Quantitative Real-Time PCR Analysis of BrBBX Genes

The cDNA samples were diluted 5 times using nuclease-free water and then 10 ng cDNA was used for each qRT-PCR analysis. The AccuPower 2X GreenStar qPCR MasterMix (Bioneer, Daejeon, Korea) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) were used for the qRT-PCR analysis. Each 20-µL reaction comprised 5 μL SYBR mix, 1 μL cDNA, 1 μL of each primer (10 μM), and 12 μL ddH₂O. The Brassica ERF1 gene was used as an internal control for normalizing expression levels (Table S17). The PCR program was as follows: 94 °C for 5 min, 40 cycles of 94 °C for 15 s, 62 °C for 20 s, and 72 °C for 20 s. A melting curve analysis was performed at the end of the qRT-PCR analysis to confirm gene-specific products were amplified. The qRT-PCR was completed using three technical replicates and relative gene expression levels were calculated according to the 2^−ΔΔCt method [84 (link)]. The heat map showed expression patterns of 51 BrBBX genes in different organs and during various developmental stages using available transcriptome data [51 (link)]. The relative expression data shown in the heat map were generated using the pheatmap package of R.