Alternative splicing (AS) and fusion transcripts produce a vast expansion of transcriptomes and proteomes diversity. However, the reliability of these events and the extend of epigenetic mechanisms have not been adequately addressed due to its limitation of uncertainties about the complete structure of mRNA. Here we combined single-molecule real-time sequencing, Illumina RNA-seq and DNA methylation data to characterize the landscapes of DNA methylation on AS, fusion isoforms formation and lncRNA feature and further to unveil the transcriptome complexity of pig. Our analysis identified an unprecedented scale of high-quality full-length isoforms with over 28,127 novel isoforms from 26,881 novel genes. More than 92,000 novel AS events were detected and intron retention predominated in AS model, followed by exon skipping. Interestingly, we found that DNA methylation played an important role in generating various AS isoforms by regulating splicing sites, promoter regions and first exons. Furthermore, we identified a large of fusion transcripts and novel lncRNAs, and found that DNA methylation of the promoter and gene body could regulate lncRNA expression. Our results significantly improved existed gene models of pig and unveiled that pig AS and epigenetic modify were more complex than previously thought.

Long intergenic non-coding RNAs (lincRNAs) play essential roles in numerous biological processes and are widely studied. The skeletal muscle is an important tissue that plays an essential role in individual movement ability. However, lincRNAs in pig skeletal muscles are largely undiscovered and their biological functions remain elusive. In this study, we assembled transcriptomes using RNA-seq data published in previous studies of our laboratory group and identified 323 lincRNAs in porcine leg muscle. We found that these lincRNAs have shorter transcript length, fewer exons and lower expression level than protein-coding genes. Gene ontology and pathway analyses indicated that many potential target genes (PTGs) of lincRNAs were involved in skeletal-muscle-related processes, such as muscle contraction and muscle system process. Combined our previous studies, we found a potential regulatory mechanism in which the promoter methylation of lincRNAs can negatively regulate lincRNA expression and then positively regulate PTG expression, which can finally result in abnormal phenotypes of cloned piglets through a certain unknown pathway. This work detailed a number of lincRNAs and their target genes involved in skeletal muscle growth and development and can facilitate future studies on their roles in skeletal muscle growth and development.

There are major differences between Yorkshire (lean-type) and Wannanhua pig (fat-type) in terms of growth performance and meat quality. Long intergenic noncoding RNAs (lincRNAs) are a class of regulators that are involved in numerous biological processes and widely identified in many species. However, the role of lincRNAs in pig is largely unknown, and the mechanisms by which they affect growth and meat quality are elusive. In this study, we used published data to identify 759 lincRNAs in porcine longissimus dorsi muscle. These putative lincRNAs shared many features with mammalian lincRNAs, such as shorter length and fewer exons. Gene ontology and pathway analysis indicated that many potential target genes (PTGs) of lincRNAs were involved in muscle growth-related and meat quality-related biological processes. Moreover, we constructed a co-expression network between differentially expressed lincRNAs (DELs) and their PTGs, and found a potential mechanism that most DELs can use to upregulate their PTGs, which may finally contribute to the growth and meat quality differences between the two breeds through an unknown manner. This work details some lincRNAs and their PTGs related to muscle growth or meat quality, and facilitates future research on the roles of lincRNAs in these two types of pig, as well as molecular-assisted breeding for pig.

Background: Placental efficiency is strongly associated with litter size, fetal weight and prenatal mortality. Together with its rapid growth during late gestation, the Large White pig breed shows a significant increase in placental size and weight, but this does not occur in the highly prolific Chinese pig breeds. To understand the molecular basis of placental development during late gestation in Chinese indigenous and Western breeds with different placental efficiency, female placental samples were collected from six pregnant Erhualian gilts at gestation day 75 (E75) and day 90 (E90) and from six pregnant Large White gilts at gestation day 75 (L75) and day 90 (L90). Two female placentas from one sow were used to extract RNA and then pooled in equal volumes. Twelve pooled samples were hybridized to the porcine Affymetrix GeneChip. Results: A total of 226 and 577 transcripts were detected that were differentially expressed between E75 and L75 and between E90 and L90 (p < 0.01, q < 0.2), respectively. Gene Ontology (GO) analysis revealed that these genes belong to the class of genes that participate in angiogenesis and development. Real-time RT-PCR confirmed the differential expression of eight selected genes. Significant differential expression of five genes in the VEGF pathway was also detected between the breeds. A search of chromosomal location revealed that 44 differentially expressed genes located to QTL regions related to reproduction. Differential expression of six candidate imprinted genes was also confirmed. Three of the six genes (PLAGL1, DIRAS3, and SLC38A4) showed monoallelic expression in the porcine placenta. Conclusion: Our study detected many genes that showed differential expression between placentas of two divergent breed of pigs, and confirmed the imprinting of three genes. These findings help to elucidate the genetic control of placental efficiency and improve the understanding of placental development.

Intramuscular fat (IMF) content is an important trait that can affect pork quality. Previous studies have identified many genes that can regulate IMF. Long intergenic non-coding RNAs (lincRNAs) are emerging as key regulators in various biological processes. However, lincRNAs related to IMF in pig are largely unknown, and the mechanisms by which they regulate IMF are yet to be elucidated. Here we reconstructed 105,687 transcripts and identified 1,032 lincRNAs in pig longissimus dorsi muscle (LDM) of four stages with different IMF contents based on published RNA-seq. These lincRNAs show typical characteristics such as shorter length and lower expression compared with protein-coding genes. Combined with methylation data, we found that both the promoter and genebody methylation of lincRNAs can negatively regulate lincRNA expression. We found that lincRNAs exhibit high correlation with their protein-coding neighbors in expression. Co-expression network analysis resulted in eight stage-specific modules, gene ontology and pathway analysis of them suggested that some lincRNAs were involved in IMF-related processes, such as fatty acid metabolism and peroxisome proliferator-activated receptor signaling pathway. Furthermore, we identified hub lincRNAs and found six of them may play important roles in IMF development. This work detailed some lincRNAs which may affect of IMF development in pig, and facilitated future research on these lincRNAs and molecular assisted breeding for pig.

Epigenetic modifications (especially altered DNA methylation) resulting in altered gene expression may be one reason for development failure or abnormalities in cloned animals, but the underlying mechanism of the abnormal phenotype in cloned piglets remains unknown. Some cloned piglets in our study showed abnormal phenotypes such as large tongue (longer and thicker), weak muscles, and exomphalos. Here we conducted DNA methylation (DNAm) immunoprecipitation and high throughput sequencing (MeDIP-seq) and RNA sequencing (RNA-seq) of muscle tissues of cloned piglets to investigate the relationship of abnormal DNAm with gene dysregulation and the unusual phenotypes in cloned piglets. Analysis of the methylomes revealed that abnormal cloned piglets suffered more hypomethylation than hypermethylation compared to the normal cloned piglets, although the DNAm level in the CpG Island was higher in the abnormal cloned piglets. Some repetitive elements, such as SINE/tRNA-Glu Satellite/centr also showed differences. We detected 1,711 differentially expressed genes (DEGs) between the two groups, of which 243 genes also changed methylation level in the abnormal cloned piglets. The altered DNA methylation mainly affected the low and silently expressed genes. There were differences in both pathways and genes, such as the MAPK signalling pathway, the hypertrophic cardiomyopathy pathway, and the imprinted gene PLAGL1; all of which may play important roles in development of the abnormal phenotype. The abnormal cloned piglets showed substantial changes both in the DNAm and the gene expression. Our data may provide new insights into understanding the molecular mechanisms of the reprogramming of genetic information in cloned animals.