Evaluation of Candidate Reference Genes for Quantitative Gene Expression Studies in Tree Peony

Abstract

Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) is a sensitive and widely used technique for gene expression analysis that depends on stability of the reference genes used for data normalization. Tree peony (Paeonia suffruticosa), known as one of the most famous traditional ornamental plants in China, is very popular in both domestic and international markets for its showy and colorful flowers. To date, no systematic studies on reference genes have been performed in tree peony with different flower colors. In this study, we evaluated the expression stability of 12 candidate reference genes in different tissues and five flower developmental stages of tree peony with six different colors by three algorithms: geNorm, NormFinder, and BestKeeper. The results showed that protein phosphatase 2A (PP2A), ubiquitin protein ligase (UPL), and ubiquitin (UBQ) were the most stable genes across all samples. Helicase, alpha-tubulin (TUA), and eukaryotic translation initiation factor 5A (EIF5A) also exhibited high expression stability in different tissues, in samples with different colors, and at different flower developmental stages. According to the geNorm analysis, the combination of two most stable reference genes was optimal for normalization in all tested sample sets in this study. To further validate the suitability of the reference genes identified in this study, the expression patterns of two putative homologs of chalcone synthase gene (PsCHS1) and chalcone isomerase gene (PsCHI1) were studied at different developmental stages of white flowers. The results provide information for transcriptional analyses in future studies of gene expression on tree peony flower development and pigmentation.