Identification Of The Causative Gene And Mutation For Increasing IMF Content At SSC12 And Its Mechanism Study

Meat quality traits are economically important in the pig industry.As most of the meat quality traits could only be measured when the animals were harvested,the dissection of these traits genetic mechanism was limited,only several major genes were identified.The intramuscular fat(IMF)content is positively correlated with the flavor and nutritional value.Higher IMF meat usually accompanied by better acceptability of meat juiciness,tenderness and fragrance.Therefor,understanding the molecular basis of inter-individual variation in IMF content may not only inform us about the genetic architecture of this trait,but also have greatly commercial value in pig breeding.We first phenotyped 18 meat quality traits and then conducted genome-wide association analyses(GWAS)for meat quality traits in Chinese purebred Laiwu pigs(N =316)covering all patenal lineages and most of the maternal blood by using Illumina porcine 60 K Beadchips.A total of 170 SNPs passing the suggestive significance level were identified on SSC1,4,12 and 15.Of the 170 SNPs,112 SNPs and 58 SNPs reached the genome-wide and suggestive significance levels,respectively.For IMF content,25 SNPs reached the genome-wide significance levels.Except the SNP ss131037242 on SSC11,all genome-wide significant SNPs were located at a 2.97 Mb region on SSC12 from 57.83 Mb to 60.80 Mb.The most significant SNP was identified at the ss131468514(SSC12:57.83 Mb)(P = 2.09 × 10-13),which accounted for 29.3%of phenotypic variance.To refine the QTL interval,we first performed a linkage and linkage disequilibrium analysis(LDLA),the most significant association was identified at 58.42 Mb(P =2.51 ×10-36).Using the drop off 2 of LOD,the critical interval was defined to a～1.60 Mb region from 58.04～59.65 Mb.Then,a total of 127 informative SNPs were genotyped and added to the analysis.We identified another SNP rs49 at 58.36 Mb showing the most significant association with IMF(P = 4.81 × 10-15)and accounting for the 34.7%of phenotypic variance.Furthermore,the critical QTL interval was refined to a 525.99 kb region by LOD drop off 2 and r2 ≥ 0.8 from the strongest SNP.We then reconstructed the haplotypes of the 525.99 kb region and performed a haplotype-sharing analysis in the 316 experimental pigs.The shared haplotypes were 128.15 kb.We also performed re-sequencing of the 525.99 kb targeted region,the genotypes of 131 SNPs were consistent with the genotypic patterns of the haplotypes.Further genotyping in all 316 Laiwu pigs and followed by further association study,we found a missense mutation in MYH4(rs222)at 58.24 Mb showing the most significant association with IMF(P = 2.86 × 10-16).This SNP accounted for 39.4%of the phenotypic variance.Also,rs222 was significantly associated with meat color,marbling,meat moisture and loin eye area.But we did not identify any significant associations for other traits(P>0.05).We tested the expression levels of all nine genes within 525.99 kb region in longissimus dorsi.Three genes were not expressed in longissimus dorsi muscle.We performed a quantitative trait transcript(QTT)analysis to test the correlation between gene expressions and IMF content.Interestingly,only the expression level of MYH4 was significantly correlated with IMF content(r =-0.39,P = 3.53 × 10-6).We further performed eQTL mapping for the above six genes with all genotyped SNPs in the 135 Laiwu pigs with gene expression data.We only detected the significant eQTL for MYH4.More interestingly,the strongest association was identified at the SNP rs222 that was exactly the strongest SNP associated with IMF content.The QTT and eQTL mapping provided evidence that MYH4 and rs222 should be the QTG and QTN for this QTL.To further validate the causality of rs222 for IMF at SSC12,we performed the association study in 419 Lulai,77 Yushan × Duroc intercross pigs and 821 F6 pigs from a heterozygote population.rs222 showed the strongest association with IMF content in both populations.Furthermore,we confirmed the causality of rs222 for IMF phenotype and the gene expressions in MYH4 rs222 knock-in mice.All results confirmed that the rs222 is the causative mutation underlying the SSC12 QTL effect on IMF.To evaluate the effects of the rs222 mutation on MYH4 gene expression,we performed DNA pull down experiment.We found the rs222 region was bind nuclear proteins regulating MYH4 expression.A total of 90 nuclear proteins showed differential bindings.To further confirm the interaction between rs222 and the pulled-down proteins,we separately constructed the expression vectors of mutant and wild-type MYH4.The results confirmed that the QTN changes the nuclear protein binding(e.g.MYL6),and then influences the MYH4 expression.Furthermore,we also observed significantly higher numbers of type Ⅰ,type Ⅱx and type ⅡA muscle fibers in mutants than that in wild-type pigs and mice.However,the mutants had the lower number of the type ⅡB muscle fibers.The QTN changes the muscle fiber composition,which increases the GLUT4 abundance through AKT pathway.GLUT4 promotes the glucose uptake,and subsequently increases the IMF content.In conclusion,this study first illustrates a mutation in MYH4 is responsible for the porcine IMF content.The results from this study provide the knowledge for selecting highly quality "snowflake meat" and breeding technology.