Genetic Dissection of Growth Traits and Identification of Candidate Genes in Japanese Flounder (Paralichthys olivaceus) Using Single- and Multi-Trait Genome-Wide Association Analyses

Genetic improvement of growth traits is a key objective in aquaculture breeding. In this study, we performed whole-genome resequencing of 282 Japanese flounder individuals from a single farmed cohort and obtained approximately 6.67 million high-quality SNPs. Single-trait and multi-trait GWAS were conducted for 11 growth traits: body weight (BW), total length (TL), body length (BL), body depth (BD), trunk length (TUL), head length (HL), snout length (SnL), caudal peduncle depth (CPD), eye diameter (ED), postorbital head length (PoL), and interorbital width (IW). Heritability estimates ranged from 0.172 (IW) to 0.487 (HL). Head length showed the highest heritability, followed by PoL (0.328), ED (0.323), BD (0.300), and CPD (0.291). Genetic correlation analysis revealed that BW was strongly positively correlated with most body size traits (rg > 0.9), indicating that simultaneous improvement of these traits is feasible. Single-trait GWAS identified a total of 55 suggestive associated loci (top five SNPs per trait, combined across all 11 traits, at p < 1 × 10−5). Notably, we observed pronounced pleiotropic effects, where a single SNP or gene influences multiple growth traits. For example, multiple SNPs on chromosome 14 were associated with more than five traits (TL, BL, BD, TUL, BW), and the fras1 gene affected both head length and post-orbital head length, demonstrating a genetic basis of pleiotropy. Additionally, fras1 was associated with head length, and nkd1 with interorbital width. Multi-trait GWAS uncovered 12 novel loci not detected in single-trait analyses, with candidate gene annotation pointing to pathways such as ECM–receptor interaction (e.g., fras1, lamb4) and Wnt signaling (e.g., nkd1), suggesting that these biological processes may underlie the observed associations. These findings are preliminary and require validation in larger independent cohorts. KEGG enrichment analysis showed significant enrichment in ECM–receptor interaction and non-homologous end-joining pathways (corrected p < 0.05). Our results provide a genetic basis for growth trait improvement and candidate markers for breeding programs in Japanese flounder.