ACT3

MYB1

bHLH1

28 !

26!

28 !

Fig. S1 Expression analyses of two transcription factor genes in CrV and CrW.

CrV CrW Cycle !no.

Additional file 2: Figure S1

RT-PCR analysis of TfMYB1 and TfbHLH1 and the β-actin gene. TfMYB1 and TfbHLH1 are suggested to be involved in anthocyanin biosynthesis in torenia. Gene names and PCR cycles are shown to the left and right of each panel, respectively.

B

! ! !I V V S R R R L P P G P T G F P !CrV ⋅⋅⋅⋅ATCGTCGTCTCCCGCAGACGGTTACCCCCCGGCCCAACGGGCTTCCCC⋅⋅⋅⋅ CrV, CrW ⋅⋅⋅⋅ATCGTCGTCTCCCGCAGACGGTTACCCCC-GGCCCAACGGGCTTCCCC⋅⋅⋅⋅

! !I V V S R R R L P P A Q R A S P 　

ΔC!

exon1!TfF3′5′H

A

! N L F A A G T D T T S !CrV ⋅⋅⋅tttttctttctgcagAACCTTTTTGCTGCGGGAACGGACACGACGTCG⋅⋅⋅!CrV,CrW ⋅⋅⋅tttttctttctgcagAACCTTTT------------GGACACGACGTCG⋅⋅⋅ !

! ! N L L D T T S  

exon3!TfF3′H

Δ12bp

exon2!

exon1!

Fig. S2 Schematic structure and mutated sequences of ThF3′H and ThF3′5′H genes.

exon1!

500bp Additional file 2: Figure S2

(A) The ThF3′H gene consists of three exons and two introns. The deduced amino acid sequence corresponding to the 12-bp deletion in CrW is shown. Small letters indicate intron sequence, and an acceptor site (ag) is boxed. (B) The ThF3′5′H gene consists of two exons and one intron. The deduced amino acid sequence corresponding to the cytosine deletion in CrW is shown.

! ! ...GCACGAACCAAGTCATTCATTTAATGATGTGAGAAAATGTCATACTTTAATTGATATTTTGGAATACATGTGGAGAAATTAGCTCATGAACTTGGCTAGTTGGAGCTGAAA! ! TSD!

!ACTGGTTGCATGGTTTTGTTTCTGGTTGTGTGAAGAATCGTCGATGCTTATGAGAAGAAAATGCAGTGGAAGAGTAAAGAGTTATAAGTGTGTTATCACTTATAAAGTTGC!!TGTTTACTATATCACGTTTTCTTTGAGAGCTGCACATGGGAACGGTTAATGAGCAGTTATTAAGCTGAAGCAAGATTGTGTGTTCTGTTAGTTCGGTTTCAAGTAGCAGTT!!TTGCAGATCTCATAACTTGGTGTGTAGTTAGCCAATTTAAGCGATTTAAATTGCGTTGAAAAGCTCACATTACATACAACACATTGTAACTCATCATACTCAATAAAATTC!!TCTCTTCGTCCCGTGGAGTAGGGCTAAAGTTAGCGCCGAACCACGTTAAATCTGTTTTCTTCTGAGACTTTTCTGATTTTCTGTTCTTATTCAATCAAGTTTCTTTCGATC!!CAAACGCGTAGTAAACATATCACAAGACTTGTTAACTTTAATCTTGAGCATCTAAGAGCAAGAATTCTCAACAAGTGGTGCGGTGAGCGTGGATAACTCGAAGATGGCTGC!

PBS ! M A A!!GAGGTTCGAAGTTGAAAAGTTCACGGGTGATAATGACTTCGGTTTGTGGAAAATGAAGATGAGGGCGTTGCTTACCCAACAAGGGCTGATAGAGGTGTTGATGGTGGAGGA!

!R F E V E K F T G D N D F G L W K M K M R A L L T Q Q G L I E V L M V E D!!TCCACCGGCCACAGTGGTGGCTGGGACTGCTCCGACGGGCCAAGAAGATGCCGCTGCTGCAGCTGTAAATGCACAAGCTGCGGCAGAGAAGAAAATCTTGGATTCGAAGGC!

!P P A T V V A G T A P T G Q E D A A A A A V N A Q A A A E K K I L D S K A!!GCATTCAGTGATCATTCTGAGTTTGGGAGATCGAGTCTTGCGTCAAGTCTCTCATGAATCAACCGCTCTTGGTCTGTGGAAGAAATTGGAAGAGCTTTACATGACAAAGTC!

!H S V I I L S L G D R V L R Q V S H E S T A L G L W K K L E E L Y M T K S!!TCTTGCCAATCGACTTTATCTCAAGCAAGCGCTCTATTCGTTCAAGATGATTGAAGAAAAGGCCATTGATGAGCAGATGGATCAGTTTATCAAGCTGATTTTGGATCTTGA!

!L A N R L Y L K Q A L Y S F K M I E E K A I D E Q M D Q F I K L I L D L E!!AAACATCGAAGTGAAGATAGAGGATGAGGATCAAGCCTTGCTCTTGGTGTGTGCTTTGCCTCGGAGTTACAACACATTCAAGGACACGTTGTTGTATGGCCGAGAGACTTT!

!N I E V K I E D E D Q A L L L V C A L P R S Y N T F K D T L L Y G R E T L!!GACTTTGAAAGAGGTGCAAGCCGCGTTGAAGTCGAAACAGCTGAACACGAGGATTGATAACAAGGCAGTGGGATCTACATCCGAGGCTCTTTATGTTAAAGGGAAAGGTGA!

!T L K E V Q A A L K S K Q L N T R I D N K A V G S T S E A L Y V K G K G E!!AGAGAAGAAAACACACAAAGAAAGAAAGAACAAGTCCAAAAAGAAGGTTAAATGTTTCTATTGCGATGAGGAGGGTCATATGTGCAAGAATTGTCCCAAGAAGGAGAGAGA!

!E K K T H K E R K N K S K K K V K C F Y C D E E G H M C K N C P K K E R D!!CAAGGGCAAGAAAGTGGAGCAAGGCGAGGCTGCAATGGCTTGTGAAAGTTATGAGTCGGCTGACGTCTTGGCCGTGACTCATGAAGATCAAGATGTGACCAAATCTGAAAA!

!K G K K V E Q G E A A M A C E S Y E S A D V L A V T H E D Q D V T K S E K!!ATCTGGTAAATGGCTACTTGATTCCGCTTCTTCTTTTCATGTCACATGTGTTAAGTCTTGGATTAAAGACTTTAAGGGTTGTGATGGTTGTCTGGTCTCTGTTGGGGAAGA!

!S G K W L L D S A S S F H V T C V K S W I K D F K G C D G C L V S V G E E!!AAAGCAATACAAAATTCTGGGCTTTGGAACGGTTAAAATAAGGCTTAAAACCGGTGGAGTAAGAATTTTGAGAAATGTGAAGTTCATTCCGGATTTGGGTAGAAACCTTAT!

!K Q Y K I L G F G T V K I R L K T G G V R I L R N V K F I P D L G R N L I!!TTCTGTAGGGTTGTTGGATGTTCAAGGCTTTAAGTGTGTTGCTGGAAATGGTGTGATGAAGGTTTTCAAAGGGTCTAAGGTGATTATGAGTGGTACTTTGCAGAAAAACAG!

!S V G L L D V Q G F K C V A G N G V M K V F K G S K V I M S G T L Q K N R!!AACTTATCATGTTACAGGATCTGAAACAGTGGTGAACTGGGCTGGTTTGGGTGCTCGTAAAATCTGCTGTGGTGATTCTGGGAGTGAGAGGTTTGTTCAGGGTAATAGTAT!

!T Y H V T G S E T V V N W A G L G A R K I C C G D S G S E R F V Q G N S I!!CCTATGTGGTGATCATGATAAGGCCGGGTTTGAGCAGGTTACACAAGGGGAGATCACAGATCAGAGGGCTCAAAGTGAGGGATCTGACACTTTATTGTCTGAGGTGGAGCA!

!L C G D H D K A G F E Q V T Q G E I T D Q R A Q S E G S D T L L S E V E Q!!GATTTGTGTGAGAGTTGGTAGAGTTCTTTGTCCAGTTTCTGGGAAAGATTTGAATTATCTGAGGAATGGGGATATTCCTGAGGCTGATTGTGGTGGCCAACTACAAGGATC!

!I C V R V G R V L C P V S G K D L N Y L R N G D I P E A D C G G Q L Q G S!!GAATCTGCAAGTGACAAAGGAAGTGGGACTTATCAAGTTTGAAAGGCTGAAAAATGTGATTTCTGAGTCGTGTTTGGTGGTGGATAACTGTGGTGCTTCGAGTTTTCCTGG!

!N L Q V T K E V G L I K F E R L K N V I S E S C L V V D N C G A S S F P G!!GGCTGCTGAAGATCAAACTCAGTTTAATCTGCAGAATGGGAGTTGGTTTACAGCTGAGGTGGAGGAAAATGAAGCAAATGGTCAGTTTTATGAATGCTCAAAATCTGATGA!

!A A E D Q T Q F N L Q N G S W F T A E V E E N E A N G Q F Y E C S K S D D!!TGGCAGCAAGCTTGGAACAACAGTGGGCAAGTAGGTACCTTTGCGAGGTTGTAGCAGGCTGTGTGGGTGGATTTTGAGGAGGCTGTGGGATGCTTCGGGTTCACATCATAT!

!G S K L G T T V G K *!!GGTACTTCAATGTTCTGGGGATTTAACTCAGGTGGAGTTAAAGAGGAATCCACAAATTAAGCTCAAAGTACTCGGTTTGCGCATATTTGGTACAGAGTTTCTGGTTTGTTT!

!!TTGGGGGCTAAAACTTGGTTTTCTGGGCTGTTAACAAGTTGTTCTTGTGCTGAGAAAGGTTGTTAGCTGTGAGGTAACCAGAGTTGTGGATGTAGAACTGAAGTTTGGACT!!TGATGTGATAGTATTTGAGGTTGATCGAGTGAATACAAGTTCGCTGAAAAATTCTCAAATTTGTGTTCGTTTGGCTGGTTTCTGGGGCGTGTTCTGGATCGTTCTTGAGGG!!AATTCAGCTGGATGTTGTTTGCTTGGGAAAGTAGGCATGGTGGACTGAAGAGGGAAGTGGAGAGTTAATGAGCTAAGGTGGAGATTTGTGGAGAAATTAGCTCATGAACTT!

! PPT!!GGCTAGTTGGAGCTGAAAACTGGTTGCATGGTTTTGTTTCTGGTTGTGTGAAGAATCGTCGATGCTTATGAGAAGAAAATGCAGTGGAAGAGTAAAGAGTTATAAGTGTGT!!TATCACTTATAAAGTTGCTGTTTACTATATCACGTTTTCTTTGAGAGCTGCACATGGGAACGGTTAATGAGCAGTTATTAAGCTGAAGCAAGATTGTGTGTTCTGTTAGTT!!CGGTTTCAAGTAGCAGTTTTGCAGATCTCATAACTTGGTGTGTAGTTAGCCAATTTAAGCGATTTAAATTGCGTTGAAAAGCTCACATTACATACAACACATTGTAACTCA!!TCATACTCAATAAAATTCTCTCTTCGTCCCGTGGAGTAGGGCTAAAGTTAGCGCCGAACCACGTTAAATCTGTTTTCTTCTGAGACTTTTCTGATTTTCTGTTCTTATTCA!!ATCAAGTTTCTTTCGATCCAAACGCGTAGTAAACATATCACAAGACTTGTTAACTTTAATCTTGAGCATCTAAGAGCAAGAATTCTCAACAATACAATTAATAGGTACTCT...!

! TSD!Fig. S3 Sequence of the 5′-upstream region of F3H in CrW. The figure shows 5 bp of the target site duplication (TSD; blue letters), 560 bp of long terminal repeats (LTRs; red letters), the primer binding site (PBS; green letters), and the polypurine tract (PPT; brown letters). The amino acid sequence deduced from the open reading frame (ORF) is indicated by black letters.

Additional file 2: Figure S3

Fig. S4 Insertion of the solo-LTR in 5′-upstream region of F3H in CrW.

3.6k ATG TfF3H

-249

TGTAT TGTAT

500bp

LTR 560bp TSD TSD

ACGT containing element !　　　(CACGT)

P recognition element !　　　(CCT/AACC)

Binding sequence of vertebrate MYB protein !　　　　(C/TAACT/GG)

+1

Additional file 2: Figure S4

A putative solo-LTR of 560 bp was identified in F3H promoter of CrW. It was flanked by 5-bp target site duplications (TSD) and the position was identical to the TORE1 insertion site.

H E X H E X CrW CrV

H E X H E X CrW CrV

Fig. S5 Southern blot analysis of F3′H and F3′5′H in CrV and CrW.

10 6

4

3

1.5

2.5

2

1

A B

Additional file 2: Figure S5

Total genomic DNAs were digested with Hind III (H), Eco RI (E), and Xba I (X) and transferred to nylon membranes as described in Methods. Membranes were probed with DIG-labeled sequences of F3′H (A) and F3′5′H (B). DNA marker sizes (kbp) are shown.

//

E H H X X E E E H

TORE1

Xba I

Eco RI

Hin dIII

10.5 kb

3.0 kb

4.5 kb 3.7 kb 4.7 kb

//

E H H X X

4.5 kb 4.9 kb

Xba I

Eco RI

Hin dIII

5.2 kb

7.0 kb

Fig. S6 Schematic diagram of the genomic structure of the F3H gene in CrV and CrW.

Probe

Probe

CrV

CrW

Additional file 2: Figure S6

Sites of restriction enzymes Xba I, Eco RI, and Hind III used in the Southern blot analysis are shown. Restriction sites at the 3′-proximal region were deduced from the results of Figure 4. Lengths following complete digestion are shown in blue, green, and red for Xba I, Eco RI and Hind III, respectively. The F3H probe used is shown in orange.

Fig. S7 HPLC analysis of flower petal anthocyanidins in GtF3H-overexpressing transgenic CrW.

1.75

1.93

2.36 2.63

2.75

12.15

14.00

14.45

16.32

16.72

17.03

17.28

18.81

19.56

21.55

21.85

22.56

24.71

0 2 4 6 8 10 12 14 16 18 20 22 24

保持時間(min)

0.00

0.05

0.10

0.15

0.20

0.25

信号強度(AU)

1.67

1.93

2.33 2.63

2.73

10.21

11.85

12.12 13.92

24.73

0 2 4 6 8 10 12 14 16 18 20 22 24

保持時間(min)

0.00

0.05

0.10

0.15

0.20

0.25

信号強度(AU)

Dp

Cy

Pg

1.76

1.95

2.35 2.64

2.76

13.96

18.13

21.87

24.65

0 2 4 6 8 10 12 14 16 18 20 22 24

保持時間(min)

0.00

0.05

0.10

0.15

0.20

0.25

信号強度(AU)

Pg

CrW

Standard

No. 4

Sign

al in

tens

ity (A

U)

Sign

al in

tens

ity (A

U)

Sign

al in

tens

ity (A

U)

Retention time

Additional file 2: Figure S7

After acid hydrolysis of petal extracts, the hydrolysates containing anthocyanidins were subjected to HPLC analysis. Dp, Cy, and Pg correspond to delphinidin, cyanidin, and pelargonidin, respectively.

Fig. S8 Example of pigment recovery in a CrW petal. (A) Whole flower. (B) Magnification of boxed red area in A.

A B

Additional file 2: Figure S8