Supplemental Data

Files in this Data Supplement:

• Supplemental Table 1 - Genetic analysis of flower colour and pattern in the genus Antirrhinum.
• Supplemental Figure 1 - Alignment of sequences of MYB-related transcription factors controlling anthocyanin biosynthesis in different plant species. Sequences were derived from Arabidopsis; At MYB75 (At1g56650), At MYB90 (At1g66390), At MYB113 (At1g66370), At MYB114 (At1g66380) and At MYB123 (TT2) (At5g35550), Petunia; Ph AN2 (AF146702), tomato; Le ANT1 (AAQ55181), grape; Vv MYBA1 (BAD18977) and Vv MYBA2 (BAD18978), maize; Zm MYBC1 (X06333), A.majus; Am Rosea1 (DQ275529), Am Rosea2 (DQ275530), Am Venosa (DQ275531), and as outliers At MYB0/GL1 (At3g27920), At MYB66/WER (At5g14750) and At MYB23 (At5g40330) from Arabidopsis. The region used for phylogenetic analysis is indicated by a solid line immediately below the sequence. Within their DNA binding domains, Ros1 differs from Ros2 by 5 out of 11 amino acids in helix 1 of R2. Not all of these differences are conservative substitutions. Within this region Ve differs from Ros1 by 3 amino acids (two of which, I/T and K/R, are conservative substitutions). In helix 2 of R2, Ros1 differs from Ve by one non-conservative change (H/I) in 9 amino acids and Ros2 differs from Ros1 by two amino acid substitutions, one conservative and one non-conservative in this region. In the third helix of R2, Ve differs from Ros1 and Ros2 by one amino acid in a total of 10 (M/L), a conservative substitution. In helix 1 of R3 there are no differences between the three proteins. In helix 2 of R3, Ros1 and Ros 2 differ from Ve by two amino acids out of 9; K/R and L/I, both conservative substitutions. In helix3 of R3, Ve differs from Ros1 and Ros2 by one amino acid out of 10; G/A, a conservative substitution. Several amino acids of helix 1 and helix 2 of R3 have been identified by Grotewold et al.,(2000) as important for interaction with BHLH proteins (L76, R79, R82, L83, G94 and R95; C1 numbering). These residues are conserved in all the anthocyanin regulatory proteins including Ros1, Ros2 and Ve except for R82 which in the Antirrhinum proteins, AN2 from Petunia and MYB90 from Arabidopsis is K, (a conservative substitution).
• Supplemental Figure 2 - Phenotypes from genetic analysis of A.graniticum, A.meonanthemum and A.latifolium. A). Phenotypes from genetic analysis of A.graniticum: Top row: A.graniticum Middle: Phenotypes from F₁ plants from crosses of A.graniticum x A.majus (WT), A.graniticum x A.majus (ros^col), A.graniticum x A.majus (ros^dor Ve), A.graniticum x A.majus (mut del). Bottom row: Phenotypes of plants segregating in the F₂ generation of A.graniticum x A.majus (WT). B). Phenotypes from genetic analysis of A.meonanthemum: Top row: A.meonanthemum Middle: Phenotypes from F₁ plants from crosses of A.meonanthemum x A.majus (WT), A.meonanthemum x A.majus (ros^dor Ve), A.meonanthemum x A.majus (mut del). Bottom row: Phenotypes of plants segregating in the F₂ generation of A.meonanthemum x A.majus (WT). The ratios for the relative numbers of each phenotype in the F₂ generation are given below the pictures. C). Phenotypes from genetic analysis of A.latifolium (Marseilles). Top row: A.latifolium (Marseilles). Bottom row: Phenotypes from F₁ plants from crosses of A.latifolium x A.majus (WT) and A.latifolium x A.majus (ros^dor).
• Supplemental Figure 3 - Expression of the gene encoding DFR in flowers with different phenotypes shown by saturating RT-PCR. DNA gel blot of RT-PCR products amplified with 40 cycles from first strand cDNA from flowers of 1). wild type, 2). del, 3). ros^col, 4). ros^dor (greenhouse grown), 5). mut/del,6). ros^dor (grown outside), 7). ros^dor Ve (grown outside) and 8). control (no DNA) samples. The blot was probed with a radioactively labelled EcoRI-BamHI fragment of the genomic DNA of the DFR (pallida) gene of A.majus. A low level of DFR expression was detected in all ros lines.