Integrative genomics decoding DNA methylation-mediated genetic control of complex traits in pigs

INTRODUCTION: DNA methylation is an important epigenetic feature associated with genetic regulation, gene expression, and phenotypic variation. However, the genetic architecture, regulatory organization, and trait relevance of the porcine skeletal muscle methylome remain poorly characterized. OBJECTIVES: This study aimed to characterize the genetic regulation of DNA methylation in porcine skeletal muscle and identify genetically supported relationships among genotype, methylation, gene expression, and complex traits. METHODS: We integrated 185 whole-genome bisulfite sequencing (WGBS) datasets, alongside integrated RNA-seq, Hi-C, and genetic association datasets of growth and meat quality traits. Cis-methylation quantitative trait loci (cis-meQTLs) were mapped at both the single-CpG and CpG-block levels. Genetically regulated methylation signals were further integrated with eQTL and GWAS results using colocalization, Mendelian randomization, mediation analysis, and chromatin interaction annotation. In addition, genotype-predicted methylation was used to construct methylation-based genetic scores (MGS), and a prioritized CpG block was functionally assessed using CRISPR-dCas9-TET1-mediated demethylation. RESULTS: We identified widespread cis-genetic control of skeletal muscle DNA methylation, revealing both single-CpG and block-level regulatory architectures. CpG-block methylation captured broader and more abundant genetic regulatory signals than single-CpG methylation, supporting methylation blocks as regional methylation features. Integrative analyses prioritized 6,894 candidate genes and highlighted genetically supported regulatory relationships linking methylation, transcription, and economically important traits. MGS showed moderate predictive performance for several carcass and meat-quality traits, including backfat thickness. Targeted demethylation of a CpG block increased SOCS3 protein abundance and altered proliferation-related phenotypes in porcine muscle satellite cells, providing proof-of-concept functional support for this prioritized locus. CONCLUSION: Porcine skeletal muscle DNA methylation is extensively influenced by cis-genetic variation. Integrating genetically regulated methylation with expression and trait-associated signals provides an interpretable framework for prioritizing candidate regulatory relationships and trait-associated loci.