Genetic Variation And Related Genes Excavation Of IMF Traits In Chinese Local Breds Cattle Crossed With Wagyu

Progeny(Wagyu crosses)of Wagyu cattle crossed with Qinchuan or Luxi cattle showed significant differences in intramuscular fat(IMF)deposition.In order to find the factors responsible for the large differences in IMF,we investigated the loci and genes associated with IMF in Chinese Local breds cattle crossed-with Wagyu.In this study,Whole Genome Sequencing was performed on Wagyu crossbred cattle(153 Qinchuan cattle × Wagyu cattle and 33 Luxi cattle× Wagyu cattle),and BSA-seq(Bulked Segregant Analysis)was used to resolve the candidate intervals and Single Nucleotide Polymorphism(SNP)and the relevant candidate genes associated with high and low IMF differences in Chinese Local breds cattle crossed with Wagyu.In addition,so as to improve the accuracy of the sequencing results,SNPs and genes associated with Backfat Thickness(BFT),Eye muscle area(EMA)and IMF were identified by Genome-Wide Association Studies(GWAS).Meanwhile,the KASP marker was developed based on the candidate regions and SNP selected by BSA and GWAS.At the same time,transcriptome sequencing analysis was performed with the longest back muscle tissue of Wagyu crossbred cattle(Qinchuan cattle ×Wagyu cattle)to find the differential genes between Wagyu crossbred cattle and Qinchuan cattle(a blank control)and compare the differential candidate genes related to high and low IMF.Finally,the expression pattern and function of bovine IMF-related gene ELOVL5 screened by genome and transcriptome in the proliferation and differentiation of bovine adipocytes were investigated.The main results were shown as follows:1.A modified BSA method was applied to analyse the loci and intervals associated with extreme high and low IMF samples(16 F1 samples,14 F2 samples and 13 F3 samples)in Wagyu hybrid cattle.The results showed that there were high density allele overlapping regions between high and low IMF populations in the F2 and F3 populations.Among all the samples,the region with the highest correlation with IMF was located between 24.8-29.6 M on chromosome 23.The SNPs associated with IMF in Wagyu hybrid cattle were located in some regions on chromosomes 7,19,21 and 23.The strong signal SNPs were associated with genes,and 17 IMF-related genes(CAST、TNXB、NSD3、ELVOL5、GSTA4、AGPAT1、GSTA2、PDE2A、WBP2、MYO9B、FGFR1、FADS6,ZFP57,BOLA-NC1、AIF1、ALDH5A1、GPLD1)were screened out.2.In this study,32 SNPs associated with IMF,EMA and BFT were detected by GWAS of 186 Wagyu crossbred cattle.Among them,22 SNPs associated with IMF were concentrated on chromosomes 13,14,19,21 and 25,and 3 SNPs associated with BFT were found on chromosome 11.7 SNPs associated with EMA appeared on chromosomes 5,8,11,19,22 and 23.A total of 23 genes were identified in these SNPs,WDR97,SCX,ZNF34,DGAT1,HGH1,CCDC43,CD300H,FANCI,ISG20,ANKRD34C,SERPINE were IMF-related candidate genes.H1FOO,PLXND1 were EMA-associated candidate genes,and ZNF598,NTSR2,SYNGR3,CHID1,LAMB1,AFAP1,CPT2,TSPAN4,SHH,MYH13 were candidate genes associated with BFT.In this study,the selected loci were verified in 8 samples,and the results showed that the CC genotype of g1955347436 was a low IMF marker,while the TT genotype was a high IMF marker.3.Using RNA-seq and bioinformatics analysis between high/low IMF Local cattle crossed with Wagyu Cattles and Qinchuan cattle,the study screened candidate expression genes related to IMF,the results showed that four candidate genes,ELOVL3,ACOT2,ND1 and ATP8(P<0.05),are mainly enriched in the Thermogenesis signalling pathway in high IMF group.FABP5,APOA1 and PLIN2(P<0.05)were screened in low IMF group,and enriched in the PPAR signaling pathway and Ferroptosis signaling pathway.No significantly differential expressed genes were found between high IMF and low IMF group.The BSA and GWAS screening of IMF-related genes were compared with the RNA-seq results,the following results were the same for CYP21(P<0.05),ELOVL5(P<0.05),CDR2L(P<0.05),PDE2A(P<0.05),PPP1R18(P<0.05).4.In this study,adult bovine adipose cells at 24 months of age were cultured.The result shown that ELOVL5 can increased adipocyte proliferation but inhibited adipocyte differentiation of bovine adipocytes.The results of western blotting,RT-qPCR,cell transfection assay showed that the over expression of ELOVL5 promoted adipocytes differentiation,the knockdown of ELOVL5 inhibited bovine adipocyte proliferation.Oil red O staining,qRT-PCR and Western Bloting experiments showed that interference with ELOVL5 inhibited the differentiation of adipocytes,and over expression of ELOVL5 promoted the differentiation of adipocytes.In summary,this study provides a BSA-based QTL localization method for the analysis of molecular markers in crossbred Wagyu cattle.Based on whole-genome resequencing combined with BSA and GWAS analysis,genetic variant loci associated with IMF in crossbred wagyu cattle were associated;and genes associated with IMF in crossbred wagyu cattle were co-localised with transcriptome sequencing.This study provides a molecular theoretical basis and reference for molecular breeding of crossbred Wagyu cattle,This study has potential economic value for the development of Local cattle crossed with Wagyu Cattles.