Expression Patterns of 12 Genes in Brassica

Using qRT-PCR, the expression patterns of 12 genes (Gene ID: Bra003517, Bra013923, Bra020878, Bra028899, Bra031485, Bra038089, Bra034061, Bra035732, Bra010802, Bra012938, Bra00944, and Bra025833) were analyzed (Primers are listed in Additional file 6: Table S3). Actin was used as the reference gene according to [81 (link)]. In brief, cDNA was synthesized using ReverseTra Ace qPCR-RT Kit (Toyobo, Japan). The reverse transcription reaction system included 0.5 μL primer mix, 2 μL RNA template, 0.5 μL RT enzyme mix, 2 μL 5 × RT buffer, 5 μL ddH₂O. In reference to the corresponding unigene sequence, gene-specific primers were designed using the online tool [GenScript Real-time PCR (TaqMan) Primer Design, https://www.genscript.com/ssl-bin/app/primer]. And the efficiency of the primer pairs were checked by serial dilutions of template cDNA as shown in Additional file 7: Figure S3. The cDNA was diluted to 100 ng μL⁻¹ and then used for qRT-PCR test with each gene-specific primers and SYBR® Green Real time PCR Master Mix (Toyobo, Japan) on the Bio-Rad iQ5 real time system. Reactions were conducted at 96 °C for 1 min, 40 cycles of 95 °C for 15 s, 60 °C for 15 s and 72 °C for 45 s.