Reference gene selection and validation by qRT-PCR during flower development and in different organs of Primula forbesii

Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) is an effective and widely used method in gene expression studies. Selection of appropriate reference genes is key to generating reliable data using this technique and there are no published studies regarding the selection of reliable reference genes in the genus Primula. In this study, 10 candidate reference genes (28S, ACT, TUA, TUB, HIS, GAPDH, RPL18a, STPP, CSD and RNA pol II), were chosen from the transcriptome database in Primula forbesii Franch., and their expression levels were assessed by qRT-PCR in different flower developmental stages and different organs. Four software programs; geNorm, NormFinder, Bestkeeper, and Ref-Finder were used to validate the stability and suitability of potential reference genes. Comprehensive results showed that GAPDH was the most stable reference gene for P. forbesii, while TUA and TUB were the most unstable ones in flower development and different organs, respectively. Finally, to illustrate the usefulness of the top-ranked reference genes, the expression profile of the P. forbesii linalool synthase/nerolidol synthase gene (PfLIS/NES) was analysed, which highlighted the importance of proper reference gene selection. This work represents the first systematic study of reference gene stability in Primula and lays the foundation for future research into Primula gene expression patterns.