Splicing retention and enhancer divergence govern the evolutionary fate of ohnologues following whole-genome duplication in rainbow trout.

Whole-genome duplication (WGD) generates gene duplicates, or ohnologues, that enhance evolutionary potential while challenging regulatory coordination and functional balance. Although gene expression divergence after WGD is well studied, the long-term evolutionary dynamics of alternative splicing (AS) remain unclear. We investigated AS evolution in rainbow trout (Oncorhynchus mykiss), which experienced a salmonid-specific WGD ~ 100 million years ago. Using a high-quality genome assembly, transcriptomes from six tissues, and ChIP-seq profiling of histone modifications, we classified ohnologue pairs by expression divergence, splicing complexity, and epigenetic signatures. Most ohnologues were retained through conservation, with a gradual decline in AS diversity over time. The study provides evidence that AS initially evolved through accelerated divergence, while also underscoring the potential role of the independent model in its long-term evolutionary trajectory. Enhancer-associated histone marks, particularly H3K27ac, diverged markedly between neofunctionalized and independently splicing pairs, suggesting that enhancer rewiring may contribute to regulatory and functional divergence. Our results indicate that AS evolution after WGD is shaped by both selective pressures and epigenetic modulation, challenging the assumption of rapid splicing loss and the negligible role of the independent model. This study provides an integrated framework for understanding the evolution of splicing and regulatory landscapes after genome duplication, with implications for vertebrate genome evolution and functional innovation.