Reference Gene Validation for Quantitative RT-PCR during Biotic and Abiotic Stresses in Vitis vinifera

Alexandre Filipe Borges, Catarina Fonseca, R.B. Ferreira, Ana Maria Ferreira da Costa Lourenço, Sara Monteiro

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest
economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it
has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative
reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform
gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference
gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the
most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were
investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C
irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using
geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide
comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created
using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference
genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment,
EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine
samples can contribute for accurate gene expression quantification in forthcoming studies.
Original languageEnglish
Article numbere111399
Number of pages9
JournalPlosOne
Volume9
Issue number10
DOIs
Publication statusPublished - 23 Oct 2014

Keywords

  • REAL-TIME PCR
  • POLYMERASE-CHAIN-REACTION
  • EXPRESSION ANALYSIS
  • HOUSEKEEPING GENES
  • INTERNAL CONTROL
  • GRAPEVINE
  • NORMALIZATION
  • SELECTION
  • PATHOGEN
  • IDENTIFICATION

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