The aim of this study was to characterize and identify causative polymorphisms in the leptin receptor (LEPR) gene responsible for the seasonal variation of reproductive traits in sheep. Three reproductive seasonality traits were studied: the total days of anoestrous (TDA), the progesterone cycling months (P4CM) and the oestrous cycling months (OCM). In total, 18 SNPs were detected in 33 ewes with extreme values for TDA and OCM. Six SNPs were non-synonymous substitutions and two of them were predicted in silico as deleterious: rs596133197 and rs403578195. These polymorphisms were then validated in 239 ewes. The SNP rs403578195, located in exon 8 and leading to a change of alanine to glycine (Ala284Gly) in the extracellular domain of the protein, was associated with the OCM trait, being the G allele associated with a decrease of 12 percent of the OCM trait. Haplotype analyses also suggested the involvement of other non-synonymous SNP located in exon 20 (rs405459906). This SNP also produces an amino acid change (Lys1069Glu) in the intracellular domain of the protein and segregates independently of rs403578195. These results confirm for the first time the role of the LEPR gene in sheep reproductive seasonality.

Sheep are very well adapted to changing environments and are able to produce and reproduce with low inputs in feed and water better than other domestic ruminants. Indeed, the ewe body condition score (BCS) and live weight (LW) play a significant role in productive and reproductive performance. This work conducts a genome-wide association study (GWAS) to detect genetic variants associated with growth traits in 225 adult ewes of the Rasa Aragonesa breed by using the genotypes from 50 k and HD Illumina Ovine BeadChip. These ewes were measured for LW, BCS and growth rate (GR) for 2 years, from January to September. Corrected phenotypes for BCS, LW and GR were estimated and used as input for the GWAS. Only one single nucleotide polymorphism (SNP) rs425509273 in chromosome 9 (OAR9), associated with the GR, overcame the genome-wise significance level. One, three and nine SNPs were associated at the chromosome-wise level (FDR 10%) for traits BCS, LW and GR, respectively. The cytochrome P450 family 7 subfamily B member 1 (CYP7B1) candidate gene, located 83 kb upstream from SNP rs425509273 in OAR9, was partially isolated and Sanger-sequenced. Fifteen polymorphisms comprising 12 SNPs, two indels and one polyC, were detected in promoter, exon 1, 3, 5, and intron 1-3 region. The SNP association analysis of the polymorphisms located close to the transcription start site (TSS) showed that a 22 bp insertion located at -58 nucleotides from the TSS (indel (-58)), a polyC (-25), and two A/G SNPs (SNP3 (-114) and SNP5 (-63)) were associated with the GR trait, whereas only the indel (-58) was associated with the BCS trait. The haplotype analysis confirmed these results. The functional characterisation of the polymorphisms at CYP7B1 gene in liver by real-time quantitative PCR analysis confirmed that the mutations in the promoter region affected CYP7B1 gene expression. Our results demonstrated the involvement of the CYP7B1 gene promoter on GR and BCS traits in Rasa Aragonesa. These findings suggest that variations in ovine CYP7B1 may serve as potential genetic markers to be used in breeding programmes to improve growth characteristics that could influence reproductive traits.

A genome-wide association study (GWAS) was used to identify genomic regions influencing seasonality reproduction traits in Rasa Aragonesa sheep. Three traits associated with either ovarian function based on blood progesterone levels (total days of anoestrus and progesterone cycling months) or behavioral signs of oestrous (oestrous cycling months) were studied. The GWAS included 205 ewes genotyped using the 50k and 680k Illumina Ovine Beadchips. Only one SNP associated with the progesterone cycling months overcame the genome-wide significance level (rs404991855). Nine SNPs exhibited significant associations at the chromosome level, being the SNPs rs404991855 and rs418191944, that are located in the CD226 molecule (CD226) gene, associated with the three traits. This gene is related to reproductive diseases. Two other SNPs were located close to the neuropeptide Y (NPY) gene, which is involved in circadian rhythms. To validate the GWAS, partial characterization of both genes by Sanger sequencing, and genotyping of two synonymous and two nonsynonymous SNPs in the NPY and CD226 genes, respectively, were performed. SNP association analysis showed that only SNP rs404360094 in the exon 3 of the CD226 gene, which produces an amino acid substitution from asparagine (uncharged polar) to aspartic acid (acidic), was associated with the three seasonality traits. Our results suggest that the CD226 gene may be involved in the reproductive seasonality in Rasa Aragonesa.

Haplotypes from sequencing data may improve the prediction accuracy in genomic evaluations as haplotypes are in stronger linkage disequilibrium with quantitative trait loci than markers from SNP chips. This study focuses first, on the creation of haplotypes in a population sample of 450 Holstein animals, with full-sequence data from the 1000 bull genomes project; and second, on incorporating them into the whole genome prediction model. In total, 38,319,258 SNPs (and indels) from Next Generation Sequencing were included in the analysis. After filtering variants with minor allele frequency (MAF< 0.025) 13,912,326 SNPs were available for the haplotypes extraction with findhap.f90. The number of SNPs in the haploblocks was on average 924 SNP (166,552 bp). Unique haplotypes were around 97% in all chromosomes and were ignored leaving 153,428 haplotypes. Estimated haplotypes had a large contribution to the total variance of genomic estimated breeding values for kilogram of protein, Global Type Index, Somatic Cell Score and Days Open (between 32 and 99.9%). Haploblocks containing haplotypes with large effects were selected by filtering for each trait, haplotypes whose effect was larger/lower than the mean plus/minus 3 times the standard deviation (SD) and 1 SD above the mean of the haplotypes effect distribution. Results showed that filtering by 3 SD would not be enough to capture a large proportion of genetic variance, whereas filtering by 1 SD could be useful but model convergence should be considered. Additionally, sequence haplotypes were able to capture additional genetic variance to the polygenic effect for traits undergoing lower selection intensity like fertility and health traits.