Spatial Expression Analysis of BraMAPK Genes

To investigate the spatial expression patterns of BraMAPK genes, the RNA-seq data were downloaded from the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE43245. The gene expression levels were quantified as FPKM (fragments per kilobase of exon per million fragments mapped) values by the TopHat/Cufflinks pipeline in the previous report [66 (link),67 (link)]. The log₂-transformed (FPKM + 1) values of the 32 BraMAPK genes were used for heat map generation.
To determine the expression profiles of BraMAPK genes in the second youngest leaves sampled from treated and control plants, quantitative real-time PCR (qRT-PCR) was performed using SYBR Premix Ex Taq II (Perfect Real Time) (TaKaRa, Dalian, China) in a CFX96 real-time PCR system (Bio-Rad, USA) with the following conditions: 95°C for 2 min and then 40 cycles of 95°C for 10 s, annealing for 30 s with designated temperature listed in S2 Table, and 72°C for 20 s. Gene-specific primers were designed using Geneious Pro 4.8.5 software with previously described parameters [59 ,68 (link)]. Using gradient PCR with the cDNA as template, the highest feasible annealing temperatures of primer pairs were determined and listed in S2 Table. To ensure primer specificity a BLASTN search against the whole B. rapa genome was performed, and agarose gel electrophoresis and melting curve analysis ensured that only one product was amplified. Three technical replicate reactions were implemented at each time point. All the qRT-PCR assays were repeated three times. Two endogenous housekeeping genes, B. rapa UBC21 (ubiquitin conjugating enzyme 21) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase), were used for normalization and quantification of fold changes of BraMAPK genes using the 2^–ΔΔCT method [69 (link)–71 (link)]. Genes with (1) ΔΔCt values of > 1 or < -1 and (2) p-value < 0.05 (determined by two-tailed Student's t-test) were considered as being significantly regulated. The sub-functionalization of duplicated members in the same BraMAPK gene subfamily were statistically measured by two-way analysis of variance (ANOVA).
To verify the consistency of expression levels of BraMAPK genes in leaves between qRT-PCR and RNA-seq methods, and compare the expression patterns of MAPK genes between B. rapa and Arabidopsis, the expression data of AtMAPK genes were obtained from AtGenExpress (http://www.weigelworld.org/resources/microarray/AtGenExpress) databases [72 (link)]. The relative expression levels of BraMAPK genes in leaves after 0 h of mock treatment in qRT-PCR analysis were calculated as Ct(IC)-Ct(X) ('Ct' stands for cycle number; 'IC' stands for two internal control genes; 'X' stands for BraMAPK genes), while those in RNA-seq analysis were calculated as Log₂ (FPMK + 1) transformed expression value ((IC)-(X)). In Arabidopsis, the relative expression levels of AtMAPK genes in leaves were calculated as Log2 transformed expression value ((IC)-(X)) ('X' stands for AtMAPK genes). All the Pearson correlation coefficients were estimated by using SPSS 21 (IBM SPSS Statistics, IBM Corporation).