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Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization.  Caragana intermedia , a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in  C. intermedia  subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but  UNK2 ,  PP2A  and  SAND  were the most stable across all tested samples.  UNK2  and  SAND  would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of  UNK2 ,  SAND  and  EF-1α  would be appropriate for salt-treated leaves.  UNK1 ,  UNK2  and  PP2A  would be appropriate for PEG-treated (osmotic) roots, whereas the combination of  TIP41  and  PP2A  was the most suitable for PEG-treated leaves.  SAND ,  PP2A  and  TIP41  exhibited the most stable expression in heat-treated leaves. In cold-treated leaves,  SAND  and  EF-1α  were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of  DREB1  and  DREB2  (homologs of  AtDREB1  and  AtDREB2 ) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in  C. intermedia  under different abiotic stress conditions, and will benefit future studies on gene expression in  C. intermedia  and other species of the leguminous genus  Caragana .

Reference Gene Selection for Quantitative Real-time PCR Normalization in Caragana intermedia under Different Abiotic Stress Conditions