Reference Gene Validation in Energy Cane Under Smut Infection and Insights into Antioxidant Gene Expression

Abstract Sporisorium scitamineum is the causal agent of smut disease affecting sugarcane and energy cane crop production. Despite the frequent use of traditional reference genes for gene expression normalization by RT-qPCR, their stability can vary across genotypes and stress conditions. Here, we evaluated the stability of seven commonly used reference genes (GAPDH, SAMDC, UBQ, 25S rRNA, αTUB, SARMp1, and ACAD) and two additional candidates (EF5A-1 and SDH1-1) identified from RNA-Seq data in two contrasting energy cane genotypes (Vertix 1, smut-susceptible; Vertix 2, smut-resistant). Stability was assessed at three infection time points (12 hours post-inoculation, 48 hpi, and 5 days post-inoculation) separately and also on a combined dataset comprising all time points, under mock and smut-inoculated conditions. Gene stability, evaluated through multiple approaches (coefficient of variation, RefFinder, GeNorm) and consolidated using RankAggreg, revealed genotype and condition-dependent variability, with no universal reference gene set for most conditions. GAPDH, commonly used in sugarcane studies, showed the highest variation in our experiments, reinforcing the need for genotype and condition-specific validation. The best-ranked reference genes were used to quantify ROS-related defense responses. Vertix 2 (Vx2) exhibited coordinated induction of SOD, POX5, and CATB, whereas Vertix 1 (Vx1) showed delayed or reduced activation of these genes alongside a compensatory up-regulation of GSTt3. By validating reliable endogenous genes from literature and RNA-Seq data, this study ensures accurate gene expression quantification under biotic stress and highlights antioxidant mechanisms as contributors to smut resistance in S. scitamineum–energy cane interactions. Competing Interest Statement The authors have declared no competing interest.