Transcriptome Analysis and Validation in Brassica

Gene ontology (GO) enrichment analysis was conducted on the DEGs using GOseq R software [25 (link)]. KOBAS software was used to perform DEG enrichment statistics in the KEGG pathway [26 (link)]. Brassica Database v3.0 was used as reference lists [19 (link)]. Twelve DEGs were randomly selected to confirm the transcriptome data by qRT-PCR analysis. Gene-specific primers were designed using Primer v5.0 (Table S2). The normalized eEF1Bα2 gene(BraA10g020830.3C.gene) was used as an internal control of gene expression [27 (link)]. The quality-assured RNA was used as template, with the synthesis of cDNA processed by a Takara PrimeScript RT reagent Kit (Takara, Nojihigashi, Kusatsu, Japan). The Bio-Rad CFX96 RT-PCR Detection system (Bio-Rad, Hercules, CA, USA) and SYBR Green II PCR Master mix (Takara) were used for the qRT-PCR reactions. The gene expression data were analyzed using the 2^−ΔΔCt method [28 (link)]. SPSS v19.0 (SPSS, Chicago, IL, USA) was used to conduct the one-way analysis of variance (ANOVA) with Duncan’s multiple range test using a significance threshold of p < 0.05. The results were visualized using SigmaPlot v10.0 (Systat Software Inc., San Jose, CA, USA).