A systems-level framework integrating evolutionary genomics and developmental transcriptomics reveals the regulatory architecture underlying seed development in makhana (Euryale ferox Salisb.).

Seed size is a complex quantitative trait governed by coordinated developmental, hormonal, and metabolic pathways, yet its regulatory architecture in makhana (Euryale ferox Salisb.) remains largely unknown. This study integrated comparative genomics with developmental transcriptomics to construct a systems-level framework for investigating seed development in makhana. Genome-wide analyses identified 543 genes across ten conserved gene families containing established regulators of seed development and related growth processes, with WGD/segmental duplication contributing substantially to their expansion and duplicated genes predominantly evolving under purifying selection. Despite extensive evolutionary constraint, only 193 genes met the expression criterion during seed development. Temporal profiling revealed dynamic stage-specific reprogramming, with NAC and WRKY genes prominently represented among development-associated expression patterns across seed maturation. Co-expression network analysis identified six positively associated development modules, with the blue module containing the largest proportion of candidate genes and showing enrichment for ubiquitin-mediated protein turnover, calcium signalling, vacuolar trafficking, and autophagy. Leave-one-sample-out (LOSO) analysis further supported hub-gene stability, with 61 of 64 development-associated hubs retaining the module-membership criterion in at least 11 of 12 runs. Integrating these LOSO-stable hubs with genes upregulated between the earliest and latest developmental stages prioritized 51 high-confidence seed-development-associated candidate genes from the NAC, WRKY, EXPANSIN, ARF, AP2, AUX/IAA, and SWEET families. This study establishes a comprehensive framework linking gene-family evolution with developmental transcriptional and co-expression dynamics in makhana, providing a prioritized resource for functional investigation of seed-development-associated candidates and future genomics-assisted molecular breeding.