Screening and detection of chromosomal copy number alterations in the domestic horse using SNP-array genotyping data.

Chromosomal abnormalities are a common cause of infertility in horses. However, they are difficult to detect using automated methods. Here, we propose a simple methodology based on single nucleotide polymorphism (SNP)-array data that allows us to detect the main chromosomal abnormalities in horses in a single procedure. As proof of concept, we were able to detect chromosomal abnormalities in 33 out of 268 individuals, including monosomies, chimerisms, and male and female sex-reversions, by analyzing the raw signal intensity produced by an SNP array-based genotyping platform. We also demonstrated that the procedure is not affected by the SNP density of the array employed or by the inbreeding level of the individuals. Finally, the methodology proposed in this study could be performed in an open bioinformatic environment, thus permitting its integration as a flexible screening tool in diagnostic laboratories and genomic breeding programs.

Sex chromosomal abnormalities associated with equine infertility: validation of a simple molecular screening tool in the Purebred Spanish Horse.

Chromosomal abnormalities in the sex chromosome pair (ECAX and ECAY) are widely associated with reproductive problems in horses. However, a large proportion of these abnormalities remains undiagnosed due to the lack of an affordable diagnostic tool that allows for avoiding karyotyping tests. Hereby, we developed an STR (single-tandem-repeat)-based molecular method to determine the presence of the main sex chromosomal abnormalities in horses in a fast, cheap and reliable way. The frequency of five ECAX-linked (LEX026, LEX003, TKY38, TKY270 and UCDEQ502) and two ECAY-linked (EcaYH12 and SRY) markers was characterized in 261 Purebred Spanish Horses to determine the efficiency of the methodology developed to be used as a chromosomal diagnostic tool. All the microsatellites analyzed were highly polymorphic, with a sizeable number of alleles (polymorphic information content > 0.5). Based on this variability, the methodology showed 100% sensitivity and 99.82% specificity to detect the most important sex chromosomal abnormalities reported in horses (chimerism, Turner's syndrome and sex reversal syndromes). The method was also validated with 100% efficiency in 10 individuals previously diagnosed as chromosomally aberrant. This STR screening panel is an efficient and reliable molecular-cytogenetic tool for the early detection of sex chromosomal abnormalities in equines that could be included in breeding programs to save money, effort and time of veterinary practitioners and breeders.

Transmission ratio distortion detection by neutral genetic markers in the Pura Raza Española horse breed.

Transmission Ratio Distortion (TRD) is a genetic phenomenon widely demonstrated in several livestock species, but barely in equine species. The TRD occurs when certain genotypes are over- or under-represented in the offspring of a particular mating and can be caused by a variety of factors during gamete formation or during embryonic development. For this study, 126 394 trios consisting of a stallion, mare, and offspring were genotyped using a panel of 17 neutral microsatellite markers recommended by the International Society for Animal Genetics for paternity tests and individual identification. The number of alleles available for each marker ranges from 13 to 18, been 268 the total number of alleles investigated. The TRDscan v.2.0 software was used with the biallelic procedure to identify regions with distorted segregation ratios. After completing the analysis, a total of 12 alleles (out of 11 microsatellites) were identified with decisive evidence for genotypic TRD; 3 and 9 with additive and heterosis patterns, respectively. In addition, 19 alleles (out of 10 microsatellites) were identified displaying allelic TRD. Among them, 14 and 5 were parent-unspecific and stallion-mare-specific TRD. Out of the TRD regions, 24 genes were identified and annotated, predominantly associated with cholesterol metabolism and homeostasis. These genes are often linked to non-specific symptoms like impaired fertility, stunted growth, and compromised overall health. The results suggest a significant impact on the inheritance of certain genetic traits in horses. Further analysis and validation are needed to better understand the TRD impact before the potential implementation in the horse breeding programme strategies.

New single nucleotide polymorphisms in the mu-calpain gene in Spanish maternal beef breeds.

Calpains play an important role in the postmortem tenderization process of meat and several SNP in the mu-calpain gene (CAPN1) have been reported to be associated with tenderness in beef cattle. Our objectives were to identify the previously reported CAPN1 331G>C SNP and to detect new polymorphisms in this gene in Spanish maternal beef breeds. A fragment (exon 8 to 10) of the bovine CAPN1 gene was sequenced and genotyped in a sample of the main Spanish maternal beef breeds including Retinta, Morucha, and Avilenã Negra-Ibérica. These breeds are characterized for their high meat quality, their adaptation to adverse environmental conditions, and their good maternal aptitude. This adaptation makes it possible to rear these breeds in the south and west of Spain, where drought and feed shortages occur frequently. Six SNP in the mu-calpain gene were found, five of which (CAPN1 80C>T, 302C>G, 310G>A, 445C>T, 524A>C) have not been reported previously. Sequences obtained for these five newly found SNP were submitted to GenBank (Accessions EU386166 to EU386183).

Genetic and environmental risk factors for vitiligo and melanoma in Pura Raza Español horses.

BACKGROUND: Vitiligo and melanoma are relatively common disorders in grey Pura Raza Español horses and other horse breeds with grey-coloured coats. OBJECTIVES: To determine the breed prevalence, environmental risks factors and estimate the genetic parameters for vitiligo and melanoma in Pura Raza Español horses. STUDY DESIGN: Retrospective cohort study. METHODS: We analysed data from a large worldwide population of Pura Raza Español horses. The database included the vitiligo and melanoma scores, on either a four- or six-point linear scale, of 11,436 horses. Genetic parameters were estimated using a Bayesian genetic animal model including the four associated environmental risk factors as systematic effects. Inbreeding was used as a covariate, and animal and residual effects were included as random effects. RESULTS: Of the horses included in the study, 2.8 and 20.5% showed some traces of vitiligo around the eyes and mouth, respectively, while 1.6% showed varying degrees of melanoma. Age, coat colour and inbreeding were significantly associated with the three outcomes studied. The estimated heritability for the whole population was 0.09 (s.d. +0.019), 0.44 (s.d. +0.031) and 0.13 (s.d. +0.037), for eye vitiligo score, nostril vitiligo score and melanoma scores respectively. The genetic correlations ranged from 0.42 (s.d. +0.084) between eye and nostril vitiligo score to 0.15 (s.d. +0.096) between nostril vitiligo and melanoma. MAIN LIMITATIONS: Vitiligo scores for the perianal regions were not collected. The veterinarian responsible for each assessment was not recorded. CONCLUSIONS: Vitiligo and melanoma are prevalent in this population and those environmental risk factors and genetics both have an effect on the clinical expression of the diseases. These findings may help to reduce prevalence through breeding programmes.

Prevalence of twin foaling and blood chimaerism in purebred Spanish horses.

Twin foaling is associated with chimaerism in several domestic species and is recognised in horses. In this study, 21,097 purebred Spanish (Pura Raza Español) horse births from the 2015 to 2016 breeding season were investigated for chimaerism. Twin foaled and chimaeric individuals were assessed on the basis of foaling records, short-tandem repeat (STR) parentage test results and a sex-linked STR-based technique. Fourteen twin pregnancies with 23 twin foals born alive were identified (0.066% twin foaling prevalence), including five blood chimaeric cases (21.7%; overall prevalence 0.011%), suggesting that this genetic condition is extremely low in horses. Furthermore, no true chimaeras were detected. This is the first large scale study analysing the occurrence of chimaerism in a horse population and the first assessment of twin foaling in purebred Spanish horses.

Prevalence, risk factors and genetic parameters of cresty neck in Pura Raza Español horses.

REASONS FOR PERFORMING STUDY: Cresty neck is a relatively common morphological defect in Pura Raza Español horses and other Baroque type horse breeds, which adversely affects the breeding industry. OBJECTIVES: To establish the within-breed prevalence, possible associated factors and heritability of cresty neck in Pura Raza Español horses. STUDY DESIGN: Cross-sectional analysis of a large worldwide database of Pura Raza Español horses. METHODS: The database included evaluations of 10,929 Pura Raza Español horses from 24 countries. Cresty neck score, on a 9 point scale, girth-to-height ratio, height at the withers, length of neck, head-neck junction and neck-body junction were recorded. A Bayesian genetic animal model included the following systematic effects: girth-to-height ratio, age, sex, coat colour and geographical area of the stud. Animal and residual effects were included as random effects. RESULTS: Within this Pura Raza Español population, 8.91% had a cresty neck score ≥5, which is the threshold for penalty or disqualification in the studbook of the breed. The age, sex, coat colour and stud geographical area were significantly associated with cresty neck score. The estimated heritability coefficient for cresty neck score was 0.37 (s.d. = 0.034) and genetic correlation between cresty neck score and other conformational traits ranged from -0.06 (height-at-withers) to -0.21 (neck-body junction). CONCLUSIONS: Cresty neck is a prevalent defect in Pura Raza Español horses, associated with age, sex, coat colour and other conformational traits, with a moderate level of heritability. Breeding to select against this condition may therefore be beneficial in this breed.

Technical note: a novel method for routine genotyping of horse coat color gene polymorphisms.

The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype using a macro freely available at http://www.igijon.com/personales/fgoyache/software_i.htm (last accessed February 26, 2007). The performance of the method is demonstrated on 156 Spanish Purebred horses.

Phylogenetic relationships among Spanish goats breeds.

We partially sequenced the mitochondrial hypervariable region 1 (HVR1) in 60 goats belonging to six Spanish breeds. The analysis of these and previously published sequences reveals a weak phylogeographical structure in the Iberian Peninsula breeds. Individuals from a single breed did not group into a single cluster. Furthermore, individuals from different breeds often shared single phylogenetic tree branches after UPGMA analysis. This could reflect the non-existence of breed isolation because of traditional seasonal pastoralism and annual long-distance migrations. Three goats belonging to the C maternal lineage were found, demonstrating a wider than previously thought distribution for this lineage.