Selection and validation of suitable reference genes for gene expression studies in Callerya speciosa (Champ. ex Benth.) Schot under different experimental conditions

Abstract The accuracy of reverse transcription quantitative real-time PCR (RT-qPCR) is strongly depended on the stability of reference gene. Callerya speciosa, as a traditionally Chinese medicine, has a long cultivation history in south China. It is essential to select the suitable reference gene to obtain reliable RT-qPCR results when gene expression changes were evaluated. However, suitable reference genes in C. speciosa have not yet been investigated for accurate gene expression quantification under different experimental conditions. In this study, eight candidate reference genes (GAPDH, 60S, ACTIN, TUA2, TUB1, TIF5, UBQ, EF2) were selected from the transcriptome databases, and their expression stability under six experimental conditions (developmental stages, tissues, MeJA treatment, GA3 treatment, CPPU and PP333 treatment) was evaluated using ΔCT, geNorm, NormFinder, BestKeeper, RefFinder programs. The results showed that GAPDH was the optimal reference gene for all different experimental conditions, whereas ACTIN showed the most stability under the hormone treatments in C. speciosa. GAPDH and EF2 were proved to be the most stable genes for developmental stages, while different genes (GAPDH and TUB1) were stable reference genes for tissues. For treatments, ACTIN was identified as the most stable gene under most of hormone treatments. TUBI and ACTIN were at the beginning of the ranking order in PP333 treatment, while GAPDH and ACTIN were adequate for normalization in MeJA and GA3 treatments. TUBI and GAPDH were the most stable genes for CPPU treatment, while ACTIN was proved to be the most stable gene for three different treatments (MeJA, GA3 and PP333). Validation of reference genes was carried out by the target gene CsMYB36, which further confirmed their reliability. These results provided a theoretical basis for subsequent research on the regulation of functional gene expression in C. speciosa.