Identification And Validation Of Candidate Genes Regulating Muscle Related Traits In Simmental Beef Cattle Based On Genotypes Imputed With Next-generation Sequencing Data

Genome-wide association studies(GWAS)have commonly been used to identify candidate genes that control economically important traits in livestock.Our objective was to detect potential candidate genes associated mainly with muscle development traits related to dimension of hindquarter and net meat weight and validate these candidate genes in cattle.A next generation sequencing(NGS)dataset to imputed to 12 million single nucleotide polymorphisms(SNPs)(from 1252 Simmental beef cattle)were used to search for genes affecting hindquarter traits using a linear mixed model.We also used haplotype and LD blocks to further support our identifications.We identified 202 significant SNPs in the BTA4 region associated with width of hind leg,based on a stringent statistical threshold(P=0.05/effective number of SNPs identified).After exploring the region around these SNPs,we found candidate genes that were potentially related to the associated markers.More importantly,we identified a region of approximately 280 kb on the BTA4 that harbored several muscle specific candidate genes,genes to be in a potential region for muscle development.However,we also found candidate gene SLC13A1 on BTA4,which seems to be associated with bone disorders(such as chondrodysplasia)in Simmental beef cattle.In net meat weight,we identified one SNP(chr4:100670823)in the BTA4 that exceeded our stringent threshold(p=8.58×10-~8),therefore,causing this SNP to be listed as candidate for putative QTL related to net meat weight trait.After checking around this SNP,we identified very promising candidate gene in Simmental beef cattle called MTPN,potentially related to the associated marker.post GWAS,the fundamental validation is required to implement,not only to make strong contention about gene function but also for verifying the biological significance of detected genes.Therefore,we validated our candidate gene using cell culture experiments in vitro.Using myogenesis markers,qRT-PCR with different concentrations of myotrophin(MTPN)was conducted.We noticed Myotrophin had stimulating effect on six muscle-related markers regulating differentiation and hypertrophy.We also investigated the effect of myotrophin on the number of nuclei,fusion index and diameter of myotube.Notably,different concentrations of protein had positive effect on mentioned experiments,leading us to conclude that myotrophin had promoted effect not only on differentiation but also hypertrophy of skeletal muscle in Simmental beef cattle.Moreover,we investigated the impact of myotrophin on proliferation of skeletal muscle by CCK-8 proliferation assay and flow cytometry.Surprisingly,myotrophin had opposite effect on muscle cells and decrease the proliferation.Taken all together,myotrophin was able to increase differentiation and hypertrophy of skeletal muscle cells,while it had opposite effect on proliferation of skeletal muscle cells Our research is attempt to identify muscle-specific candidate genes in Simmental beef cattle by combining GWAS analysis and molecular experiment validation.We strongly hope our finding provide useful information for meat science industry as well as helpful information for further studies.