Screening And Identification Of Candidate Genes Related To Bovine Meat Quality By Conjoint Analysis With RNA-seq And Whole Genome Methylation

Due to the abundance of protein,iron,zinc,B vitamins and essential polyunsaturated fatty acids,beef is regarded as an ideal source of high-quality nutrition for humans.In China,with the improvement of modern living standards and health awareness of the consumers,the quantity and quality of the demand for beef is increasing year by year.The meat quality traits are complex economic traits which can be varied within one breed even under the same living condition.Therefore,molecular genetic has been considered as a vital factor that can control the development of muscle and adipose tissues by a large number of genes.However,the information on the genetic markers and functional genes of meat quality traits is very limited,which seriously blocks the way of breeding for excellent varieties of beef cattle.So far,to make a breakthrough in the process of cattle breeding,the key points of the research include three aspects: screening the genetic markers,searching functional genes and analysis regulation network.In this study,Japanese black(Wagyu)and Chinese Red Steppes cattle which had been reported with significant differences on meat quality traits and fat deposition were used to analysis differentially expressed genes(DEGs)and differentially methylated regions(DMRs)by RNA-seq and Whole Genome Bisulfite Sequencing(WGBS),respectively.The partial candidate genes and regions of meat quality traits were functional and analyzed.The WGBS results showed that a total of 23,150 DMRs were identified which were located in 8,596 genes that significantly enriched in 1,046 GO terms(p<0.05).KEGG analysis suggested that the DMRs were distributed among 276 pathways but no pathway significantly enriched(p>0.05).Furthermore,the RNA-seq results revealed that between two breeds there were 388 DEGs(Log2FC>0.585 or <-0.585,FDR <0.05)enriched significantly in 475 GO terms(p<0.05);among them,205 genes were higher and 183 genes were lower expressed in Japanese black cattle than Chinese Red Steppes cattle(p<0.05)..KEGG analysis indicated that the up-regulated genes and down-regulated genes were significantly enriched in 7 pathways and 20 pathways(p<0.05),respectively.The expression levels of DEGs were verified by fluorescence quantitative PCR and Western Blot.The results were consistent with the sequencing analysis outcomes.Additionally,genomic methylation and transcriptome analysis showed that the methylation of 331 DMRs were negatively correlated with the expression levels of DEGs.And there are 21 DMRs located in the promoter regions of DEGs.In the four populations of cattle and pigs,the CG island distribution and genetic polymorphism in promoter regions of five negative correlation genes were analyzed,and this result will lay the foundation for further study of methylation in promoter.To further elucidate the function and regulation mechanism of candidate genes on adipogenesis and fatty acid metabolism,overexpression of HSL,CD44,KLF5,CRYAB,ANKRD2,ALDH9A1 and EHHADH in bovine fetal muscle fibroblasts(BFF)were separately performed to analyze the effect of selected genes on the intracellular fat accumulation and fatty acid contents,and regulation on expression level and protein interaction of genes involved in fat and fatty acid metabolism were detected and analyzed using q PCR array and STRING,respectively.The results of triglyceride tests indicated that overexpression of CRYAB,ANKRD2,ALDH9A1 and EHHADH genes can lead to a decrease of triglyceride content in BFF(p<0.05),while the overexpression of HSL,CD44 and KLF5 resulted in an increase of triglyceride content(p<0.05).Furthermore,the analysis of fatty acid content showed that the overexpression of HSL gene can cause for an increase of intracellular hexanoic acid,octanoic acid,palmitic acid,stearic acid and total fatty acid,but a reduction of linoleic acid and Cis-4,7,10,13,16,19-docosahexaenoic acid(p>0.05);overexpressed CRYAB gene can give rise to a higher concentration of hexanoic acid,but a lower content of other fatty acid(p>0.05);The overexpression of ANKRD2 gene can affect each fatty acid content and the total fatty acid were lower than that of the control group(p>0.05);Contents of fatty acids in groups overexpressed with ALDH9A1,CD44 and KLF5 were higher than those in the control group,but no significant difference between the overexpression group and the control group(p>0.05).Moreover,the result of q PCR array demonstrated that when HSL is overexpressed,the expression levels of CPT1 C,FABP2,BDH2,PECR,PRKAB2,EHHADH were increased(FC>1.5,p<0.05);In CD44 overexpression group,the expression levels of HMGCS2 and CPT1 C were up-regulated(FC>1.5,p<0.05),while ELHADH,FABP4,GK2 and HSL was down-regulated(FC<1/1.5,p<0.05);For KLF5 overexpression group,the expression levels of HMGCS2 were lifted(FC>1.5,p<0.05),but 12 genes,such as ACOT12,ACOT6,ACSBG2,were reduced(FC<1/1.5,p<0.05);In cells overexpressed with CRYAB,the expression of GPD1 were elevated(FC>1.5,p<0.05)whereas ACSL6,MCEE and PRKAG1 were decreased(FC<1/1.5,p<0.05);When ANKRD2 is overexpressed,the m RNA level of HMGCS2 and MUT gene were rose(FC>1.5,p<0.05),while GK2 was declined(FC<1/1.5,p<0.05);with the overexpression of ALDH9A1,the expression of HMGCS2,FABP5,SLC27A1,ACADSB,ACAD11,MUT,FABP4,CRAT,PECR,ACSL5 gene were up-regulated(FC>1.5,p<0.05),but HSL was down-regulated(FC<1/1.5,p<0.05);In EHHADH overexpression group,there are 33 genes,like ACAA1,ACAD10,ACAD11,ACADM,ACADS,ACADSB that were up-regulated or down-regulated.In addition,the analysis of protein interaction showed that CD44,KLF5,CRYAB and ANKRD2 had no association with the genes that were up-regulated and down-regulated after overexpression,while it demonstrated that HSL,ALDH9A1,EHHADH could interact with several genes.In summary,a large number of DMRs and DEGs between Japanese black(Wagyu)and Chinese Red Steppes were obtained;the promising candidate genes of meat quality traits were screened.To study the regulation of SNPs in promoter on the DNA methylation and gene transcription,the SNPs,transcription factor binding site(TFBS)and CG islands locus were analyzed and predicted in different populations and to comprehensive elucidate,the influence and regulation mechanism of key genes on fat and fatty acid metabolism,the functional gene verification were performed by gene overexpression in vitro.This study will lay the theoretical foundation to further excavating the functional genes of meat quality and improving regulated networks of fat and fatty acid metabolism.In addition,the functional genes and regions with important value in breeding will cultivate new varieties of transgenic cattle and its industrialization to provide genetic resources and technical support.