The Molecular Evolutionary Pattern And Methylation Pattern Of LincRNA In Pig Liver

Pig is an important domestic animal and is the main source of meat protein in China.The domestic pig Sus scrofa domesticus originated from the wild boar S.scrofa about 10,000 years ago.During domestication and breed differentiation,drastic morphological,physiological,and behavioral changes occurred in domestic pigs under artificial and natural selection.Long non-coding RNA（lncRNA）has become a hot research topic in recent years.More and more studies have shown that lncRNA is associated with mammalian traits,such as H19 play an important role in regulating growing and muscle development.With the development of high-throughput sequencing technology,more and more lncRNAs have been identified,but the lincRNA database of pigs is not perfect and the function research of pig lincRNA is quite a few.In this study,the RNA-seq data of pig liver tissue were used to identify lincRNAs.And combining with BS-seq of pig liver,identified lincRNAs and pig re-sequencing data,the molecular characteristics,methylation and expression and molecular evolutionary patterns of lincRNAs were analyzed.In addition,the molecular evolutionary and methylation patterns of H19,the important imprinting lncRNA,were also analyzed.The main results are as follows:（1）Tophat alignment,cufflinks assembly,cuffmerge de-redundancy and filtration were performed by RNA-seq data reads of liver tissue,and 861 transcripts were obtained,corresponding to 713 lincRNA genes,of which 403 lincRNAs have no overlap with currently annotated genes and are new identified lincRNA genes.Combining with the identified lincRNA genes,the biology function and process of potential target genes of lincRNAs,which were located in upstream 10 kb or downstream 10 kb region of lincRNAs,were predicted,and we found that these potential target genes of lincRNAs mainly involved in transcriptional regulation,substance metabolism,muscle cell differentiation.（2）Then the expression pattern of lincRNAs were analyzed by RNA-seq data and the expression level of linRNAs are significant lower than that of coding genes at the whole genome level.Besides,there were no significant difference in the expression level of all lincRNA genes in liver tissue among Enshi black pig（ES）,Large white（LW）and Guizhou wild pig（GZWB）,and the same result was obtained by comparing the expression level of all lincRNA genes in different tissues.（3）72 lincRNA genes were found to be differentially expressed in liver tissue among three pig breeds by differential expression analysis,accounting for 12.2% of the total differentially expressed lincRNAs,in which 40 lincRNAgenes were liver-specific expressed.Then we found that potential target genes of differential expressed lincRNAs may play an important role in metabolic metabolism,cell proliferation by gene ontology（GO）analysis.（4）Then the methylation pattern of lincRNA genes was analyzed by BS-seq data and we found that the methylation level of lincRNA genes in different functional regions from high to low was in the order of intron,exon,downstream 2 kb of transcriptional terminal site（TTS）and promoter region,in which the methylation level of lincRNA genes in promoter and the 2 kb region of TTS is significantly lower than coding genes.The methylation level of lincRNA genes and coding genes is significantly reduced across the transcriptional start sites（TSSs）and TTSs,but the methylation level of lincRNA genes is higher than coding genes across TSSs and TTSs.（5）261 differentially methylated lincRNA genes were identified among three pig breeds by differential methylation analysis.The potential target genes of differentially methylated lincRNA genes were found to be mainly involved in transcription process and protein binding process by GO analysis.（6）The relationship among promoter methylation of lincRNA genes,expression of lincRNAs and expression of potential target genes were integrated analyzed,and we found that there was a negative between the promoter methylation level and the expression level of lincRNA genes,and the expression of lincRNA genes is positively correlated with the expression of their potential target genes.（7）The selected lincRNA genes were screened by re-sequencing data of Eurasian domestication pig and wild boar,and 10 lincRNA genes were found to be under selection during the process of domestication and differentiation of pigs.Combing with lincRNA genes which were differentially methylated,differentially expressed and liver-specific expressed,lincRNA XLOC₀32274 were screened and may play a crucial role in liver metabolism during the process of domestication and breed differentiation of pigs.（8）Then the detailed species evolutionary analysis of H19,an important imprinting lncRNA,was preformed,and we found that H19 is highly conserved among species,especially its 5‘ terminal is higher conserved than its 3‘ terminal.Molecular evolutionary analysis showed that the nucleotide diversity of domestication pig is significant higher than that of wild pig.Molecular micro-evolutionary analysis showed that H19 have high nucleotide diversity during the pig breed differentiation.MeDIP data analysis showed that the methylation level of H19 gene,upstream and downstream of H19 have differences among pig breeds and also have differences among tissues.Last,the relationship between promoter methylation level and expression of H19 was analyzed using the BS-seq and RNA-seq,and we found that the methylation level of promoter of H19 may affects its expression.There may have relationship between imprinting and methylation differences among different species by ASM analysis.