Identification Functional Genes And Mutations Related To Economics Traits In Pigs Using Integrated Omics Approach

Due to the long-term direction of artificial selection and environmental differences,the genetic background of Chinese domestic pig and Western domestic pig is different,which results in differences in the phenotypes of economic traits such as growth rate and lean meat rate between Chinese domestic pig and Western domestic pig.The study of selection signal contributes to resolve the corresponding genetic mechanisms behind these differential phenotypes,it is also an effective strategy for identifying gene involved in economic traits in pigs.However,due to the linkage disequilibrium and the functional annotation of pig is incomplete,it is difficult to identify the major genes and functional mutations affecting economic traits.In order to overcome these difficulties,we used an integrated omics strategy system analysis to identify artificially selected genes and functional mutation in pigs,and we also analyzed their molecular mechanisms and the impact on economic traits.The main results of this study are:(1)Identification of seletive sweep in Chinese domestic and Western domestic pig and localization the artificialselection regions:SNPs were called from 64 individuals of 13 breeds,and their population structure and volutionary relationships were analyzed by population genetics.Fst was calculated using sliding windows method,1296 Western domestic pig selected genes,169 Chinese domestic pig selected genes and 1420 highly differentiated genes between Chinese domestic pig and Western domestic pig were identified.Basis on this,using the distribution characteristic of selective signal and the differentiation signal,original genetic background were removed,390 Western domestic pig artifically selected gene and 62 Chinese domestic pig artificially selected gene were identified,while these genes significantly enriched in the growth,body size,muscle development and immune system related phenotypes.The enrichment results were closely related to the phenotypic differences between Chinese domestic pig and Western domestic pig.(2)Genome wide identify allelic specific expression genes:We established the hybrid F1 popultion through crossbreed the Large White and Meishan/Enshi.Through haplotype phasing technology in 38 tissues from embryonic stage and adult stage,499 allele specific expression genes were identified.The haplotype phasing results showed that the phasic haplotype constructed by the F1 hybrid heterozygous population strategy of Chinese and Western domestic pig breeds cover 79%of genome.Allelic specific expression gene analysis results showed that allele specific expression was tissue-specific in pigs.These tissue-specific allele specific expressed genes are closely related to the biological processes of tissue.At the same time,this study also found that 75 of the 390 artifically selected genes in Western domestic pig were allele specific expressed.The discovery of these genes is very important for the study of phenotypic differences between Chinese and Western domestic pig breeds.For example,the CPE gene that is artificially selected gene and associated with obesity,in the 95-day brain tissue of the embryo,the expression level of the western domestic pig haplotype is much higher than that of the Chinese domestic pig haplotype.(3)Genome wide predict and validate cis regulatory elements of pig:We identified 1.33G homologous fragments by co-linear analyisis between pig and human genome,Annotated the homologous fragments by the map of the human regulatory element which were generated by ENCODE projects and ROADMAP epigenetic projects.A total of 82,522 potential regulatory elements were predicted in the pig genome,of which 64%of potential regulatory elements were validated in ChIP-seq data from pig liver tissue.The results of the joint analysis with the artificial selection region indicated that the extreme difference SNP sites between Chinese and Western domestic pig around the artificially selected genes were more enriched in cis regulatory elements regions than the other SNP sites.In this study,112,167 extreme SNP between Chinese and Western domestic pig were located in cis regulatory elements were found in the artificial selection region.The above results predicted the genome-wide level of the regulatory elements of pigs,which laid the foundation for further identification of functional SNPs,and also proved that the regions where selection occurred were more likely to enriched in regulatory elements regions.(4)Identification of regulatory elements of pig liver tissue:We used public data to analyze the histone ChIP-seq data of pig liver tissue,and identified 61,570 enhancers,14,007 active promoters,1,711 super enhancers and 1,989 broad H3K4me3 domain.This provides the functional annotation of pig genome and reference materials for molecular function studies related to pig liver tissue.We further analyzed the conservation of regulatory elements between species.The results showed that the functionally conserved proportion of active promoters between species(77%)was significantly higher than that of enhancers(30%),while only sequence-conserved enhancers can play an enhancer role in other tissues(78%),and it also demonstrates that the regulation of super-enhancers in the liver is also highly conserved among species(P<1.66e-15).These results indicate that the rest and activation of enhancers are restricted by tissue and space-time specificity,and that the regulatory elements are highly conserved among species.(5)Identification of artificially selected causative mutations:We combined the analysis results of potential regulatory elements of pigs and allele specific expression with artificially selected genes,and locked 1,617 of them by MOTIF sequence characteristics to induce or destroy transcription factor core Motif sequence.On this basis,80 candidate SNPs were further locked by co-expression analysis of transcription factors and genes,including the IGF1R intron G>A mutation that alters MYOD binding,the IL1R1 intron A>T mutation that alters CEBPB binding,and the CD36 intron T>C mutation that alters JUN binding.Association analysis in the F2 population indicated that these sites were significantly associated with daily gain,corpus callosum,and immune traits.These sites provide an important reference for revealing genetic changes after phenotypic changes,and also provide candidate markers for molecular breeding.(6)Molecular mechanism of artificial selection G/A mutation in IGF1R gene intron:IGF1R luciferase activity assay and Westernblot results indicate that IGF1R is regulated by MYOD,and G allele will up-regulate 1GF1R activity.Gel migration(EMSA)results showed that the G allele was significantly more able to bind to nuclear proteins than the A allele.When the MYOD binding site of the G/A allele is knocked out,IGF1R is down-regulated.These results demonstrate that the G/A haplotype regulates the expression of IGF1R gene by altering the binding efficiency with MYOD.