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[…] The paired sequence reads were trimmed for adapter removal with FASTQ Quality Trimmer [] to a minimum of 80% of the original sequence length, poor quality reads were eliminated using a minimum Phred score of 32. For each seed coat cDNA library, the Illumina sequence reads (in FASTQ format) were mapped to the genomic sequence of the P. vulgaris G19833 reference genome (assembly version 1.0; [, ]) with Bowtie2 using default parameters, including a maximum sum of mismatch qualities across the alignment of 70. The data was analyzed for exon-exon junctions in TopHat as described previously []. Transcriptome assemblies were generated in Cufflinks, and annotation was performed with Cuffcompare. Differentially expressed genes were identified with Cuffdiff, and transcript abundance was reported as FPKM, using cummeRbund in R [].A cluster analysis was performed to identify genes with similar expression patterns in the seed coat transcriptome. To this end, raw read counts for all differentially expressed genes were obtained from Binary Alignment/Map (BAM) files using samtools [] v0.1.17 and HTSeq v0.6.1p2 []. Clustering of genes was performed with the HTSCluster v2.0 package [] in R [] with the number of clusters ranging from 1 to 50. A model containing 14 clusters was selected a posteriori using the model selection criterion Dimension jump []. Thereafter, GO enrichment analysis was performed on the gene cluster model conducted using the Singular Enrichment Analysis tool available on AgriGO v1.0 [] with a significance level of 5% using Fisher statistical testing and Yekutieli multi-test adjustment.A TFBS enrichment analysis was performed for all differentially expressed genes. To this end, we downloaded the Phaseolus vulgaris genome assembly (Pvulgaris_218_v1.0.fa) and its annotation (Pvulgaris_218_v1.0.gene.gff3) from Phytozome [, ]. All scaffolds were removed from the genome assembly, and chromosomal sequences were retained. To investigate groups of genes for transcription factor binding sites, gene start positions were isolated from the .gff3 file. Differentially expressed genes with no annotated sequence in the bean genome were excluded from the analysis. For each gene group, we extracted sequence 500 bp upstream from each transcription start site, excluded Ns (and nucleotides upstream of Ns), and searched the sequence and its reverse complement for one or more motif binding sites. The analysis searched the following sites: C[AGCT]GTT[AG] and CACGTG, where [AGCT] indicates any single nucleotide, and quantified the number of genes within each group of differentially expressed genes with at least one binding site. All analyses were performed with custom perl scripts.PCA was performed in R [] to determine whether there was an association between RNA-seq transcript profiles and proanthocyanidin accumulation patterns in cranberry bean seed coats. In order to generate scores for the PCA, transcript levels of the differentially expressed genes (expressed as FPKM) corresponding to each of the 18 seed coat replicates were converted to uncorrelated variables using an orthogonal linear transformation. Thereafter, the components accounting for 95% of the cumulative variance were considered for the correlation analysis. A correlation analysis was performed between the selected PCs and the seed coat total extractable proanthocyanidin levels in R. A score plot was generated for the PCs that were highly correlated with seed coat proanthocyanidin levels. Finally, transcripts with the highest contribution for each of these PCs were identified with a loading plot analysis. [...] Recombinant PvANR1 activity was assayed in vitro at 30 °C in a final volume of 400 μL. PvANR1 activity was directly proportional to the amount of recombinant enzyme added (within the 10–90 μg range) to the assay mixture and time (2 to10 min). Unless otherwise mentioned, assays included 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (pH 7.0), 800 μM NADPH, 100 μM cyanidin chloride (Extrasynthese), 50 μg recombinant PvANR1, and 5% (v/v) methanol. Cyanidin chloride was freshly prepared in 96% (v/v) methanol containing 0.4 mM of methane sulfonate buffer (pH 2.0), and reactions were initiated by their addition to the assay mixture and incubated for 10 min at 30 °C. All assays were terminated by the addition of ethyl acetate (500 μL), and reaction products were partitioned into the organic phase by vortexing for 30 s and centrifugation at 18800 x g for 1 min. Upon removal of the organic phase, the aqueous phase was re-extracted with ethyl acetate as described above. The organic layers from the successive extractions were pooled and dried under a stream of argon gas at room temperature. As a control, assays were performed in the absence of recombinant PvANR1.The dried reaction residue was resuspended in 100 μL of methanol, filtered with 0.45 μm polytetrafluoroethylene syringe filter (Mandel Scientific Company Inc.), and 5 μL injections were separated on a Kinetex pentafluorophenyl column (100 × 4.6 mm, 2.6 μm Phenomenex, Torrence, California, USA) coupled to an Agilent 1200 HPLC-DAD system. The reaction products were eluted with a gradient of solvent B (CH3CN:C2HF3O2, 99.9:0.1, v/v) in solvent A (H2O:CH3CN:C2HF3O2, 90:9.9:0.1, v/v/v) of 0–25%, 0–10 min; 25–81.8%, 10–20 min; and 81.8–100%, 20–25 min at a flow rate of 0.8 mL min−1. In assays containing cyanidin, peaks corresponding to catechin (retention time = 3.9 min) and epicatechin (retention time = 4.7 min) were detected at 280 nm. In either case, retention times and UV spectra were compared to known amounts of authentic (+)-catechin and (−)-epicatechin standards (both from Extrasynthese). Kinetic parameters were estimated using non-linear regression models available in SigmaPlot (version 12.3) Enzyme Kinetics Module. […]

Pipeline specifications

Software tools Bowtie2, TopHat, Cufflinks, CummeRbund, SAMtools, HTSeq, HTSCluster, agriGO, SigmaPlot
Databases Phytozome
Applications Miscellaneous, RNA-seq analysis, Transcriptome data visualization
Chemicals Catechin, NADP, Quinones, Proanthocyanidins