Abstract
Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.
Highlights
Real-time reverse transcription PCR (RT-qPCR) has emerged as the most widely used method to quantify changes in gene expression profiles in response to developmental transitions and environmental changes in plants
Identification of candidate citrus reference genes In order to identify suitable citrus reference genes, 15 candidates were chosen from three sources: traditional housekeeping genes frequently used for transcript normalization in citrus; citrus homologues to superior reference genes selected from Arabidopsis transcriptome microarray data [21], and reference genes tested in Swingle citrumelo under drought stress [38]
The stability of expression of the candidate genes was assessed by RT-qPCR in a set of 38 samples grouped into six experiments
Summary
Real-time reverse transcription PCR (RT-qPCR) has emerged as the most widely used method to quantify changes in gene expression profiles in response to developmental transitions and environmental changes in plants. Statistical algorithms such as geNorm [25] and NormFinder [26] have been recently used to identify the best reference genes for RT-qPCR data normalization in a given set of biological samples These algorithms have been used for assessing the expression stability of candidate reference genes across a variety of tissues and organs, developmental stages, biotic and abiotic stresses and cultivars in many plant species such as grapevine [27]; rice [28,29]; tomato [30]; soybean [31]; coffee [32]; brachiaria grass [33]; cotton [34]; eucalyptus [35]; cucumber [36] and petunia [37]. These reference genes will enable more accurate and reliable RTqPCR normalization for gene expression studies in citrus
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