Abstract
Quantitative Polymerase Chain Reaction (qPCR) is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring the use of appropriated reference genes for data normalization. To accurately estimate the relative expression of target tomato (Solanum lycopersicum L.) genes responsive to several virus species in reverse transcription qPCR analysis, the identification of reliable reference genes is mandatory. In the present study, ten reference genes were analyzed across a set of eight samples: two tomato contrasting genotypes (‘Santa Clara’, susceptible, and its near-isogenic line ‘LAM 157’, resistant); subjected to two treatments (inoculation with Tomato chlorotic mottle virus (ToCMoV) and its mock-inoculated control) and in two distinct times after inoculation (early and late). Reference genes stability was estimated by three statistical programs (geNorm, NormFinder and BestKeeper). To validate the results over broader experimental conditions, a set of ten samples, corresponding to additional three tomato-virus pathosystems that included tospovirus, crinivirus and tymovirus + tobamovirus, was analyzed together with the tomato-ToCMoV pathosystem dataset, using the same algorithms. Taking into account the combined analyses of the ranking order outputs from the three algorithms, TIP41 and EF1 were identified as the most stable genes for tomato-ToCMoV pathosystem, and TIP41 and EXP for the four pathosystems together, and selected to be used as reference in the forthcoming expression qPCR analysis of target genes in experimental conditions involving the aforementioned tomato-virus pathosystems.
Highlights
Tomato (Solanum lycopersicum L.) belongs to the Solanaceae family and is one of the most consumed and economically important vegetable crops in the world [1]
Recent gene expression studies involving interactions between the begomovirus TYLCV and tomato [35, 36] and Pepper golden mosaic virus (PepGMV) and pepper [37] have used TUB as reference gene for expression quantification of several target genes putatively involved in the pathosystems
Taking into account the results presented here, we have selected TIP41 and EF1 to be used in combination as reference genes in the forthcoming expression Quantitative Polymerase Chain Reaction (qPCR) analysis of target genes for ‘Santa Clara’- and ‘LAM 157’-Tomato chlorotic mottle virus (ToCMoV) interaction studies
Summary
Tomato (Solanum lycopersicum L.) belongs to the Solanaceae family and is one of the most consumed and economically important vegetable crops in the world [1]. Major constraints to PLOS ONE | DOI:10.1371/journal.pone.0136820. Reference Genes for Tomato-Begomovirus Interaction provided support in the form of salaries for authors [LNF; LSB; SGR and ACMB], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section
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