| Body length(BL)is one of the most important economic indicators for evaluating the performance of pigs.Understanding the genetic mechanism underlying BL is still a hotspot and difficulty in the field of international animal genetics.Previously,Zhou et al found one major QTL for BL at 35 Mb on SSC7 and refined it to a 300 Kb of confidence interval by perfoming a genome-wide association study(GWAS)in a White Duroc × Erhualian F2 intercross,as well as Chinese Erhualian and Bamaxiang pigs.In addition,they inferred HMGA1 as the candidate gene for the QTL.The aim of this study was to reveal the causal gene(QTG)and causitive mutation(QTM)for the QTL and clarify the relevant molecular mechanism.Via the increment of the marker density,the regional association analysis and the haplotype analysis in 7 pig populations including Bamaxiang,Erhualian,heterogeneous stock F3 and F5,Duroc × Lingao,Ertongliangtouwu and Lingao pigs,we further restricted the QTL interval to 16 Kb that was shared among all the populations.After that,individuals with known QTL genotypes and different haplotypes were selected for whole genome resequencing or targeted re-sequencing(60 Kb).Within the QTL region,there were only three varients that were completely co-segregatting with the QTL allele and thus named as candidate QTM1,QTM2 and QTM3 repectively.QTM1 and QTM2 are located in the promoter and 3' UTR of the HMGA1 gene,respectively.QTM3 is located in the promoter of the ENSSSCG00000023160 gene.Then,we used the bioinformatics methods(such as PMC A)to predict the function of QTMs and found that QTM1 and QTM2 were located in the conserved cis-regulatory modules of transcription factor families HESF and MYOD,respectively.Afterwards,the electrophoretic mobility shift assay(EMSA)demonstrated that both QTM1 and QTM3 could affect the binding affinity of upstream transcription factor but it is not the case for QTM2.However,QTM1 resulted in a lower binding affinity,while the QTM3 strengthened the binding effect of its regulatory element.To determine the QTG for BL,all genes within the candidate region of 600 Kb were studied.The mRNA expression levels of GRM4,HMGA1,ENSSSCG00000023160,NUDT3,RPS1 O,PACSIN1,SPDEF and C6orf106 genes were analysed of 30 Erhualian individuals with different QTL genotypes(6 CC,17 CT,7 TT)in the liver.Qnly HMGA1 had significant difference in expression levels among QTL genotypes(P <0.05).In addition,the expression levels of the two transcripts(HMGAla and HMGAlb)of HMGA1 were also significantly different among different genotypes.The gene expression level in QQ individuals was significantly higher than that in qq individuals(P <0.01),but the proportion of transcript expression was not significantly different(P> 0.05),suggesting that the QTL does not alter the proportion of the amounts of the two transcripts.Furthermore,we performed e QTL and QTT analysis using liver samples from F2 individuals(N=148)and F6 heterogeneous stock F6 individuals(N=260).A cis-e QTL was detected for HMGA1 and its e SNP was in a strong linkage disequilibrium with the GWAS top SNP(r2 ? 0.44).Moreover,the expression level of HMGA1 showed a moderately or strongly positive correlation with BL(r = 0.23,P = 2.23E-04 in F2;r = 0.73,P = 6.82E-03 in HS-F6),while such results were not obtained for other candidate genes.By RNA-seq of the articular cartilage tissue from F6 heterogeneous stock pigs we also found a significant difference in HMGA1 m RNA levels among three QTL genotypes.There were 52 differentially expressed genes(DEGs)co-expressing with HMGA1,including a few growth-related genes such as BMP6 and PLAG1.GO functional analysis showed that these DEGs were enriched in those functional annotations of the bone minerlization,regulation of hormone levels,developmental growth,lipid phosphorylation,positive regulation of hormone secretion,regulation of small GTPase mediated signal transduction and epithelial cell development process.In addition,the expression level of transcription factor BHLHE40 is higher than that of any other genes of the HESF family in the liver and articular cartilage tissues.Forthermore,BHLHE40 transcription factor is closely related to the differentiation of chondrocytes.Therefore,we deduced that BHLHE40 would be the most likely upstream transcription factor of the cis-regulatory module spanding QTM1.Taken together,we infer that QTM1 and HMGA1 are the most likely causal mutation and causal gene,respectively.Here,through a systemic genetics study,the first major gene affecting swine BL and its favorable alleles uniquely derived from the Chinese indigenous pig breeds were identified.The results will not only deepen people's understanding of the genetic mechanism for body length in pigs,but also promote the development of molecular breeding techniques for Chinese and foreign pigs.An increasing attention has been paid to meat quality by consumers and producers.However,the genetic mechanism of most quality traits is little known.To determine the genetic basis of pork eating quality traits and cooking loss,we herein performed a genomewide association study(GWAS)for tenderness,juiciness,oiliness,umami,overall liking and cooking loss by using whole genome sequences of 836 heterogeneous stock F6 pigs which were generated by crossing 4 typical western pig breeds(Duroc,Landrace,Large White and Pietrain)and 4 typical Asian pig breeds(Erhualian,Laiwu,Bamaxiang and Tibetan).We identified 50 trait-associated loci(QTLs),of which 42 were detected for the first time.Seven loci also showed pleiotropic associations with different traits.In addition,we identified multiple promising candidate genes for these traits,including PAK1 and AQPll on SSC9 for cooking loss,EP300 on SSC5 for tenderness,SDK1 on SSC3 for juiciness,FITM2 on SSC17 and 5-linked MYH genes on SSC12 for oiliness,and TNNI2 and TNNT3 on SSC2 for overall liking.Our results provide not only a better understanding of the genetic basis for meat quality,but also a potential application in future breeding for these complex traits. |
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