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

Abstract

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 Genes for RT-qPCR Analysis of Environmentally and Developmentally Regulated Gene Expression in Alfalfa

“…It should be noted that Rer1 and ATPase genes that were included as candidates on the basis of their stability of expression in a differential macroarray hybridization of alfalfa ESTs had not been previously tested as putative reference genes in other species. New reference genes identified by microarray analysis in Arabidopsis thaliana [33] and soybean (Glycine max) [34] were shown to outperform commonly used housekeeping genes in these species and other plant systems as well [21]. It is also noteworthy that eEF-1α consistently maintained an M value well below the 0.5 geNorm threshold across all treatments and genetic sources and is another candidate to consider in the validation of reference genes in RT-qPCR analysis of gene expression in alfalfa.…”

“…This is in stark contrast with the highly stable expression of UBL-2a and 18S-rRNA under these experimental conditions. Act has been previously shown to be unstable under various experimental conditions and was deemed unsuitable to normalize stress-induced gene expression in several species [12] [21] [32] including alfalfa [30]. Rapacz et al [18] concluded that the stability of Act in barley (Hordeum vulgare) exposed to abiotic stress could depend on the developmental stage.…”

“…It has been well documented that the selection of reference genes can markedly affect the accuracy of RT-qPCR analysis and that no single gene or genes combination should be arbitrarily used without validation under specific experimental conditions and varied sources of genetic material [9]. For that reason, reference gene candidates have been recently tested in a wide array of plant species and experimental conditions [10]- [21]. These studies clearly demonstrate the need to validate reference gene candidates under the experimental conditions specific to each study in order to avoid erroneous conclusions.…”

The evolution of determinate and indeterminate nodules within the Papilionoideae subfamily

“…8 of these 13 genes are similar to genes that in other plant species have been reported to be upregulated under abiotic stresses. These are the isoflavone reductase-like protein in grapefruit induced in response to UV irradiation, in coffee during a stress-response in leaves, and in rice induced by gibberellic acid (Lers et al, 1998;Brandalise et al, 2009;Wen et al, 2010); vacuolar processing enzyme in radish involved in floral bud abortion under heat stress, and in Malus hupehensis and Arabidopsis in response to high temperature stress (Zhang et al, 2013;Su et al, 2015); cytochrome P450 induced by salt and mannitol treatments in apple . Further, the sulphate transporter gene, which can be induced by both sulphur starvation and mycorrhiza formation in Lotus (Giovannetti et al, 2014).…”

“…Reactions were performed in triplicate. Relative expression values were normalized to IaPTB, IaSAND, and IaUNK1 reference genes (Kakar et al, 2008;Zhu, 2013), which are stable expressed in different organs (Fig. S1).…”