Identification Of Candidate Genes For Meat Production Traits By Integrating Multi-omics Information And Fuctional Validation In Beef Cattle

Currently,China’s beef consumption demand is growing,and the situation of beef supply shortage is relatively severe.In the case that the stock of beef cattle can not increase rapidly,it is crucial to improve the meat production performance of beef cattle to increase the supply and profitability of beef.Improved breeds of livestock and poultry contribute more than 40% to the development of animal husbandry.The key to improve the core competitiveness of improved breeding is seed industry technology.The Global Functional Annotation of Animal Genomes(FAANG)Program,led by the United States,was launched in 2015.Genomes resequencing data,transcriptome data,and epigenome data are available in FAANG.It has set off a boom in integrating multi-omics information to mine key functional and regulatory mechanisms in animal genomes.Therefore,in this study,multiple omics data were used to identify candidate variants and functional genes related to meat production traits in the Huaxi cattle population,and functional verification was conducted in skeletal muscle satellite cells.The main results are as follows:(1)A total of 18239 significant cis-eQTLs(FDR < 0.05)and 1837 Ensembl genes were detected by performing cis-eQTLs analysis on the longissimus dorsi muscle tissue of 120 Huaxi cattle.The candidate genes were functionally enriched and found to be mainly enriched in the items of catalytic activity,small molecule metabolic process and cytoplasm.Also,gene-based GWAS analysis was performed based on the imputation data of 1233 Huaxi cattle(from 770 K chip to sequence level)and the phenotypic data of live weight before slaughter,net meat weight,dreesing percentage and net meat percentage.A total of2196 candidate genes were associated with live weight,1047 for net meat weight,70 for slaughter percentage and 9 for net meat percentage.(2)Ten genes(DLGAP3,PON1,ATP8B1,PRKG1,ZZF227,STXBP5,RBM19,PON3,RMDN1 and PRIM2)were associated with meat production traits by integrating gene-based GWAS,cis-eQTL and previous GWAS results.PON3 and PRIM2 were further identified by SNP codomain analysis as candidate genes related to meat production traits.PON3 and PRIM2 overexpression vectors were constructed and transfected into bovine skeletal muscle satellite cells for functional verification.The results showed that overexpression of PON3 and PRIM2 genes significantly promoted the proliferation of bovine skeletal muscle satellite cells,and significantly decreased the m RNA expression level of PON3 and PRIM2 genes during the myoblast differentiation of bovine skeletal muscle satellite cells.This finding further proved that PON3 and PRIM2 as candidate genes can affect the meat production traits.(3)ANXA4 was identified as a candidate gene for meat production by integrating GWAS,cis-eQTL,ATAC-seq and QTL databases.Knockdown of ANXA4 gene can significantly inhibit the proliferation,migration and myogenic differentiation of skeletal muscle satellite cells,and promote the apoptosis of skeletal muscle satellite cells.The analysis of the ANXA4 promoter region shows that there is an obvious CTCF peak in the promoter region of ANXA4,and the shorter the fragment,the stronger the transcription activity.And the upstream of the initiation codon is the main inhibitory transcription region from-2000 bp to-1000 bp.The interaction between YBX1 and ANXA4 was demonstrated by combining the results of different expression genes from RNA-seq,the transcription factors binding site prediction of ANXA4 promoter region,the dual luciferase reporting assay,and the overexpression of YBX1.The overexpression of YBX1 can inhibit the transcriptional activity of ANXA4 genes and downregulate the expression of ANXA4 genes.Furthermore,YBX1 expression was significantly up-regulated when ANXA4 gene was interfered.Inaddition,the knockout of ANXA4 gene was performed on bovine primary fibroblasts using CRISPR/Cas9.After flow cytometry,PCR identification,and off-target detection,we successfully obtained ANXA4 knockout cell lines,and ANXA4 knockout significantly reduced the cell proliferation rate.In summary,this study integrated multi-omics information to mine candidate genes related to meat production traits in the Huaxi cattle population and verified their functions in skeletal muscle satellite cells.Moreover,systematic and in-depth study on ANXA4 gene may provide an important theoretical basis for the subsequent selection and breeding of meat traits in beef cattle.