The cDNA sequences of the candidate WRKY genes were retrieved from GenBank and analyzed using Beacon Designer Software (Biorad) to design gene-specific qPCR primers with an amplicon size of 80e150 bp and a melting temperature (Tm ) of 60C ± 1C. In order to prevent off-target amplification, BLAST was used to identify the ten most homologous sequences to the WRKY TF cDNA sequence. These sequences were then aligned using ClustalW2 (EMBL-EBI; http://www.ebi.ac.uk/Tools/msa/clustalw2/), and the software suggested primers were compared to the alignments to select the primers with the least homology to off-target sequences.