Screening and Validation of Stable Reference Genes for Real-Time Quantitative PCR in Indocalamus tessellatus (Munro) P. C. Keng Under Multiple Tissues and Abiotic Stresses

Indocalamus tessellatus (Munro) P. C. Keng is a bamboo species with significant economic and ecological value, and exhibits considerable resistance to abiotic stresses. However, systematic evaluation of reference genes for gene expression analysis in this species is lacking. Analysis of multi-tissue transcriptomes yielded 3801 relatively stable genes; from these, we selected eleven new candidates along with nine widely adopted reference genes. We then evaluated these candidates under four conditions: drought (15% PEG-6000), salt (200 mM NaCl), waterlogging (root submergence in water), and a multi-tissue panel (leaf, leaf sheath, culm, shoot, and root). Under stress, early and sustained time points were sampled to capture dynamic transcriptional responses. Expression stability was assessed using geNorm, NormFinder, BestKeeper, and ΔCt, and results were integrated with RefFinder to generate comprehensive stability rankings for each condition. The most stable reference genes were condition-dependent: MD10B and PP2A under drought, eIF1A and Ite23725 under salt stress, PP2A and eIF4A under waterlogging, and 60S and UBP1 across different tissues. Notably, commonly used genes such as UBI and Actin7 were less stable. Peroxidase (POD) was used as a validation marker because it is a known stress-responsive gene, providing a sensitive readout of normalization accuracy. Validation confirmed that selecting suitable reference genes is essential for dependable expression quantification. These findings provide a robust set of reference genes for qRT-PCR studies in I. tessellatus, supporting future molecular and functional research in bamboo.