Identification of genes controlling grain size in barley through RNA-seq and weighted gene co-expression network analysis

To elucidate the molecular mechanisms underlying grain size formation in barley, this study selected two distinct barley cultivars with significant differences in grain dimensions: the large-grained cultivar 'jianglinhuang' and the small-grained cultivar 'heiseyeqingke'. We conducted a comprehensive comparative analysis by integrating phenotypic measurements with RNA-seq data to elucidate molecular-level differences between barley varieties with distinct grain sizes. The study focused on three critical grain developmental stages. Dynamic phenotypic monitoring revealed that grain length and thickness exhibited significant differences across all three stages, whereas grain width only showed significant variation during the mid- and late-filling stages. Transcriptome analysis revealed that the number of Differentially Expressed Genes(DEGs) peaked during the mid-filling stage. Subsequent Weighted Gene Co-expression Network Analysis (WGCNA) revealed a brown module highly associated with grain size traits. By intersecting the module's hub genes with pan-stage DEGs, we obtained 13 candidate genes. Following homology-based screening and functional annotation, five of these candidate genes were found to be orthologs of known regulators of grain size in rice and other crops, and were therefore designated as the core candidate genes for this study. The RT-qPCR validation results further confirmed the stage and cultivar-specific expression patterns of these genes, supporting their potential as molecular targets for barley grain improvement. Through an integrated multi-omics approach, this study has identified a set of key genes potentially governing grain size and yield potential in barley, providing novel insights into the molecular mechanisms of grain formation and laying the groundwork for future genetic improvement.