Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions

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.

Selection of reference genes for quantitative real-time PCR normalization in creeping bentgrass involved in four abiotic stresses

“…UPL7 was confirmed as a reference gene for Citrus spp. infected with viruses (Mafra et al 2012). In our study, UPL7 expression exhibited the highest stability in salt-stressed roots and cold-and heat-stressed leaves, while the lowest stability was in heat-stressed roots ( Table 2).…”

The ARF, AUX/IAA and GH3 gene families in citrus: genome-wide identification and expression analysis during fruitlet drop from abscission zone A

“…Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) using SAND gene (Mafra et al 2012) as normalizer was performed. RT-PCR was carried out using Eppendorf Mastercycler (Serial No.…”

Selection of reference genes for expression analyses of red-fleshed sweet orange (Citrus sinensis)

“…Mafra et al (2012) analyzed the stability of 15 candidate reference genes in various tissues and organs of Citrus plants infected with several pathogens, including C. liberibacter and Xylella fastidiosa. FBOX (F-box family protein, which regulates cellular processes such as cell cycle transition, transcriptional regulation, and signal transduction), SAND (SAND protein family, which regulates intracellular vesicle transport), GAPC2 (GAPDH C2, which participates in the metabolism of glucose), and UPL7 (ubiquitin ligase 7 protein, which functions in protein trafficking) were demonstrated to be the most stable in all of the conditions and samples tested by Mafra et al (2012). When the analysis was conducted with samples from C. liberibacter-infected plants, TUB was the least stable gene, which is similar to what we observed in the present study.…”

“…The instability of reference gene expression may result in an over-or under-estimation of target gene expression . Gene expression studies in animals (Small et al, 2008) and plants, including Citrus species (Kou et al, 2012;Mafra et al, 2012;Yan et al, 2012), have demonstrated that even genes considered to have a constitutive expression exhibit variation in expression under different experimental conditions, which requires an a priori determination of the stability of reference gene candidates, depending upon the conditions to be evaluated.…”