Studies On Genome Wide Indels And Identification Of Key Genes For Milk Production Traits In Chinese Holstein

In this study,we sequenced the genomes of eight Chinese Holstein bulls with high and low estimated breeding values(EBVs)of milk protein percentage(PP)and fat percentage(FP)using Illumina re-sequencing and detected the insertions and deletions(indels)[1-49 base pairs(bp)].By integrated the indels that were polymorphic between the high and low individuals across the four families,previously reported quantitative trait loci(QTL),single nucleotide polymorphisms(SNPs)identified by genome-wide association studies(GWASs)and function of genes,we identified the candidate genes and indels,detected association between these indels with milk production traits in Chinese dairy cattles and find the key indels and genes for further functional verification.Section 1:Indels detection and candidate genes identificationWe sequenced the genomes of eight Holstein bulls from four half-or full-sib families,with high and low EBVs of PP and FP using Illumina sequencing.Over 0.9 million nonredundant short indels were obtained.Among them,3,625 indels with the same allelic distribution directions were polymorphic between the high and low groups of bulls were revealed.Follow-up validation assays confirmed that most(70%)of the randomly selected indels represented true variations.By integrated indels,QTL,GWASs and function of genes,we identified a total of 29 promising candidate genes and 51 indels.These genes were consist of FCGR2B,CENPE,RETSAT,ACSBG2,NFKB2,TBC1D1,NLK,MAP3K1,SLC30A2,ANGPT1,UGDH IL22RA1,SCD5,EEF2,SEC24D,SCARBl,SAA1,NR1D2,RICTOR,ELOVL6,ADAM17,GPAM,XAF1,SLC01A2,CD1B,FA2H,ACADL,ERBB2 and PARD6A.Section 2:Genetic effects analysis of indels on milk production traits in Chinese dairy cattles and functional verification of key indelsWe performed association studies on an independent dairy cattle population to evaluate whether such 51 indels were associated with milk production traits.Through pooled DNA of 40 Chinese Holstein bulls,totally 36 indels were verified.Among them,a total of 1093 Chinese Holstein individuals were genotyped for the 24 indels using MALDI-TOF-MS.The associtaiton results revealed that these identified indels were significantly associated with at least one trait(P<0.05 or P<0.01).Aming at detecting the significant genetic effects for four key indels,we used the MatInspector and Enhancer Element Locator(EEL)software to predict the transcription factor binding sites(TFBSs)and potential enhancer motifs,respectively,the promoter/enhancer activity assays,the gel shift assay(EMSA)and super-shift EMSA to verify the function of the four indels.The results showed that rs383700527 located upstream of ACSBG2 gene altered the TFBSs,while the promoter activity was consistent with prediction,and detected the ZNF350 transcription factor(TF)utilizing the super-shift EMSA.Luciferase assays demonstrated that the genomic region harboring ss2419489560 located the downstream of SEC24D gene acted as an enhancer by binding different TFs,and MYBL2 transcription factor was detected through EMAS and super-shift EMSA.Meanwhile,the promoter activities of ss2019489552 located upstream of IL22RA1 gene and rs380213439 in the intron of FCGR2B gene were not changed significantly.Section 3:Functional verification of ACSBG2 geneBased on the conding sequence(CDS)of ACSBG2 gene,we designed siRNA to knock down ACSBG2 gene in transient transfection into MAC-T cells,the interference efficiency of ACSBG2 gene was up to 60%.After transfection of ACSBG2 for 48h,we identified mRNA expression levels of fat-related genes by real-time PCR.The results revealed significant higer expression of SCD,DBI,CD36,VLDLR and SREBF1(P<0.01 or P<0.05),and the expression of INSIG1,ACOX1 and CT1B were significantly down-regulated(P<0.01 or P<0.05).Pathway analysis demonstrated that silenceing ACSBG2 gene may affect milk fat by influencing the expression of related genes,SCD and CD36 through reducing fatty acid oxidation process in dairy cattle.Accordingly,bovine ACSBG2 was considered to be functional gene for milk production traits,these results provided powerful evidence for further analysis.