Reference Gene Selection for qPCR Analysis in Tomato-Bipartite Begomovirus Interaction and Validation in Additional Tomato-Virus Pathosystems

• DOI: 10.1371/journal.pone.0136820
• Publication Date: 2015-08-28T18:22:20Z
• Authors: Ana L. M. Lacerda; Leonardo N. Fonseca; Rosana Blawid; Leonardo S. Boiteux; Simone G. Ribeiro; Ana C. M. Brasileiro
• Keywords: 2. Zero hunger; 0303 health sciences; Genes, Viral; Science; Q; R; Reference Standards; Genes, Plant; Real-Time Polymerase Chain Reaction; 3. Good health; 03 medical and health sciences; Solanum lycopersicum; Begomovirus; Host-Pathogen Interactions; Medicine; Research Article

Quantitative Polymerase Chain Reaction (qPCR) is currently the most sensitive technique used for absolute and relative quantification of a target gene transcript, requiring use appropriated reference genes data normalization. To accurately estimate expression tomato (Solanum lycopersicum L.) responsive to several virus species in reverse transcription qPCR analysis, identification reliable mandatory. In present study, ten were analyzed across set eight samples: two contrasting genotypes ('Santa Clara', susceptible, its near-isogenic line 'LAM 157', resistant); subjected treatments (inoculation with Tomato chlorotic mottle (ToCMoV) mock-inoculated control) distinct times after inoculation (early late). Reference stability was estimated by three statistical programs (geNorm, NormFinder BestKeeper). validate results over broader experimental conditions, samples, corresponding additional tomato-virus pathosystems that included tospovirus, crinivirus tymovirus + tobamovirus, together tomato-ToCMoV pathosystem dataset, using same algorithms. Taking into account combined analyses ranking order outputs from algorithms, TIP41 EF1 identified as stable pathosystem, EXP four together, selected be forthcoming analysis conditions involving aforementioned pathosystems.