Integrative Multi‐Omics Analysis Identifies Novel Enhancer Variants on SSC2 Associated With Backfat Thickness in Landrace Pigs

Backfat thickness is an economically important trait in pig production, yet the functional regulatory variants underlying it remain poorly characterized. Here, we performed a genome-wide association study (GWAS) for backfat thickness at 100 kg (BF100) in a large Landrace population (n = 5923) using 13.46 million imputed SNPs. We identified two significant quantitative trait loci (QTLs) on SSC2 (1.21-3.69 Mb; explaining 2.3% of phenotypic variance) and SSC12 (51.70-52.88 Mb; explaining 0.8% of phenotypic variance). While nonsynonymous SNPs were limited (16 variants), we prioritized functional non-coding variants by integrating high-resolution Hi-C interaction maps, epigenomic marks, and previously published enhancer and promoter annotation results from public backfat datasets. This multi-omics strategy revealed that the SSC2 QTL functions as an active three-dimensional regulatory hub, with over 20 enhancer-promoter loops physically engaging the promoters of IGF2, CTSD, TSPAN32, and TSSC4. Similarly, the SSC12 QTL formed more than 10 long-range interactions with the promoters of ASGR1, YBX2, GPS2, MDPU1, and TP53. Focusing on SSC2, we prioritized two tightly linked SNPs (2-1280617 and 2-1280654) located within a putative enhancer, representing two major haplotypes. Dual-luciferase reporter assays in PK15 and 3T3-L1 cells confirmed that the GG haplotype drives significantly higher transcriptional activity than the AT haplotype (p < 0.001). Consistently, pigs carrying the GG haplotype exhibited significantly lower backfat thickness. By integrating multi-omics and functional assays, this study not only decodes the regulatory architecture of two backfat QTLs but also provides new molecular markers for genetic improvement in Landrace breeding.