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Background  Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from  Triticum aestivum  cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways.    Findings  Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus:  TaFNRII  (ferredoxin-NADP(H) oxidoreductase; AJ457980.1),  ACT2  (actin 2; TC234027), and  rrn26  (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes:  CYP18-2  (Cyclophilin A, AY456122.1) and  TaWIN1  (14-3-3 like protein, AB042193) were most consistently stably expressed.  Furthermore, we showed that  TaFNRII, ACT2 , and  CYP18-2  are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus.    Conclusions  This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production.

Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions