Thoroughbred stallion fertility is significantly associated with FKBP6 genotype but not with inbreeding or the contribution of a leading sire.

This is a follow-up study to validate the previously detected association of the FKBP6 gene with stallion subfertility. Using a select cohort of 150 Thoroughbred stallions with detailed breeding records, we confirm significant association (P < 0.0001) between low per-cycle pregnancy rates (≤50%) and a combined A/A-A/A genotype of SNPs chr13:11 353 372G>A and chr13:11 353 436A>C in FKBP6 exon 5. We also show that stallion subfertility and the combined genotype A/A-A/A are not associated with the level of genetic diversity based on 12 autosomal microsatellite markers, or with pedigree-based inbreeding rate, or the extent of contribution of a leading Thoroughbred sire, Northern Dancer, in a stallion's pedigree. We develop a TaqMan allelic discrimination assay for the two SNPs to facilitate accurate and high-throughput genotyping. We determine allele, genotype and combined genotype frequencies of FKBP6 exon 5 SNPs in a global cohort of 518 Thoroughbreds (76% stallions or geldings and 24% mares) and show that the frequency of the A/A-A/A genotype is 4%. Because there is no similar association between the FKBP6 exon 5 genotype and stallion subfertility in Hanoverians, we suggest that the two SNPs are not causative but rather tagging a breed-specific haplotype with genetic variants unique to Thoroughbreds. Further WGS-based research is needed to identify the molecular causes underlying the observed genotype-phenotype association in Thoroughbred stallions.

The evolutionary history of the DMRT3 'Gait keeper' haplotype.

A previous study revealed a strong association between the DMRT3:Ser301STOP mutation in horses and alternate gaits as well as performance in harness racing. Several follow-up studies have confirmed a high frequency of the mutation in gaited horse breeds and an effect on gait quality. The aim of this study was to determine when and where the mutation arose, to identify additional potential causal mutations and to determine the coalescence time for contemporary haplotypes carrying the stop mutation. We utilized sequences from 89 horses representing 26 breeds to identify 102 SNPs encompassing the DMRT3 gene that are in strong linkage disequilibrium with the stop mutation. These 102 SNPs were genotyped in an additional 382 horses representing 72 breeds, and we identified 14 unique haplotypes. The results provided conclusive evidence that DMRT3:Ser301STOP is causal, as no other sequence polymorphisms showed an equally strong association to locomotion traits. The low sequence diversity among mutant chromosomes demonstrated that they must have diverged from a common ancestral sequence within the last 10 000 years. Thus, the mutation occurred either just before domestication or more likely some time after domestication and then spread across the world as a result of selection on locomotion traits.

Genetic diversity and admixture among Canadian, Mountain and Moorland and Nordic pony populations.

As part of the requirements of the Convention on Biological Diversity, Canada has been investigating the genetic diversity of its native equine and pony populations. Along with examining four indigenous Canadian equine populations (Canadian horse, Lac La Croix pony, Newfoundland pony and Sable Island population), another 10 Mountain and Moorland, three Nordic, four horse and two feral equine populations (thought to have influenced some pony breeds) were also investigated. In total, 821 individuals were genotyped at 38 microsatellite loci. Results of the analysis of molecular variance indicated that 13.3% of genetic diversity was explained by breed differences, whereas 84.6% and 2.1% of diversity came from within and among individuals, respectively. The average effective number of alleles and allelic richness was the lowest in the Eriskay (2.51 and 3.98) and Lac La Croix (2.83 and 4.01) populations, whereas it was highest in the New Forest (4.31 and 6.01) and Welsh (4.33 and 5.87) breeds, followed closely by the Newfoundland-CDN (4.23 and 5.86) population. Expected heterozygosities varied from 0.61 in the Lac La Croix to 0.74 in the Welsh and in Newfoundland. Observed heterozygosities ranged from 0.57 in the Exmoor and 0.58 in the Sable Island herd to 0.77 in the Kerry Bog and 0.76 in the New Forest breeds. Structure and admixture analyses revealed that the most likely number of clusters was 21, although some substructure was also observed when K = 16, compared with the 24 predefined populations. Information gathered from this study should be combined with other available phenotypic and pedigree data to develop, or amend, a suitable conservation strategy for all populations examined.

A microsatellite analysis of five Colonial Spanish horse populations of the southeastern United States.

The domestic horse (Equus caballus) was re-introduced to the Americas by Spanish explorers. Although horses from other parts of Europe were subsequently introduced, some New World populations maintain characteristics ascribed to their Spanish heritage. The southeastern United States has a history of Spanish invasion and settlement, and this influence on local feral horse populations includes two feral-recaptured breeds: the Florida Cracker and the Marsh Tacky, both of which are classified as Colonial Spanish horses. The feral Banker horses found on islands off the coast of North Carolina, which include, among others, the Shackleford Banks, the Corolla and the Ocracoke, are also Colonial Spanish horses. Herein we analyse 15 microsatellite loci from 532 feral and 2583 domestic horses in order to compare the genetic variation of these five Colonial Spanish Horse populations to 40 modern horse breeds. We find that the Corolla horse has very low heterozygosity and that both the Corolla and Ocracoke populations have a low mean number of alleles. We also find that the Florida Cracker population has a heterozygosity deficit. In addition, we find evidence of similarity of the Shackleford Banks, Marsh Tacky and Florida Cracker populations to New World Iberian horse breeds, while the origins of the other two populations are less clear.

Genetic analysis of the Venezuelan Criollo horse.

Various horse populations in the Americas have an origin in Spain; they are remnants of the first livestock introduced to the continent early in the colonial period (16th and 17th centuries). We evaluated genetic variability within the Venezuelan Criollo horse and its relationship with other horse breeds. We observed high levels of genetic diversity within the Criollo breed. Significant population differentiation was observed between all South American breeds. The Venezuelan Criollo horse showed high levels of genetic diversity, and from a conservation standpoint, there is no immediate danger of losing variation unless there is a large drop in population size.

Incidence of the mask phenotype M264V mutation in Labrador Retrievers.

The introduction of SNP (Single Nucleotide Polymorphism) chips allows for the rapid typing of multiple markers for many individuals at one time. Our lab routinely types dogs using a custom designed combined panel of SNPs for parentage verification and a number of genes for diagnostic tests using an OpenArray platform manufactured by BioTrove (Woburn, MA, USA). By utilizing the same SNP panel across a wide array of canine breeds it is possible to detect trait-associated SNPs in breeds not thought to carry those traits. We genotyped 245 Labrador Retrievers on the canine SNP chip and found 13 animals heterozygous for the M264V mutation associated with autosomal dominant mask trait, and one animal homozygous for this trait. The color genotypes for these animals were further examined. In standard colored Labradors (black, chocolate, and yellow), the mask phenotype would never be distinguishable. As illustrated by this example, we feel this SNP panel is a valuable method for discovering traits not known to exist in a breed.

Teste de marcadores bioquímicos na identificação de prováveis indivíduos homozigotos dominantes para o gene tobiano em cavalos Pampa / Biochemical markers test to identify probable homozygous dominant for the tobiano gene in Pampa horses

Utilizaram-se 195 cavalos Pampa e um grupo-controle de 41 cavalos da raça Paint, provenientes de plantéis de várias regiões brasileiras, com o objetivo de avaliar a eficiência do teste mediante uso de marcadores bioquímicos: albumina (Al) e proteína de ligação da vitamina D (Gc), para identificação dos possíveis indivíduos homozigotos dominantes para o padrão de pelagem tobiano nos cavalos Pampa. Não foram encontrados genótipos AlBB e GcSS, revelando indício de quebra de ligação gênica entre tais locos e o loco tobiano e a ineficácia do teste bioquímico na detecção dos prováveis indivíduos homozigotos dominantes para o padrão de pelagem tobiano nos cavalos Pampa

One hundred and ninety five Pampa horses and a control group of 41 Paint Horses originated from herds located in different Brazilian regions were used to objective of evaluate the efficiency of the biochemical markers albumin (Al) and vitamin D binding protein (Gc) to identify the probable homozygous dominant for the tobiano coat color pattern in Pampa horses. It was not found any genotype AlBB and GcSS, indicating a possible break of the genetic linkage between these loci and the locus Tobiano, as well as the inefficacy of the biochemical test in the detection of the probable homozygous dominant for the tobiano color pattern in Pampa horses

Genetic diversity and relationships of Portuguese and other horse breeds based on protein and microsatellite loci variation.

There are three native Portuguese horse breeds: Lusitano, Sorraia and Garrano. This study compares diversity patterns of 17 protein and 12 microsatellite markers in these three as well as 30 other breeds to infer relationships among the breeds and to compare levels of polymorphism of these breeds for use in conservation efforts. The Garrano and the Lusitano showed a high level of genetic diversity, similar to that observed for most of the other analysed breeds, while the Sorraia and Friesian breeds showed low levels of variation for both genetic marker types. The combined protein and microsatellite data produced a tree that fit historical records well and with greater confidence levels than those for either data set alone. The combined genetic diversity and relationship information provides important baseline data for future breed conservation efforts, especially for a critically endangered breed such as the Sorraia.

A lost Sorraia maternal lineage found in the Lusitano horse breed.

A common female founder individual of the Portuguese horse breeds Sorraia and Lusitano was found while conducting research on the variation of the Lusitano mitochondrial DNA lineages in relation to studbook information. We obtained 416-bp control region sequences from 16 descendents of a female Sorraia founder (Pomba) still represented in the living population of the Lusitano, according to the most recent edition of this breed's studbook. The same haplotype was found for all analysed samples and belongs to the haplogroup described by several authors as having predominantly Iberian, South American and North African haplotypes bringing new insights on the relationship between the Sorraia and the other Iberian breeds. This work illustrates how weak the boundary of breed establishment can be, especially at the same geographical region. Using the same founders in different breeds is surely one of the explanations to frequently shared haplotypes among recent breeds, resulting in a lack of consistency between mtDNA sequences and breeds and/or geographical regions.

Major histocompatibility complex locus DRA polymorphism in the endangered Sorraia horse and related breeds.

The major histocompatibility complex (MHC) genes play well-defined roles in eliciting immune responses and combating infectious diseases. This genetic system is among the most polymorphic. The extent of genetic variation within a population has been directly correlated with fitness for many traits. The MHC class II locus DRA polymorphism was analysed in the endangered Sorraia horse, two other Portuguese and four New World horse breeds considered to be historically close to the Sorraia. Comparison of the Sorraia with other breeds demonstrated less MHC variation among Sorraia horses. If DRA polymorphism provides greater disease resistance, selective breeding to increase MHC polymorphism may increase fitness of this population.

The epitheliogenesis imperfecta locus maps to equine chromosome 8 in American Saddlebred horses.

Epitheliogenesis imperfecta (EI) is a hereditary junctional mechanobullous disease that occurs in newborn American Saddlebred foals. The pathological signs of epitheliogenesis imperfecta closely match a similar disease in humans known as Herlitz junctional epidermolysis bullosa, which is caused by a mutation in one of the genes (LAMA3, LAMB3 and LAMC2) coding for the subunits of the laminin 5 protein (laminin alpha3, laminin beta3 and laminin gamma2). The LAMA3 gene has been assigned to equine chromosome 8 and LAMB3 and LAMC2 have been mapped to equine chromosome 5. Linkage disequilibrium between microsatellite markers that mapped to equine chromosome 5 and equine chromosome 8 and the EI disease locus was tested in American Saddlebred horses. The allele frequencies of microsatellite alleles at 11 loci were determined for both epitheliogenesis imperfecta affected and unaffected populations of American Saddlebred horses by genotyping and direct counting of alleles. These were used to determine fit to Hardy-Weinberg equilibrium for control and EI populations using Chi square analysis. Two microsatellite loci located on equine chromosome 8q, ASB14 and AHT3, were not in Hardy-Weinberg equilibrium in affected American Saddlebred horses. In comparison, all of the microsatellite markers located on equine chromosome 5 were in Hardy-Weinberg equilibrium in affected American Saddlebred horses. This suggested that the EI disease locus was located on equine chromosome 8q, where LAMA3 is also located.

Equine epitheliogenesis imperfecta in two american saddlebred foals is a lamina lucida defect.

Necropsy of two American Saddlebred fillies diagnosed with epitheliogenesis imperfecta (EI) revealed missing patches of epithelium of the skin and oral mucosa as well as dental abnormalities. Examination of the digestive tract did not reveal signs of pyloric atresia in either foal. Histopathologic examination revealed separation of the epidermis from the dermis. In both foals a division within the lamina lucida of the basal lamina was observed by transmission electron microscopy. In comparison with an age-specific control, the ultrastructure of intact skin from the EI-affected foals showed abnormal hemidesmosomes, which lacked a subbasal plate. The morphological and ultrastructural defects observed in the EI-affected American Saddlebred foals were similar to those observed in Herlitz junctional epidermolysis bullosa-affected human newborns, which is caused by a defect in one of the subunits of laminin-5. The close similarity of lesions of the human and equine diseases suggests that EI may be caused by a laminin-5 defect.

Genetic diversity of Cheju horses (Equus caballus) determined by using mitochondrial DNA D-loop polymorphism.

We used sequence polymorphism of the mitochondrial DNA D-loop (968 bp excluding the tandem repeat region) to determine genetic diversity of horses inhabiting Cheju (a southern island of Korea). Seventeen haplotypes with frequencies from 1.5 to 21.5% were found among 65 Cheju horse samples. Genetic diversity (h) of the 17 haplotypes was calculated to be 0.91, indicating that the extant Cheju horse population consists of diverse genetic groups in their maternal lineage. Phylogenetic analysis showed that 17 types of Cheju (D-loop sequences determined), 5 Mongolian, 6 Arabian, 3 Belgian, 2 Tsushima, 2 Yunnan, 1 Przewalskii, and 3 Thoroughbred horses (published sequences for the latter seven breeds) showed that Cheju horses were distributed into many different clusters in the tree. Four Mongolian horses clustered with separate Cheju horse groups, showing that some Cheju horses are clearly of Mongolian origin. The analysis of partial sequences (284 bp) of the D-loop of 109 horses showed that Thoroughbred, Mongolian, Lipizzan, and Arabian breeds are as diverse as Cheju horses. Our data together with others suggest that most horse breeds tested with reasonably sufficient numbers of samples are diverse in their maternal lineages and also are not uniquely different from each other.

Genetic variation in the feral horses of the Namib Desert, Namibia.

Genetic variation at 7 blood-group and 10 biochemical genetic loci was examined in 30 horses from a feral herd from the Namib Desert of Namibia, Africa. The observed genetic variability was extremely low compared with that found in domestic horse breeds. The low variation was most probably a result of recent small population size and a small founding population size. Genetic comparison of the Namib horses, which were of unknown origins, to domestic horse breeds, showed that the Namib horses had the highest genetic similarity to Arabian type horses, although they did not closely resemble this type of horse in conformation.

Linked markers exclude KIT as the gene responsible for appaloosa coat colour spotting patterns in horses.

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 in KIT. However, KIT is located on horse chromosome 3 close to albumin (ALB), serum carboxylesterase (ES), vitamin D-binding protein (GC) and microsatellite markers ASB23, LEX007, LEX57, and UCDEQ437. Indeed, KIT and ASB23 were localized to ECA3q21-22.1 and 3q22.1-22.3, respectively, by fluorescent in situ hybridization. Family studies were conducted to investigate linkage of Lp to these markers using eight half-sib families in which Appaloosa stallions were mated to solid coloured mares. Linkage of Lp to the chromosome region containing ES, ALB, GC, ASB23, UCDEQ437, LEX57, and LEX007 was investigated by a multipoint linkage analysis using the computer program GENEHUNTER. LOD scores over the interval under investigation ranged from -4.28 to -12.48, with a score of -12.48 at the location for ASB23. Therefore, it was concluded that appaloosa (Lp) is not linked to any of the tested markers on ECA3, and thus Lp is unlikely to be the product of KIT.

Characterization of expressed sequence tags generated from skin cDNA clones of Equus caballus by single pass sequencing.

A cDNA library was built using RNA extracted from the skin tissue of an adult horse. The library was primed with oligo (dT) and sequences were directionally inserted in order to produce an expression library. The library has 5.8X 10(5) plaque forming units with 99.6% recombinant phage. The average insert size is 1.3 Kbp. Three hundred and thirteen expressed sequence tags (ESTs) were generated from sequencing of the 5 prime end of randomly selected skin cDNA clones. The ESTs were sequenced on an ABI 377 using Big-Dye chemistry. A similarity search was performed on each EST using the NCBI non-redundant protein database and 206 ESTs were putatively identified. Twenty six percent of the identified ESTs were redundant. The ESTs were categorized by function. The most frequently identified functional class was translational proteins.

Report of the International Equine Gene Mapping Workshop: male linkage map.

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 for all families for which the sire was heterozygous. Genealogies and typing data were sent for analysis to the INRA laboratory (Jouy-en-Josas, France) according to a specific format and entered into a database with input verification and output processes. Linkage analysis was performed with the CRIMAP program. Significant linkage was detected for 124 loci, of which 95 were unambiguously ordered using a multipoint analysis with an average spacing of 14.2 CM. These loci were distributed among 29 linkage groups. A more comprehensive analysis including synteny group data and FISH data suggested that 26 autosomes out of 31 are covered. The complete map spans 936 CM.

Polymorphism of Old Kladruber horses, a surviving but endangered baroque breed.

Analysis of MHC class I and class II polymorphism, as well as data from other polymorphic systems (non-MHC lymphocyte alloantigen, blood groups systems, biochemical polymorphisms and microsatellite loci), was used to characterize the extent and distribution of the genic polymorphism of Kladruber horses. A breed-characteristic distribution of the MHC polymorphism was found. The repertoire of defined MHC class I specificities was restricted, especially in the grey subpopulation and in stallions, but a high frequency of blanks suggests the possible existence of undetected specificities. Despite the small population size and a relatively high degree of inbreeding, high heterozygosity in MHC haplotypes has been conserved. The extent of polymorphism and the degree of heterozygosity in other loci were also relatively high. A comparison of the two existing subpopulations, grey and black, at all the loci tested, including RAPD markers, characterized them as genetically distinct, although clearly related. The genetic distances between them were of the same order of magnitude as between distinct breeds. The results may be useful in defining short-term and long-term breeding policy within the breed and for further studies of associations with disease and other traits.