Supplemental Table S1. Overview of MYB transcription factor genes analyzed for expression in red and pink tomato fruit. MYB Primer pairs TC AtMYB Forward Reverse TC199266 MYB12 AGGCTCTTGGAGGTCGTTACC CAACTCTTTCCGCATCTCAATAATC TC193361 MYB4 TCGTCATCATCAACGATCAA TCACTTCCACAACTGCAATCT TC15914 MYB6 CCGGAAGATTACCTGGAAGA TGTTGAGTGGACGGTGAGTT TC17639 MYB14 CAAGGATTCCAAAGCTCCTT GATGACAATTGTGGCGAGTT TC194241 MYB17 CCGCAATAGCAGCAAGATTAC TTGGAGGATTCAGAAGTTGTAGG TC157319 MYB16 GGAAGGAATTTGGGAACTCA TCCATGTTGTGTCCATTGGT TC172451* MYB13 AGCAAGATTACCCGGAAGAA GCTCCGATTGGGTTTCTTTA TC159371 MYB15 CATGGGATCACGAAGAAGAC ATTCATCCACCTCAATCTGC TC192683 MYB3 TCATGTGATGGGACTCCAAA TGACTCAAATGCTTCCATCAA TC169296* MYB95 GCTACTAAGATGTGGCAAGAG TGATGTCATTGTCTGTTCTACC TC169247 MYB94 AATACTGGTTTGCTTAGATG TTTGTGGGAGATACGAAG TC16745* MYB36 GTGGTAATTGGATTGCTCTTC GGTCCTCCCTGGTAACTG TC157442 MYB43 CCAACGACTCACAATCCAAT CGTCGTTCCAATGTTCTTGT BG13455 MYB49 AAATCATCTTGAACCTCCATGCTC GATCTTCCTGGTAAGTGAATTGCAA AI77179* MYB111 CAAACGAGGACGCAGTAGAA TCGTCATGCCATAACATCTG AY34887* MYB113 GGATCTCATTTTGAGGCTTCATAAG GGAAGTCTACCAGCAATAAGTGACC TC166799* MYB67 GCATCTTGTTCCTGCTAGAGCTG GTCTGCAACTCTTTCGACATCG Abbreviations: TC, TIGR ID of the tomato cdna; AtMYB: ID of most closely-related Arabidopsis MYB transcription factor; *, cdnas showing no significant expression in tomato fruit Supplemental Table S1. Overview of MYB transcription factor genes analyzed for expression in red and pink tomato fruit.
Supplemental Table S2. Overview of bhlh transcription factor genes analyzed for expression in red and pink tomato fruit. bhlh Primer pairs TC AtbHLH Forward Reverse AJ785529 bhlh14, bhlh42, bhlh9 ATTCTACGCTCTGCGTTCTG TTCCAATTCAGCAACTTTCG TC199635 bhlh21, bhlh33, bhlh116 TCTATCCTCTAACACCAACTG GCTCTTCCTTCTCTTGCC TC194613 bhlh27, bhlh35 ATCACCACCATCTTCTTCC AAACACTTCACAAACTTTAACC TC21452 bhlh4, bhlh5, bhlh6, bhlh28 ATGGGATGCAATGATTCGTA TTCACTGTGGCTTGTTGGAT TC15569 bhlh61, bhlh93 CCACACTGGAGGCTTTAGGT TCCTCTCTGCTCCATTTCCT TC16976 bhlh3, bhlh17 GAGGCAACGGAGGGAGAAG ATGTCAGTGATGTGAGCAATGG TC199386 bhlh1 ACAAGTGAAAGTGAAGAAGACC GCACAGCCAAACAGTTCC TC196492 bhlh6 ATCTCCATGACCCAATCGTT CAGTGTGAAGCTGCCAATTT TC166822* bhlh29 GACCGGTCCAGAACTTTGAT TCTCCAATGATGGAGGCTTT Abbreviations: TC, TIGR ID of the tomato cdna; AtbHLH: ID of most closely-related Arabidopsis bhlh transcription factor; *, cdnas showing no significant expression in tomato fruit. Supplemental Table S2. Overview of bhlh transcription factor genes analyzed for expression in red and pink tomato fruit.
Supplemental Table S3. Correlation analysis of expression levels of MYB and bhlh transcription factor and flavonoid genes in several segregating breeding S. lycopersicum population for pink and red fruit. Genes CHS1 CHS2 F3H FLS CHS1 CHS2.844 F3H.887.94 FLS.643.754.851 MYB TC199266.634.641.781.847 TC193361 -.127 -.186 -.68.152 TC15914.4 -.154 -.45 -.65 TC17639 -.95 -.111 -.68 -.152 TC194241 -.258 -.313 -.314 -.43 TC157319 -.135 -.193 -.26.197 TC172451* TC159371 -.99 -.1 -.28 -.38 TC192683 -.214 -.233 -.127 -.22 TC169296* TC169247 -.195 -.268 -.185 -.15 TC16745* TC157442.259.465.718.448 BG13455 -.99 -.124 -.8 -.41 AI77179* AY34887* TC166799* bhlh AJ785529 -.242 -.296 -.335 -.45 TC199635.252.266.161 -.84 TC194613 -.283 -.361 -.373 -.412 TC21452 -.432 -.58 -.361 -.492 TC15569 -.24 -.244 -.273 -.362 TC16976 -.37 -.42 -.369 -.57 TC199386 -.268 -.337 -.34 -.222 TC196492 -.524 -.655 -.513 -.638 TC166882* Note: *, cdnas showing no significant expression in tomato fruit Supplemental Table S3. Correlation analysis of expression levels of MYB and bhlh transcription factor and flavonoid genes in several S. lycopersicum breeding population segregating for pink and red fruit.
Supplemental Table S4. Oligo nucleotides of phenylpropanoid and flavonoid genes used for SYBR Green RT-PCR analysis. Gene TC/Acc.n. Primer pairs Forward Reverse Bibliography PAL M83314 ATTGGGAAATGGCTGCTGATT TCAACATTTGCAATGGATGCA C4H TC2912 CCAATGGCAACGACTTCAGA TCTGCACCAAACGTCCAATG (present article) 4CL TC193457 TGTGAGGAACGCAGAGATG CAATATCGCCAGTGTGTAACC (present article) HQT TC181911 GTGTTTTGTTTGTTGAGGCTGA TGATGAAGTGGATGGATGAGAG (Luo et al., 28) C3H TC183733 TCTCAGTTTACTTCGGTTCAC TTCAATACTTGTTTCGCTAA (present article) HCT TC173823 AGGTGAAAAACTCAACGATGGT ACACTAGGCGTGTGGAAATTAG (Luo et al., 28) CHS1 X55194 GTTCCGTGGACCCAGTGAAT AAAAGGGCTTGGCCTACCA (O'Neill et al., 199) CHS2 X55195 GGCCGGCGATTCTAGATCA TTTCGGGCTTTAGGCTCAGTT (O'Neill et al., 199) CHI-1 TC198728 GAAGCAGTGCTCGATTCCATAAT GTTTTTCACAAACCAACAGTTCTGAT CHI-like TC17875 GCGATAGAAGGTAAGGA AGCCAAAGAAGAAATAGTTGT (Butelli et al. 28) F3H TC171483 CACACCGATCCAGGAACCAT GCCCACCAACTTGGTCTTGTA F3 H TC175149 GCACCACGAATGCACTTGC CGTTAGTACCGTCGGCGAAT FLS TC1728 GAGCATGAAGTTGGGCCAAT TGGTGGGTTGGCCTCATTAA 3GT TC176549 CGAACGACGAAACACTGTTGA TGCAGCATAGATGGCATTGG Supplemental Table S4. Oligo nucleotides of phenylpropanoid and flavonoid genes used for SYBR Green RT-PCR analysis.
3525 3537 FLAVONOIDS 15 1 5 5 4 3 2 1 Naringenin chalcone Rutin 8 6 4 2 Lycopene CAROTENOIDS 1 8 6 4 2 3. 2. 1.. β-carotene Lutein Supplemental Figure S1. of flavonoids and carotenoids in peel of tomato fruit of two populations (3525 and 3537) segregating for pink (empty symbols) and red (black symbols) fruit colour, harvested at three different stages of ripening.
1 2 3 4 5 6 7 8 9 1 EU419748 ATGGGAAGAACACCTTGTTGTGAAAAAGTGGGCATCAAGAGAGGCAGATGGACTGCAGAAGAAGATCAAATTCTCACTAATTATATTATTTCTAATGGAG 1 Moneyberg ATGGGAAGAACACCTTGTTGTGAAAAAGTGGGCATCAAGAGAGGCAGATGGACTGCAGAAGAAGATCAAATTCTCACTAATTATATTATTTCTAATGGAG 1 IL1b ATGGGAAGAACACCTTGTTGTGAAAAAGTGGGCATCAAGAGAGGCAGATGGACTGCAGAAGAAGATCAAATTCTCACTAATTATATTATTTCTAATGGAG 1 11 12 13 14 15 16 17 18 19 2 EU419748 AAGGCTCTTGGAGGTCGTTACCTAAAAATGCCGGATTATTGAGATGCGGAAAGAGTTGTAGACTACGATGGATTAATTATTTGAGGTCTGATCTCAAGAG 2 Moneyberg AAGGCTCTTGGAGGTCGTTACCTAAAAATGCCGGATTATTGAGATGCGGAAAGAGTTGTAGACTACGATGGATTAATTATTTGAGGTCTGATCTCAAGAG 2 IL1b AAGGCTCTTGGAGGTCGTTACCTAAAAATGCCGGATTACTGAGATGCGGAAAGAGTTGTAGACTACGATGGATTAATTATTTGAGGTCTGATCTCAAGAG 2 21 22 23 24 25 26 27 28 29 3 EU419748 AGGGAACATTACTTCTCAAGAGGAAGATATAATTATAAAGTTACATGCAACTTTGGGTAACAGATGGTCTCTTATAGCAGAACATTTATCAGGTAGAACA 3 Moneyberg AGGGAACATTACTTCTCAAGAGGAAGATATAATTATAAAGTTACATGCAACTTTGGGTAACAGATGGTCTCTTATAGCAGAACATTTATCAGGTAGAACA 3 IL1b AGGGAACATTACTTCTCAAGAGGAAGATATAATTATAAAGTTACATGCAACTTTGGGTAACAGATGGTCTCTAATAGCAGAACATTTATCAGGTAGAACA 3 31 32 33 34 35 36 37 38 39 4 EU419748 GACAATGAGATAAAAAACTATTGGAACTCTCATCTAAGTCGAAAAGTTGATAGCTTAAGGATACCAAGCGATGAGAAGTTACCTAAAGCCGTAGTTGATT 4 Moneyberg GACAATGAGATAAAAAACTATTGGAACTCTCATCTAAGTCGAAAAGTTGATAGCTTAAGGATACCAAGCGATGAGAAGTTACCTAAAGCCGTAGTTGATT 4 IL1b GACAATGAGATAAAAAACTATTGGAACTCTCATCTAAGTCGAAAAGTTGATAGCTTAAGGATACCAAGCGATGAGAAGTTACCTAAAGCCGTAGTTGATT 4 41 42 43 44 45 46 47 48 49 5 EU419748 TGGCTAAAAAAGGTATACCGAAGCCAATTAAAAAATCATCGATTAGTCGACCAAAAAATAAAAAGTCAAACTTATT------------------------ 476 Moneyberg TGGCTAAAAAAGGTATACCGAAGCCAATTAAAAAATCATCGATTAGTCGACCAAAAAATAAAAAGTCAAACTTATT------------------------ 476 IL1b TGGCTAAAAAAGGTATCCTGAAGCCAATTAAAAAATCATCGATTAGTCGATCGAAAAATAAAAAATCAAACTTATTTGAAGCTAAAGAAAATAATACTAG 5 51 52 53 54 55 56 57 58 59 6 EU419748 ---------------------------------------------AGAAAAAGAAGCATTGTGTTGTACAAATATGCCAGCTTGTGATAGTGCCATGGAA 531 Moneyberg ---------------------------------------------AGAAAAAGAAGCATTGTGTTGTACAAATATGCCAGCTTGTGATAGTGCCATGGAA 531 IL1b TGGGGCTTTGATTGAAATTGTTCCTATGCCTTCAACACCAAACATAGAAAAAGAAGCATTGTGTTGTACAAATATGCCAGCTTGTGATAGTGCCATGGAA 6 61 62 63 64 65 66 67 68 69 7 EU419748 TTAATGCAAGAAGATCTAGCAAAGATAGAGGTGCCAAATTCTTGGGCAGGACCTATAGAGGCCAAGGGAAGCCTTAGTTCAGATAGTGATATCGAATGGC 631 Moneyberg TTAATGCAAGAAGATCTAGCAAAGATAGAGGTGCCAAATTCTTGGGCAGGACCTATAGAGGCCAAGGGAAGCCTTAGTTCAGATAGTGATATCGAATGGC 631 IL1b TTAATGCAAGAAGATCTAGCAAAGATAGAGGTGCCAAATTCTTGGGCAGGGCCTATAGAGGCCAAGGGAAGCCTTAGTTCAGATAGTGGTATGGAATGGC 7 71 72 73 74 75 76 77 78 79 8 EU419748 CAAGACTCGAGGAGATTATGCCAGACGTGGTGATTGATGATGAAGATAAGAACACAAATTTCATATTGAATTGTTTCAGAGAAGAAGTAACGAGCAATAA 731 Moneyberg CAAGACTCGAGGAGATTATGCCAGACGTGGTGATTGATGATGAAGATAAGAACACAAATTTCATATTGAATTGTTTCAGAGAAGAAGTAACGAGCAATAA 731 IL1b CAAGACTCGAGGAGATTATGCCAGACGTGGTGATCGATGATGAAGATATGAACACAAATTTCATATTGAATTGTTTCGAAGAAGAAGTAACGAGCAATAA 8 81 82 83 84 85 86 87 88 89 9 EU419748 TGTAGGGAATAGTTATTCATGTATCGAGGAAGGTAATAAAAAGATATCAAGCGACGATGAAAAAATCAAATTATTAATGGATTGGCAAGATAATGATGAG 831 Moneyberg TGTAGGGAATAGTTATTCATGTATCGAGGAAGGTAATAAAAAGATATCAAGCGACGATGAAAAAATCAAATTATTAATGGATTGGCAAGATAATGATGAG 831 IL1b TGCGGGGAATAGTTATTCATATATCGAGGAAGGTAATAAAAAAATATCAAGCGACGATGAAAAAATCAAATTATTAATGGATTGGCAAGATAATGATGAG 9 91 92 93 94 95 96 97 98 99 1 EU419748 TTAGTATGGCCAACGTTACCATGGGAATTAGAAACGGATATAGTTCCCAGTTGGCCACAATGGGACGATACTGACACTAACTTACTTCAAAATTGCACCA 931 Moneyberg TTAGTATGGCCAACGTTACCATGGGAATTAGAAACGGATATAGTTCCCAGTTGGCCACAATGGGACGATACTGACACTAACTTACTTCAAAATTGCACCA 931 IL1b T T AGT AT GGCCAACGT T ACCAT GGGAAT T AGAAACGGAT AT AGT T CCT AGT T GGCCACAAT GGGACGAT ACT GGCACT AAT T T ACT T CAAAAT T GCACCA 1 11 12 13 14 15 16 17 18 19 11 EU419748 AT GAT AAT AAT AAT T AT GAAGAAGCAACAACAAT GGAAAT T AAT AACCAAAAT CAT AGT ACCAT T GT AT CT T GGCT T T T GT CT T AGAAAT AT AAT AAT AT 131 Moneyberg AT GAT AAT AAT AAT T AT GAAGAAGCAACAACAAT GGAAAT T AAT AACCAAAAT CAT AGT ACCAT T GT AT CT T GGCT T T T GT CT T AGAAAT AT AAT AAT AT 131 IL1b AT GAT AAT AAT - - - T AT GAAGAAGCAACAACAAT GGAAAT T AAT AACCAAAAT CAT AGT GCCAT T GT CT CT T GGCT T T T GT CT T AGAAAT AT AAT AAT AT 197 111 112 113 114 115 116 117 118 119 12 EU419748 GACAT T AT AT AT T GCT T T T GAAT AT AT T ACT CAACT CT T T T T GT T T CGT T T T AT AT T T GGAAT GT GGGAAT T AGAAT GACT AGT T T AT GT ACAT AT T T T A 1131 Moneyberg GACAT T AT AT AT T GCT T T T GAAT AT AT T ACT CAACT CT T T T T GT T T CGT T T T AT AT T T GGAAT GT GGGAAT 112 IL1b GACAT T AT AT AT T GCT T T T GAAT AT AT T ACT CAACT CT T T T T GT T T CGT T T T AT AT T T GGAAT GT GGGAAT 1168 EU419748 Moneyberg IL1b 121 122 123 124 125 126 127 128 129 13 AGT T T CGT T AGAAAT AT CGT CAAGT CAGAT T AAAAT AT GT AT GAGT T GAT GT AGT AAT AAAT GT T AT T GT T AT T ACT T T T T T T GAT GT AAAAAAAAAAAA 1231 112 1168 EU419748 Moneyberg IL1b 131 AAAAAAAAAAAAAAAAA 1248 112 1168 Supplemental Figure S2. Sequence alignment of tomato MYB12 genes, including sequences of tomato cv Moneyberg (acc. no. FN555126), IL1b (acc. no. FN555127)and the published sequence EU419748. Nucleotide differences in one of the three sequences are boxed in grey.
A Control ptrv2-del/ros1 Test ptrv2-del/ros1-slmyb12 B 6 4 2 Naringenin chalcone Del/Ros1-S Red Del/Ros1-NS Purple Del/Ros1-M-S Pink Del/Ros1-M-NS Red 6 4 2 Rutin 5 Chlorogeninc Chlorogenic acid acid 4 3 2 1 Test Control Pink sectorsred sectors Supplemental Figure S3. SlMYB12 gene silencing in purple transgenic Microtom tomatoes expressing the Delila and Rosea1 transcription factor (Del/Ros1) genes. A. Peel of tomato fruit agroinjected with ptrv1 + ptrv2-del/ros1-slmyb12 (Del/Ros1-M-S) showing the pink phenotype (right) or ptrv1 + ptrv2-del/ros1 (Del/Ros1-S) showing a red phenotype (left) in the silenced sectors. Non-silenced sectors (NS) remained purple. B. Effect of SlMYB12 gene silencing in Del/Ros1 Microtom tomato fruit. Content of naringenin chalcone, rutin and chlorogenic acid in peel of six independent silenced tomato fruit (ptrv1 +ptrv2-del/ros1- SlMYB12) and in peel of six control fruit agroinjected with ptrv1 + ptrv2-del/ros1.