that caused by the rump-white (Rw) gene in the mouse. In the mouse Rw colour pattern is the result of an inversion involving the proto-oncogene c-kit (KIT). Therefore, we investigated KIT as a candidate gene that encodes the appaloosa coat colour gene (Lp) in horses. KIT plays a critical role in haematopoiesis, gametogenesis, and melanogenesis and encodes a transmembrane tyrosine kinase receptor that belongs to the PDGF/CSF-1/c-KIT receptor subfamily. Half-sib families segregating for Lp were uninformative for a reported polymorphism...
Bjørnstad G, Røed KH.Population demarcation of eight horse breeds was investigated using genotype information of 306 horses from 26 microsatellite loci. The breeds include the indigenous Norwegian breeds Fjord Horse, Nordland/Lyngen Horse, Døle Horse and Coldblooded Trotter together with Icelandic Horse, Shetland Pony, Standardbred and Thoroughbred. Both phylogenetic analysis and a maximum likelihood method were applied to examine the potential for breed allocation of individual animals. The phylogenetic analysis utilizing simple allele sharing statistics revealed clear demarcation among the breeds; 95% of the in...
Lindgren G, Swinburne JE, Breen M, Mariat D, Sandberg K, Guérin G, Ellegren H, Binns MM.A horse bacterial artificial chromosome (BAC) library was screened for 19 microsatellite markers from unassigned or non-oriented linkage groups. Clones containing 11 (AHT20, EB2E8, HMS45, LEX005, LEX014, LEX023, LEX044, TKY111, UCDEQ425, UCDEQ464 and VIASH21) of these were found, which were from eight different linkage groups. The BAC clones were used as probes in dual colour FISH to identify their precise chromosomal origin. The microsatellite markers are located on nine different horse chromosomes, four of which (ECA6, ECA25, ECA27 and ECA28) had no previously in situ assigned markers.
Mariat D, Oustry-Vaiman A, Cribiu EP, Raudsepp T, Chowdhary BP, Guérin G.In order to increase the number of markers on the horse cytogenetic map and expand the integration with the linkage map, an equine BAC library was screened for genes and for microsatellites. Eighty-nine intra-exon primers were designed from consensus gene sequences in documented species. After PCR screening, 38 clones containing identified genes were isolated and FISH mapped. These data allowed us to refine the available Zoo-FISH results, to define ten new conserved cytogenetic segments and expand two others, thus leading to the identification of a total of 26 conserved segments between horse ...
Tozaki T, Mashima S, Hirota K, Miura N, Choi-Miura NH, Tomita M.We performed efficient cloning and genotyping methods for isolation of a large number of polymorphic microsatellites. The methods contain the time-efficient cloning method of constructing microsatellite-enriched libraries and the economic genotyping method of fluorescent labeling of PCR products. Eighty novel equine microsatellites cloned were efficiently isolated from the enrichment library and analyzed for genotype polymorphism. Of these, 72 microsatellites were analyzed with a good resolution. The average heterozygosity of all loci was 0.52, and the number of alleles ranged from one to 9 wi...
Swinburne J, Gerstenberg C, Breen M, Aldridge V, Lockhart L, Marti E, Antczak D, Eggleston-Stott M, Bailey E, Mickelson J, Røed K, Lindgren G....Two 3-generation full-sibling reference families have been produced and form a unique resource for genetic linkage mapping studies in the horse. The F(2) generations, now comprising 61 individuals, consist of 28- to 32-day-old embryos removed nonsurgically from two pairs of identical twin mares. The same stallion sired all F(2)s such that the two full-sibling families are half-sibling with respect to each other. The families are crossbred to maximize levels of heterozygosity and include Arabian, Thoroughbred, Welsh Cob, and Icelandic Horse breeds. Milligram quantities of DNA have been isolated...
Tozaki T, Inoue S, Mashima S, Ohta M, Miura N, Tomita M.Microsatellites are useful tools for the construction of a linkage map and parentage testing of equines, but only a limited number of equine microsatellites have been elucidated. Thus, we constructed the equine genomic library enriched for DNA fragments containing (CAG)n repeats. The enriched method includes hybridization-capture of repeat regions using biotin-conjugated oligonucleotides, nucleotide substrate-biased polymerase reaction with the oligonucleotides and subsequent PCR amplification, because these procedures are useful for the cloning of less abundant trinucleotide microsatellites. ...
Shiue Y-L , Millon LV, Skow LC, Honeycutt D, Murray JD, Bowling AT.The hypothesis that the conservation of sex-chromosome-linked genes among placental mammals could be extended to the horse genome was tested using the UCDavis horse-mouse somatic cell hybrid (SCH) panel. By exploiting the fluorescence in-situ hybridization (FISH) technique to localize an anchor locus, X-inactivation-specific transcript (XIST) on the horse X chromosome, together with the fragmentation and translocation of the X- and Y-chromosome fragments in a somatic cell hybrid panel, we regionally assigned 13 type I and 13 type II (microsatellite) markers to the horse X- and Y-chromosomes. T...
Cañon J, Checa ML, Carleos C, Vega-Pla JL, Vallejo M, Dunner S.Partition of the genetic variability, genetic structure and relationships among seven Spanish Celtic horse breeds were studied using PCR amplification of 13 microsatellites on 481 random individuals. In addition, 60 thoroughbred horses were included. The average observed heterozygosity and the mean number of alleles were higher for the Atlantic horse breeds than for the Balearic Islands breeds. Only eight percentage of the total genetic variability could be attributed to differences among breeds (mean FST approximately 0.08; P < 0.01). Atlantic breeds clearly form a separate cluster from th...
Lagneaux D, Pomarici AM, Sattler M, Bruneau B, Duchamp G, Camillo F, Palmer E.Day 6.5 equine embryos (n=30) were frozen in a medium containing glycerol (2.5-10.0%) supplemented with 0, 20 or 100 mmol L-glutamine 1(-1). After thawing, the embryos were tested individually, using 4',6'-diamidino-2-phenylindole (DAPI) staining to evaluate cell death. Three embryos (one frozen at each L-glutamine concentration) were transferred together into individual recipient mares. Pregnancy diagnosis was performed at day 12 (age of embryo). Embryos were collected at day 14 (age of embryo) and were identified by PCR amplified microsatellite analysis. Nine of ten recipient mares that rece...
Guérin G, Bailey E, Bernoco D, Anderson I, Antczak DF, Bell K, Binns MM, Bowling AT, Brandon R, Cholewinski G, Cothran EG, Ellegren H, Förster M....The goal of the First International Equine Gene Mapping Workshop, held in 1995, was the construction of a low density, male linkage map for the horse. For this purpose, the International Horse Reference Family Panel (IHRFP) was established, consisting of 12 paternal half-sib families with 448 half-sib offspring provided by 10 laboratories. Blood samples were collected and DNA extracted in each laboratory and sent to the Lexington laboratory (KY, USA) for dispatch in aliquots to 14 typing laboratories. In total, 161 markers (144 microsatellites, seven blood groups and 10 proteins) were tested f...
Kalashnikov V, Khrabrova L, Blohina N, Zaitcev A, Kalashnikova T.The Thoroughbred (TB) horse has hugely impacted the development of horse breeding around the world. This breed has unique genetic qualities due to having had a closed studbook for approximately 300 years. In Russia, TBs have been bred since the second half of the 18th century. Here, we analyzed the genetic diversity and the inbreeding level in TB horses (n = 9680) for the period from 1990 to 2018 using polymorphisms of 17 microsatellite loci. We found that the genetic structure of the TB breed in Russia is represented by 100 alleles of panel STR (short tandem repeat) loci and has been stable f...
Yoshihara T, Kikuchi M, Akiba Y, Horiguchi M, Takasu M, Tozaki T.The Taishu horse in Tsushima is one of eight Japanese native breeds. The breed is on the verge of extinction due to a rapid decrease in numbers since the 1960s owing to motorization in Japan. In this study, we aimed to confirm the pedigree information of 52 horses by genotyping 31 microsatellites in order to avoid inbreeding. Parentage verification failed to identify genetic contradictions among trios (sires, dams, and foals) registered with the Japan Equine Affairs Association (JEAA). Pedigree information registered at the JEAA was obtained and adequately understood. Additionally, the genetic...
Gupta AK, Chauhan M, Bhardwaj A.For studying the genetic diversity and bottleneck problem in Bhutia and Manipuri pony breeds of India, we analysed DNA samples of 34 Bhutia and 50 Manipuri, true to breed, ponies using 47 polymorphic microsatellite markers. All the microsatellites were observed to be highly polymorphic in nature in both Bhutia and Manipuri breeds with mean no. of alleles as 8.702 ± 0.0493 and 8.416 ± 0.0548 respectively. Genetic diversity values in terms of heterozygosity values within individual breeds were also high with very low inbreeding (Fis 0.102 and 0.055 in Bhutia and Manipuri ponies, respective...
Schelling C, Hagger C, Pieńkowska A, Siegfried JP, Stranzinger G.A population of Baudet du Poitou donkeys was genetically characterized using microsatellites. The results were used to verify the pedigrees and to estimate the genetic variability. It could be confirmed that a equine parentage test kit works well for donkeys and that by using 13 microsatellites more than 99% of wrong pedigree informations would be detected. The genetic variability was comparable to a representative group of Baudet du Poitou donkeys in France.
Zhao CJ, Qin YH, Lee XH, Wu Ch.An alleged male foal of a female mule, whose sire and grandparents were unknown, was identified for its pedigree. Parentage testing was conducted by comparing polymorphism of 12 microsatellite DNA sites and mitochondrial D-loop sequences of the male foal and the female mule. Both the sequence analysis of species-specific DNA fragments and a cytogenetic analysis were performed to identify the species of the foal and its parents. The results showed that the alleged female mule is actually a hinny, and the male foal, which possesses 62 chromosomes, qualifies as an offspring of the female hinny an...
Troyer D, Howard D, Leipold HW, Smith JE.Southern blot analysis of equine DNA's digested with the restriction endonuclease Hinfl hybridized with a 32 PdCTP labeled human VNTR probe revealed a highly polymorphic pattern of restriction fragments upon autoradiography. The horses were unrelated individuals of the quarter horse breed. This heterologous probe can thus be used in the equine species for individual identification and pedigree analysis.
Díaz S, Kienast ME, Villegas-Castagnasso EE, Pena NL, Manganare MM, Posik D, Peral-García P, Giovambattista G.In order to detect switching and/or manipulation of samples, the owner of a stallion asked our lab to perform a DNA test on a positive doping urine sample. The objective was to compare the urine DNA profile versus blood and hair DNA profiles from the same stallion. At first, 10 microsatellite markers were investigated to determine the horse identity. No results were obtained when horse specific markers were typed in the urine sample. In order to confirm the species origin of this sample we analyzed the mitochondrial cytochrome b gene. This analysis from blood and hair samples produced reproduc...
Feofilov AV, Bardukov NV, Glazko VI.Using ISSR-PCR marker data, comparative analysis of the gene pools of Altaic and trotting horse breeds was carried out. Horse groups of different origin demonstrated differences in amplification spectra of DNA fragments flanked by inverted repeats of four microsatellites. Combinations of certain DNA fragments present in these profiles reproducibly distinguished genomes of the Altaic breed from the trotting breeds. Genetic differentiation between some trotting breeds, based on Nei genetic distance values, was found to be comparable to that between the groups of horses of Altaic breed from two d...
Chen JW, Uboh CE, Soma LR, Li X, Guan F, You Y.To evaluate whether urine supernatant contains amplifiable DNA and to determine factors that influence genotyping of samples from racehorses after storage and transportation. Methods: 580 urine, 279 whole blood, and 40 plasma samples obtained from 261 Thoroughbreds and Standardbreds. Methods: Genomic DNA was isolated from stored blood and urine samples collected from racehorses after competition. Quantified DNA was evaluated to determine whether 5 equine microsatellite loci (VHL20, HTG4, AHT4, HMS6, and HMS7) could be amplified by use of PCR techniques. Fragment size of each amplified locus wa...
Yarali C, Özsensoy Y, Kösemann A, Seker I, Toprak B, Zengin K.This study aimed to determine the effectiveness of parentage verification in Arabian and Thoroughbred horses in Türkiye using microsatellite markers. A total of 813 Arabian and 959 Thoroughbred horses were genotyped using a total of 17 microsatellite markers. The mean effective number of alleles was 3,34 and the mean number of alleles was 7,41 in Arabian horses. It was calculated that the mean He and Ho values in Arabian horses were 0,677 and 0,680, respectively. The mean effective number of alleles was 3,55 and the mean number of alleles was 6,59 in Thoroughbred horses. It was calculated tha...
An J, Tseveen K, Oyungerel B, Kong HS.Mongolian horses are one of the oldest horse breeds, and are very important livestock in Mongolia as they are used in various fields such as transportation, food (milk, meat), and horse racing. In addition, research and preservation on pure Mongolian breeds are being promoted under the implementation of the new Genetics of Livestock Resources' act in Mongolia. However, despite the implementation of this act, genetic research on Mongolian horses using microsatellites (MS) has not progressed enough. Therefore, this study was conducted to analyze the genetic polymorphism of five breeds (Gobi shan...
Lopes MS, Diesterbeck U, da Câmara Machado A, Distl O.Navicular disease or podotrochlosis is one of the main causes of progressive forelimb lameness in warmblood horses. The objective of this study was to refine a quantitative trait locus on horse chromosome 2 for radiological alterations in the contour of the navicular bone (RAC) in Hanoverian warmblood horses. Genotyping was performed in 192 Hanoverian warmblood horses from 17 paternal half-sib groups. The marker set was extended to 58 informative microsatellites including nine newly developed microsatellites. QTL for RAC could be delineated at 32.50-43.13 Mb and a further new QTL for RAC could...
Amano T, Tozaki T, Takasu M, Onogi A, Yamada F, Kawai M, Ueda J.We investigated whether regular changes of the sire in a breeding farm of Hokkaido Native Horses (HKDs) enables the DNA-level genetic variation of the produced animals to be maintained. The genotypes of 31 microsatellite markers were identified and analyzed in 207 animals produced in a breeding farm in which the sire was replaced every 3 to 5 years. The mean allele number indicating the degree of genetic variation was 5.97 and was similar to those reported previously. The mean observed heterozygosity was 0.74 and was higher than the expected heterozygosity, 0.69; F was -0.07, indicating that ...
Tozaki T, Hirota K, Hasegawa T, Ishida N, Tobe T.The identification of candidate genes for significant traits is crucial. In this study, we developed and tested effective and systematic methods based on linkage disequilibrium (LD) for the identification of candidate regions for genes with Mendelian inheritance and those associated with complex traits. Our approach entailed the combination of primary screening using pooled DNA samples based on DeltaTAC, secondary screening using an individual typing method and tertiary screening using a permutation test based on the differences in the haplotype frequency between two neighbouring microsatellit...
Ghosh S, Kjöllerström J, Metcalfe L, Reed S, Juras R, Raudsepp T.We present cytogenetic and genotyping analysis of a Thoroughbred foal with congenital neurologic disorders and its phenotypically normal dam. We show that the foal has non-mosaic trisomy for chromosome 26 (ECA26) but normal 2n = 64 diploid number because two copies of ECA26 form a metacentric derivative chromosome der(26q;26q). The dam has normal 64,XX karyotype indicating that der(26q;26q) in the foal originates from errors in parental meiosis or post-fertilization events. Genotyping ECA26 microsatellites in the foal and its dam suggests that trisomy ECA26 is likely of maternal origin and tha...
Machado FB, de Vasconcellos Machado L, Bydlowski CR, Bydlowski SP, Medina-Acosta E.Validation of parentage and horse breed registries through DNA typing relies on estimates of random match probabilities with DNA profiles generated from multiple polymorphic loci. Of the twenty-seven microsatellite loci recommended by the International Society for Animal Genetics for parentage testing in Thoroughbred horses, eleven are located on five chromosomes. An important aspect in determining combined exclusion probabilities is the ascertainment of the genetic linkage status of syntenic markers, which may affect reliable use of the product rule in estimating random match probabilities. I...
Cozzi MC, Valiati P, Longeri M, Ferreira C, Abreu Ferreira S.The Lusitano Horse (LH) originates from Portugal, but is reared worldwide. Since 1994, the University of Milan has routinely tested the LHs bred in Italy for parentage control. This study aims to assess the genetic variability of the LH reared in Italy using 16 microsatellites markers. Moreover, the genetic variability changes over the years in the total population (n.384) and in unrelated horses (n.47) were evaluated. Horses were grouped according to their date of birth (1975-1990, 1991-2000, 2001-2010, 2010-2019). Standard genetic diversity parameters, including observed (Ho) and expected (H...
Lopes MS, Diesterbeck U, Machado Ada C, Distl O.Navicular disease is characterized by a progressive degenerative alteration of the equine podotrochlea. In this study, we refined a previously identified quantitative trait locus (QTL) on horse chromosome 10 for the abnormal development of canales sesamoidales (DCS) of the navicular bone in Hanoverian warmblood horses. Genotyping was done in 192 Hanoverian warmblood horses from 17 paternal half-sib groups. The whole marker set comprised 45 markers including seven newly developed microsatellites and 13 single nucleotide polymorphisms (SNPs) within positional candidate genes. Chromosome-wide sig...
Chen JW, Uboh CE, Soma LR, Li X, Guan F, You Y, Liu Y.A novel multiplex of independent dinucleotide tandem repeat (DTR) loci was previously described that is capable of not only discriminating human and equine DNA, but of identifying a single equine source. We report a case in which a bloodstained syringe and two needles were found during inspection of a barn by inspectors of the Pennsylvania Racing Commissions. Using the multiplex and single-locus detection, all 21 equine DTR markers were detected in a suspect horse and two evidence samples, indicating the evidence samples came from the suspect animal. Only six markers were detected in the third...
