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

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

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

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

Equine gene model of <i>PLCz1</i> and polymorphisms detected.

<p>Sequence analyses of <i>PLCz</i> 1 revealed four transcripts of which the full-length variant (JX545317) is shown. Translated exons are shown as solid black boxes, untranslated exons as open boxes. Numbers above indicate the exon number. Continuous lines represent introns, numbers below the exons and introns indicate the sizes in base pairs. The predicted translation start and stop and the polyadenylation site is indicated. The highly associated single nucleotide polymorphism (SNP) BIEC2-952439 identified in a genome-wide association study (GWAS) is given in bold. The dashed lines below represent the coverage of the complementary DNA (cDNA) primers.</p

Jiří Haller in the contexts of Czech linguistics

This study tries to examine the position of Jiří Haller in the context of Czech linguistics, or more precisely in the context of the development of Czech research of language culture. The first chapter analyzes the work of Jan Gebauer, Josef Zubatý and Václav Ertl, Le. the work of Haller's predecessors. The goal is to find joint aspects of theirs approach to the language culture. The second chapter is concerned with the background of Haller's professional work, mainly with the study entitled "Problém jazykové správnosti", which summarizes Haller's early theoretical attitude. The third chapter analyzes the dispute over literary language; it offers a complex survey of those debates and tries to critically evaluate particular polemic appearances. The fourth part examines other works of Jiří Haller, Le. his activities after 1932. The main goal of the study is to revise traditional opinions on the personality of Jiří Haller and to present a more realistic view on his linguistic appearance and his work

Additional file 6: of Screening of whole genome sequences identified high-impact variants for stallion fertility

Distribution of high-impact variants contributing to stallion fertility in discovery breeds compared to validation breeds. The discovery breeds include horses of the breeds Arabian (AR), Hanoverian (HA), Standardbred (SB), Icelandic (IS), Norwegian fjord (NO), and the non-breed horses Dülmen Horse (DU) and Sorraia (SO), and the wild horse Przewalski (PR). The validation breeds include stallions of the pure breeds Arabian (AR), and Thoroughbred (TH), German sport horses including Hanoverian (HA), Holstein (HO), Oldenburg (OL), Westphalian (WE) Rhinelander (RH) and German Riding Pony (GE). Next, cold blood breeds, represented by Black Forest Horses (BF), Mecklenburger Cold Blood (MC), Rhenish German Cold Blood (RG), Saxon Thuringia Cold Blood (ST), Schleswig Cold Blood (SC) and Southern German Cold Blood (SG), further Dülmen Horse (DU) and Sorraia (SO). Further, robust horses and non-breed horses were represented by Norwegian fjord (NO), Konik (KO) and Tarpan (TA). (DOCX 30 kb

Additional file 7: of Screening of whole genome sequences identified high-impact variants for stallion fertility

Number of stallions genotyped (n) and genotypic means with their standard errors (SE) and P-values (P) of the de-regressed estimated relative breeding values of the paternal component for the pregnancy rate per estrus (EBV-PAT) in 216 Hanoverian stallions for high-impact variants, their nominal (P) and for multiple tests Bonferroni-corrected P-values (Pmultiple) for differences among the genotypes. The de-regressed EBV-PAT values were standardized onto a scale of 100 ± 20. (DOCX 20 kb

Additional file 8: of Screening of whole genome sequences identified high-impact variants for stallion fertility

Distribution of fertile stallions genotyped for high-impact variants. Fertile stallions have a minimum number of 10 progeny. (DOCX 14 kb