Selection and validation of reference genes for normalization of qRT-PCR gene expression in wheat (Triticum durum L.) under drought and salt stresses

Eight candidate housekeeping genes were examined as internal controls for normalizing expression analysis of durum wheat (Triticum durum L.) under drought and salinity stress conditions. Quantitative real-time PCR was used to analyse gene expression of multiple stress levels, plant ages (24 and 50 days old), and plant tissues (leaf and root). The algorithms BestKeeper, NormFinder, GeNorm, the delta Ct method and the RefFinder were applied to determine the stability of candidate genes. Under drought stress, the most stable reference genes were glyceraldehyde-3 phosphate, ubiquitin and β-tubulin2, whereas under salinity stress conditions, eukaryotic elongation factor 1-α, glyceraldehyde-3 phosphate and actin were identified as the most stable reference genes. Validation with stress-responsive genes NAC29 and NAC6 demonstrated that the expression level of target genes could be determined reliably with combinations of up to three of the reference genes. This is the first report on reference genes appropriate for quantification of target gene expression in T. durum under drought and salt stresses. Results of this investigation may be applicable to other Triticum species.