Genome-wide DNA methylation analysis in pigs using long-read sequencing unveils high-altitude adaptation and...

The advent of third-generation sequencing, particularly Oxford Nanopore Technologies (ONT), has revolutionized epigenetic studies by enabling direct detection of DNA methylation modifications and single-base resolution profiling of methylation patterns. While this technology has been predominantly utilized in human and bacterial research, its applications in livestock and poultry remain limited. In this study, we employed ONT sequencing to construct comprehensive 5-methylcytosine modification maps for ten representative pig breeds, explored the mechanism of high altitude adaptive methylation and allele-specific methylation events in these pigs.

Through genome-wide integration of sequencing data, we identified 27,857,021 CpG sites, with 71.5% (19,836,456) shared across pigs. Comparative differential methylation analysis between high-altitude and low-altitude pigs revealed four candidate genes (CALM1, HBB, PRKCQ and RAMP1) and the Sp1 transcription factor as potential key regulators of hypoxic adaptation. Notably, Tibetan pigs exhibited promoter hypomethylation patterns at the CALM1 locus, correlating with its consistently elevated expression confirmed by public transcriptomic databases. Allele-specific methylation (ASM) analysis integrated with transcriptomic profiles demonstrated significant enrichment of ASM events in promoters or exons of allele-specific expression (ASE) genes, suggesting synergistic regulatory mechanisms between epigenetic modifications and allelic expression patterns.

Our results provided a high-resolution DNA methylation atlas based on long-read sequencing encompassing ten representative pigs across Eurasia, and identified hypoxic adaption-related genes (CALM1, etc.) and a transcription factor (Sp1) based on the unique physiological characteristics of Tibetan pigs. Further, combined with transcriptome data, it was demonstrated ASM and ASE events are synergistic and expressions of ASE genes may be regulated by ASM. This study offers valuable insights into the epigenetic mechanisms underlying adaptation and gene regulation in pigs.