Identification of the most suitable reference genes for gene expression normalization in safflower (Carthamus tinctorius L.) treated with gibberellin

Safflower (Carthamus tinctorius L., Asteraceae) is an important oil crop and medicinal plant which is of considerable interest for the expression of agronomically important genes. Semi-quantitative reverse transcription PCR (semi-qRT-PCR) is a powerful tool for studying gene expression, but it requires appropriate reference genes that can reduce variability. To identify safflower reference genes, four candidate housekeeping genes were investigated: CtPP2A (serine/threonine-protein phosphatase), CtE1F4A (eukaryotic initiation factor 4A), CtUBI (ubiquitin) and Ct60S (60S acidic ribosomal protein). 32 samples of safflower seedlings were treated with different gibberellin (GA) concentrations (0 ppm, 50ppm, 100ppm and 150 ppm; eight replicates each). Expression stability across samples was then investigated using semi-qRT-PCR. GA at 150ppm is observed as an optimal concentration for promoting safflower growth with superior plant heights, stem diameters, numbers of branches and flowers per plant. Additionally CtUBI and Ct60S were identified as the most suitable reference genes for evaluating the expression of gibberellin-biosynthesis genes including GA20-oxidase (GA20ox), GA 3-oxidase (GA3ox) and GA 3-beta-hydroxylase (GA4). The results indicate that both genes are appropriate as internal controls to normalize safflower gene expression.