Gene Expression Analysis in Citrus Flavedo

RNA extraction, cDNA synthesis and RT-qPCR were performed following previously described well-established protocols [50 (link)]. Total RNA was isolated from flavedo samples, and 2 µg were used for the first-strand cDNA synthesis with the “Maxima H Minus First Strand cDNA Synthesis kit with dsDNase” (Thermo Scientific). The specific primer pairs for the genes of interest (CsCER3, CsCER4/FAR3, CsCER6/KCS6, CsSQS, CsABCG11/WBC11, CsABCG12/WBC12, CsCD2 and CsCER7) and those employed for data normalization (CsACT and CsTUB) (Supplementary Materials Table S1) were mixed with SYBR Green to monitor cDNA amplification in a LyghCycler480 System (Roche Diagnostic). Amplicon specificity was determined by a melting curve analysis. Fold change relative gene expression values of the target genes were obtained by the Relative Expression Software Tool (REST, rest.gene-quantification.info), as previously described in [50 (link)]. Three independent biological replicates and two technical replicates were performed per sample.