Relative expression levels of <i>LCORL</i> in relation to the BIEC2-808543 genotype in 13 Hanoverian horses.

<p>Using the T/T genotype as standard, the expression of horses with the C/T genotype is decreased by 40% and for the genotype C/C (P = 0.024) by 56%. The expression differences were accounted for using the ΔΔCT method.</p

Averaged height at the withers plotted against the frequency of the allele T in ponies, small horses, light coldblood and heavy coldblood horses.

<p>The results show that the allele frequency corresponds to the average height.</p

Distribution of genotypes of the associated single nucleotide polymorphism (SNP) BIEC2-808543 in 214 Hanoverian warmblood horses and the means of the breeding values with their standard errors per genotype.

<p>The SNP BIEC2-808543 is located at 105.55 Mb on horse chromosome (ECA) 3 with a minor allele frequency (MAF) of 0.45. The additive effect of the BIEC2-808543 polymorphisms amounted to 16.3 and the dominance effect to 0.6.</p

Relative expression levels of <i>LCORL</i> in relation to the BIEC2-808543 genotype across five different breeds.

<p>In comparison with the T/T genotypes, the expression of horses with the C/T genotype (P = 0.016) is decreased by 44% and for the genotype C/C (P<0.001) by 54%. The expression differences were accounted for using the ΔΔCT method.</p

Genome-wide association analysis shows a highly significant peak on equine chromosome (ECA) 3 in Hanoverian.

<p>(A) Manhattan-plot of the −log₁₀P-values from genome-wide association analysis (MLM) of body size in Hanoverian warmblood horses. The highest peak is located at 105 Mb on ECA3. (B) Q-Q plot of observed versus expected −log₁₀P-values from a genome- wide association study (GWAS) in Hanoverian warmblood horses. The expected distribution (solid line) and the observed −log₁₀P-values plotted against the expected −log₁₀P-values (black dots) are shown. The peak value (BIEC2-808543) is located on horse chromosome 3 at 105.55 Mb.</p

Comparison of equine <i>PLCz1</i> transcript variants with and human <i>PLCz1</i>.

<p>Transcript variants (1) ENSECAT00000012304, (2) ENSECAT00000012372, (3) XM_001497766.3, (4) JX545317, (5) JX545319, (6) ENST00000318197, (7) NR_073075.1, (8) JX545318, and (9) JX545320 are presented. (1–4) Human and equine variants without a premature termination codon (PTC) in sequences are indicated. (5–9) A PTC is activated within human and equine variants at similar positions (c.136). The PTC region is highlighted by a red box.</p

Haplotype blocks within <i>PLCz1</i> significantly associated with estimated breeding values for the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) in Hanoverian stallions.

<p>The start and end of the haplotype block in base pairs (POS), the variance explained (R²) by each haplotype block, significantly associated SNPs within haplotype blocks (SNP) and P-values (P) are given.</p><p>Haplotype blocks within <i>PLCz1</i> significantly associated with estimated breeding values for the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) in Hanoverian stallions.</p

Genome-Wide Association Study Identifies <i>Phospholipase C zeta 1 (PLCz1)</i> as a Stallion Fertility Locus in Hanoverian Warmblood Horses

<div><p>A consistently high level of stallion fertility plays an economically important role in modern horse breeding. We performed a genome-wide association study for estimated breeding values of the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) in Hanoverian stallions. A total of 228 Hanoverian stallions were genotyped using the Equine SNP50 Beadchip. The most significant association was found on horse chromosome 6 for a single nucleotide polymorphism (SNP) within <i>phospholipase C zeta 1 (PLCz1)</i>. In the close neighbourhood to <i>PLCz1</i> is located <i>CAPZA3</i> (<i>capping protein (actin filament) muscle Z-line</i>, <i>alpha 3</i>). The gene <i>PLCz1</i> encodes a protein essential for spermatogenesis and oocyte activation through sperm induced Ca²⁺-oscillation during fertilization. We derived equine gene models for <i>PLCz1</i> and <i>CAPZA3</i> based on cDNA and genomic DNA sequences. The equine <i>PLCz1</i> had four different transcripts of which two contained a premature termination codon. Sequencing all exons and their flanking sequences using genomic DNA samples from 19 Hanoverian stallions revealed 47 polymorphisms within <i>PLCz1</i> and one SNP within <i>CAPZA3</i>. Validation of these 48 polymorphisms in 237 Hanoverian stallions identified three intronic SNPs within <i>PLCz1</i> as significantly associated with EBV-PAT. Bioinformatic analysis suggested regulatory effects for these SNPs via transcription factor binding sites or microRNAs. In conclusion, non-coding polymorphisms within <i>PLCz1</i> were identified as conferring stallion fertility and <i>PLCz1</i> as candidate locus for male fertility in Hanoverian warmblood. <i>CAPZA3</i> could be eliminated as candidate gene for fertility in Hanoverian stallions.</p></div

Transcript variants detected in the equine <i>PLCz1</i>.

<p>Variants were detected in cDNA sequences of testis tissues from six Hanoverian stallions. The size of the untranslated exon 1, mRNA and coding sequence (CDS) in base pairs, the location of the premature termination codon (PTC) and the predicted number of amino acids are given.</p><p>Transcript variants detected in the equine <i>PLCz1</i>.</p

Chromatograms of transcripts detected in equine <i>PLCz1</i>.

<p>(A) The sequences of all animals tested show both adjacent splice sites of the cDNA. Both, forward and reverse sequences show ambiguous double traces when elongation passes exon boundary. (B, C) Visualization of single trace sequence is used to portray each single transcript. (B) For the transcripts without a premature termination codon (PTC), exon 4a starts with the residues GAT, (C) whereas PTC-containing transcript variants harbor an extended exon 4b and start with the residues TAG. (D) Genomic sequence was used to illustrate usage of adjacent acceptor splice sites AG-TA for PTC-containing transcripts and AG-GA for non-PTC-containing transcripts. Vertical lines mark the exon/exon boundary or intron/exon boundary, respectively. The black open boxes indicate specific nucleotide residues used.</p