Selection of Suitable Reference Genes for Gene Expression Normalization Studies in Dendrobium huoshanense

Dendrobium huoshanense is a kind of precious herb with important medicinal and edible value in China, which is widely used in traditional Chinese medicine for various diseases. Recent studies have paid close attention to the genetic expression of the biosynthetic pathway of the main active components (polysaccharides, alkaloids, and flavonoids), and real-time polymerase chain reaction (qPCR) is one of the most widely used methods for doing so. However, so far, no reference gene selections have been reported in D. huoshanense. In this study, 15 reference gene candidates (GAPDH, eIF, EF-1α, PP2A, UBCE, RPL5, TBP, APT1, MDH, PTBP3, PEPC, CYP71, NCBP2, TIP41, and F-box) were selected and evaluated for their expression stability in D. huoshanense under various experimental conditions, including in different tissues (root, stem, and leaf), abiotic stresses (oxidative, drought, cold, and UV), and hormone treatment (methyl jasmonate) using three statistical programs (geNorm, NormFinder, and BestKeeper). Then, the RefFinder program was employed to comprehensively validate the stability of the selected reference genes. Finally, the expression profiles of the CESA and GMPP genes were further analyzed, and these results indicated that TBP, NCBP2, and CYP71 were the top three most stable reference genes after comprehensive comparison, which could be used as stable reference genes for normalizing the genes expression in D. huoshanense. This study described here provides the first data regarding on reference gene selection in D. huoshanense, which will be extremely beneficial for future research on the gene expression normalization in D. huoshanense.