Differences in DNA and mRNA expression.

<p>(A) The insertion location is shown in gray rectangles in the ORF of the <i>BrTT8</i>. The black rectangles represent the exons, and TL1, TL2, TR1 and YCR1 are the primers that were developed from the corresponding exon sequences and insert sequence. The arrows are used to indicate the directions of the primers. The insertion sequences and flanking <i>BrTT8</i> intron 2 are shown in red and black, respectively. The conserved sequences at the termini of the element are underlined. Palindromic sequences that are capable of forming a hairpin are shown in blue. (B) The amplification products of the genomic DNA of the yellow-seeded (1) and black-seeded line (2) using the primers TL1 and TR1. (C) The primers TL2, TR1 and YCR1 amplified the genomic DNA from the three genotypes: (1) the homozygous yellow-seeded, (2) heterozygous black-seeded, (3) homozygous black-seeded plants. (D) mRNA levels in the immature seeds of the yellow-seeded line (three on the left) and black-seeded line (three on the right). The numbers 10, 20, and 30 signify the number of days after pollination. (E) 18S control.</p

BrTT8 shows features of a bHLH DNA-Binding domain protein.

<p>(A) Amino acid comparison of the bHLH domain of <i>BrTT8</i>, <i>B. napus</i> (ABY59772.1), <i>B. rapa</i> (AEA03281), <i>B. oleracea</i> (ADP76654.1), <i>Arabidopsis</i> (NP_192720.2) and <i>Populus</i> (XP_0023067). (B) Dendrogram of the relationships among the bHLH domains from several bHLH-related proteins. For the construction of the tree, the BrTT8 protein sequence and other selected bHLH-related proteins were used. The sequence similarity was calculated using the MEGA programme to generate a branching pattern. The numbers below the branches indicate the percentages of bootstrap support after 1000 replicates. The sequences used are <i>Brassica rapa</i> AEA03281, <i>Arabidopsis</i> NP_192720.2(AtTT8), <i>Brassica napus</i> ABY59772.1, <i>Brassica oleracea</i> ADP76654.1, <i>Populus</i> XP_002306769.1, <i>Vitis vinifera</i> CBI32369.3, <i>Lotus</i> BAH28881.1(LjTT8), <i>Raphanus</i> AEO53065.1, <i>Pisum sativum</i> ADO13282.1, <i>Perilla</i> BAC56998.1(F3G1), <i>Ricinus</i> XP_002520758.1, <i>Malus</i> AEI84807.1, <i>Petunia</i> AAG25927.1(AN1), <i>Nicotiana</i> AEE99260.1, <i>Nicotiana</i> AEE99258.1(NtAN1b), <i>Hordeum vulgare</i> BAJ92594.1, <i>Lilium</i> BAE20058.1, <i>Sorghum</i> XP_002448313.1, <i>Dahlia</i> BAJ33515.1, <i>Oryza</i> NP_001053530.2, <i>Zea mays</i> NP_001105706.1, <i>Cornus</i> AAR21675.1, <i>Cornus</i> AAS86268.1, <i>Oryza</i> EEC77782.1, <i>Mimulus</i> ACA04013.1, and <i>Gynura bicolor</i> BAJ17663(GbMYC1).</p

Expression of flavonoid biosynthetic genes in developing seed.

<p>Seeds were obtained from the yellow-seeded and black-seeded plants 10 days after pollination. The expression of the different genes was detected by quantitative Real-time PCR. Transcripts for two flavonoid EBGs <i>BrTT6</i> (encode F3H, flavanone 3-hydroxylase) and <i>BrTT7</i> (encode F3′H, flavanone 3′-hydroxylase), three flavonoid LBGs <i>BrDFR</i> (encode dihydroflavonol reductase), <i>BrBAN</i> (encode anthocyanidin reductase) and <i>BrLDOX</i> (encode LDOX, leucoanthocyanidin dioxygenase) in <i>B. rapa</i>. The comparative Ct method was used to calculate the levels of transcripts relative to black-seeded plant. (“B” in the legend is the black seed and “Y” is the yellow seed).</p

Seed coat structure of the <i>B. rapa</i>.

<p>(A) The black seeds of the <i>B. rapa</i>. (B) The whole black seed treatment with Safranine O and Fast Green. Arrowheads show the Phenolic compounds are localised in the seed coat of immature seed (20 days after flower). (C) Magnified image of (B). Phenolic compounds stain red, formed granules are localised in the endothelium cell of the ii1 layer (circle in C). (D) The yellow seeds of the <i>B. rapa</i>. (E) The whole yellow seed treatment with Safranine O and Fast Green (20 days after flower). Phenolic compounds are absent in the endothelium of the yellow seed (arrowhead in E). (F) Magnified image of (E). There are only unknown fragmented structures in the region of the endothelium cell layer (circle in F). EM, embryo; EN, endosperm; ii, inner integument; Oi, outer integument. Bar(N) = 100 µm for (B) and (E); 50 µm for (C) and (F).</p

Mapping of the <i>BrTT8</i> gene.

<p>(A) The genetic linkage map of the <i>BrTT8</i> gene. The markers gsr23, gsr44, and gsr29 are derived from Scaffold000135. (B) BLAST analysis with the <i>Arabidopsis</i> genome showed that Scaffold000135 shared similarity with a region on chromosome 4. The rectangles containing <i>Arabidopsis</i> genes present several representative genes (E<10⁻³⁰) in this region.</p