1. Supplemental Fig. S1. Orange- and yellow-flowered cultivars of calendula used in this study. Alice OrangeOrange StarPompom OrangeOrange Gem Alice YellowGold StarPompom YellowGolden Gem

2. Supplemental Fig. S2. Breeding process used to obtain calendula F 2 progenies from crosses between orange- and yellow-flowered lines. ‘Alice Orange’ X ‘Orange Star’ (Orange-flowered) ‘Alice Yellow’ X ‘Golden Gem’ (Yellow-flowered) Orange-flowered progenyYellow-flowered progenyX F 1 Progenies (6 individuals, yellow-flowered) F 2 Progenies (146 individuals)

3. Supplemental Fig. S3. ClustalW tree analysis of calendula CRTISO homologs in various plant species. Numbers at branch points indicate bootstrap values (1000 replicates). AtCRTISO (Arabidopsis thaliana, AT1G06820), CmCRTISO (Chrysanthemum morifolium, AB205043), DcCRTISO (Daucus carota sp. sativus, DQ192188), IpomoeaCRTISO (Ipomoea sp., AB499053), SlCRTISO (Solanum lycopersicum, AF416727), OncidiumCRTISO (Oncidium Gower Ramsey, AY973633), ZmCRTISO1 (Zea mays, FJ603466), ZmCRTISO2 (Zea mays, FJ465413), and SynechocystisCRTISO (Synechocystis sp. PCC 6803, gene sll0033). SlCRTISO ZmCRTISO2 AtCRTISO CmCRTISO IpomoeaCRTISO SynechocystisCRTISO CoCRTISO4 CoCRTISO3 CoCRTISO2 CoCRTISO1-ORb CoCRTISO1-ORa CoCRTISO1-Y 0.1 DcCRTISO OncidiumCRTISO 758 1000 977 995 944 996 361 932 359 572 677 TRICHOTOMY 1000 ZmCRTISO1

4. AB 1 2 3 4 5 CoCRTISO1-Y CoCRTISO1-ORaCoCRTISO1-YCoCRTISO1-ORa 202 116 98 47 (kDa) 202 116 98 47 (kDa) 1 2 3 4 5 Supplemental Fig. S4. Expression of CoCRTISO-MBP fusion proteins in E. coli carrying pMAL- CoCRTISO. The arrowhead shows putative size (100 kDa) of the fusion protein. A, Proteins were separated by using SDS-PAGE and were stained with Coomassie Brilliant Blue. B, Western blotting with MBP antibodies. Lane 1, insoluble fraction (pellet) of E. coli lysate under noninducing conditions; lane 2, insoluble fraction under inducing conditions; lane 3, soluble fraction (supernatant) under noninducing conditions; lane 4, soluble fraction under inducing conditions; lane 5, affinity- purified CoCRTISO polypeptide (approximately 1 μg in A and approximately 0.2 μg in B).

5. CoCRTISO1-Y CoCRTISO1-ORa CoCRTISO1-ORb CoCRTISO2 CoCRTISO3 CoCRTISO4 Control Absorbance at 475 nmAbsorbance at 450 nm 0 20 40 60 80 100 [min] Retention time 0 20 40 60 80 100 [min] Retention time CoCRTISO1-Y CoCRTISO1-ORa CoCRTISO1-ORb CoCRTISO2 CoCRTISO3 CoCRTISO4 Control A substrate: (5Z,9Z)- and (5Z,9Z,5’Z)-lycopene B substrate: (5’Z)-γ-carotene 1 2 Supplemental Fig. S5. HPLC analysis of the in vitro assay products of CoCRTISO1, -2, -3, and -4. Percentages of total peak area are shown in Supplemental Tables S11, S12, and S13. A, (5Z,9Z)- and (5Z,9Z,5Z)-lycopene were used as substrates. B, (5Z)-γ-carotene was used as a substrate. C, (5Z)- rubixanthin was used as a substrate. Peak 1, (5Z,9Z,5Z)-lycopene; peak 2, (5Z,9Z)-lycopene; peak 3, (all-E)-lycopene; peak 4, (all-E)-γ-carotene; peak 5, (5Z)-γ-carotene; peak 6, (all-E)-rubixanthin; and peak 7, (5Z)-rubixanthin. CoCRTISO1-Y CoCRTISO1-ORa CoCRTISO1-ORb CoCRTISO2 CoCRTISO3 CoCRTISO4 Control Absorbance at 450 nm 0 20 40 60 80 100 [min] Retention time C substrate: (5’Z)-rubixanthin 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 4 5 5 5 5 5 5 5 4 6 7 7 7 7 7 7 7