The Research Of Long Non-coding RNAs In Pigs And Dogs

Domestic animals are significant animal models in genetics studies,due to thousands of years of artificial selection upon domestic animals creating considerable genetic diversity.The pig and dog are important domestic animals.Till now,many protein-coding mutation with major effects on the domestication of pigs and dogs have been identified,such as a G->A mutation in the third intron of IGF2 effect on muscle growth in the pig,a SINE repetition in IGF1 associated with small size in the dog.Recent studies demonstrated that the number of long intergenic non-coding RNA(lincRNA)genes may be far more than that of protein-coding genes in the genome.But,the lincRNAs of domestic animals need to be identified in genome-wide scale and it is unclear whether they are involved in the domestication in the similar manner as protein-coding genes.In recent years,the high-throughput sequencing technology has been considerable developed and more and more RNA-seq,methylation sequencing,re-sequencing data have been generated from domestic animals.Therefore,it is feasible to study lincRNAs in the genome of domestic animals and to investigate the relationship between lincRNA genes and the domestication of domestic animals using multi-omics methods.In our studies,we identified lincRNA genes in pig and dog genomes and investigated the relationship between lincRNA DNA methylation and the domestication of the pig.To facilitate identification of the SNPs associated with phenotype and identify causal mutations,we build a database,focusing on pig SNPs and their positive selection signatures.1.Long intergenic noncoding RNA genes are potential association with domestication in pigsThousands of long intergenic noncoding RNAs(lincRNAs)have been identified in the human and mouse genomes,some of which play important roles in fundamental biological processes.The pig is an important domesticated animal,however,pig lincRNAs remain poorly characterized and it is unknown if they were involved in the domestication of the pig.Here,we used available RNA-seq resources derived from 93 samples and expressed sequence tag data sets,and identified 6,621 lincRNA transcripts from 4,515 gene loci.Among the identified lincRNAs,some lincRNA genes exhibit synteny and sequence conservation,including linc-sscg2561,whose gene neighbor Dnmt3a is associated with emotional behaviors.Both linc-sscg2561 and Dnmt3a show differential expression in the frontal cortex between domesticated pigs and wild boars,suggesting a possible role in pig domestication.This study provides the first comprehensive genome-wide analysis of pig lincRNAs.2.The genetic diversity of the dog contributes to the functional research of lincRNA genesRecent studies show that the number of lincRNA genes in human genome is more than that of mRNA genes.Domesticated dog is the first animal that has been domesticated by human.Due to thousands of years’ artificial selection,the dog has produced huge genetic diversity.Domesticated dog differs from its relative ancestors in behavioral traits.Thus,the research of dog lincRNAs in genomics scale may provide important clues for the lincRNA functional research in nervous system.Here,we integrated RNA-seq,EST and RefSeq datasets,and identified 5,590 lincRNA genes encoded 8,670 lincRNAs.Among these lincRNA genes,we found a lincRNA gene,named linc-4682,which showed differential frontal cortex expression between domesticated dogs and gray wolves,and exhibits synteny conservation between human,mouse and dog,and sequence conservation between vertebrates.Therefore,we conducted functional research for linc-4682.We used T98G and U251 cells as models and found cell apoptosis after knock down linc-4682 from them.Linc-4682 is interacted with KRT1 and participates KRTl’s transport process from nucleus to cytoplasm.Above all,this study illustrates that the genetic diversity of dogs may prompt the functional research of lincRNA genes.3.DNA methylation signatures of long intergenic noncoding RNAs in porcine adipose and muscle tissuesLong intergenic noncoding RNAs(lincRNAs)are one of the major unexplored components of genomes.Here we re-analyzed a published methylated DNA immunoprecipitation sequencing(MeDIP-seq)dataset to characterize the DNA methylation pattern of pig lincRNA genes in adipose and muscle tissues.Our study showed that the methylation level of lincRNA genes was higher than that of mRNA genes,with similar trends observed in comparisons of the promoter,exon or intron regions.Different methylation pattern were observed across the transcription start sites(TSS)of lincRNA and protein-coding genes.Furthermore,an overlap was observed between many lincRNA genes and differentially methylated regions(DMRs)identified among different breeds of pigs,which show different fat contents,sexes and anatomic locations of tissues.We identify a lincRNA gene,linc-sscg3623,that displayed differential methylation levels in backfat between Min and Large White pigs at 60 and 120 days of age.We found that a demethylation process occurred between days 150 and 180 in the Min and Large White pigs,which was followed by remethylation between days 180 and 210.These results contribute to our understanding of the domestication of domestic animals and identify lincRNA genes involved in adipogenesis and muscle development.4.PigSPS:a database for pig SNPs and signatures of positive selectionNext-generation sequencing technology has been used to generate hundreds of pig genome sequences,however a single resource compiling the variations found in these genomes is not available.To address the scarcity of single nucleotide polymorphism(SNP)data in dbSNP and to unify the criteria for SNP calling,we used GATK to identify 47.3 million and 36.8 million high-quality whole-genome SNPs in 184 pigs and 7 individuals from 6 outgroup species,respectively.Putative selected loci were identified using these SNPs through five different statistical tests on the evolutionary attributes for these SNPs.To combine the list of non-redundant SNPs and the scores of the evolutionary attributes of these SNPs,we build the first pig-specific SNP database,PigSPS(http://res.xaut.edu.cn/pigsps/),which is a web-based,open-access resource.In addition,PigSPS integrates related information including whether the SNPs are located within protein-coding or long intergenic non-coding RNA(lincRNA)genes,cause synonymous or non-synonymous changes,are ancestral or the derived state,and the sample location and breed information of these SNPs.This database enables the exploration and comprehensive analysis of SNPs in pig-related studies.Among the above research,we provided the first comprehensive genome-wide identification of pig and dog lincRNAs and found they have several features which differ from protein-coding genes,such as their length,number of exons and level of expression.We characterized the DNA methylation pattern of pig lincRNA genes in adipose and muscle tissues,and observed several different characteristics in DNA methylation between lincRNA genes and protein-coding genes.To facilitate the identification of SNPs that affect phenotype of domesticated pigs,we built the first pig SNP and their positive selection score database,which provide clues for identification of the lincRNA genes and protein-coding genes regulating the phenotype diversity of the pig.