| The commercial crossbred Duroc×（Landrace×Yorkshire）（DLY）pigs generate a large bulk of meat products in the world,whereas their meat quality are often impaired by low ultimate p H（p Hu）and heavy drip loss.Muscle glycogen content at death is closely related to its p Hu,drip loss and meat technological quality.However,the genetic basis of the trait remains elusive.To study the molecular genetic mechanisms for glycogen content,we first conducted genome-wide association analyses（GWAS）for 5 catagories of meat quality traits related to GP in three hundred and thirty six Chinese Erhualian and 610 DLY pigs using the Illumina Porcine SNP60K Beadchip.We detected 28 and nine suggestive SNPs that surpassed the significance level for meat quality in Erhualian and DLY pigs,respectively.Taking advantage of the relatively lower extent of linkage disequilibrium in DLY crossbred compared to its founder purebreds,fine mapping is relatively easy.Therefore,we will further decipher the genetic basis of GP,p H,drip loss and related meat quality traits in DLY population.Our initial GWAS demonstrated three genomic loci（SSC3,4 and 15）associated with residual glycogen and glucose（RG）concentration in longissimus muscle at 45min post-mortem.Among them,SSC15 QTL achieves a genomic significance level（P=2.54 E-11）,and this site overlaps with QTL that affects meat color redness（a*value）and meat color score.The most significant SNP rs326377357 was located close to PRKAG3（RN）gene,which have reported to affect RG in muscle.Through the target resequencing,fine mapping,haplotype and replication analysis,and gene-expression and AMPK-activity assay,we verified that the strongest association with RG was resulted from a low-frequency（MAF=0.014）missense mutation R200Q in PRKAG3.After conditioning on R200Q,re-association analysis revealed three quantitative trait loci（QTLs）for RG.Of these,one major QTL on SSC3 was most likely caused by a splice mutation（g.8283A>C）in the PHKG1 gene that we identified before.Based on functional annotation,two genes（TMCO1 and CKB）could be regarded as promising candidate genes for the other two loci.There were significant interaction effects of the GWAS tag SNPs at those two loci and PRKAG3 R200Q on RG.Additionally,a number of common variants with potentially smaller effect on RG(P<10-4)were uncovered by a second conditional GWAS adjusting for the two causal SNPs R200Q and g.8283A>C.Subsequently,we conducted a meta-analysis of glycogen content traits of DLY,F2 and Sutai populations.The results found that there were significant signals on SSC3,5,15 and23,and their strong candidate genes were:PHKG1,SMUG1,PRKAG3 and G6PD.Additionally,for the SSC15 signal,R200Q has been confirmed to separate in DLY population,however,it showed fixed in F2 and Sutai pigs.So we performed a meta-analysis of glycogen content in these two breeds,the signals on SSC15 still existed,suggesting that other causative variants influencing meat quality exists in the region of PRKAG3 or other causative genes on SSC15 that affect muscle glycogen content.We conducted the analysis of quantitative trait transcript（QTT）,GO enrichment,integrative system approach（GWAS,e QTL and QTT）to identify candidate genes and weighted gene co-expression network analysis（WGNCA）to reveal biological networks for22 meat quality traits including three categories of p H,drip loss and GP related components,from 493 liver and longissimus dorsi muscle（LM）samples in a White Duroc×Erhualian F2 population.The QTT results found that FOS-JUNB constitutes a gene pathway that affects glycogen metabolism.Integration analysis of GWAS,e QTL and QTT identified GALNT15 and HTATIP2 were potential candidate genes for drip loss and p Hdrop＿45min＿24h.WGNCA further identified a module significantly related to glycogen metabolism and energy metabolic pathways in muscle.Finally,based on the genomic resequencing data,we conducted a genomic association analysis of glycogen content in the embedded F6 population.The results were SSC2,3,7,9,13,15 and 18 have signals affecting glycogen content.Among them,a SNP rs343877498（126.01 Mb,P=1.06E-09）on SSC15 reached a genomic significant level,which close to the known acid gene PRKAG3.In addition,we detected several key candidate genes:EPB41L4A,PKDCC,RIN3,FAM163A,NEK10 and CLDN18.In summary,we detected the QTLs for glycogen content on SSC2,3,4,5,7,9,13,15and 18 using 4 large genetic analysis populations,and excavated or validated some important candidate genes.It was revealed that muscle glycogen content and PSE meat occurrence in DLY pigs are regulated by two low-frequency causal variants（PRKAG3 R200Q and PHKG1g.8283C>A）of large-effect and multiple common variants of small effects.This study has deepened the understanding of molecular basis of meat quality traits,and benefits the development of molecular markers for MAS and further identification of causative genes or causative mutations for these traits. |
