Identification and validation of reference genes for qRT-PCR analyses under different experimental conditions in <i>Dendrobium nobile</i>

<i>Dendrobium nobile</i> L. (<i>D. nobile</i>), a perennial epiphytic herb belonging to the Orchidaceae family, is widely utilized in traditional Chinese medicine. Identifying stable reference genes (RGs) in <i>D. nobile</i> under various experimental conditions is essential for accurate quantitative real-time polymerase chain reaction (qRT-PCR) analysis. In this study, eight genes were selected as the candidate RGs based on the transcriptome data from different tissues of <i>D. nobile.</i> The qRT-PCR experiments were conducted under diverse experimental conditions, including various tissues and developmental stages, temperature stress, drought stress, and methyl jasmonate (MeJA) stress. The software programs GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were employed to evaluate the stability of the candidate RGs. The results indicated that <i>Actin</i> was the most stable reference gene (RG) across different tissues, <i>UBCE2</i> was the most stable RG under MeJA stress, while <i>ADF7</i> exhibited the highest stability in various developmental stages, low-temperature stress, and drought stress. The reference gene that demonstrated the most stable expression under high-temperature stress was <i>60SRPL26</i>. The experiment also validated the selection of optimal reference genes by assessing the expression levels of the farnesyl pyrophosphate synthase gene (<i>FPPS</i>) in different tissues and developmental stages of <i>D. nobile</i>. Furthermore, the expression levels of <i>DnFPPS</i> were analyzed under various stress conditions using qRT-PCR with the most stable reference genes, and the results were consistent with RNA-seq data. These findings provide valuable guidance for future gene expression studies involving qRT-PCR in <i>D. nobile</i>.