| Intramscular Fat(IMF)content is a key factor evaluating pork quality,affecting flavor,tenderness and juiciness.A high level of marbling or Intramscular Fat content has a positive impact on the eating quality of pork,and IMF is also a key indicator for improving pork quality traits in breeding.In recent years,with the continuous development of genotyping technology platforms and the declining price of high-density SNP chips,genome-wide Association Study(GWAS)has also been widely used in livestock and poultry genetics and breeding studies.Using THE GGP Porcine 50 K chip,GWAS analysis of IMF in longissimus dorsi muscle of 598 Ningxiang pigs from China was performed to search for genetic variation associated with Porcine INF traits.The main experimental results are as follows:1.IMF content in muscle tissues of 598 Ningxiang pigs was measured in this study.The average IMF content was 3.6%,and the variation was 25.5%,which was in line with normal distribution.Based on SNP data,GCTA software was used to estimate the heritability of IMF.IMF estimated the heritability to be 0.57,which was in line with IMF as a quantitative genetic trait and showed medium-high heritability.2.The MLM and Farm CPU models were used to conduct GWAS analysis on NINGxiang pig IMF.Principal component analysis and fixed effect were added to the two correlation analysis models to correct the false positive correlation results.The SNP detection efficiency of Farm CPU model is better than that of MLM model.As shown in the Manhattan diagram,a total of 14 SNPS significantly correlated with IMF were screened out from the two models.The SNPS significantly associated with IMF were annotated by Ensembl and NCBI,and 13 candidate genes related to IMF were screened out.Six SNPS were located on introns of RGS17,LDHC,CNTN1,MX2,EBF3 and WDR17 genes,respectively.Other significant SNPS were located in the spacer regions of ARL5 B,C1orf174,PTPN,AKR1E2,NALF1,WDFY1 and BMPER.3.EBF3 and WDFY1 genes were finally determined as key candidate genes based on GO analysis results and NCBI website search for relevant literatures.Bioinformatics analysis of EBF3 and WDFY1 genes and according to their physicochemical properties,protein secondary structure,N-glycosylation and homology evolution showed that EBF3 and WDFY1 genes were related to pork quality traits.4.By real-time quantitative PCR,the expression trend of candidate genes EBF3 and WDFY1 in ningxiang pig muscle tissue was consistent,and the expression level of high IMF group was significantly higher than that of low IMF group.The polymorphic sites of candidate genes are searched through Ensembl.The polymorphism site at 282 bp of exon 13 of WDFY1 gene was also the restriction site of Hinfl enzyme(G^ANTC),and the polymorphism site at 862 bp of exon 1 of EBF3 gene was found.EBF3 and WDFY1 genes were genotyped by sequencing and enzyme digestion in the validation population.Finally,the BB genotype of WDFY1 gene was determined to have the highest IMF content by association analysis with traits,and the difference between WDFY1 gene and the other two genotypes was significant.Because the polymorphic loci of EBF3 gene were low-frequency mutations and the number of validation populations was small,only 5 individuals with mutations were detected,which was not of genetic statistical significance.Subsequently,EBF3 gene can be studied by expanding the number of validation populations.The function of EBF3 gene was verified at the cellular level by using ningxiang pig primary fat progenitor cells isolated in the early stage of this project.The results showed that EBF3 gene was not only involved in regulating the differentiation of porcine preadipocytes into adipocytes,but also promoted the proliferation of preadipocytes and accelerated lipid deposition.In conclusion,EBF3 and WDFY1 genes can be used as important candidate genes related to IMF in Ningxiang pigs,and the B allele of WDFY1 gene can be used as reference markers in breed selection aiming at improving IMF in later stage. |
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