Identification Of Key Candidate Genes For Reproductive Traits In Large White Pigs Based On Genomic Variation Information

The pig industry is an important pillar of my country’s livestock industry,and my country is the world’s largest pig producer and consumer.However,due to the low reproductive efficiency of breeding pigs and dependence on imported breeding sources,the development of my country’s pig industry has been subject to certain constraints.As one of the main introduced breeding breeds,the reproductive traits of Large White pigs have always attracted much attention.However,to fully understand and optimize reproductive traits requires the construction of a high-quality reference genome and in-depth exploration of their genetic basis.At the same time,ovarian tissue is a key reproductive organ,and its ovulation number directly affects the number of litters.Studying the regulation of oocyte maturation is of great significance to improve the reproductive performance of sows.Therefore,this study was based on a population of 560 Dan-lined large white sows in Shunxin,Shaanxi Province,and collected and compiled 9 reproductive traits,including total number born(TNB),number born alive(NBA),Litter birth weight(LBW),Number of weaned(NW),Number of mummies(MUM),Number of stillborn(NSB),Number of born deformity(NBD),number of born weak(NBW)and gestation length(GL).First,the genetic parameters of the reproductive traits of the population were estimated,and then combined with the large white pig reference genome assembly at the chromosome level,and the population resequencing data were used to classify single nucleotide polymorphism(SNP),insertion-deletion(In Del)and structural variant(SV),and genome-wide association analysis(Genome-wide association study(GWAS),screened out some new important candidate genes related to reproductive traits,and compared the impact of different models and mutation types on the accuracy of genomic selection(GS);finally,the effect of the candidate gene METAP2 on eggs was studied Effects of mother cell maturation.The main research results are as follows:1.The GL trait of Large White pigs is a trait with medium heritability,and the other eight traits are all traits with low heritability.The heritability of GL is 0.3301,and the heritability of the remaining eight traits is between 0.0028-0.1601.There is a positive genetic correlation and a positive phenotypic correlation between the reproductive traits TNB,NBA,LBW and NW.Among the reproductive traits,NBW and TNB traits showed positive phenotypic correlation and positive genetic correlation,while LBW traits showed negative genetic correlation and negative phenotypic correlation.The reproductive performance of Large White pigs is best in spring when there are 3 to 5 litters.2.Successfully assembled and annotated a large white pig genome at the chromosome level.The size of the assembled genome is 2.6Gb,the number of Scaffolds is144,the Scaffolds N50 level reaches 140.1Mb,and 26,357 genes and 42.87% repeated sequences are annotated,which is better than the previously published large white pig genome.Compared with the reference genome,SVs related to reproductive traits were identified as mainly involved in biological processes such as meiosis,oxytocin signaling pathway,estrogen signaling pathway,and TGF-β signaling pathway.Three selection signal analyzes based on high-and low-yield pig breeds jointly identified 250 positive selection genes(PSG),which are mainly highly expressed in the brain,pituitary gland,ovary and uterine tissues,and participate in the gonads,embryos and mesoderm.development process.3.Based on SV GWAS,more candidate genes related to reproductive traits can be identified.NBA and LBW jointly identified four candidate genes PTPRN2,LINGO2,MNX1 and UBE3C based on SV GWAS.NBW based on SNP GWAS and TNB based on In Del GWAS jointly identified a candidate gene,DGCR8.Based on In Del GWAS,NW and MUM jointly identified a candidate gene,DNMT3 L.TNB and NBA identified a candidate gene,MFSD14 B,based on SV GWAS.Six genes were co-located between the candidate genes identified by GWAS and PSG: MYC,PTPRN2,XYLT1,LRRC4 C,BRINP1 and PRKCE.4.The genome prediction(Deep Neural Network Genomic Prediction,DNNGP)model based on SV information and deep neural network can greatly improve the accuracy of genome selection for reproductive traits.The accuracy of genomic selection of DNNGP on nine reproductive traits including TNB,NBA,LBW,NW,GL,NBW,NBD,NSB and MUM were 0.44,0.44,0.41,0.43,0.46,0.41,0.48,0.46 and 0.50 respectively.The accuracy is the highest among the five models in each trait,especially in low heritability traits.Based on the comparison of different variant information,DNNGP achieved the highest accuracy in genome selection using the SV dataset in LBW,MUM,NBW,NSB,NBA and NW,and the In Del dataset achieved the highest accuracy in GL and TNB.5.METAP2 knock-down can significantly reduce the maturation rate of oocytes.GWAS based on SNP,In Del and SV for LBW traits jointly identified the candidate gene METAP2.METAP2 gene haplotypes are inherently different between Eastern and Western pigs,and are highly expressed in various stages of human oocyte maturation and embryonic development.In order to further verify the effect of METAP2 on oocyte maturation,si RNA targeting this gene was microinjected into porcine oocytes at the GV stage.Western blot showed that the METAP2-KD histone protein level was significantly lower than that in the control group(P < 0.05),the first polar body excretion rate in the METAP2-KD group was significantly lower than that in the control group(P < 0.0001).In summary,this study based on the assembly of high-quality Large White pig reference genome(Large White)typing to obtain a variety of variant information can improve the detection ability of GWAS;combined with the DNNGP deep learning model can greatly improve the accuracy of GS,and finally combined with egg Mother cell maturation experiments verified that the candidate gene METAP2 is a key regulatory gene affecting oocyte maturation.This study identified many previously unreported candidate genes related to pig reproduction,providing molecular markers for pig genetic improvement and providing a reference for pig genome breeding work.