Supplementary Table S1 Summarized TCP binding consensus
Binding Consensus Class GGNCCCNC I & II TGGGC(C/T) I & II
GAGGGACCCT I
TTGGGACCTC I
GTGGGAACCA I
GTGGGNCC I
GTGGNCCC II
tGGKMCCa II
GGACCA II
tGGGtCCAC II
TGGKGCC II
The consensus was summarized from the TCP binding sequences found in ‘TCP Transcription Factors: Evolution, Structure, and Biochemical Function’ (González-Grandío and Cubas, 2016)
Supplementary Table S2 Primer list for the isolation of S. speciosa ‘Espirito Santo’ transcription factor coding sequence
Primer Sequence
Supplementary Table S3 Primer list for qRT-PCR confirmation of dorsal-expressed transcription factor of S. speciosa ‘Espirito Santo’
Primer Sequence
Supplementary Table S4 Primer list for the isolation of S. speciosa ‘Espirito Santo’5’ regulatory region
Primer Sequence
SsRL2_reg_F TTAGATCACAGGTTATAACCCGATCTAATTTC
SsERF3_reg_F1 GAGGAAGTAAAACGTGTGGGGTTCTC SsERF3_reg_F2 GTTTGGACGACTTTAAACCACCAG SsERF3_reg_R CTGCGGTCCCTTCTTTACGTAAA SsOFP6_reg_F AGAGCATCGCTATATTTGTGGCT
SsOFP6_reg_R GTGGCAATAAGGGAAACTGAAGTC
SsCYC_reg_F TCCCTGCAAGAACGTATAGGAATC
SsCYC_reg_R GGTCAACCAAAGAAGTAGAGGCA
SsCIB2_reg_F1 ATCGCGCGTCACGTTTACTTA SsCIB2_reg_R1 CTTCACTACAATTCAGTCCATTCC SsCIB2_reg_F2 ATTTCCACCACAAAGCTGGGAAG SsCIB2_reg_R2 CTCCTTATCCATCATCAAGATTCC SsNGAL1_reg_F CAGAGATGGTGTGGTCACAGGGAATC
SsERF1_reg_F GAATCACTGGGATAACTCAGCCATCTGCAG
Supplementary Table S5 Thermal Asymmetric Interlaced PCR (TAIL-PCR) primer list for the isolation of S. speciosa ‘Espirito Santo’ 5' regulatory region
Primer Sequence
SsERF17_TAIL1 CGTGCTTCGACGCAGCAACCTGAATCTGC SsERF17_TAIL2 TAAGTGTCACCGAAGGTCCACGTAAGC SsERF17_TAIL3 CTGTTGGGTTGTCGAACCTCTG
SsERF17_TAIL4 GCGATTCCTTTCTCGATTGCGGTTTCACC
AD1 NGTCGASWGANAWGAA
AD2 TGWGNAGSANCASAGA
AD3 AGWGNAGWANCAWAGG
AD4 STTGNTASTNCTNTGC
AD5 NTCGASTWTSGWGTT
AD6 WGTGNAGWANCANAGA
Supplementary Table S6 Self-Formed Adaptor PCR (SEFA-PCR) primer list for the isolation of S. speciosa ‘Espirito Santo’ 5' regulatory region
Primer Sequence
SsRL2_SP1 CCTTCTCACTTCCTCTGCTGATTTACCAGTAACC
SsRL2_SP2 CCTTGTCGTACATAGCCAGAGCTTCTTCG
SsRL2_SP3 ACGTGAAGATGACATGGANNNNNNNNGGTGAC
SsRL2_SP4 GAGATCGACCAAGAAACCGAGAAACGAAGACG
SsRL2_SP5 CTTAGGTGTCCATGAAGAGGACG
SsNGAL1_SP1 GCAGTCAACTGTTCATCGACCACTAATTCTTCCTC
SsNGAL1_SP2 GGAACCACCAGCTCCACCTCCTTCTACGG
SsNGAL1_SP3 AGGATTCCATCATCATATNNNNNNNNGGCCAG
SsNGAL1_SP4 GTGGGCTTCTGGAATCTGGTCTGAAGAGTAGTGG
SsNGAL1_SP5 CCCAGAAACTAGAGTTCGTAGTATTAG
SsERF1_SP1 GCGGAGAACTCACGTATTCCGTTTTCACCG
SsERF1_SP2 GGCTCAATTTTACATCTCGTCTTGGGTTTCACG
SsERF1_SP3 GACCGCAAATTACCATANNNNNNNNATCATC
SsERF1_SP4 TTTAACGGCAAGTCTCCCCAAGTTTCCGTC
SsERF1_SP5 AAACGGCGTCCATCCATCATTAACC
Supplementary Table S7 Primer list for the construction of dual-luciferase vector
Primer Sequence
SsRL2_F_BamHI GGATCCTTAGATCACAGGTTATAACCCGATC
SsRL2_R_Sal1 GTCGACGAGATCGACCAAGAAACCGAG
SsERF17_F_BamHI GGATCCAGCTTGGTCGAGTGA
SsERF17_R_SalI GTCGACTTTTTCATAGCCTCTGCAAA
SsERF3_F_HincII GCAGTCGACTCTAGAGGGGAT
SsERF3_R_NcoI CCATGGTTGGTACCTTTTGCTGAGC
SsCYCA_F_BamHI GGATCCTCCCTGCAAGAACGTATAG
SsCYCB_F_BamHI GGATCCTTCCTGCAAGAACGTATAG
SsCYC_R_SalI GTCGACTTTTCTTTTTTGGGAGAGGG
SsCIB2_F_BamHI GGATCCATTTCCACCACAAAGCT
SsCIB2_R_Sal1 GTCGACCATCAAGATTCCAAAAAACAA
SsNAGL1_F_BamH1 GGATCCCAGAGATGGTGTGGTCAC
SsNGAL1_R_Sal1 GTCGACCTCAGTGCGTGTAGTGTG
SsERF1_F_HincII GTCGACGAATCACTGGGATAACT
SsERF1_R_NcoI CCATGGGAAGAATTGATCAATTGAAGTAA
Supplementary Table S8 RNA-seq and BLASTx annotation result of dorsal expressed genes encoding cell wall loosening agent
RPKM = Reads Per Kilobase per Million mapped reads (Source: Pan, Z.J., unpublished work)
Supplementary Figure S1 The role of CYC in floral zygomorphy
(A) Genetic regulation of floral symmetry in Antirrhinum. CYCLOIDEA (CYC), DICHOTOMA (DICH) and RADIALIS (RAD) specify dorsal petal identity, whereas DIVARICATA (DIV) determines ventral petal identity. CYC and DICH activate RAD, which in turn represses DIV activity in the dorsal and lateral regions. (B) Alteration in spatial CYC expression patterns can generate different flower symmetry. No CYC expression in any petals can create radial flowers, and ubiquitous CYC expression in all petals can also make radial flowers. Dorsal or ventral expression of CYC generates bilateral flowers. (C–E) Changes in CYC2 expression coincide with flower symmetry evolution. (C) In Dipsacales, CYC2 duplication and its expression change from ubiquitous to differential (only dorsal/lateral), coinciding with the radial to bilateral transition. (D) Similarly, in Malpighiaceae and its related basal families such Centroplacaceae and Elatinaceae, a progression of CYC2 expression (absent- ubiquitous − dorsal/lateral − dorsal) plays an important role in the radial to bilateral transition. (E) When dorsal CYC2 expression was lost or reverted to the ubiquitous status, radial symmetry was regained in Plantago major flowers. [CYC2 expression in red, D; dorsal petal, L; lateral petal, v;
ventral petal] (Spencer and Kim, 2018).
Supplementary Figure S2 Phylogeny of TCP proteins from Sinningia speciosa, Solanum lycopersicum and Arabidopsis thaliana
The phylogenetic tree was constructed using Neighbor-Joining method with 1000 bootstrap support indicated at each node. SsTCP: TCPs of S. speciosa; SlTCP: TCPs of S. lycopersicum; AtTCP: TCPs of A. thaliana. Proteins marked with stars have mRNA’s containing an R domain. Those with circles containing putative target sites for miR319.
SsTCP22 is the SsCYC, Antirrhinum majus CYC orthologue which in the tree showed to have only a single copy. (Source: Ye, 2018, unpublished work).
Supplementary Figure S3
Thermal Asymmetric Interlaced PCR (TAIL-PCR) (a.) Schematic representation of primer binding sites in TAIL-PCR and TAIL-PCR products. (b.) The TAIL-PCR result of SsERF17 5’ regulatory region from first to third PCR reaction using each of 6 AD primers. The red circle shows the isolated band.
Supplementary Figure S4 Self-Formed Adaptor PCR (SEFA-PCR)
(a.) Schematic representation of primer binding sites in SEFA-PCR and SEFA-PCR products. (b.) The SEFA-PCR result of SsRL, SsNGAL1, and SsERF1 5’ regulatory region from the first to third reaction. The red circle shows the isolated band.
Supplementary Figure S5 Reporter and internal control construct for dual-luciferase assay
The PJD301-luciferase was constructed following Luehresen et al. (1995). (a.) Reporter construct in which the 5’ regulatory region of interest was used to replace the CAMV 35s promoter to drive the firefly (F)-luciferase transcription. (b.) Internal control construct using renilla (R)-luciferase as the signal.
Supplementary Figure S6 Construct of SsCYC tag GFP (by Yu-An, Shi)
Supplementary Figure S7 Construct of 35s-GFP (by Yu-An, Shi)
Supplementary Figure S8 Sequence alignment for the 5’regulatory region of S.
speciosa ‘Espirito Santo’ and “Avenida Niemeyer’
Sequence Alignment was done by Clustal MUSCLE tool (http://www.ebi.ac.uk/Tools/msa/muscle/) to compare the 5’regulatory region of S.
speciosa ‘Espirito Santo’ (SsES) and ‘Avenida Niemeyer’ (SsAN) of the TFs SsRL2, SsERF17, SsERF3, SsOFP6, SsCYC, SsCIB2, SsNGAL1, and SsERF1. The TCP binding sites found at the positive strand were marked with red box. The TCP binding sites found at the negative strand were marked with green box. The allelic variations of SsCYC regulatory region were marked with blue box.
SsRL2
SsERF17
SsERF3
SsOFP6
SsCYC
SsCIB2
SsNGAL1
SsERF1
Supplementary Figure S9 Expression profile of SsDIV in S. speciosa 'Espirito Santo' (SsES)
(a.) SsDIV expression in the floral bud stage 3 and 5 of SsES. (b.) SsDIV expression during the floral developmental stages of SsES. (Source: Pan Z.J., unpublished data).
Supplementary Figure S10 Dorsal and ventral petals observation of S. speciosa 'Espirito Santo' (SsES)
(a.) Sectioning of SsES flower bud stage 5 for scanning electron microscope. (b.) Comparison of total cell area within the proximal, middle, central and lobe of SsES flower bud stage 5, observed using SEM. (Source: Pan Z.J. and Nien, Y.C., unpublished data).
Supplementary Table S9 Full coding sequences of TFs containing TCP binding sites at their 5’ regulatory regions
The sequences were isolated from S. speciosa ‘Espirito Santo’
> SsABF2
TTTGACTCCATCTGTAGAATAAATTAAAAAAAACCTCAGTCCATTATCATTGGAATATGG
AGATTCATCACTAACTGTAATTTTGAATCTGTCACTTCCTCTGGTAAAGGAGTAGGATCCTCCA
ATCCGGCTTTGCTTATAGTTAGTTGCAATATTTTGCAAGTCCTTGGTGAAGATCTGTTTAAGATG
GAGGAGAAGGTGGTGGTGGTGGTTTCCCATGAGCTGGATGAGGCGGGGGAGTGGTGGTGGT
CCAGATTTTTTATGAATTTTCAGTAATTTCTTACTACTCTTTTTAACTTTTTTACCTTGATTGATT
>SsNGAL1
Supplementary Table S10 5’ Regulatory region sequence of dorsal-expressed TFs of S. speciosa ‘Espirito Santo’
The 5’ regulatory region sequences were referred to the sequence before translation start site (ATG)
GGCGATCGAGTTTGACTCTTTAAAAATAATATATTTTTTAAGAAAGTAACCGTATTAGAGAATAT
CCAGATCAAAAGATGGATGGATTCAGCTGCGCTGCATACATTTCCAAAATTGTTGTGAAATTAC
GCCCCATTACAAAAGCAAAGAAAATGAAGTTAAATTTGTCCATTGCGTAGGGTGATAGACAAA
CCTAACTACACACACTCACACACCAAACTAAGAGAAATTCACACTTCATAACCCTCTCTCTCT
GTTATTTCAAGAAAGGTTGGACATTTTGGTGGTTTGACGTCTAAGGCCCGGCATTTAACCCTTT
CGCACTGAG