Grapevine Gene Expression Profiling

Expression levels of 16 genes were analyzed by qRT-PCR. This gene set included 12 defense-related genes and four genes involved in sugar transport or metabolism (Supplementary Material S2). Primers were designed using the Primer3 tool (Untergasser et al., 2012 (link)) and tested for their specificity and efficiency (>95% for all primers). Real-time quantitative PCR was carried out using the GoTaq qPCR MasterMix (Promega) and a Mastercycler realplex2 instrument (Eppendorf). In this work, four V. vinifera reference genes were tested, elongation factor EF1γ and EF1α, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin, described in several previous reports (Reid et al., 2006 (link); Nicolas et al., 2013 (link); Camps et al., 2014 (link)). The stability of all these genes was tested in our biological conditions. GAPDH was finally selected as the best reference gene. Gene expression results were normalized to GAPDH as an internal standard (Supplementary Material S2). Results were expressed as relative gene expression using the 2^–ΔCt method (Schmittgen and Livak, 2008 (link); Cardot, 2017 ). For all qRT-PCR analyses, the number of independent biological replicates is specified in the legend caption. For each independent experiment, three technical replicates have been performed.