Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions
Open Access
- 9 February 2012
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 7 (2), e31263
- https://doi.org/10.1371/journal.pone.0031263
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.Keywords
This publication has 51 references indexed in Scilit:
- RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalizationBMC Genomics, 2011
- Identification and Validation of Reference Genes for Normalization of Transcripts from Virus-Infected Arabidopsis thalianaMolecular Plant-Microbe Interactions®, 2011
- Transcriptome analysis of a spontaneous mutant in sweet orange [Citrus sinensis (L.) Osbeck] during fruit developmentJournal of Experimental Botany, 2009
- Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCRMolecular Breeding, 2009
- Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of referencesJournal of Experimental Botany, 2009
- Transcriptional analysis of the sweet orange interaction with the citrus canker pathogensXanthomonas axonopodispv.citriandXanthomonas axonopodispv.aurantifoliiMolecular Plant Pathology, 2008
- The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription‐polymerase chain reaction (RT‐PCR) analysis in plantsPlant Biotechnology Journal, 2008
- Citrus GenomicsInternational Journal of Plant Genomics, 2008
- A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck)Journal of Experimental Botany, 2007
- Quantification of mRNA using real-time RT-PCRNature Protocols, 2006