Genome-wide identification of RNA editing in seven porcine tissues by matched DNA and RNA high-throughput sequencing

Background: RNA editing is a co/posttranscriptional modification mechanism that increases the diversity of transcripts, with potential functional consequences. The advent of next-generation sequencing technologies has enabled the identification of RNA edits at unprecedented throughput and resolution. However, our knowledge of RNA editing in swine is still limited.Results: Here, we utilized RES-Scanner to identify RNA editing sites in the brain, subcutaneous fat, heart, liver,muscle, lung and ovary in three 180-day-old Large White gilts based on matched strand-specific RNA sequencing and whole-genome resequencing datasets. In total, we identified 74863 editing sites, and 92.1% of these sites caused adenosine-to-guanosine(A-to-G) conversion. Most A-to-G sites were located in noncoding regions and generally had low editing levels. In total, 151 A-to-G sites were detected in coding regions(CDS), including 94 sites that could lead to nonsynonymous amino acid changes. We provide further evidence supporting a previous observation that pig transcriptomes are highly editable at PRE-1 elements. The number of A-to-G editing sites ranged from 4155(muscle) to 25001(brain) across the seven tissues. The expression levels of the ADAR enzymes could explain some but not all of this variation across tissues. The functional analysis of the genes with tissuespecific editing sites in each tissue revealed that RNA editing might play important roles in tissue function.Specifically, more pathways showed significant enrichment in the fat and liver than in other tissues, while no pathway was enriched in the muscle.Conclusions: This study identified a total of 74863 nonredundant RNA editing sites in seven tissues and revealed the potential importance of RNA editing in tissue function. Our findings largely extend the porcine editome and enhance our understanding of RNA editing in swine.

The landscape of chromatin accessibility in skeletal muscle during embryonic development in pigs

Background:The development of skeletal muscle in pigs during the embryonic stage is precisely regulated by transcriptional mechanisms,which depend on chromatin accessibility.However,how chromatin accessibility plays a regulatory role during embryonic skeletal muscle development in pigs has not been reported.To gain insight into the landscape of chromatin accessibility and the associated genome-wide transcriptome during embryonic muscle development,we performed ATAC-seq and RNA-seq analyses of skeletal muscle from pig embryos at 45,70 and 100 days post coitus(dpc).Results:In total,21,638,35,447 and 60,181 unique regions(or peaks)were found across the embryos at 45 dpc(LW45),70 dpc(LW70)and 100 dpc(LW100),respectively.More than 91%of the peaks were annotated within−1 kb to 100 bp of transcription start sites(TSSs).First,widespread increases in specific accessible chromatin regions(ACRs)from embryos at 45 to 100 dpc suggested that the regulatory mechanisms became increasingly complicated during embryonic development.Second,the findings from integrated ATAC-seq and RNA-seq analyses showed that not only the numbers but also the intensities of ACRs could control the expression of associated genes.Moreover,the motif screening of stage-specific ACRs revealed some transcription factors that regulate muscle developmentrelated genes,such as MyoG,Mef2c,and Mef2d.Several potential transcriptional repressors,including E2F6,OTX2 and CTCF,were identified among the genes that exhibited different regulation trends between the ATAC-seq and RNA-seq data.Conclusions:This work indicates that chromatin accessibility plays an important regulatory role in the embryonic muscle development of pigs and regulates the temporal and spatial expression patterns of key genes in muscle development by influencing the binding of transcription factors.Our results contribute to a better understanding of the regulatory dynamics of genes involved in pig embryonic skeletal muscle development.

Genome-wide search for candidate genes determining vertebrae number in pigs

Longer porcine carcasses may be expected to have more vertebrae. Therefore, vertebrae number in pigs is an economically important trait. To examine the genetic basis of this trait, we genotyped 578 F-2 Large White x Minzhu pigs using the Porcine SNP60K BeadChip. A genome-wide association study (GWAS) identified 36 significant single nucleotide polymorphisms (SNPs) on the chromosomes SSC1 (294.28-300.32 Mb) and SSC7 (102.22-109.39 Mb). A 6.04-Mb region that contained all 13 significant SNPs on SSC1 also contained the gene NR6A1, previously reported to influence the number of vertebrae in pigs. However, the reported putative casual mutation of NR6A1 c. 748C > T showed no genome-wide significant association with the trait, suggesting it was not a causal mutation in our population. The remaining 23 significant SNPs on SSC7 were concentrated in a 7.17-Mb region, which was within a quantitative trait locus interval for number of vertebrae. TMED10 was the closest gene to the most significant SNP and might be a candidate. Haplotype sharing and block analysis refined the QTL to an interval of about 3 Mb containing 29 candidate genes. Of these 29 genes, the previously reported possible casual mutation of VRTN g. 19034A > C was not found to be a causal mutation in our population. Exploration of these genes via additional genetic and functional studies in mammals revealed that TGF beta 3 could be a good candidate on SSC7. A mutation of TGF beta 3 c. 1749G > A was detected by GWAS and could be proposed as a candidate causal mutation, or as closely linked to a causal mutation, for the number of vertebrae in pigs.