Genome-wide search of candidate genes associated with «depth of chest» parameter in Severocavcazskaya sheep breed

Increasing the meat productivity of existing sheep breeds is one of the most important tasks of sheep breeding. One of the modern and most effective methods for searching for polymorphisms and genes associated with economically significant traits is the Genome-Wide Association Study (GWAS). Sheep are characterized by extremely high ecological plasticity. As a result, genetic markers of productivity may differ between breeds, which is associated with the characteristics of the environmental conditions where they were bred. In this regard, it is relevant to search for new candidate genes in sheep breeds adapted to local conditions. One of these breeds is the Severocavcazskaya sheep breed, bred in the arid steppes of Southern Russia. For her character, the meat productivity is quite high for her class. Thus, breeding rams have an average weight of more than 100 kg, and bright ones - 60 kg. The dispersion of the phenotype of meat forms among representatives of the breed indicates genetic diversity and the possibility of further selection in the direction of increasing meat productivity. One of the important parameters of meat productivity is depth of chest, which reflects the degree of development of the chest. Thus, the purpose of this study was to search for Single Nucleotide Polymorphism (SNP) and candidate genes associated with depth of chest in Severocavcazskaya sheep breed. The object of the study were rams (n = 50) of the Severocavcazskaya sheep breed at the age of 12 months. The animals were clinically healthy, kept in optimal conditions that met zootechnical standards and zoohygienic requirements, and were not shorn. Genomic DNA was obtained from blood samples collected aseptically from the jugular vein using the Pure Link Genomic DNA MiniKit (Invitrogen Life Technologies, USA) according to the manufacturer's protocol. Animal genotyping was carried out using Ovine Infinium HD BeadChip 600K DNA biochips (Illumina Inc., California, USA) in accordance with the manufacturer's protocol. Initial processing of genotyping results was carried out using the Genome Studio 2.0 program (Illumina Inc., California, USA). Quality control of genotyping was performed using the PLINK V.1.07 program. Samples with the number of detected SNPs (Call Rate) more than 0.95 were included in data processing. Of the 606.006 SNPs, 562.549 polymorphisms were used for further analysis. Genome-wide association study were performed using PLINK V.1.07 software, function - assoc. Differences were considered significant when -log10(p) > 5. Visualization and plotting were carried out using the “QQman” package in the R programming language. The search for the nearest candidate genes was performed in an area of 250,000 bp. around SNPs that showed significant differences in occurrence among animals of the studied groups. Mapping of single nucleotide substitutions was carried out using the Oar_v3.1 genome assembly. Gene annotations were performed using the Ensemble Genome Browser (www.ensembl.org) and Genome Data Viewer (www.ncbi.nlm.nih.gov). As a result of a genome-wide association study, it was possible to identify polymorphisms associated with the “depth of chest” parameter. As a result, 14 SNPs were found that passed the significance threshold of -log10(p) = 5 (See Fig. 1). These polymorphisms are found on chromosomes 1, 5, 9 and 15. SNPs with significant associations were selected for candidate gene searches. One of the polymorphisms is localized in an exon, four in introns, another in the upsream region of genes, and the remaining seven in intergenic regions (See Table 1). On chromosomes 1 and 9, we identified 7 substitutions in intergenic regions (5 on chromosome 1, two on chromosome 9); the genes closest to them belong to the lincRNA group (long non-coding intergenic RNA). At the same time, the distance from genes to SNP varied from 13025 nucleotide pairs in rs416093141 to 222747 in rs426975931. There are 4 such genes in total: ENSOARG00000025606, ENSOARG00000025607, ENSOARG00000025510 and ENSOARG00000026528. LincRNAs are involved in the regulation of gene expression, epigenetic mechanisms, and cell differentiation, although they themselves do not encode any proteins. In sheep, the influence of this group of genes on lipid metabolism and, most importantly, on muscle growth during embryonic development and in the postembryonic period has been revealed. In addition to five substitutions in intergenic regions on chromosome 1, 4 more SNPs were identified that were reliably associated with the trait under study. Polymorphism rs401698065, which is located in the intron of the SSBP3 gene. The SSBP3 expression product is capable of binding to DNA, participating in the regulation of transcription. The closest gene to SNP rs408075804 is SATB1. It is a homeobox gene involved in chromatin organization and transcription. Two polymorphisms on chromosome 1 are associated with the SLC44A3 gene: rs421246568 is located in the upsream region of this gene, and rs414249944 is in the intron. The protein encoded by SLC44A3 is presumably localized in the plasma membrane of cells and is a transport protein. In previous studies, it was indicated as associated with important productive traits in sheep, such as slaughter weight, birth weight, muscle eye width and some others. On chromosome 5, the rs414906974 substitution was identified, located in the intron of the ADGRV1 gene. The product of this gene is a membrane receptor and, according to some researchers, is associated with the regulation of autophagy. Two SNPs were identified on chromosome 15: rs409835265 and rs421657104. Both polymorphisms are located in the MS4A14 gene in the intron and exon, respectively. The MS4A gene group encodes membrane hydrophobic proteins. MS4A14 in humans is expressed mainly in the testes and spleen. The exact function of this gene has not yet been identified. As a result of our research, we can propose 9 new candidate genes associated with sheep depth of chest. Among them are 4 lincRNA genes with as yet unknown functions. The remaining 5 genes encode proteins: SSBP3, SATB1, SLC44A3, ADGRV1 and MS4A14. The 14 SNPs we discovered can be used as molecular markers in the selection of sheep of the Severocavcazskaya sheep breed. The article contains 3 Figures, 2 Table, 35 References. The Authors declare no conflict of interest.

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