Expression Analysis by Reverse Transcribed Quantitative PCR

The relative gene expression of selected DEGs was analysed by RT-qPCR on a Light Cycler^® 480 System, using the LightCycler^® 480 SYBR Green I Master protocol. PCR amplification efficiencies were tested for all primers for target and reference genes using cDNA two-fold dilution series. As reference genes, β-tubulin, photosystem I P700 apoprotein A2, γ-tubulin, chromodomain helicase DNA-binding protein, and histone H2A.2, previously described by Almeida et al. (2015) and Santos et al. (2018), were tested. Using the geNorm and NormFinder software packages from the GenEx v.5 software (MultiD, Goteborg, Sweden), two reference genes were selected for the gene relative expression analysis. Thermo cycling reactions were carried out following the described conditions: denaturation step at 90°C for 5 min; 45 cycles of amplification at 95°C for 10 s; 10 s at 60°C and 10 s at 72°C. For each reaction, a melting curve (dissociation stage) was performed to detect non-specific PCR products and/or contaminants. A non-template control (NTC), without cDNA, was also included for each primer mix to detect possible contaminations.

Relative expression levels (Fold change, FC) were calculated using the Pfaffl method (Pfaffl, 2001) compared with expression levels of the reference genes (β-tubulin and γ-tubulin) and using the susceptible parental line BGE008277 as a calibrator. Finally, FC data were transformed into a logarithmic scale (base 2) to meet the data normality assumptions for statistical analysis and graphical representation.

Since the number of biological replicates varied from 1 to 3, the absence of significant differences between biological replicates was confirmed by ANOVA using the Genstat software (Genstat^® for Windows 19th edition), considering the genotypes represented by three biological replicates. Therefore, the average of relative expression levels per RIL was used as a metric for eQTL detection.