An epistatic effect of KRT25 on SP6 is involved in curly coat in horses.

Curly coat represents an extraordinary type of coat in horses, particularly seen in American Bashkir Curly Horses and Missouri Foxtrotters. In some horses with curly coat, a hypotrichosis of variable extent was observed, making the phenotype appear more complex. In our study, we aimed at investigating the genetic background of curly coat with and without hypotrichosis using high density bead chip genotype and next generation sequencing data. Genome-wide association analysis detected significant signals (p = 1.412 × 10-05-1.102 × 10-08) on horse chromosome 11 at 22-35 Mb. In this significantly associated region, six missense variants were filtered out from whole-genome sequencing data of three curly coated horses of which two variants within KRT25 and SP6 could explain all hair phenotypes. Horses heterozygous or homozygous only for KRT25 variant showed curly coat and hypotrichosis, whereas horses with SP6 variant only, exhibited curly coat without hypotrichosis. Horses with mutant alleles in both variants developed curly hair and hypotrichosis. Thus, mutant KRT25 allele is masking SP6 allele effect, indicative for epistasis of KRT25 variant over SP6 variant. In summary, genetic variants in two different genes, KRT25 and SP6, are responsible for curly hair. All horses with KRT25 variant are additionally hypotrichotic due to the KRT25 epistatic effect on SP6.

Relationships among stallion fertility and semen traits using estimated breeding values of German Warmblood stallions.

A high quality of stallion semen is of particular importance for maximum reproductive efficiency. In the present study, we estimated the relationships among estimated breeding values (EBVs) of semen traits and EBVs for the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) for 100 German Warmblood stallions using correlation and general linear model analyses. The most highly correlated sperm quality trait was total number of progressively motile sperm (r = 0.36). EBV-PAT was considered in three classes with stallions 1 SD below (<80), around (80-120), and above (>120) the population mean of 100. The general linear model analysis showed significant effects for EBVs of all semen traits. EBVs of sperm quality traits greater than 100 to 110 were indicative for EBV-PAT greater than 120. Recommendations for breeding soundness examinations on the basis of the assessments of sperm quality traits and estimation of breeding values seem to be an option to support breeders to improve stallion fertility in the present and future stallion generation.

Genome-wide association study for semen quality traits in German Warmblood stallions.

We performed a genome-wide association study for semen quality traits in 139 German Warmblood stallions. Stallions were genotyped using the Illumina equine SNP50 Beadchip. Traits analysed were de-regressed estimated breeding values (EBVs) for gel-free volume, sperm concentration, total number of sperm, progressive motility and the total number of progressively motile sperm. The GWAS revealed 29 SNPs on 12 different chromosomes as genome-wide significantly associated with semen quality traits. For ten genomic regions we could retrieve candidate genes influencing stallion fertility. Among the candidate genes, we could find the genes encoding cysteine-rich secretory proteins (CRISP1, CRISP2 and CRISP3). This was the first GWAS in horses performed for semen quality traits.

Screening of whole genome sequences identified high-impact variants for stallion fertility.

BACKGROUND: Stallion fertility is an economically important trait due to the increase of artificial insemination in horses. The availability of whole genome sequence data facilitates identification of rare high-impact variants contributing to stallion fertility. The aim of our study was to genotype rare high-impact variants retrieved from next-generation sequencing (NGS)-data of 11 horses in order to unravel harmful genetic variants in large samples of stallions. METHODS: Gene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human und mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologous genes were searched in whole genome sequence data of seven stallions and four mares and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen 2 software. All genetic variants with the missing homozygous mutant genotype were genotyped on 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis was employed for an association analysis with de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). RESULTS: We screened next generation sequenced data of whole genomes from 11 horses for equine genetic variants in 1194 human and mice genes involved in male fertility and linked through common gene ontology (GO) with male reproductive processes. Variants were filtered for high-impact on protein structure and validated through SIFT and Polyphen 2. Only those genetic variants were followed up when the homozygote mutant genotype was missing in the detection sample comprising 11 horses. After this filtering process, 17 single nucleotide polymorphism (SNPs) were left. These SNPs were genotyped in 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. An association analysis in 216 Hanoverian stallions revealed a significant association of the splice-site disruption variant g.37455302G>A in NOTCH1 with the de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). For 9 high-impact variants within the genes CFTR, OVGP1, FBXO43, TSSK6, PKD1, FOXP1, TCP11, SPATA31E1 and NOTCH1 (g.37453246G>C) absence of the homozygous mutant genotype in the validation sample of all 337 fertile stallions was obvious. Therefore, these variants were considered as potentially deleterious factors for stallion fertility. CONCLUSIONS: In conclusion, this study revealed 17 genetic variants with a predicted high damaging effect on protein structure and missing homozygous mutant genotype. The g.37455302G>A NOTCH1 variant was identified as a significant stallion fertility locus in Hanoverian stallions and further 9 candidate fertility loci with missing homozygous mutant genotypes were validated in a panel including 19 horse breeds. To our knowledge this is the first study in horses using next generation sequencing data to uncover strong candidate factors for stallion fertility.

Analysis of breed effects on semen traits in light horse, warmblood, and draught horse breeds.

In the present study, systematic effects on semen quality traits were investigated in 381 stallions representing 22 breeds. All stallions were used for AI either at the Lower Saxon National Stud Celle or the North Rhine-Westphalian National Stud Warendorf. A total of 71,078 fresh semen reports of the years 2001 to 2014 were edited for analysis of gel-free volume, sperm concentration, total number of sperm, progressive motility, and total number of progressively motile sperm. Breed differences were studied for warmblood and light horse breeds of both national studs (model I) and for warmblood breeds and the draught horse breed Rhenish German Coldblood from the North Rhine-Westphalian National stud (model II) using mixed model procedures. The fixed effects of age class, year, and month of semen collection had significant influences on all semen traits in both analyses. A significant influence of the horse breed was found for all semen traits but gel-free volume in both statistical models. Comparing warmblood and light horse stallions of both national studs, we observed highest sperm concentrations, total numbers of sperm, and total numbers of progressively motile sperm in Anglo-Arabian stallions. The draught horse breed Rhenish German Coldblood had the highest least squares means for gel-free volume, whereas all other investigated semen traits were significantly lower in this breed compared to the warmblood stallions under study. The variance components among stallions within breeds were significant for all semen traits and accounted for 40% to 59% of the total variance. The between-breed-variance among stallions was not significant underlining the similar size of the random stallion effect in each of the horse breeds analyzed here. In conclusion, breed and stallion are accounting for a significant proportion of the variation in semen quality.

Ricinus communis cyclophilin: functional characterisation of a sieve tube protein involved in protein folding.

The phloem translocation stream of the angiosperms contains a special population of proteins and RNA molecules which appear to be produced in the companion cells prior to being transported into the sieve tube system through the interconnecting plasmodesmata. During this process, these non-cell-autonomous proteins are thought to undergo partial unfolding. Recent mass spectroscopy studies identified peptidyl-prolyl cis-trans isomerase (PPIases) as potential molecular chaperones functioning in the phloem translocation stream (Giavalisco et al. 2006). In the present study, we describe the cloning and characterisation of a castor bean phloem cyclophilin, RcCYP1 that has high peptidyl-prolyl cis-trans isomerase activity. Equivalent enzymatic activity was detected with phloem sap or purified recombinant (His)(6)-tagged RcCYP1. Mass spectrometry analysis of proteolytic peptides, derived from a 22 kDa band in HPLC-fractionated phloem sap, immunolocalisation studies and Western analysis of proteins extracted from castor bean tissues/organs indicated that RcCYP1 is an abundant protein in the companion cell-sieve element complex. Microinjection experiments established that purified recombinant (His)(6)-RcCYP1 can interact with plasmodesmata to both induce an increase in size exclusion limit and mediate its own cell-to-cell trafficking. Collectively, these findings support the hypothesis that RcCYP1 plays a role in the refolding of non-cell-autonomous proteins after their entry into the phloem translocation stream.