To quantify expression levels, RNA was isolated from samples of leaf, flower, and root tissue of two A. majus plants using the RNeasy Plant Minikit (Qiagen). cDNA was prepared from 1 μg of total RNA using the iScript cDNA synthesis kit (Bio-Rad). qRT-PCR was performed on a CFX96 real-time PCR detection system (Bio-Rad) using SSO Advanced SYBR Green Supermix (Bio-Rad). For each of the four candidates, gene-specific oligonucleotides were designed (supplemental Table S3) to amplify a 100-bp long section of the open reading frame, and their individual efficiency was tested. For comparative analysis of the expression of each gene in leaf, flower, and root, the detected transcript levels were compared with the tissue with the highest expression level using the Δ CT method (36).