Genome Sequencing And Research Of Bama Mini-pig

Swine have served as an important biomedical model for humans for decades.The Bama Xiang pig（BX pig）,2 males and 14 females,have been inbred for more than 30 years（1987-）to establish a stably genetic inbred line--Bama mini-pig（BM pig）.Owing to a variety of unique characteristics ideal in an experimental animal（small adult body size,low cost of maintenance,resemblance to human within anatomy and physiology）and features of inbred line（clear genetic background,high homozygosity,high inbreeding endurance）,the BM pig has been made attractive as animal models for different biomedical analyses in recently years.Despite marked progress in using BM pig as an animal model,BM pigs are studied only in a handful of laboratories worldwide,partially because a large number of obstacles to furthering these studies remain,especially the lack of a high-quality genome and an overall view of gene expression profiling.In this study,we generated a high-quality and chromosome level genome sequence for the BM pig using Illumina,10× genomics,Pacbio and Hi-C,firstly.And then,we reported a primary genome of BX pig and generated re-sequencing analysis of BM pigs and BX pigs.Through whole genome discovery of variants analysis,the effects of long-term inbreeding on the genome of BM pigs were uncovered markedly.We used comparative genome analysis to reveal the evolutionary mechanism of some special traits of BM pig.At last,we use induced BM and Duroc pigs by high-fat and high-carbohydrate diet and study the relationship between LncRNAs and susceptibility of diabetes using transcriptom sequencing.1.We presented a chromosome level genome sequence of a male BM pig using 153.59×Illumina、71.97×10×genomics、20×Pacbio and 106×Hi-C data.The total length of the BM pig genome is aroud 2.46 Gb,close to the size of the pig genome reported previously.The contig N50 and scaffold N50 of BM pig genome reach to 1.03Mbp and 140.06 Mbp.The tatal sequencing depth was staggering 900 × which guarantees very high single-base correctness of BM pig genome.Through further assessments by CEGMA and BUSCO,the assembled genome were identified to covered 234（94.35%of total 248）core eukaryotic genes and 3878（94.5%of total 4104）complete single-copy BUSCOs,which indicates the high completeness of assembly of BM pig genome.To aid the gene annotation of the BM pig genome,we carried a transcriptome sequencing of ten tissues embracing the brain,liver,heart,spleen,lung,kidney,pancreas,stomach,skeletal muscle and adipose collected from a BM pig.The BM pig genome contains 20,988 protein-encoding genes,averaging 8.61 exons and 1,509.03 bps CDS per gene,of whose 94.8%function are predicted.Moreover,repetitive elements comprise 37.04%of the BM pig genome and the genome encompasses ncRNA as 0.0312%miRNA,0.0136%tRNA,0.0014%rRNA and 0.0084%snRNA,respectively.2.The analysis of variants between BM pigs and BX pigs by re-sequencing showed that all PIs（nucleotide divergence rate）of BM pigs were acquired below the counterparts of BX pigs in all autosomes and X-chromosome,which indicates that BM pig,under long-term inbreeding,had already became a relatively stable genetical animal herd,due in part to low degree of genetic diversity.Furthermore,we obtained the first comprehensive inbred variants discovery of these 2 swine population,including SNP（single nucleotide polymorphism）,Indel（Insertion and Deletion）,SV（Structural Variation）and CNV（Copy Number Variation）events using an assembly-versus-assembly approach by BM pig genome and a simple assembly of BX pig genome（contig N50 size of 11.87Kb,a scaffold N50 size of 99.2 Kb and a total of length of 2.57 Gb）.The analysis of these genetic variants revealed that a large number of long-fragment variants between BM pig and BX pig were located in genes related to energy metabolism,smell and immunity,suggesting that long-term inbreeding has led to a greater evolutionary divergence in these areas of these 2 stains.The abundant genetic diversity between BX and BM pig strains could considerably facilitate the exploration of genetic diversity for animal inbreeding programs and population genetics studies,and the phylogeographic structure of these two Bama pig strains.In addition,our results also provide references for the conservation and utilization of these two Bama pig strains.3.We engineered the comparative genome analysis of BM pig and other mammals,especially other pig.Homologous clustering results showed that the number of BM pig single-copy homologous genes was almost the closest to that of humans compared to other mammals.The BM pig specific genes not only show that it’s better for BM pig than other experimental animals to be chose as a model when studying diseases involved in these genes but suggest that these genes participate in the specific features of BM pig,like sexual precocity and the low resistance to the diabetic environment.The branch lengths in the phylogenetic tree illustrate rapid evolution in Duroc pig albeit BM pig and Duroc pig clustered in a clade,which has given rise to less genetic divergences between humans and BM pig than between humans and Duroc pig.In comparing protein sequences between Duroc pig and BM pig,we found that the number of BM pig proteins close to human proteins is more than the number of Duroc pig and the enriched GO terms and KEGGs among the specific genes of which BM pig is closer to human than the Duroc pig be encompass extensive different biology processes,indicating that greater availability of using BM pig for different biomedical research than Duroc pig.In the comparisons of gene sets of 8 common human diseases,all cases,we sought out numerous encoded human proteins to be better conserved in BM pig than in Duroc pig,revealing that many BM pig proteins relevant to human diseases have probably evolved to conserve similar molecular functions as their human protein orthologs.4.For the expansion and contraction of gene families,BM pig performs conspicuously substantial contractions of many subfamilies belong to cytochrome P450（CYP）superfamily,including CYP 3A,CYP 4F,CYP 4A,CYP 2J and CYP 2E,which are known to influence Bone formation,sperm testicular development,water reuptake,drug metabolism.It is believed that hese contractions are related to the formation of some features of BM pig,like small body size,sexual precocity,adaption to high temperature environment and degenerative drug metabolism.Furthermore,we identified 3 positively selected genes in Duroc pig（HOXB3,HOXD11 and LCORL）not in BM pig,which explains the elongation of back from increased number of vertebrae of Duroc pig compared with BM pig.In the contrary to Duroc pig,the CHAD of BM pig has been undergone positive seletion,which may be one of the important factors shortening the fibula bone of BM pig to result the lower body height of BM pig than Duroc pig.The sexual precocity of BM pig was related to the positive selection of CDK7 gene.In accordance with previously reported Duroc pig genome,plenty of PSGs in Duroc pig have enriched in a large number of GO terms and KEGG pathways relevant to energy metabolism categories,while the BM pig PSGs were less enriched in energy metabolism categories than Duroc pig.Meanwhile,most of these duroc pig PSGs show less similarity to human counterparts than the genes of BM pig.These results illustrate the less genetic divergences of metabolism system and diseases between humans and BM pig than between humans and Duroc pig,which has been probably caused by the rapid evolution in Duroc pig.Central nervous system（CNS）functions in metabolic control to resist diabetic environment by a reduction in food intake and an increase in energy expenditure when CNS senses dietary excess.There is a significant difference of PSGs involved in CNS and energy control between BM pig and Duroc pig,which probably resulted in the lower resistance of BM pig to diabetic environment.5.To deepen our understanding of the relationship between lncRNAs and susceptibility of diabetes,we induced diabetes in BM and Duroc pigs and carried out the LncRNA transcriptom.According to the standards to select LncRNA and their target genes,the fulfilled pairs involving 6 lncRNAs and 2 target genes were detected.3 differentially expressed LncRNAs（XLOC₀06422,XLOC₀26958,XLOC₀44402）in skeletal muscle and other 3 differentially expressed LncRNAs（XLOC₀03015,XLOC₀23027,XLOC₀26564）in liver affect diabetes susceptibility by acting on two differentially expressed target mRNAs（PGC-1α and PEPCK）.In addition,WGCNA results showed that PGC-1α and PEPCK interact with a large number of genes related to diabetes in different tissues.Therefore,the 6 lncRNAs are in the expression correlation（trans action）relationships to PGC-1α and PEPCK genes to probably affect the expression of a large number of genes related to diabetes and ultimately regulate the susceptibility to diabetes.In summary,we assembled the BM pig genome by multipile sequencing technologies and performed the comprehensive annotation of BM pig genom.Then,the whole genome resequencing of BM and BX pigs indicate the high inbreeding degree of BM pig in the whole genome level.Meanwhile,the genome sequencing and simple assembly of BX pig were carried out,and the details of long genome vatiation among three kinds of pigs were obtained by using the BM pig genome,BX pig genome and Duroc pig genome.In addition,the comparative genomic analysis revealed the detailed information of BM pig gene family clustering,evolutionary tree,species divergenece time,gene family expansion and contraction,positive selection,which indicated the advantage of BM pig perfoming as an experimental animal in the genomic level.Finally,the effects of lncRNA on the occurrence and development of diabetic susceptibility were further analyzed by transcriptome analysis using the BM and Duroc pig diabetes model.