Evaluation of candidate reference genes for gene expression studies in Cymbidium kanran

• The first report of a systematic Evaluation of candidate reference genes for gene expression studies in Cymbidium kanran.
• ACT and TUB exhibited the most stable expression pattern when all samples were taken together.
• We recommend that RPS3 and RpoB could serve as the reliable internal control in different tissues.
• RPS19, 18S and PsaB performed poorly in all sample pools, and it should be used with caution as internal control in RT-qPCR analysis.

Quantitative real-time polymerase chain reaction (RT-qPCR) is a preferred method for rapid and reliable quantification of gene expression study. However the transcription levels of target genes may be misinterpreted due to differential expression of the reference gene which was thought to express stably. Thus, a systematic evaluation on the expression stability of nine frequently used reference genes across various tissues (including leaf, root, stem, sepal, peal, lip and gynostemium) and treatments (including phytohormone and stress treatments) in Cymbidium kanran was performed in this study. The results demonstrated that expression stability of the candidate genes varied greatly across different sample pools. Based on geNorm and NormFinder analysis, Tubulin (TUB), Actin (ACT) and Ribosomal protein S3 (RPS3) were considered to be the most suitable reference genes when all 25 samples were taken together. In addition, ACT and TUB were also the suitable internal controls in C. kanran with ABA, NaCl, cold and CuSO4 treatments. RPS3 and TUB exhibited the most stable expression pattern in NAA, 6-BA and GA treatments, while ACT and RNA polymerase beta (RpoB) could be used as the reliable internal control genes under PbAC stress. In different tissues, RPS3 and RpoB could be served as the reliable internal controls, while RpoB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were ranked top in heat treatment. On the other hand, 18S ribosomal RNA (18S), ribosomal protein S19 (RPS19) and photosystem I P700 apoprotein B (PsaB) performed poorly in expression stability analysis across all sample pools, and they could not be used as internal controls for RT-qPCR studies in C. kanran.