Feed efficiency (FE) is one of the main factors that determine the production costs in the pig industry. In this study, RNA Sequencing (RNA-seq) was applied to identify genes and long intergenic non-coding RNAs (lincRNAs) that are differentially expressed (DE) in the adipose tissues of Yorkshire pigs with extremely high and low FE. In total, 147 annotated genes and 18 lincRNAs were identified as DE between high- and low-FE pigs. Seventeen DE lincRNAs were significantly correlated with 112 DE annotated genes at the transcriptional level. Gene ontology (GO) analysis revealed that DE genes were significantly associated with cyclic adenosine monophosphate (cAMP) metabolic process and Ca²⁺ binding. cAMP, a second messenger has an important role in lipolysis, and its expression is influenced by Ca²⁺ levels. In high-FE pigs, nine DE genes with Ca²⁺ binding function, were down-regulated, whereas S100G, which encodes calbindin D9K that serve as a Ca²⁺ bumper, was up-regulated. Furthermore, ATP2B2, ATP1A4, and VIPR2, which participate in the cAMP signaling pathway, were down-regulated in the upstream of lipolysis pathways. In high-FE pigs, the key genes involved in the lipid biosynthetic process (ELOVL7 and B4GALT6), fatty acid oxidation (ABCD2 and NR4A3), and lipid homeostasis (C1QTNF3 and ABCB4) were down-regulated. These results suggested that cAMP was involved in the regulation on FE of pigs by affecting lipid metabolism in adipose tissues.

The domestic pig Sus scrofa domesticus originated from the wild boar S. scrofa about 10,000 years ago. During domestication, drastic morphological, physiological, and behavioral changes developed between domestic pigs and wild boars through artificial and natural selection. The long non-coding RNA (lncRNA) H19, which is located within the imprinting gene cluster H19-IGF2, plays an important role in regulating muscle development in humans and mice. This study systematically analyzed the molecular evolution of H19 and its possible epigenetic changes during pig domestication and breeding to explore the genetic and epigenetic contributions of H19 to pig domestication. The molecular evolution of H19 was initially analyzed on a large phylogenetic scale. Results showed that the gene was highly conserved within a broad range, especially in the 5′ terminal sequence. The molecular evolution of the gene was then analyzed using published re-sequencing data of 30 wild boars from Tibet, 3 wild boars from Sichuan, and 15 native pigs from other regions in China. Eight polymorphic sites were identified, and the nucleotide diversity (π) value within the H19 gene body was significantly higher (Z-test, P < 0.05) in domesticated pigs than in wild pigs. However, no significant divergence occurred between domesticated and wild pigs. Single nucleotide polymorphisms in the 3′ terminal sequence were surveyed in other Chinese local breeds and foreign pig breeds. We observed a consistently higher diversity in domesticated pigs than in wild pigs. The methylation pattern of the H19 gene in pigs was subsequently analyzed using published methylated DNA immunoprecipitation data and an unpublished single-base resolution liver methylome. Analysis results showed distinct methylation levels in some tissues. Among the samples surveyed, Landrace showed the lowest methylation level, followed by the Guizhou wild boar, whereas the Enshi pig exhibited the highest methylation level in the 2 kb upstream region of the H19 gene. Liver transcriptome data suggested that Landrace harbored the highest expression of the H19 gene, followed by the Guizhou wild boar, whereas the Enshi pig harbored the lowest expression of the gene. Differential methylation sites (DMSs) among the three breeds were mainly identified in the 2 kb upstream region of the H19 gene. In the Enshi pig, we detected allele-specific methylation (ASM) regions in the 2 kb upstream region of the H19 gene. Most of the DMSs in the upstream 2 kb region of the gene were also located in the ASM region in this breed. Molecular analyses suggest that the H19 gene was highly conserved during large-scale evolution and exhibited genotype differentiation during domestication and breed differentiation. The drastic diversity pattern between domestic and wild pigs in the H19 gene body, which was highly conserved during large-scale evolution, suggests that this gene might have played roles in the breed differentiation of domestic pigs. Methylation analysis indicates an opposite epigenetic regulation direction between Chinese and European pig (EU) domestication, which resulted in opposite expression changes in this gene between the two domesticated groups. Our preliminary analyses on DMSs among different pig breeds and ASM imply that imprinting was associated with methylation differences. This study systematically demonstrates the genetic and epigenetic patterns of H19 during pig domestication and provide valuable cues and basis for further research on the function of H19 in pig domestication.

The liver plays a key role in metabolism and affects pig production. However, the functional annotation of noncoding regions of the pig liver remains poorly understood. We revealed the landscape of cis-regulatory elements and their functional characterization in pig liver. We identified 102,373 cis-regulatory elements in the pig liver, including enhancers, promoters, super-enhancers, and broad H3K4me3 domains, and highlighted 26 core transcription regulatory factors in the pig liver as well. We found similarity of cis-regulatory elements among those of pigs, humans, and cattle. Despite the low proportion of functionally conserved enhancers (~30%) between pig and human liver tissue, ~78% of the pig liver enhancer orthologues sequence could play an enhancer role in other human tissues. Additionally, we observed that the ratio of consistent super-enhancer-associated genes was significantly higher than the ratio of functionally conserved super-enhancers. Approximately 54% of the core regulation factors driven by super-enhancers were consistent across the liver from these three species. Our pig liver annotation and functional characterization studies provide a system and resource for noncoding annotation for future gene regulatory studies in pigs. Furthermore, our study also showed the high level functional conservation of cis-regulatory elements in mammals; it also improved our understanding of regulation function of mammal cis-regulatory elements.