Screening And Application Of Key Genes For Fragrant Pork Quality Traits Based On EQT

Gene expression is one of the major determinants of meat quality traits,which is regulated by genomic genetic variants.In order to select and identify the crucial genes（e Genes）and variants（e Variants）related to our target traits,the eQTL（Expression quantitative trait locus）analysis was performed using the whole genome sequencing（WGS）,and RNA sequencing（RNA-seq）data from dermal tissues,white adipose tissues and longissimus dorsi tissues.These data were obtained from 19 hybrid individuals（sow:Congjiang Xiang pig×boar:Large White pig）and the samples were all F2 generation in one pedigree.In this research,the e Variant and e Gene affecting porcine meat quality traits were deeply detected from the novel multi-tissue perspective,and the mainly conclusions were as follows:1.A total of 179,388 single nucleotide polymorphism（SNPs）,23,476 small insertions and deletions（small In Del）and 28,369 structural variants（SV）were detected and filtered by WGS analysis,and all of genomic variants were genotyping to construct a（0,1,2）genotype matrix.Subsequently,the expression matrix was constructed with the expression of 12,553 protein-coding genes which extracted from normalized and trimmed RNA-seq in three tissues,respectively.Then,the genotype matrix was associated with the expression matrix from each tissue separately,which performed using Matrix EQTL v2.3,and gender was included as covariates simultaneously.Combining WGS with RNA-seq,and using multiple types of variants for eQTL analysis was practical,with a total of 150,980 cis-eQTLs were detected in the three tissues(P_FDR<0.01).e Variants with cis-acting were annotated,resulting 66,217 e SNPs,8,642 e In Del and 9,632 e SV.These variants were overwhelmingly located in the intron and intergenic region,which mainly regulate gene expression through regulatory elements rather than change the coding sequence directly.Ultimately,based on gene region annotations,2,444 variants in dermal tissue,4,001 variants in white adipose tissue and 3,741 variants in longissimus dorsi tissue were identified and selected as candidate e Variant which correlated with meat quality traits.2.In longissimus dorsi tissue,a candidate functional molecular marker rs319855910（Chr11:27,905,634,C>G）was associated with adipose androstenone amount,which was validated by RFLP-PCR and Sanger sequencing.The candidate e SNP rs319855910 is located in the 5’UTR of the mitochondrial intermediate peptidase（MIPEP）gene,and correlated with MIPEP gene expression levels significantly(P_FDR=8.86E-03).The effect of candidate e SNP genotype on MIPEP gene expression was determined using q PCR,and there was a significant difference（*P=0.0326<0.05）in the expression of MIPEP gene between the wild homozygote（CC）and homozygous mutant（GG）,which was consistent with the trend in the normalized RNA-seq data.The transcription factors（TF）bound to 20 bp upstream and downstream of the locus were predicted by Animal TFDB database,with difference in the ability and type of TF bound to allele C and allele G.We hypothesize that the mutation of C allele to the G allele might affect TF binding,resulting in a down-regulation of MIPEP gene expression levels.3.The second candidate functional molecular marker in longissimus dorsi tissue was validated based on PCR and Sanger sequencing.This cndidate e SV is a 164 bp deletion（Chr1:215,901,358-215,901,522）,located in the 3’UTR of the interleukin 33（IL33）gene.The candidate e SV was significantly associated with IL33 gene expression levels(P_FDR=6.65E-03),and the normalized RNA-Seq data indicated that a highly significant difference（**P=0.0029<0.01）in IL33 gene expression between the wild homozygote（WW）and the heterozygous（WD）.A total of 9 mi RNAs and 91 RBPs were predicted to bind to the deletion sequence by mi Rnada software and RBPsuite website for the 164 bp deletion variant of IL33 3’UTR.It’s implying that the candidate e SV may change the binding of mi RNA as well as RBP,leading to the down-regulation of IL33 gene expression levels.4.The third candidate functional e Variant in longissimus dorsi tissue was also validated based on PCR and Sanger sequencing.It’s a 223 bp deletion（Chr2:102,435,683-102,435,905）,which is located in the muscle cells redox metabolism associated e Gene glutaredoxin（Grx1）Intron/3’UTR.This candidate e SV was significantly associated with Grx1 gene expression levels(P_FDR=9.70E-03),and the normalized RNA-Seq data indicated that a significant difference（*P=0.0380<0.05）in Grx1 gene expression between the genotypes with no deletion has occurred（II/IN/NN）and the deletion genotype（ID）.A total of 5 mi RNAs and 138 RBPs were predicted to bind with the 223 bp deletion fragment.It’s hinted that this candidate e SV may change the binding of mi RNA and RBP,resulting the up-regulation of Grx1 gene expression levels.5.The fourth candidate functional molecular marker in longissimus dorsi tissue was validated based on PCR and Sanger sequencing.It’s a 100 bp deletion（Chr6:703,105,08-703,106,09）,which is located in the 3’UTR of the nicotinamide nucleotide adenylyltransferase 1（NMNAT1）correlated with the physiological function of skeletal muscle contraction and oxidative respiration.There is a significant association between candidate e SV and NMNAT1 gene expression(P_FDR=9.00E-03),and the normalized RNA-Seq data showed a 1-fold difference in the average of NMNAT1 gene expression between wild homozygote（WW）and homozygous mutant（DD）,but no statistical difference（P=0.0660>0.05）.Subsequently,3 mi RNAs and 113 RBPs were predicted to bind with the 100 bp deletion sequence,hinting that the candidate e SV may change the binding of mi RNA and RBP,resulting the down-regulation of NMNAT1 gene expression levels.This study performed eQTL analysis using WGS and RNA-seq with multiple tissues,and aimed to comprehensively identify functional markers associated with meat quality traits.In this research,expression levels of protein-coding genes in multiple tissues were separately correlated with multi-type variants detected in genome.Subsequently,considerable loci which significantly regulate the expression of candidate e Genes for meat quality traits were revealed.Results in this article provide the theoretical evidences for resolving the molecular mechanisms of differential regulation among tissues,and shed light on the biological functions of trait causal genes,and can reveal the genetic basis of traits.Ultimately,a total of four functional molecular markers（one SNP molecular marker and three SV molecular markers）were reported.They are essential for pork phenotype investigation and can be widely applicated in the selection and genetic improvement of pig breeds.