Decoding epigenetic drived heterosis in upland cotton (Gossypium hirsutum L.) through Multi-omics integration

Leaves, the primary photosynthetic organ in plants, provide critical insights into the genetic and epigenetic mechanisms underlying heterosis. This study systematically investigated the leaf phenotypic characteristics, gene expression profiles, and epigenetic regulation through methylation patterns, miRNAs and lncRNAs in two contrasting cotton hybrids, thereby establishing a novel multi-omics framework for heterosis analysis. Our primary innovation is the elucidation of expression level dominance (ELD) and its differential influence on heterotic performance. The Z41S hybrids display superior phenotypic heterosis, which correlates with heightened over-dominant (OD), under-dominant (UD), and paternal expressions (ELD-P). In contrast, the B985S hybrids are characterized by a predominance of maternal expression patterns (ELD-M). We identified CHH-type methylation variations as the major epigenetic regulators influencing dominant gene expression across both hybrids. Differentially methylated regions (DMRs), similarly methylated regions (SMRs), and transposable element distributions significantly impacted heterosis-related gene regulation, particularly through gene body modifications. Furthermore, the joint analysis of multi-omics data revealed that, nine genes including serine/threonine-protein kinase gene, zinc finger BED domain-containing protein RICESLEEPER 3/2, protein tyrosine kinase myosin-6 (Myh6), AGO2, RGA3, LRR and NB-ARC, subjected to multiple epigenetic regulation by miRNAs, lncRNAs, or DNA methylation, are poised to play a pivotal role in the manifestation of strong heterosis in cotton hybrids. These findings establish three fundamental contributions: (1) a comprehensive model integrating genomic and epigenetic regulation of cotton heterosis, (2) identification of parent-specific ELD patterns as molecular predictors of hybrid performance, and (3) characterization of CHH methylation as a critical epigenetic determinant in heterosis manifestation. Our results provide a transformative theoretical framework for optimizing hybrid breeding strategies through targeted manipulation of both genetic and epigenetic factors.