INTRODUCTION: Cibotium barometz is a medicinal fern species with high economic value, Chinese people produce the traditional medicine herb “Gou-ji” using its robust rhizome. To balance the conservation of wild plant resources with the sustainable development of herbal medicine, much more effort is needed in scientific research on C. barometz, which might be crucial to mitigate conflicts between wild resource protection and the development of the traditional medicine industry. Real-time quantitative PCR (RT-qPCR) is a commonly used method for characterizing plant gene molecular function, and an appropriate reference gene is pivotal for this technique. Currently, limited literature focuses on the gene function studies for C. barometz, and little validated reference genes is available. 

RATIONALE: Previous studies have demonstrated that the expression stability of reference genes is influenced by multiple factors, including species, tissue type, developmental stage, stress treatment, and sequence specificity. Consequently, distinct reference genes are required for different research subjects. When employing RT-qPCR to analyze gene expression pattern in C. barometz tissues, it is essential to pre-validate the stability of reference genes within the novel experimental system. This validation process ensures the accuracy and reliability of experimental results by maintaining proper data normalization. 

RESULTS: In this study, 12 commonly used plant reference genes were identified as candidate genes from transcriptome data of C. barometz rhizome. The amplification efficiency of candidate reference genes was calculated by template gradient dilution method, and eight pairs of reference gene primers with high amplification efficiency were screened out. The Ct values of these eight reference genes in ten tissues of C. barometz were measured using RT-qPCR method; the software RefFinder and Normfinder determined that the best reference genes are CbUBC4 and CbEF1A, respectively. If studying the gene expression patterns of C. barometz at different developmental stages, it was suggested that CbEF1A and CbUBC4 should be used as optimal reference genes. The reference genes CbUBC4 and CbUBC28 were recommended under mechanical injury and waterlogging conditions. 

CONCLUSION: CbUBC4, CbEF1A and CbUBC28 were validated as stable reference genes in C. barometz across distinct experimental contexts, demonstrating their suitability for normalization in gene expression studies for RT-qPCR assay. From a methodological rigor perspective, we recommended that the stability of reference genes should be evaluated prior to conducting qRT-PCR experiments, particularly when analyzing samples from diverse tissues or distinct developmental stages of a specific tissue.