Reference gene stability and stress marker expression during drought and senescence in forage and malting barley

Abstract

Reference genes are essential for accurate normalisation in qPCR-based gene expression studies, yet their stability can vary widely across tissues, developmental stages, genetic backgrounds and stress conditions. Fourteen candidate reference genes previously reported as stable in barley were evaluated in forage and malting varieties across senescence, grain development, and drought stress. Seven genes satisfied specificity and amplification-efficiency criteria and were subsequently assessed for expression stability using the ΔCt, BestKeeper, NormFinder, and geNorm algorithms integrated through a comprehensive stability index. Across all tissues, EF1α consistently emerged as the most stable reference gene, whereas MALD, UPL, and HSP70 provided reliable alternatives depending on the tissue type, variety, or stress condition. In contrast, widely used housekeeping genes such as TUBα showed high variability and were unsuitable as internal controls. To validate the selected reference genes, eight drought- and senescence-responsive marker genes were analysed. Expression profiling normalised with EF1α and MALD revealed both conserved and variety-specific patterns: HSP17 and GPR were strongly induced under drought, whereas WRKY1 showed mild repression. During senescence, GS2 and RBCS were sharply downregulated, while AGTX and SAG12 displayed contrasting induction between varieties. These findings identify a robust set of reference genes for barley and reveal distinct stress-response strategies between forage and malting backgrounds, providing a validated framework for qPCR normalisation and stress-adaptation studies.