Expression levels of LCORL are associated with body size in horses.

Body size is an important characteristic for horses of various breeds and essential for the classification of ponies concerning the limit value of 148 cm (58.27 inches) height at the withers. Genome-wide association analyses revealed the highest associated quantitative trait locus for height at the withers on horse chromosome (ECA) 3 upstream of the candidate gene LCORL. Using 214 Hanoverian horses genotyped on the Illumina equine SNP50 BeadChip and 42 different horse breeds across all size ranges, we confirmed the highly associated single nucleotide polymorphism BIEC2-808543 (-log(10)P = 8.3) and the adjacent gene LCORL as the most promising candidate for body size. We investigated the relative expression levels of LCORL and its two neighbouring genes NCAPG and DCAF16 using quantitative real-time PCR (RT-qPCR). We could demonstrate a significant association of the relative LCORL expression levels with the size of the horses and the BIEC2-808543 genotypes within and across horse breeds. In heterozygous C/T-horses expression levels of LCORL were significantly decreased by 40% and in homozygous C/C-horses by 56% relative to the smaller T/T-horses. Bioinformatic analyses indicated that this SNP T>C mutation is disrupting a putative binding site of the transcription factor TFIID which is important for the transcription process of genes involved in skeletal bone development. Thus, our findings suggest that expression levels of LCORL play a key role for body size within and across horse breeds and regulation of the expression of LCORL is associated with genetic variants of BIEC2-808543. This is the first functional study for a body size regulating polymorphism in horses and a further step to unravel the mechanisms for understanding the genetic regulation of body size in horses

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 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

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

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

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