Integrative transcriptomics and machine learning reveal key regulatory genes for meat quality traits in pigs

Meat quality traits are typically regulated by multiple genes, each contributing a small effect. In this study, to pinpoint candidate genes involved in meat quality traits, we performed transcriptome profiles of porcine longissimus dorsi (LD) muscle and applied machine learning (ML) models to analyze RNA-seq data. We also carried out Gene Set Enrichment Analysis (ssGSEA), Weighted gene co-expression network analysis (WGCNA) and functional validation of putative target genes to better support the biological relevance of our findings. In this study, LD muscle samples were collected from 142 Huoshou Black (HSH) pigs and 191 Anqing Six-end-white (AQLB) pigs. Based on results of the estimated breeding values (EBV) analysis, of meat quality traits, we selected 101 HSH pigs and 99 AQLB pigs for transcriptomic analysis. Using an integrative analytical framework that combined ssGSEA and WGCNA, we identified 197 candidate genes 197 candidate genes. These genes were significantly associated with various metabolic pathways, including fatty-acid elongation and metabolism, amino-acid catabolism, protein turnover, and biosynthetic processes. To further refine the identification of key regulatory genes, we systematically evaluated ten ML models, ultimately selecting XGBoost, Random Forest, and Lasso Regression for subsequent analysis. This approach pinpointed CYSLTR1 and LPCAT2 as the key regulatory genes. To investigate the functional roles of CYSLTR1 and LPCAT2 in intramuscular fat (IMF) deposition, we established a porcine intramuscular adipocyte model via siRNA-mediated knockdown of either CYSLTR1 or LPCAT2. RNA-Seq analysis identified 339 differentially expressed genes (DEGs) in the siLPCAT2 group and 2,376 DEGs in the siCYSLTR1 group relative to the control. Heatmap analysis indicated that genes involved in triacylglycerol (TAG) biosynthesis were upregulated in the siCYSLTR1 group, but downregulated in the siLPCAT2 group. KEGG pathway enrichment analysis further demonstrated that LPCAT2-associated DEGs were predominantly enriched in the MAPK signaling pathway, mTOR signaling pathway, biosynthesis of unsaturated fatty acids, and glycerolipid metabolism—pathways closely linked to cell proliferation, nutrient sensing, and lipid remodeling. In contrast, CYSLTR1-associated DEGs were significantly enriched in lipid metabolism, atherosclerosis, and adipocytokine signaling pathways—processes directly implicated in adipocyte differentiation, lipid storage, and inflammatory crosstalk within adipose tissue. Collectively, these findings elucidate distinct yet complementary regulatory roles for CYSLTR1 and LPCAT2 in intramuscular adipogenesis and provide mechanistic support for targeting these genes to modulate IMF content and improve pork quality traits. In conclusion, CYSLTR1 and LPCAT2 were identified as pivotal regulatory genes governing IMF deposition. Functional enrichment and pathway analyses revealed that both genes exert their effects on IMF accumulation through the coordinated regulation of lipid metabolism–associated pathways—including fatty acid synthesis, triglyceride assembly, and phospholipid remodeling. These findings offer mechanistically grounded evidence and actionable biological insights for improving pork quality traits, particularly marbling and tenderness.