Selection of Suitable Reference Genes for Quantitative Real-time Polymerase Chain Reaction in Prunus mume during Flowering Stages and under Different Abiotic Stress Conditions

Abstract

Mei ( Prunus mume ) is widely cultivated in eastern Asia owing to its favored ornamental characteristics and its tolerance for low temperatures. Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for gene expression analysis, requiring carefully selected reference genes to ensure data reliability. The aim of this study was to identify and evaluate reference genes for qRT-PCR in mei. Ten candidate reference genes were chosen, and their expression levels were assessed by qRT-PCR in four sample sets: 1) flowering mei; 2) mei undergoing abiotic stress; 3) different genotypes of Prunus species; and 4) all mei samples. The stability and suitability of the candidate reference genes were validated using commercially available software. We found that protein phosphatase 2A-1 ( PP2A-1 ) and PP2A-2 were suitable reference genes for flowering with ubiquitin-conjugating enzyme E2 ( UBC ) also being suitable for different genotypes of Prunus species. UBC and actin ( ACT ) were most stably expressed under abiotic stress. Finally, the expression of an AGAMOUS homolog of Arabidopsis thaliana ( PmAG ) and a putative homolog of Group 2 late embryogenesis abundant protein gene in A. thaliana ( PmLEA ) were assessed to allow comparisons between selected candidate reference genes, highlighting the importance of careful reference gene selection.