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Legends for Supplementary Material
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Figure S1. Comparison of Brassica TT16 genomic sequences. Exons and introns were
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labelled with black and white backgrounds respectively. Bn, B. napus; AA, B. rapa; CC,
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B. oleracea. The TT16 genomic DNA sequences cloned in this study were submitted to
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GenBank as follows: BnTT16.1, JF970611; BnTT16.2, JF970612; BnTT16.3, JF970613;
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BnTT16.4, JF970614; BoTT16.1, JF970615; BoTT16.2, JF970616; BoTT16.3, JF970617;
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BoTT16.4, JF970618; BrTT16.1, JF970619; BrTT16.3, JF970620; BrTT16.4, JF970621.
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Figure S2. Schematic diagram of BnTT16 overexpression constructs used to complement
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the Arabidopsis tt16-6 mutant phenotype.
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Figure S3. Localization of proanthocyanidins using p-dimethylaminocinnamaldehyde
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staining in developing seeds of complemented Arabidopsis lines. (a) wild-type
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Arabidopsis Ws-3 as a positive control; (b) Arabidopsis transparent testa 16-6 (Attt16-6)
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mutant as a negative control; (c) representative complemented line expressing Brassica
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napus BnTT16.1 (Attt16-6/35S::BnTT16.1); (d) representative complemented line
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expressing BnTT16.2 (Attt16-6/35S::BnTT16.2); (e) representative complemented line
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expressing BnTT16.3 (Attt16-6/35S::BnTT16.3); (f) representative complemented line
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expressing BnTT16.4 (Attt16-6/35S::BnTT16.4). Scale bar = 100 µm. The arrows indicate
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the endothelium containing PA.
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Figure S4. RNAi-mediated silencing of BnTT16 expression. (a) Schematic diagram of
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the construct utilized for RNAi silencing of BnTT16 homologs in B. napus. (b) The
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overall level of expression of BnTT16 genes in open flowers (0-DAP) was down-
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regulated in Bntt16 RNAi lines. (c) The expression level of B. napus AGAMOUS-LIKE 6
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(BnAGL6) exhibited no significant difference between wild-type (WT) and Bntt16 RNAi
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lines (RNAi) (p < 0.05). The data in (b) and (c) were presented as the relative expression
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levels (2-ΔCT) of target genes compared with reference genes (Table S1). 4-1, 10-3, 12-4,
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and 23-4 are 4 representative Bntt16 RNAi lines.
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Figure S5. Transcriptional expression (2-ΔCT) of each individual BnTT16 gene in open
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flowers (0 day after pollination) was down-regulated in RNAi plants. (a) BnTT16.1; (b)
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BnTT16.2; (c) BnTT16.3; (d) BnTT16.4. 4-1, 10-3, 12-4, and 23-4 are 4 representative
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Bntt16 RNAi lines. The data were presented as the relative expression levels of each
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BnTT16 gene compared with reference genes listed in Table S1.
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Figure S6. Vanillin staining of whole developing seeds (14 day after pollination)
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indicated that proanthocyanidin content was less in Bntt16 RNAi plants than wild-type
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plants. (a) Wild-type whole seeds; (b) halved wild-type seeds; (c) whole seeds from
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RNAi plants; (d) halved seeds from RNAi plants. Bars = 1 mm.
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Figure S7. Localization of proanthocyanidins with vanillin in developing seeds of B.
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napus wild-type (WT) and RNAi lines. Arrows indicate PAs that have accumulated in the
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endothelium. (a) WT 15-DAP; (b) RNAi 15-DAP; (c) WT 20-DAP; (d) RNAi 20-DAP;
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(e) WT 24-DAP; (f) RNAi 24-DAP; (g) WT 31-DAP; (h) RNAi 31-DAP. RNAi, Bntt16
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RNAi plants; DAP, days after pollination. Scale bar = 100 µm.
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Figure S8. Global similarity of the eight microarray samples for wild-type B. napus and
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RNAi plants.
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Sample clustering was used to assess the overall similarity of the eight samples. The four
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replicates of wild-type plants (sample 1-4) clustered with one another, while the four
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replicates of RNAi plants formed a separate group (sample 5-8).
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Figure S9. Validation of microarray data (fold-difference) using qRT-PCR. (a) Candidate
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genes involved in flavonoid biosynthesis and regulation. (b) Candidate genes involved in
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seed coat development. Data include the mean relative transcript levels ± standard error
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of four biological replicates and four technical replicates.
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Figure S10. Effects of Bntt16 silencing on the gene expression profile of B. napus (n=4).
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(a) Biological process. (b) Molecular function. (c) Cellular component. GO, gene
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ontology. Unknown and ambiguous genetic programming categories were not included.
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Figure S11. Vanillin staining of representative dissected developing seeds. Samples were
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harvested at 15 day after pollination (15-DAP, cotyledon stage) from wild-type B. napus
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plants. (a) embryo; (b) endosperm; (c) inner integument; (d) epidermis. Bar = 1 mm.
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Briefly, we dissected 15-DAP developing seed coats into three contiguous components:
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inner fraction composed primarily of developing endosperm, middle fraction containing
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inner integument and palisade cells, and outer fraction composed of epidermal and sub-
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epidermal cells (Jiang and Deyholos 2010). To detect which fraction contained the
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endothelium, we stained these three fractions and embryos with vanillin. The middle
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fraction highly stained to red, and the inner fraction was only slightly stained. Since
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almost all PA accumulated in the endothelium at 15-DAP (Figure 7), we confirmed that
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the middle fraction contained virtually all the endothelium. For convenience, we named
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the four sub-tissue fractions as embryo, endosperm, inner integument and epidermis in
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this study.
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Table S1. Primers utilized in this study.
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Table S2. Microarray data
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Table S3. Proteins used for phylogenetic analysis.
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