Characterization of Extrachromosomal Circular DNA Associated with Meat Quality Traits in Porcine Muscle through RNA-seq and Circle-seq Analysis

Pork quality is an important trait in pig breeding. Studies have shown that extrachromosomal circular DNA (eccDNAs) play a key role in the musculoskeletal system. However, the characteristics and roles of eccDNAs in pork quality remain unclear. Yunong Black (YN) pigs (n = 3) and Yunong Black × Landrace (YL) hybrid pigs (n = 3) were randomly selected for meat quality determination of the longissimus dorsi (LM) muscle. Multiple types of eccDNA were detected in the muscle tissues of both pig groups using Circle sequencing (Circle-seq). To further explore the mechanism of action, we performed RNA sequencing (RNA-seq) to analyze the differentially expressed genes (eccDEGs) associated with eccDNAs. The results showed that the intramuscular fat content in the YN group was significantly higher than that in the YL group (p < 0.01). In the YN and YL groups, 1,325 and 1,304 differentially expressed eccDNAs were identified, ranging from 0.11 kb to 1 kb and distributed across multiple genomic functional regions. The two sets of differential eccDNAs were annotated to 1,274 and 1,366 protein-coding genes, respectively. Combined analysis of the Circle-seq and RNA-seq results revealed 19 and 27 differentially expressed eccDNAs (eccDEGs) in the YN and YL groups, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses of these genes enriched several lipid-related pathways, including chemokine signaling, ADP metabolic process, Apelin signaling, blood lipids and atherosclerosis, and arachidonic acid metabolism. By constructing a regulatory network, several miRNAs, including ssc-miR-10391, ssc-miR-133a-3p, and ssc-miR-1343, were identified as interacting with differential eccDNAs and eccDEGs. Finally, we found that multiple networks, such as ecc_sus_8665/ssc-miR-212/ADMTS16 and ecc_sus_22708/ssc-miR-432-5p/OLFM2 may regulate pork quality. In summary, we elucidated the first eccDNA profile relating to pork quality and identified several potential eccDNAs that may regulate pig muscle. The results of this study not only offer a new perspective for breeding selection of pork texture-related traits, but also provide insights into the role of eccDNA in the LM of pigs.