Comprehensive Identification and Validation of Reliable Reference Genes in <scp> <i>Eucommia ulmoides</i> </scp>

Eucommia ulmoides Oliv., commonly known as the "Chinese gutta percha tree," is a vital medicinal and industrial resource in China, with irreplaceable applications in both industrial production and daily life. In recent years, the study of gene function has become a crucial aspect of E. ulmoides breeding programs. However, few reliable reference genes have been systematically evaluated for real-time quantitative polymerase chain reaction (RT-qPCR) in E. ulmoides, which may affect the accuracy of gene expression analysis. Here, we conducted a transcriptome-wide identification of reference genes using 159 datasets, comprising 81 tissue samples, 31 samples from diverse cultivars, and 47 stress-treated samples. A total of 20 stably expressed genes were identified for each group. Functional enrichment analysis revealed significant enrichment in pathways related to metabolism, genetic information processing, cellular processes, and organismal systems. Nine candidate genes from each group were selected and validated using RT-qPCR with 63 samples. We employed geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder to assess the stability of candidate genes. The most stable reference genes were PAB2 and UBC for tissues, GAPDH and ACTIN for cultivars, and UBC28 and RD19A for stress treatments. To validate their suitability, we analyzed relative expression patterns of DIR, CHS, and 4CL across tissues, cultivars, and stress conditions. When unstable reference genes were used for normalization, expression patterns showed significant bias compared with those obtained using optimal combinations. Overall, our study establishes a robust foundation for accurate gene expression analysis in E. ulmoides, thereby facilitating more precise functional genomics research and improved breeding strategies.