Comparison of DMRT3 genotypes among American Saddlebred horses with reference to gait.

Horse owners choose whether or not to train American Saddlebred horses (ASHs) to perform the 4-beat gaits called rack and slow gait. The rack and slow gait are similar to ambling gaits shown to be associated with variation in the DMRT3 gene in other breeds but are trained rather than naturally occurring gaits. A premature stop codon in the DMRT3 gene (DMRT3_Ser301STOP) caused by the Ch23:g.22999655C>A SNP has an effect on the pattern of locomotion in horses and allows for the pacing gait and strong association with performance of ambling gaits in diverse breeds. We used horse show records to identify ASHs trained to perform as 5-gaited horses and compared them to other Saddlebreds for the prevalence of the A allele of the Ch23:g.22999655C>A SNP of DMRT3. Genomic DNA was typed using a PCR-RFLP technique for 5-gaited ASHs (n = 55), 3-gaited or harness ASHs (n = 64) and ASHs not shown (n = 128). We did not observe differences in the frequencies of the Ch23:g.22999655C>A SNP of DMRT3 when comparing 5-gaited ASHs to other ASHs (P > 0.22). These results suggest that DMRT3 alleles do not affect the choice of breeders to train and show ASHs as 5-gaited horses.

Inbreeding in the Thoroughbred horse.

Changes in the inbreeding coefficient, F, in the Thoroughbred horse over the past 45 years have been investigated by genotyping 467 Thoroughbred horses (born between 1961 and 2006) using the Illumina Equine SNP50 bead chip, which comprises 54,602 SNPs uniformly distributed across the equine genome. The Spearman rank correlation coefficient, r, between the year of birth and F was estimated. The results indicate that inbreeding in Thoroughbreds has increased over the past 40 years, with r = 0.24, P < 0.001 demonstrating that there is a highly significant, though relatively weak correlation between the year of birth and inbreeding coefficients. Interestingly, the majority of the increase in inbreeding is post-1996 and coincides with the introduction of stallions covering larger numbers of mares.

Morphological and chemical influences on alumina-supported palladium catalysts active for the gas phase hydrogenation of crotonaldehyde.

A series of five alumina-supported palladium catalysts have previously been prepared and characterised by a combination of CO chemisorption and infrared spectroscopy. The reactive attributes of these catalysts are examined using the hydrogenation of crotonaldehyde as a test reaction, using a modified infrared gas cell as a batch reactor. Periodic scanning of the infrared spectrum of the gaseous phase present over the Pd/Al(2)O(3) catalysts was used to construct reaction profiles. Four of the catalysts were able to facilitate a 2-stage hydrogenation process (crotonaldehyde → butanal → butanol), whilst one catalyst was totally selective for the first stage hydrogenation process (crotonaldehyde → butanal). Rate coefficients for the first and second stage hydrogenation processes are normalised to the number of surface palladium atoms for the particular catalyst. Correlation of these kinetic parameters as a function of mean particle size indicates the first stage process to be structure insensitive, whilst the second stage hydrogenation is structure sensitive. Chlorine residues associated with the preparative process of one of the catalysts is seen to selectively poison the second stage hydrogenation process for that catalyst. Structure/activity relationships are considered to explain the observed trends.

Molecular heterogeneity of XY sex reversal in horses.

Male-to-female 64,XY sex reversal is a frequently reported chromosome abnormality in horses. Despite this, the molecular causes of the condition are as yet poorly understood. This is partially because only limited molecular information is available for the horse Y chromosome (ECAY). Here, we used the recently developed ECAY map and carried out the first comprehensive study of the Y chromosome in XY mares (n=18). The integrity of the ECAY in XY females was studied by FISH and PCR using markers evenly distributed along the euchromatic region. The results showed that the XY sex reversal condition in horses has two molecularly distinct forms: (i) a Y-linked form that is characterized by Y chromosome deletions and (ii) a non-Y-linked form where the Y chromosome of affected females is molecularly the same as in normal males. Further analysis of the Y-linked form (13 cases) showed that the condition is molecularly heterogeneous: the smallest deletions spanned about 21 kb, while the largest involved the entire euchromatic region. Regardless of the size, all deletions included the SRY gene. We show that the deletions were likely caused by inter-chromatid recombination events between repeated sequences in ECAY. Further, we hypothesize that the occurrence of SRY-negative XY females in some species (horse, human) but not in others (pig, dog) is because of differences in the organization of the Y chromosome. Finally, in contrast to the Y-linked SRY-negative form of equine XY sex reversal, the molecular causes of SRY-positive XY mares (5 cases) remain as yet undefined.

Equine clinical cytogenetics: the past and future.

Cytogenetic analyses of horses have benefited the horse industry by identifying chromosomal aberrations causing congenital abnormalities, embryonic loss and infertility. Technical advances in cytogenetics enabled the identification of chromosome specific aberrations. More recently, advances in genomic tools have been used to more precisely define chromosome abnormalities. In this report we review the history of equine clinical cytogenetics, identify historical landmarks for equine clinical cytogenetics, discuss how the current use of genomic tools has benefited this area, and how future genomics tools may enhance clinical cytogenetic studies in the horse.

Three autosomal chromosome translocations associated with repeated early embryonic loss (REEL) in the domestic horse (Equus caballus).

Repeated early embryonic loss (REEL) represents a considerable economic loss to the horse industry. Mares that experience REEL may be overlooked as potential carriers of a chromosome abnormality. Here we report three different autosomal translocations in Thoroughbred mares presented for chromosome analysis because of REEL. The karyotypes were 64,XX,t(1;21), 64,XX,t(16;22), and 64,XX,t(4;13), respectively. In order to confirm the chromosomes involved in the translocations, to map the breakpoints, and to determine if the translocations were reciprocal, genes surrounding the breakpoints were identified using existing maps and from the newly assembled horse genome sequence. Bacterial artificial chromosomes containing the genes of interest were identified and mapped to the translocation chromosomes by fluorescence in situ hybridization (FISH). FISH confirmed that the t(16;22) and t(4;13) translocations were reciprocal, while the t(1;21) was not. The breakpoints on horse chromosomes 1 and 16 appear to be the same or near breakpoints previously identified in translocations. These breakpoints are at the fusion boundary of human chromosomes 10 and 15 on horse chromosome 1 and at human chromosome 3p and 3q on horse chromosome 16. These sites may represent ancient breakpoints reused during equid evolution. Overall, chromosome abnormalities may have a greater influence on mare fertility than previously known. Thus, it is important to karyotype subfertile mares exhibiting REEL.

A chromosome inversion near the KIT gene and the Tobiano spotting pattern in horses.

Tobiano is a white spotting pattern in horses caused by a dominant gene, Tobiano(TO). Here, we report TO associated with a large paracentric chromosome inversion on horse chromosome 3. DNA sequences flanking the inversion were identified and a PCR test was developed to detect the inversion. The inversion was only found in horses with the tobiano pattern, including horses with diverse genetic backgrounds, which indicated a common genetic origin thousands of years ago. The inversion does not interrupt any annotated genes, but begins approximately 100 kb downstream of the KIT gene. This inversion may disrupt regulatory sequences for the KIT gene and cause the white spotting pattern.

Structure of equine 2'-5'oligoadenylate synthetase (OAS) gene family and FISH mapping of OAS genes to ECA8p15-->p14 and BTA17q24-->q25.

Mammalian 2'-5' oligoadenylate (2-5A) synthetases are important mediators of the antiviral activity of interferons. Both human and mouse 2-5A synthetase gene families encode four forms of enzymes: small, medium, large and ubiquitin-like. In this study, the structures of four equine OAS genes were determined using DNA sequences derived from fifteen cDNA and four BAC clones. Composition of the equine OAS gene family is more similar to that of the human OAS family than the mouse Oas family. Two OAS-containing bovine BAC clones were identified in GenBank. Both equine and bovine BAC clones were physically assigned by FISH to horse and cattle chromosomes, ECA8p15-->p14 and BTA17q24--> q25, respectively. The comparative mapping data confirm conservation of synteny between ungulates, humans and rodents.

F-box protein Fbxl18 mediates polyubiquitylation and proteasomal degradation of the pro-apoptotic SCF subunit Fbxl7.

Fbxl7, a subunit of the SCF (Skp-Cul1-F-box protein) complex induces mitotic arrest in cells; however, molecular factors that control its cellular abundance remain largely unknown. Here, we identified that an orphan F-box protein, Fbxl18, targets Fbxl7 for its polyubiquitylation and proteasomal degradation. Lys 109 within Fbxl7 is an essential acceptor site for ubiquitin conjugation by Fbxl18. An FQ motif within Fbxl7 serves as a molecular recognition site for Fbxl18 interaction. Ectopically expressed Fbxl7 induces apoptosis in Hela cells, an effect profoundly accentuated after cellular depletion of Fbxl18 protein or expression of Fbxl7 plasmids encoding mutations at either Lys 109 or within the FQ motif. Ectopic expression of Fbxl18 plasmid-limited apoptosis caused by overexpressed Fbxl7 plasmid. Thus, Fbxl18 regulates apoptosis by mediating ubiquitin-dependent proteasomal degradation of the pro-apoptotic protein Fbxl7 that may impact cellular processes involved in cell cycle progression.

Homologous fission event(s) implicated for chromosomal polymorphisms among five species in the genus Equus.

The genus Equus is unusual in that five of the ten extant species have documented centric fission (Robertsonian translocation) polymorphisms within their populations, namely E. hemionus onager, E. hemionus kulan, E. kiang, E. africanus somaliensis, and E. quagga burchelli. Here we report evidence that the polymorphism involves the same homologous chromosome segments in each species, and that these chromosome segments have homology to human chromosome 4 (HSA4). Bacterial artificial chromosome clones containing equine genes SMARCA5 (ECA2q21 homologue to HSA4q31. 21) and UCHL1 (ECA3q22 homologue to HSA4p13) were mapped to a single metacentric chromosome and two unpaired acrocentrics by FISH mapping for individuals possessing odd numbers of chromosomes. These data suggest that the polymorphism is either ancient and conserved within the genus or has occurred recently and independently within each species. Since these species are separated by 1-3 million years of evolution, this polymorphism is remarkable and worthy of further investigations.

FISH analysis comparing genome organization in the domestic horse (Equus caballus) to that of the Mongolian wild horse (E. przewalskii).

Przewalski's wild horse (E. przewalskii, EPR) has a diploid chromosome number of 2n = 66 while the domestic horse (E. caballus, ECA) has a diploid chromosome number of 2n = 64. Discussions about their phylogenetic relationship and taxonomic classification have hinged on comparisons of their skeletal morphology, protein and mitochondrial DNA similarities, their ability to produce fertile hybrid offspring, and on comparison of their chromosome morphology and banding patterns. Previous studies of GTG-banded karyotypes suggested that the chromosomes of both equids were homologous and the difference in chromosome number was due to a Robertsonian event involving two pairs of acrocentric chromosomes in EPR and one pair of metacentric chromosomes in ECA (ECA5). To determine which EPR chromosomes were homologous to ECA5 and to confirm the predicted chromosome homologies based on GTG banding, we constructed a comparative gene map between ECA and EPR by FISH mapping 46 domestic horse-derived BAC clones containing genes previously mapped to ECA chromosomes. The results indicated that all ECA and EPR chromosomes were homologous as predicted by GTG banding, but provide new information in that the EPR acrocentric chromosomes EPR23 and EPR24 were shown to be homologues of the ECA metacentric chromosome ECA5.

Genetic mapping of GBE1 and its association with glycogen storage disease IV in American Quarter horses.

Comparative biochemical and histopathological data suggest that a deficiency in the glycogen branching enzyme (GBE) is responsible for a fatal neonatal disease in Quarter Horse foals that closely resembles human glycogen storage disease type IV (GSD IV). Identification of DNA markers closely linked to the equine GBE1 gene would assist us in determining whether a mutation in this gene leads to the GSD IV-like condition. FISH using BAC clones as probes assigned the equine GBE1 gene to a marker deficient region of ECA26q12-->q13. Four other genes, ROBO2, ROBO1, POU1F1, and HTR1F, that flank GBE1 within a 10-Mb segment of HSA3p12-->p11, were tightly linked to equine GBE1 when analyzed on the Texas A&M University 5000 rad equine radiation hybrid panel, while the GLB1, MITF, RYBP, and PROS1 genes that flank this 10-Mb interval were not linked with markers in the GBE1 group. A polymorphic microsatellite (GBEms1) in a GBE1 BAC clone was then identified and genetically mapped to ECA26 on the Animal Health Trust full-sibling equine reference family. All Quarter Horse foals affected with GSD IV were homozygous for an allele of GBEms1, as well as an allele of the most closely linked microsatellite marker, while a control horse population showed significant allelic variation with these markers. This data provides strong molecular genetic support for the candidacy of the GBE1 locus in equine GSD IV.

Disorders of sexual development in the domestic horse, Equus caballus.

Abnormalities of sexual development causing infertility in horses have been investigated since the early 1970's. Conventional cytogenetic analysis by karyotyping has been the primary tool used to investigate these horses. Abnormalities have a broad range, from a phenotypically normal mare with gonadal dysgenesis to a horse with ambiguous external genitalia and internal male and female organs. Cytogenetic analysis can determine genetic sex but cannot identify mutations or deletions of genes involved in the sex determination pathway. Molecular technologies have been developed to confirm cytogenetic results and to aid in identifying the genetic causes of abnormal sex determination in horses. In this paper, we review the historical development of methods used to understand abnormal sexual development in the horse as well as summarize cases reported over the last 40-50 years.

Equine disorders of sexual development in 17 mares including XX, SRY-negative, XY, SRY-negative and XY, SRY-positive genotypes.

We described the clinical, cytogenetic and molecular findings of 17 clinical equine cases presented for abnormal sexual development and infertility. Six horses with an enlarged clitoris had an XX, SRY-negative genotype, which displayed male-like behavior (adult individuals). Bilateral ovotestes were noted in 2 of those cases, while another case showed increased levels of circulating testosterone. Six horses with a female phenotype, including normal external genitalia, had an XY, SRY-negative genotype. These individuals had small gonads and an underdeveloped internal reproductive tract. Four horses with normal appearing external genitalia had an XY, SRY-positive genotype, 3 of them had hypoplastic testes and male-like behavior. In addition, one young filly with enlarged clitoris and hypoplastic testes had the same genotype but did not show male-like behavior due to her age. Three of these horses were related with 2 being siblings. These findings demonstrate the diversity of disorders of sexual development seen in the horse. Furthermore, they emphasize the need for further research to identify genes involved in abnormal sex determination and differentiation in the horse.