| Different geographical environments and artificial selection directions will lead to differences in the genetic background of Asian and European pigs,resulting in phenotypic changes with different characteristics of different breeds.Structural variation（SV）is widely distributed on the genome and can directly or indirectly affect gene expression through different mechanisms,leading to phenotypic variation among individuals or breeds.The construction of the structural variation map of the pig genome can be used to study the underlying mechanisms behind the diversity of pig breeds and important traits,and provides a theoretical basis for genetic improvement of pigs.In this study,chromosome-level genome assemblies of five representative breeds and short-read sequencing（SR）data of 402 pigs worldwide were used to discover SVs（Deletions and Insertions）with a length of ≥ 50 bp,and then completed the following Research content in five aspects: a)Screening of SV detection process for long-read sequencing and comparison of different SV callers;b)Using five chromosome-level genome assemblies and 402 short-read data to construct a high-quality pig SVs set and analyze the genomic characteristics of SVs;c)Mining potential functional SVs through population differentiation FST statistics and Ohana’s adaptive signal analysis;d)Multi-omics joint analysis of the regulatory role of functional SVs,and verified the regulatory activity of key SVs and their correlation with the expression of target genes through experiments;e)combined with allele-specific expression（ASE）analysis to screen key SVs that affect the differential expression of ASE genes.Below are key research findings:1)In this study,different types of SVs were simulated on pig chromosome 1,and the SV detection and genotyping efficiencies among four long-read SV callers（PBSV,cute SV,Sniffles,SVIM）were compared.Among them,all had the highest detection and genotyping efficiency for deletions（DELs）and insertions（INSs）,among which PBSV and Sniffles had the best detection and genotyping effects.PBSV can detect more SVs alone,and cute SV and svim can detect more larger SVs（100 kb-1 Mb）.Therefore,PBSV,Sniffles and cute SV were jointly applied to the detection and genotyping of DELs and INSs in follow-up studies.2)Based on the graphic genotyping strategy,we re-genotyped all SVs detected by chromosome-level genome assemblies and SR data in 402 SR genomes,and finally successfully genotyped 190,832 SVs,including 89,831 DELs and 101,001 INSs.Comparison of different SV detection strategies（genome assembly,short-read data and graph-based typing method）on the genome assembly and SR from the same Ningxiang pig,compared with manta + paragraph（short-read Data）method,based on graphgenotyping strategy can increase nearly 40% SV detection efficiency.In 402 pig populations,37% of SVs were identified as common variants,34% of SVs were identified as low-frequency variants,29% of SVs were identified as rare variants,and most of the SVs were low-frequency in the population.The annotation of SVs in the genome shows that nearly 98% of SVs are distributed in the non-coding regions of the genome,and 1.45% of SVs are located in the exons of genes,which may lead to loss of function（LOF）of the genes.3)According to the genotype information of DEL,INS and SNP,DEL,INS and SNP have the same population structure characteristics through Principal Component Analysis（PCA）,and 402 pigs were geographically divided into European domestic pigs,European wild boars,Chinese southern pigs,Chinese northern pigs,Tibetan pigs and Asian wild boars.Nearly one-third of SVs were in low LD with neighboring SNPs,implying that a considerable part of the genetic diversity was contributed by SVs.Through the linkage disequilibrium analysis of 465 GWAS-related SNPs,143 SVs were identified to be linked to GWAS-related SNPs,among which the LD of SNPs related to feed conversion efficiency was the highest,including the pig FCR candidate gene METTL1 B 3’UTR and contained The two variants（433-bp INS,chr480856797-433;278-bp DEL,chr480874390-278）had the highest linkage value（r2 >= 0.997）,which may be related to pig feed conversion efficiency.4)This study used FST statistics to analyze selection signature and found that the high differentiation of European domestic and wild pigs may be related to the strict selection of European domestic pigs.In European pigs,multiple highly differentiated SVs wereannotated adjacent to genes related to economic traits（body length,number of vertebrae,intramuscular fat）,such as PLAG1,VRTN,NR6A1,PTCP;in Asian pigs,multiple Highly differentiated SVs were annotated adjacent to genes related to nutrient metabolism and dietary absorption,behavior（domestication）,such as CYBRD1,ASS1,FAM35 A,TBX19,WIF1.5)According to the SNP genotypes of 402 pigs,this study used Ohana’s mixed scanning model to analyze the ancestry components,and found that it can further subdivide European pigs into three subgroups: Duroc pigs,Large White pigs,other European domestic pigs and European wild boar.Some lineage-specific SVs screened by adaptive signature analysis were closely related to the phenotypic characteristics of the corresponding populations.For example,some lineage-specific SVs were identified in the Large White pig population（ancestral component two）located in or near reproductive-related genes（NOTCH2,ACVR2 A,BAMBI）,which may be related to the high fecundity of Large White pigs.6)Combined with multi-omics to mine the regulatory role of functional SVs on the genome,a total of 2,004 functional SVs were identified in the active common promoters（Typical promoter）,Epromoter and enhancers on the genome.And two candidate SVs（296-bp DEL,chr7: 52,172,101-52,172,397;278-bp DEL,SE: chr18: 23,819,971-23,848,669）related to muscle development were verified experimentally,which may block muscle-specific enhancer function,thereby affecting the expression of muscle development-related genes such as LMOD2,WASL and FSD2.7)This study identified a number of genes related to muscle development and early embryonic development that had allelic differential expression,such as FLNC,MYH8,UHRF2 and TOP2 B.Among them,the MYH8 gene is related to pig muscle development and meat quality traits.MYH8 is a maternal（Min pig）gene,which is highly expressed in paternal（Yorkshire pig）.The 307-bp DEL in the intron of the MYH8 gene（307-bp DEL,chr12: 55,148,400-55,148,707,maternal）is highly linked to the multiple differential expression sites of MYH8 genes,and the 307-bp DEL may affect the low expression of the MYH8 gene,thereby affecting pig muscle development.In summary,SVs detected by long-read and short-read were retyped in 402 samples based on the graphic typing strategy,and a high-quality porcine SV collection was constructed.A molecular network of genetic variation-regulatory elements-genes and association analysis of candidate variations were constructed through multi-omics integration analysis,and a series of key genes and mutations related to the phenotypic differences among domestic wild boars and different groups were identified,and preliminary experiments were carried out.Verify the regulatory relationship of some SVs target genes.The research results will provide a scientific basis for the genetic analysis of important economic traits of pigs,and also provide new genetic resources for genetic improvement of pigs. |
