Exploring physiological and genetic variation of digital cushion thickness in Holstein and Jersey cows and bulls.

The objectives of our study were to expand phenotypic characterization of digital cushion thickness (DCT) to the Jersey breed and include mature bulls and to identify breed-specific quantitative trait loci (QTL) for DCT within the Jersey or Holstein breeds and common QTL across breeds and sexes to better understand biological regulation and guide future use of marker-assisted selection. In a cohort of 698 cows and 85 bulls (Holstein and Jersey) from 8 farms in New York State, data were collected on DCT, body condition score (BCS), withers height, and sacral height. All animals underwent sonographic examination of the digital cushion evaluated at the sole ulcer site for the right front and hind feet. Linear mixed models were conducted on DCT separately for cows and bulls with fixed effects of time point, breed, age, digit, and BCS group. The models included random effects to control for the random subset of animals per farm, repeated measures, and multiple measurements from each animal. The phenotypic results indicated that DCT varied by time point, breed, age, digit, and BCS group for cows and by breed, age, digit, and BCS group for bulls. For the genotypic study, 616 cow DNA samples were genotyped on the Illumina BovineHD 777K BeadChip (Illumina Inc., San Diego, CA), whereas 76 bull DNA samples were genotyped on different platforms ranging from 5K to 150K. Multiple genome-wide association studies were conducted to highlight pertinent phenotyping parameters and genetic markers for genomic selection. Data were separated into 8 data sets based on different combinations of breed and sex. Each data set was assessed for quality of markers and samples before conducting genome-wide association studies for DCT, testing the inheritance models and genetic variation of digit, foot, and average thickness. Ten markers passed the Bonferroni correction threshold and 9 passed false discovery rate from 10 genome-wide association studies using a combination of the covariates breed, sex, genotyping batch plate, age, BCS, withers height, and sacral height. Of the 43 candidate genes, 8 novel biologically plausible genes were identified on Bos taurus autosomes 3, 4, 7, and 9: SFRS18 and LRRFIP1 function in fat deposition, whereas AHR, BZW2, EFNA5, USP45, and VAV3 effect bone growth, and SOSTDC1 is related to epidermal keratinocyte function. The genetic markers associated with DCT in this study were explored for variation between cows and bulls within and across breeds for their potential use in marker-assisted selection.

Phenotypic characterization and genome-wide association studies of digital cushion thickness in Holstein cows.

The bovine digital cushion is a compression pad between the distal phalanx and sole and has been associated with claw horn disruption lesions. Digital cushion thickness (DCT) is estimated to be moderately heritable. Therefore, the objectives of our study were to examine influences of management and environment on DCT and to identify genetic markers and candidate genes associated with DCT. In a cohort of 502 Holsteins from 5 farms in New York State, DCT and body condition score (BCS) were collected twice, at <137 d prepartum and from 86 to 127 d in milk, corresponding to periods when the digital cushion is thickest and thinnest, respectively, as determined by previous research. Cows underwent sonographic examination of the digital cushion evaluated at the typical sole ulcer site for the right front and hind foot. Linear mixed models were conducted on DCT with the fixed effects of time point, digit, wither height, sacral height, BCS group, and multiple farm system variables separately and included random effects to control for the random subset of cows per farm, repeated measures, and multiple measurements from each cow. The phenotypic results indicated that DCT varied by sample time point, sacral height, parity, digit, BCS group, and wither height. For the genotypic study, 447 DNA samples were genotyped on the Illumina BovineHD 777K BeadChip (Illumina Inc., San Diego, CA). Quality assessment of markers and samples provided a final data set of 431 samples and 579,449 markers. Genome-wide association studies were conducted for DCT testing inheritance models and genetic variation of digit, foot, time point, and average thickness. One marker passed the Bonferroni correction threshold and 26 passed false discovery rate from 4 genome-wide association studies with covariates of sequencing batch plate, parity group, BCS, wither height, and sacral height. Ten candidate genes were identified, with 2 genes on Bos taurus autosomes 24 and 29 involved in biological functions related to the digital cushion: MC4R and DLG2 were related to fat deposition and bone growth, respectively. The genetic markers discovered in this study have the opportunity to be used in breeding programs using genomic selection to select against claw horn disruption lesions and lameness due to associations between the markers and DCT. Further studies on the biologically plausible candidate genes may identify causative genetic variants and how they relate to DCT through gene regulation, expression, structure, or copy number variation.

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.

Loci impacting polymorphic gait in the Tennessee Walking Horse.

Following domestication, man selected the horse primarily for the purpose of transportation rather than consumption; this selective strategy created divergent traits for locomotion. At intermediate speeds, beyond the flat walk, the horse can perform a range of diagonal and lateral 2-beat or 4-beat gait patterns. The Tennessee Walking Horse (TWH) is the only U.S. breed able to perform an even-timed 4-beat gait (the "running-walk") at intermediate speeds; however, within the breed, there is remaining variation in gait type. To investigate the contribution of genetics to this unique trait, blood or hair samples for DNA and gait information were collected from 129 TWH and genotyping was performed at approximately 60,000 loci using the Illumina Equine SNP70 beadchip at GeneSeek Inc. (Lincoln, NE). Case-control association tests identified suggestive regions for gait type on equine chromosome (ECA) 19 (-value of 1.50 × 10 after 1 million permutations; PLINK version 1.07). Haplotype analysis identified 2 significant haplotypes on ECA19 and ECA11 (-values of 3.7 × 10 and 3.92 × 10, respectively). Genes within these suggestive regions play roles in developmental processes and biological regulation, indicating there may be variant differences in the neurobiology and regulation of horses with a polymorphic gait.

Genome-wide association mapping of heritable temperament variation in the Tennessee Walking Horse.

Temperament is a key criterion in the selection of horses for both leisure and competitive riding to ensure optimal performance and safety. The Tennessee Walking Horse (TWH) is described as a calm, docile breed and is often used as a trail, show and pleasure horse. However, among horse owners and caretakers, there are anecdotes supporting familial and disciplinal typical behaviors and personalities. To investigate the contribution of genetics to temperament, we collected a behavior questionnaire, brief training history and identifying information for 276 TWH, as well as blood or hair samples for DNA. Factor analysis was conducted on the 20-item questionnaire for the set of 216 horses that met inclusion thresholds. Factor analysis identified four temperament factors in TWH: 'anxious', 'tractable', 'agonistic' and 'gregarious'. These four factors account for 64% of the total trait variance. DNA from 113 TWHs were selected and genotyped using the Equine SNP70 bead chip for three separate genome-wide association studies (GWAs) using the factor 1-anxious, factor 2-tractable and factor 3-agonistic scores as the phenotype. Quantitative association analysis identified significant candidate loci for each factor that warrant further investigation.

Skeletal variation in Tennessee Walking Horses maps to the LCORL/NCAPG gene region.

Conformation has long been a driving force in horse selection and breed creation as a predictor for performance. The Tennessee Walking Horse (TWH) ranges in size from 1.5 to 1.7 m and is often used as a trail, show, and pleasure horse. To investigate the contribution of genetics to body conformation in the TWH, we collected DNA samples, body measurements, and gait/training information from 282 individuals. We analyzed the 32 body measures with a principal component analysis. Principal component (PC)1 captured 28.5% of the trait variance, while PC2 comprised just 9.5% and PC3 6.4% of trait variance. All 32 measures correlated positively with PC1, indicating that PC1 describes overall body size. We genotyped 109 horses using the EquineSNP70 bead chip and marker association assessed the data using PC1 scores as a phenotype. Mixed-model linear analysis (EMMAX) revealed a well-documented candidate locus on ECA3 (raw P = 3.86 × 10(-9)) near the LCORL gene. A custom genotyping panel enabled fine-mapping of the PC1 body-size trait to the 3'-end of the LCORL gene (P = 7.09 × 10(-10)). This position differs from other reports suggesting single nucleotide polymorphisms (SNPs) upstream of the LCORL coding sequence regulate expression of the gene and, therefore, body size in horses. Fluorescent in situ hybridization analysis defined the position of a highly homologous 5 kb retrogene copy of LCORL (assigned to unplaced contigs of the EquCab 2.0 assembly) at ECA9 q12-q13. This is the first study to identify putative causative SNPs within the LCORL transcript itself, which are associated with skeletal size variation in horses.

DMRT3 is associated with gait type in Mangalarga Marchador horses, but does not control gait ability.

The Mangalarga Marchador (MM) is a Brazilian horse breed known for a uniquely smooth gait. A recent publication described a mutation in the DMRT3 gene that the authors claim controls the ability to perform lateral patterned gaits (Andersson et al. 2012). We tested 81 MM samples for the DMRT3 mutation using extracted DNA from hair bulbs using a novel RFLP. Horses were phenotypically categorized by their gait type (batida or picada), as recorded by the Brazilian Mangalarga Marchador Breeders Association (ABCCMM). Statistical analysis using the plink toolset (Purcell, 2007) revealed significant association between gait type and the DMRT3 mutation (P = 2.3e-22). Deviation from Hardy-Weinberg equilibrium suggests that selective pressure for gait type is altering allele frequencies in this breed (P = 1.00e-5). These results indicate that this polymorphism may be useful for genotype-assisted selection for gait type within this breed. As both batida and picada MM horses can perform lateral gaits, the DMRT3 mutation is not the only locus responsible for the lateral gait pattern.

Effect of prolactin, beta-lactoglobulin, and kappa-casein genotype on milk yield in East Friesian sheep.

The effect of prolactin (PRL), beta-lactoglobulin (beta-LG), and kappa-casein (CSN3) on milk yield was estimated in an East Friesian dairy sheep population from Old Chatham Sheepherding Company, New York. Genotypes were determined by PCR amplification followed by digestion with HaeIII and RsaI for PRL and beta-LG, respectively, and by PCR amplification for CSN3. Monthly milking records and pedigree information were used to evaluate the effect of each polymorphism on milk yield. Results indicated that PRL genotype had a significant effect on milk yield. Ewes carrying one A allele produced 110.6g more milk per day than ewes with no A alleles. There was no statistical difference between ewes with only one A allele and ewes with 2 A alleles. No association among polymorphisms at the beta-LG and CSN3 loci and milk yield was found. The results presented in this study indicate that the PRL gene is a potential marker that could be used in selection programs for improving milk yield in dairy sheep.