Transcriptomic Analysis of Flavonoid Genes in Brassica juncea

To validate the transcriptome data and to characterize the flavonoid genes that were differentially expressed in the developing seeds of yellow- and dark-seeded B. juncea, total RNA was isolated from the samples using a DNA away RNA Mini-Prep Kit (Sangon Biotech, Shanghai, China). Subsequently, the cDNAs were synthesized using an RNA PCR Kit (AMV, v3.0) based on the manufacturer’s protocols (Takara, Dalian, China). The cDNA was subjected to RT-qPCR analysis using SYBR qPCR SuperMix Plus (NovoStart) on a Bio-Rad CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA), as previously described [13 (link)]. Tonoplastic intrinsic proteins-41 (TIPS-41) was used as a reference gene to normalize the gene expression levels via the 2^−∆∆Ct method [90 (link)]. Three biological replicates were performed for all experiments. The specific primer sequences used in this study were obtained from the RT-qPCR Primer Database [91 (link)] and are listed in Supplementary Table S10.