Comprehensive qRT-PCR Analysis of BnWRKY Genes

We extracted the total RNA using the RNAPrep Pure plant kit (Tiangen Biotech, Beijing, China). The complementary DNA was synthesized by the GoScript reverse transcription system (Promega, Madison, WI, USA). qRT-PCR analysis was conducted via an iQ5 multicolor real-time PCR system (Bio-Rad, Hercules, CA, USA). Here we analyzed the expression of 25 BnWRKY genes from three subgroups [23 (link)]. The primers (Table S1) applied to qRT-PCR were generated from the Primer3 website (http://primer3.ut.ee/, accessed on 1 June 2015) and were further evaluated by Oligo 7 (Molecular Biology Insights Inc., Cascade, CO, USA). The EF1A gene was selected as a reference gene. Three biological and technical replicates were performed, and the relative expression levels were calculated based on the comparative cycle threshold (2^−∆∆Ct) values [46 (link)]. Heatmap Illustrator 1.0 was used to generate a comprehensive presentation of gene expression under the different tested conditions.