Abstract
BACKGROUND: Tenacious glume a key trait limiting threshing efficiency in synthetic hexaploid wheat (SHW), represents an ancestral defense mechanism inherited from wild progenitors. Despite its evolutionary importance, the molecular basis of this domestication-associated trait remains poorly understood. To address this, we performed a comprehensive transcriptomic analysis of developing spikes from wild-type (tenacious glume)- and EMS-induced mutant (softglume) at 0, 5, and 10 days post-heading (DPH). RESULTS: Multi-factorial analysis identified 676 genes exhibiting significant developmental stage × glume type interactions, highlighting 5 DPH as a critical temporal window for trait establishment. Weighted gene co-expression network analysis (WGCNA) revealed a strongly correlated module (r = 0.92) that functions as the core transcriptional regulon underlying glume hardness. This module drives a dual-layered structural reinforcement program. Coordinated upregulation of CAD (Cinnamyl alcohol dehydrogenase) and COMT (Caffeic acid O-methyltransferase) in the phenylpropanoid pathway promotes lignin deposition, increasing mechanical rigidity, while activation of cuticle biosynthesis, lipid modification, and glycine-rich protein genes strengthens the epidermal barrier. This coordinated process is regulated by MYB and MIKC_MADS transcription factors acting as central hubs, and the co-enrichment of flavonoid biosynthesis and photoprotection pathways indicates that glume hardness is linked to integrated structural and chemical defense mechanisms. CONCLUSION: Together, these findings delineate the molecular and temporal architecture of glume persistence in SHW. The identified transcriptional regulators provide potential genetic targets f to separate the tenacious glume phenotype from beneficial wild alleles, thereby facilitating the improvement of threshing efficiency without compromising adaptive traits. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-026-12766-z.