# Initial Screening of Extrachromosomal Circular DNA Candidates for Pork Meat Quality Traits Using Circle-Seq and RNA-Seq Analysis

## TL;DR

This study explores how extrachromosomal circular DNA (eccDNA) influences pork meat quality by comparing two pig populations using advanced sequencing techniques.

## Contribution

The first comprehensive analysis of eccDNA profiles in porcine muscle, linking eccDNA to lipid metabolism pathways and meat quality traits.

## Key findings

- Significant differences in eccDNA abundance, size distribution, and genomic locations were found between two pig populations.
- EccDNA-associated differentially expressed genes were enriched in lipid metabolism pathways, suggesting a regulatory role in pork quality.
- Potential regulatory networks involving eccDNA and miRNAs were identified, offering new targets for breeding optimization.

## Abstract

The rising global demand for high-quality animal protein underscores the need for sustainable advancements in pork production. While meat quality traits are influenced by multiple genetic factors, their molecular regulatory mechanisms remain incompletely understood. Emerging evidence indicates that extrachromosomal circular DNA (eccDNA)—double-stranded circular DNA derived from chromosomal DNA—participates in muscle biology regulation, yet its functional relevance in swine remains uncharted. Here, we performed the first comprehensive analysis of eccDNA profiles in porcine longest dorsal muscle using Circle-seq technology, comparing two pig populations. Our preliminary analyses revealed significant differences in eccDNA abundance, size distribution patterns, and genomic locations. Through integrated RNA-seq analysis, we identified eccDNA-associated differentially expressed genes (eccDEGs) enriched in lipid metabolism pathways and uncovered potential regulatory networks linked to pork quality traits. These findings provide novel insights into the eccDNA-mediated genetic regulation of muscle development and offer new targets for breeding optimization.

Yunong Black (YN) pigs and Yunong Black × Landrace (YL) hybrid pigs exhibit significant differences in meat quality characteristics. Studies have suggested that extrachromosomal circular DNA (eccDNA) may play a regulatory role in muscle development. In order to study the differences in eccDNA between two groups with different meat quality traits and their potential biological significance, this study used the Circle-seq method to detect eccDNA in the longest dorsal muscle (LDM) of Yunong Black pigs (YN) (n = 3) and Yunong Black × Landrace hybrid pigs (YL) (n = 3). EccDNA-related differentially expressed genes (eccDEGs) were then analyzed in combination with RNA-seq to explore the mechanisms by which eccDNA affects meat quality. The results showed that 1325 and 1304 differentially expressed eccDNAs were identified in the YN and YL groups, varying in size and distributed across multiple genomic functional regions. These eccDNAs were also annotated according to several protein-coding genes. Combined analysis with RNA-seq results revealed 19 and 27 eccDEGs in the YN and YL groups. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis enriched many lipid-related pathways, such as chemokine signals and ADP metabolic processes. By constructing a regulatory network, several potential regulatory networks that might be related to pork quality, for example, ecc_sus_8665/ssc-miR-212/ADAMTS16, were identified. In summary, we identified several potential eccDNAs that may regulate pig muscle, offering insights into the regulation of pig muscle traits for breeding.