Systems-level discovery of housekeeping and tissue-specific genes reveals core cellular and specialized transcriptional networks in makhana (Euryale ferox Salisb.).

Housekeeping genes (HKGs) and tissue-specific genes (TSGs) play essential roles in constitutive cellular maintenance and tissue specialization. However, their transcriptome-wide characterization remains largely unexplored in makhana (Euryale ferox Salisb.), an economically and nutritionally important aquatic crop. In this study, a comprehensive transcriptomic analysis identified 652 HKGs and 5,378 TSGs across diverse tissues and developmental stages of Euryale ferox Salisb. Functional enrichment analyses revealed that HKGs were predominantly associated with conserved cellular processes, including ATP metabolism, ribosome function, oxidative phosphorylation, RNA processing, and proteostasis, whereas TSGs were enriched in developmental regulation, signaling pathways, secondary metabolism, and stress-responsive functions. Protein-protein interaction network analysis further showed that HKG-associated proteins occupied highly interconnected central positions, while TSG-associated proteins formed more specialized peripheral networks. Comparative analyses indicated that many traditional internal reference genes lacked stable constitutive expression across the analyzed biological conditions. To address this limitation, an RSI-based prioritization framework combined with biological functional curation enabled the computational identification of robust candidate reference genes for transcript normalization. Taken together, this study provides the first comprehensive systems-level characterization of HKGs and TSGs in Euryale ferox Salisb. and establishes a valuable computational resource for transcript normalization, functional genomics, and future experimental validation.