Selection and validation of reference genes for target gene analysis with quantitative real-time PCR in the leaves and roots of Carex rigescens under abiotic stress

Carex rigescens is an ornamental turfgrass in northern China which has a relatively low maintenance cost and robust tolerance to many adverse environmental conditions, so it could be considered a new material for researching into plant stress resistance. However, suitable reference genes are vacant for obtaining reliable results in quantitative real-time PCR (qRT-PCR) analysis of C. rigescens in adversity research. In this study, we tested the expression stability of nine potential reference genes in leaves and roots under five different abiotic stress conditions, including cold, salt, heat, osmotic and cadmium (Cd). We then selected the best reference genes according to the analysis results calculated by three algorithmic programs (geNorm, NormFinder and BestKeeper) and used the RankAggreg package to merge the outputted data. The results showed that combinations of at least two reference genes should be used for reliable normalization except in heat-treated root samples, which require three reference genes. eIF-4 alpha, GADPH, SAND and PEPKR1 and their combination were found to be the most stably expressed reference genes, while SAM, TUA-alpha and UPL7 were the three least stable reference genes among most of experimental samples. In addition, five stress-induced genes (Cu-Zn SOD, P5CS, LEA, GST, and APX) were chosen to verify the stability of the selected reference genes in various tissues and under various stress conditions. The results of this study will provide an important fundamental basis both for gene expression verification for transcriptomic and proteomic analyses and for gene expression analysis for future gene function research in C. rigescens.