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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188
NATURE PLANTS | www.nature.com/natureplants 1
Floral organ identity determination in Nigella damascena
(Ranunculaceae), a species with spiral flowers
Peipei Wang1,2,*, Hong Liao1,2,*, Wengen Zhang1,2,*, Xianxian Yu1,2, Rui Zhang1,
Hongyan Shan1, Xiaoshan Duan1,2, Xu Yao1,2, Hongzhi Kong1
1State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany,
Chinese Academy of Sciences, Beijing 100093, China 2University of Chinese Academy of Sciences, Beijing 100049, China *These authors contributed equally to this work
Author for correspondence:
Hongzhi Kong
Tel: +86 010 62836489 Email: [email protected]
This PDF file includes:
Supplementary Methods Supplementary Tables 1 to 10 Supplementary Figures 1 to 12
Flexibility in the structure of spiral flowers and its underlying mechanisms
2 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
Supplementary Methods
Scanning electronic microscopy. Floral buds were fixed in fresh FAA. Images were
captured using a Hitachi S-4800 scanning electron microscope. Yeast two-hybrid (Y2H). The GAL4-based MATCHMAKER Two-Hybrid System
(Clontech) was employed to determine the interactions among proteins encoded by
the floral MADS-box genes. For each gene, a full-length coding sequence was
amplified from the floral cDNA using gene-specific primers in which the appropriate
restriction recognition sites was introduced (Supplementary Table 10). The PCR
product was digested with the corresponding restriction enzymes and ligated into
the GAL4 activation domain (AD) expression vector pGADT7 and the GAL4-binding
domain (BD) expression vector pGBKT7 using the T4 DNA ligase (Promega). The
capability of auto-activation was tested by co-transforming the gene-specific
recombinant pGBKT7 vector and the pGADT7 empty vector into yeast
(Saccharomyces cerevisiae strain AH109) competent cells through the Small-Scale
LiAc Yeast Transformation Procedure, following the manufacturer’s instructions. For
genes capable of auto-activation, truncated sequences lacking the C-terminal regions
were introduced into the pGBKT7 vector. For testing protein-protein interactions, the
corresponding pGADT7 and pGBKT7 plasmids were introduced into the yeast
competent cells. Transformants carrying both AD and BD recombinant plasmids were
confirmed by PCR and their growth on the selective synthetic dropout (SD) medium
lacking leucine and tryptophan (SD/–Leu/–Trp) at 22°C for 7 days. Positive
transformants were diluted serially and incubated on selective SD medium lacking
leucine, tryptophan, histidine and adenine but containing 5 mM
3-amino-1,2,4-trizole (SD/–Leu/–Trp/–His/–Ade/ +5 mM 3-AT) at 22°C for 7 days for
His and Ade reporter gene expression test, and LacZ reporter gene expression
induced by X-Gal was assessed through the colony-lift filter method. Growth was
confirmed up to 14 days at 22°C. For each combination, at least three technical
replicates were performed. Transformants carrying the empty pGADT7 and/or
pGBKT7 vectors were used as negative controls.
NATURE PLANTS | www.nature.com/natureplants 3
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188
Supplementary Table 1. Genes and accession number information
Gene lineage Gene number Gene name Accession number
AP1 2 NdFRUITFULL1 (NdFL1)
NdFRUITFULL2 (NdFL2)
AP3 3
NdAPETALA3-1 (NdAP3-1)
NdAPETALA3-2 (NdAP3-2)
NdAPETALA3-3 (NdAP3-3)
PI 2 NdPISTILLATA1 (NdPI1)
NdPISTILLATA2 (NdPI2)
AG 2 NdAGAMOUS1 (NdAG1)
NdAGAMOUS2 (NdAG2)
SEP 3
NdSEPALATA1 (NdSEP1)
NdSEPALATA2 (NdSEP2)
NdSEPALATA3 (NdSEP3)
AGL6 1 NdAGAMOUS-LIKE6 (NdAGL6)
Total 13
4 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
Supplementary Table 2. Primers used for amplification of cDNA
Primer name Primer (5’-3’) cDNA synthesis
polyT CCGGATCCTCTAGAGCGGCCGC(T)17
Nested primers
FL_F1 GCTCATGARATNTCNRTKCTTTG
FL_F2 CTKTGYGAYGCTGAWGTTGCT
B_F1 AACAGGCAGGTSACCTAYTC
B_F2 TYACTGTTCTMTGTGATGCT
AG1_F1 CARGTSACCTTYTGCAARCG
AG1_F2 TCYGTKCTYTGTGATGCWG
AG2_F1 TKCBAARAGAAGAAATGG
AG2_F2 THTGYGATGCTGARGTTGC
SEP_F1 CTTTGTGATGCTGAAGTTG
SEP_F2 GAAAACAAGATAAATCGWCAAG
AGL6_F1 GAGAACAAGATYAAYMGDCAAG
AGL6_F2 TGYGATGCYGARGTTGSTCT
Universal reverse primer
AP CCGGATCCTCTACAGCGGCCGC
ANS-specific primers
ANS_F CTTGTSAACCATGGMATCC
ANS_R CCAATGTGCAYGATKATWGAG
NATURE PLANTS | www.nature.com/natureplants 5
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188
Supplementary Table 3. Information on genes used in this study
Taxon and species Gene name Accession number Source
Brassicaceae
Arabidopsis thaliana AP1 At1g69120 TAIRa
CAL At1g26310 TAIR
FUL At5g60910 TAIR
AGL79 At3g30260 TAIR
AP3 At3g54340 TAIR
PI At5g20240 TAIR
AG At4g18960 TAIR
SHP1 At3g58780 TAIR
SHP2 At2g42830 TAIR
STK At4g09960 TAIR
SEP1 At5g15800 TAIR
SEP2 At3g02310 TAIR
SEP3 At1g24260 TAIR
SEP4 At2g03710 TAIR
AGL6 AT2G45650 TAIR
AGL13 At3g61120 TAIR
Ranunculaceae
Aquilegia coerulea AqcoFL1A JX680252.1 ref. 1b
AqcoFL1B JX680253.1 ref. 1
AqcoFL2 Aquca_006_00408.1 Phytozome v10.2.1c
AqcoAP3-1 Aquca_006_00074.1 Phytozome v10.2.1
AqcoAP3-2 Aquca_006_00072.1 Phytozome v10.2.1
AqcoAP3-3 Aquca_007_00336.1 Phytozome v10.2.1
AqcoAP3-3b HQ694798 ref. 2
AqcoPI Aquca_014_00308.1 Phytozome v10.2.1
AqcoAG1 Aquca_136_00009.1 Phytozome v10.2.1
AqcoAG1-2 Aquca_136_00010.1 Phytozome v10.2.1
AqcoAG2 Aquca_022_00039.1 Phytozome v10.2.1
AqcoSEP1 Aquca_006_00411.2 Phytozome v10.2.1
AqcoSEP2 Aquca_002_00916.1 Phytozome v10.2.1
AqcoAGL6 Aquca_013_00477.1 Phytozome v10.2.1
Aquilegia vulgaris AqvuAP3-1 EF489478 ref. 3
AqvuAP3-2 EF489477 ref. 3
AqvuAP3-3 EF489476 ref. 3
Nigella sativa NisaFL1 KF500137.1 ref. 4
NisaFL2 KF500169.1 ref. 4
NisaAP3-2 HQ694795.1 ref. 2
NisaAP3-3 HQ694794.1 ref. 2
NisaPI1 HQ694797.1 ref. 2
NisaPI2 HQ694796.1 ref. 2
6 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
Taxon and species Gene name Accession number Source
Helleborus x hybridus HehyFL1 KF500135.1 ref. 4
HehyFL2 KF500171.1 ref. 4
Helleborus orientalis HeorAG1a AY464109.1 ref. 5
HeorAG1b AY464108.1 ref. 5
Ranunculus bulbosus RabuFL1 AY306179.1 ref. 6
RabuFL2 AY306180.1 ref. 6
RabuFL3 AY306182.1 ref. 6
RabuFL4 AY306183.1 ref. 6
Ranunculus sceleratus RascFL1 AB473875.1 —b
RascFL2 AB473876.1 —
RascFL3 AB473877.1 —
Ranunculus ficaria RafiAG1 AY464114.1 ref. 5
RafiAG2 AY464115.1 ref. 5
Hydrastis canadensis HycaFL1 KF500136.1 ref. 4
HycaFL2 KF500172.1 ref. 4
Eranthis hyemalis ErhyFL1 KF500138.1 ref. 4
ErhyFL2 KF500140.1 ref. 4
Clematis marmoraria x Clematis
paniculata (cultivar Joe) CljoFL1 KF500139.1 ref. 4
Clematis integrifolia ClinAG1 AY464113.1 ref. 5
ClinAG2 AY464112.1 ref. 5
Delphinium exaltatum DeexAP3-1 EU481804.1 ref. 7
DeexAP3-2 EU481803.1 ref. 7
DeexAP3-3 EU481802.1 ref. 7
DeexPI EU481805.1 ref. 7
Delphinium ajacis DeajPI1 AF052862.1 ref. 8
Cimicifuga racemosa CiraAP3-1 AY162862.1 ref. 9
CiraAP3-2 AY162863.1 ref. 9
CiraAP3-3 AY162864.1 ref. 9
CiraPI1 AY162865.1 ref. 9
CiraPI2 AY162867.1 ref. 9
CiraPI3 AY162868.1 ref. 9
Anemone sylvestris AnsyFL1 KF500141.1 ref. 4
Anemone nemorosa AnneAP3-1 AY162841.1 ref. 9
AnneAP3-2 AY162842.1 ref. 9
AnneAP3-3_1 AY162843.1 ref. 9
Aconitum sinomontanum AcsiPI1 EU481819.1 ref. 7
AcsiPI2 EU481820.1 ref. 7
Thalictrum thalictroides ThthAG1 JN887118.1 ref. 10
ThthAG2 AY867879.1 ref. 11
Berberidaceae
Berberis thunbergii BethunFL1 KF500149.1 ref. 4
Berberis gilgiana BegiFL1 KF500145.1 ref. 4
BegiFL2 KF500142.1 ref. 4
BegiFL3 KF500143.1 ref. 4
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188
Taxon and species Gene name Accession number Source
BegiFL4 KF500144.1 ref. 4
BegiAP3-1 AY162857.1 ref. 9
BegiAP3-2_1 AY162858.1 ref. 9
BegiAG AY464106.1 ref. 5
Berberis bealei BebeFL JN593334.1 —
Epimedium grandiflorum EpgrAP3-1 EU481793.1 ref. 7
EpgrAP3-3 EU481794.1 ref. 7
Epimedium sagittatum EpsaAGL2-2 JN590219.1 —
EpsaAGL2-1 JN590218.1 —
Menispermaceae
Menispermum dauricum MedaAP3-2 EU481784.1 ref. 7
MedaAP3-3 EU481783.1 ref. 7
MedaAP3-3_1 EU481786.1 ref. 7
Cocculus trilobus CotrAP3-1 HQ694788.1 ref. 2
CotrAP3-2 HQ694789.1 ref. 2
CotrAP3-3 HQ694790.1 ref. 2
Lardizabalaceae
Sinofranchetia chinensis SichFL1 DQ 656565.1 ref. 12
SichFL2 DQ656566.1 ref. 12
SichAP3-1 JQ806394.1 ref. 13
SichAP3-2 JQ806395.1 ref. 13
SichAP3-3 JQ806396.1 ref. 13
Akebia trifoliata AktrFL1 AY627632.1 ref. 14
AktrFL2 GU357459.1 ref. 15
AktrAP3-1 AY627630.1 ref. 14
AktrAP3-2 AY627631.1 —
AktrAP3-3 DQ303124.1 ref. 14
AktrAG1 AY627635.1 —
AktrSEP1_1 GU357447.1 ref. 15
AktrSEP1_2 GU357448.1 ref. 15
Akebia quinata AkquAP3-1_1 AY162835.1 ref. 9
AkquAP3-2_1 AY162839.1 ref. 9
Papaveraceae
Dicentra eximia DiexAP3 AF052875.1 ref. 8
Eschscholzia californica EscaFL3 KF500168.1 ref. 4
EscaFL1 HM592297.1 —
EscaFL2 HM592298.1 —
EscaDEF1 EF378697.1 ref. 16
EscaDEF2 EF378698.1 ref. 16
EscaAG1 DQ088996.1 ref. 17
EscaAG2 DQ088997.1 ref. 17
Papaver somniferum PasoFL1 AY306177.1 ref. 6
PasoFL2 AY306178.1 ref. 6
Lamprocapnos spectabilis LaspFL1 KF500114.1 ref. 4
LaspFL2 KF500152.1 ref. 4
8 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
Taxon and species Gene name Accession number Source
Sanguinaria canadensis SacaFL2 KF500157.1 ref. 4
SacaAP3 AF130868 ref. 18
Eupteleaceae
Euptelea pleiosperma EuplFL1 DQ656558.1 ref. 12
EuplFL2 DQ656559.1 ref. 12
EuplAG1 GU357452.1 ref. 15
EuplAG2 GU357453.1 ref. 15
EuplSEP1 GU357461.1 ref. 15
Euptelea polyandra EupoAP3 EU481781.1 ref. 7
Trochodendraceae
Trochodendron aralioides TrarAP3 DQ453774.1 ref. 19
Sabiaceae
Meliosma dilleniifolia MediAP3-1 AY436709.1 ref. 20
Buxaceae
Pachysandra procumbens PaprAP3-1 DQ479360.1 ref. 19
Magnoliaceae
Magnolia grandiflora MagrAP1 AY821777.1 ref. 21
MagrAGL2 AY821781.1 ref. 21
Magnolia praecocissima MaprMADS7 AB050649.1 —
MaprMADS2 AB050644.1 —
Michelia figo MifiFL AY306159.1 ref. 6
Chloranthaceae
Chloranthus spicatus ChspAP1 AY316311.1 ref. 22
ChspAP3 AF230701.1 ref. 23
ChspAG AY464100.1 ref. 5
Nymphaeaceae
Nuphar advena NuadAP1 AY936223.1 —
NuadSEP2 GU048648.1 —
Amborellaceae
Amborella trichopoda AmtrAP1 XM_006856294.1 —
AmtrAG AY936231.1 —
AmtrAGL2 AY936232.1 — aTAIR: The Arabidopsis Information Resource, https://www.arabidopsis.org/ bNCBI: National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/ cPhytozome v10.2.1: http://phytozome.jgi.doe.gov/pz/portal.html
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188
Supplementary Table 4. Primers used for qRT-PCR
Primer name Forward primer (5’-3’) Reverse primer (5’-3’) FL1_RT CAAATCAAAGTCAGAGCTCAATG ATAACTTCCAAATCTCTTACCTGT
AP3-1_RT GAGGATCTGAATATAGCCGAG GGTGGTGTTGGGTTGGTTG
AP3-2_RT AAGCTTTCGATAGAGTTCGC TCCTGTTCAACCAAAGCATAG
AP3-3_RT GTGTTGATGACTTGACTTTTG CCCAGTCGTAAATTGTAATGT
PI1_RT GCCTTAACTATATCTTGCTCAAG GCTTAATAGCAGTAATACTTTGTAG
PI2_RT ACTACATCTTGCTTAAAACTCAAC AGGAGTTTCCAGCAATGAAC
AG1_RT TAAAGGGTCTCGAGAAAAAGAT ATTCGTTTGTTGGCATCAGAC
AG2_RT TGAAGCAGCAAATTGAAATC CATGGCCTCGTATTCATTC
SEP1_RT GCTTCAGAGCAAGGAAATTACGT GAAGGATCAGAACCTGGACG
SEP2_RT CATCACCCTGTTCGAACTG AGTTTCAAGGATAATCACATGT
SEP3_RT CCTTGAGAGGCAATTAGACT TGTTGCCTACCATATCCCAC
AGL6_RT AACTTCGTAGAAAGGAACGTC CAATCTGAAGGGTGGGCTC
ANS_RT CAACCAGACTGAGATCCCTAGTG CGATGTGCACGATGATAGAGTC
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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
Supplementary Table 5. Primers used for in situ hybridization
Primer name Forward primer (5’-3’) Reverse primer (5’-3’) Probe length T7_adapter TAATACGACTCACTATAGGG
FL1_IS GAGCAACCAAATCAAAGTC GCCACTACCTCCAATATACAG 340bp
FL2_IS CTAAGCGATACTTGTCAATCC CTGACATATGCACACGCTC 360bp
AP3-1_IS AGAATTGAGGAAGCTTATGC GCCTTGAGAAGGTTGATGAG 309bp
AP3-2_IS GATCTCTTTCGTGAAATTGAAG GGTGAAATATTAATAGTGCAGG 296bp
PI1_IS GAGACTATCCTTCACAGATGC GTCGCAGGGTAAGAGAAAC 284bp
PI2_IS GGGAATGCTAGAGACTTGG GATGGCACAAATATGGTATC 324bp
AG1_IS GCTGAGAATGAAAGAACC CCTGGAGCTTTACTAAACAG 347bp
AG2_IS GGAGATTGACTTGCATAATG GGACGTCAAATATACCTAATC 367bp
SEP1_IS CTGCAATTAGGATACAACCG GTTGACAGTTATGTAGAGCC 291bp
SEP2_IS CAGGATGAGAGGAGTATGG TCAACCCATATGACATACAG 381bp
SEP3_IS CTTCCACCCTCTTGATTGTG GAGACCAACTTCAGCATCAC 334bp
AGL6_IS TGGAAACAACAATTTCTCTG GATTCATTTCCATGCTTGG 313bp
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188
Supplementary Table 6. Primers used for VIGS construction
Primer name Forward primer (5’-3’) Reverse primer (5’-3’) FL_EcoRIF GCGAATTCAATCAAGGGAGATCAAATGGAG
FL_XbaIR GCTCTAGAAACCACTACCTCCAATATACAG
FL2_BamHIF ATGGATCCATCTAAGCGATACTTGTCAATC
FL2_KpnIR ACGGTACCAGCTGACATATGCACACGCTC
AP3-1_KpnIF ATGGTACCATGAAGAAAGAATTGAGGAAGC
AP3-1_XhoIR AACTCGAGATGAAGAATCCAGTAGATAAGA
AP3-2_BamHIF TTGGATCCTTCGATTGTCACTATGCTTTGG
AP3-2_KpnIR GTGGTACCTTACTAGACAGATATTTAGGTG
AP3-3_XbaIF GGTCTAGAGTTCGTGATCGGAAGTAT
AP3-3_BamHI AAGGATCCAACCCAGTCGTAAATTGTAATG
PI1_KpnIF AAGGTACCTTCCAGAACACTAGAGACTATC
PI1_XhoIR2 AACTCGAGATAGAAACCAATAACATCAGTG
PI2_EcoRIF ACGAATTCAGACACAATAGAAGGGATTATC
PI2_XbaIR GGTCTAGATTGATGGCACAAATATGGTATC
AG1_BamHIF AAGGATCCAATTGCTGAGAATGAAAGAACC
AG1_KpnIR ATGGTACCTTCCTGGAGCTTTACTAAACAG
AG2_KpnIF AAGGTACCAATGAGAACGAGCGAGCCCAAC
AG2_XhoIR AACTCGAGCTGACGTCAAATATACCTAATC
SEP1_KpnIF ATGGTACCGACTGCAATTAGGATACAACCG
SEP1_XhoIR GACTCGAGACGTTGACAGTTATGTAGAGCC
SEP2_EcoRIF ATGAATTCATATGCAGTAATACAGTTCCAG
SEP2_XbalIR GATCTAGATGAACTGTCTTTATTCTCGTTG
SEP3_BamHIF TTGGATCCTTACCCTCTTGATTGTGAACC
SEP3_KpnIR-2 TAGGTACCTTCAAGTCACCATTGTTCAAAC
AGL6_BamHIF AAGGATCCTTGTACCACCAATATGTTTCGG
AGL6_KpnIR TCGGTACCTCTCTATTATTGATTCATTTCC
AGL6_KpnIF TAGGTACCATGAGCCCACCCTTCAGATTG
AGL6_XhoIR CGCTCGAGATTCTATTATTGATTCATTTCC
ANS_XbaIF AATCTAGAGTGAGGAGTTGAAGAAGTGC
ANS_BamHIR ATGGATCCCTGGTTGTATACTCGCTTG
Bold font indicates introduced restriction enzyme recognition site.
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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
Supplementary Table 7. Computational estimation of off-target silencing (sliding window size=21bp, step length=1bp)
NdFL1 NdFL2 NdAP3-1 NdAP3-2 NdAP3-3 NdPI1 NdPI2 NdAG1 NdAG2 NdSEP1 NdSEP2 NdSEP3 NdAGL6
NdFL1 81.0%# 71.4% 71.4% 71.4% 76.2% 76.2% 81.0% 71.4% 71.4% 76.2% 71.4% 76.2%
NdFL2 81.0% 71.4% 76.2% 76.2% 71.4% 71.4% 71.4% 71.4% 71.4% 76.2% 71.4% 71.4%
NdAP3-1 76.2% 76.2% 85.7% 85.7% 71.4% 71.4% 76.2% 76.2% 76.2% 76.2% 81.0% 76.2%
NdAP3-2 71.4% 71.4% 85.7% 90.5% 85.7% 76.2% 71.4% 76.2% 66.7% 71.4% 76.2% 71.4%
NdAP3-3 71.4% 76.1% 85.7% 90.5% 76.2% 76.2% 71.4% 66.7% 66.7% 71.4% 66.7% 71.4%
NdPI1 76.2% 71.4% 71.4% 85.7% 76.2% 81.0% 71.4% 71.4% 71.4% 71.4% 71.4% 71.4%
NdPI2 76.2% 71.4% 81.0% 76.2% 81.0% 81.0% 71.4% 76.2% 71.4% 71.4% 71.4% 76.2%
NdAG1 81.0% 71.4% 76.2% 71.4% 66.7% 71.4% 71.4% 85.7% 76.2% 71.4% 71.4% 76.2%
NdAG2 71.4% 71.4% 76.2% 76.2% 71.4% 81.0% 81.0% 85.7% 71.4% 71.4% 85.7% 66.7%
NdSEP1 71.4% 71.4% 71.4% 71.4% 81.0% 71.4% 66.7% 71.4% 71.4% 85.7% 76.2% 76.2%
NdSEP2 76.2% 71.4% 76.2% 71.4% 71.4% 71.4% 71.4% 66.7% 71.4% 85.7% 71.4% 71.4%
NdSEP3 71.4% 71.4% 71.4% 76.2% 76.2% 81.0% 81.0% 71.4% 71.4% 81.0% 71.4% 81.0%
NdAGL6 76.2% 71.4% 76.2% 71.4% 76.2% 76.2% 76.2% 76.2% 71.4% 76.2% 76.2% 71.4%
# indicates the highest percentage identity between sequences in the sliding windows of each pair of VIGS region and cDNA
sequences.
cDNA VIGS region
NATURE PLANTS | www.nature.com/natureplants 15
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188
Supplementary Table 8. Information on VIGS experiments
VIGS treated plants BatchPlants Flowers
Treated Survived Recorded Strong Moderate Weak
TRV2–NdAP3-3
1st 155 95 32 67 2 4
2nd 110 99 66 68 34 3
total 265 194 98 135 36 7
TRV2–NdAP3-1–NdANS 1st 60 24 5
TRV2–NdAP3-2–NdANS 1st 65 20 10
TRV2–NdAP3-1–NdAP3-2
1st 91 33 5 0 5 0
2nd 65 32 30 6 49 10
3rd 60 41 22 17 25 14
total 216 106 57 23 79 24
TRV2–NdAP3-1–NdAP3-2–NdAP3-3
1st 101 63 27 35 15 14
2nd 82 75 50 75 12 15
total 183 138 77 110 27 29
TRV2–NdPI1–NdPI2
1st 13 5 4 6 0 3
2nd 66 34 34 124 9 17
total 79 39 38 130 9 20
TRV2–NdAG1–NdANS 1st 80 56 13
TRV2–NdAG2–NdANS 1st 80 46 25
TRV2–NdAG1–NdAG2
1st 56 45 17 9 5 9
2nd 99 60 47 129 93 76
total 155 105 64 138 98 85
TRV2–NdFL1–NdFL2–NdANS
1st 77 30 23 28 26 28
2nd 74 53 50 57 67 56
total 151 83 73 85 93 84
TRV2–NdAGL6
1st 45 18 9 7 8 18
2nd 150 36 25 14 18 9
3rd 107 58 58 9 38 30
total 302 112 92 30 64 57
TRV2–NdFL1–NdFL2–NdAGL6 1st 79 26 24 11 43 16
TRV2–NdSEP1–NdSEP2–NdSEP3
1st 92 55 42 10 39 69
2nd 96 53 14 5 12 26
total 188 108 56 15 51 95
ndap3-3 TRV2–NdAP3-1–NdAP3-2 1st 51 33 9 21 0 0
ndap3-3 TRV2–NdPI1–NdPI2
1st 18 18 11 30 4 6
2nd 1 1 1 3 0 0
total 19 19 12 33 4 6
ndap3-3 TRV2–NdAG1–NdAG2 1st 31 30 23
ndap3-3 TRV2–NdAGL6
1st 1 1 1 0 3 0
2nd 4 4 4 1 4 2
total 5 5 5 1 7 2
16 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
Supplementary Table 9. Numbers of flowers used for statistical analysis
Plants Number of flowers
1st 2nd 3rd
Wild-type 129 129 129
ndap3-3 82 82 82
Mock 105 76 46
TRV2–NdAP3-3 82 45 19
TRV2–NdAP3-1–NdAP3-2 9 6 5
TRV2–NdAP3-1–NdAP3-2–NdAP3-3 38 31 26
TRV2–NdPI1–NdPI2 27 24 25
TRV2–NdAG1–NdAG2 10 25 23
TRV2–NdFL1–NdFL2–NdANS 25 42 33
TRV2–NdAGL6 54 19 10
TRV2–NdFL1–NdFL2–NdAGL6 7 14 13
TRV2–NdSEP1–NdSEP2–NdSEP3 14 11 8
ndap3-3 TRV2–NdAP3-1–NdAP3-2 9 4 4
ndap3-3 TRV2–NdPI1–NdPI2 8
ndap3-3 TRV2–NdAGL6 2
NATURE PLANTS | www.nature.com/natureplants 17
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188
Supplementary Table 10. Primers used for Y2H
Primer name Forward primer (5’-3’) Reverse primer (5’-3’) NdFL1_NdeI TCGCCCATATGGGTAGAGGTAG
NdFL1_EcoRI CATCAGAATTCTTACTACTCGTTAAC
NdFL2_NcoI GCATCCATGGGTAGAGGTAGAG
NdFL2_EcoRI GTAGGAATTCTTCATTACAACTCATTTATG
NdAP3-1_NdeI CAGACATATGGGAAGAGGAAAG
NdAP3-1_PstI TTCTGCAGATTAACCTAAACGGAG
NdAP3-1ΔC_NdeI CAGACATATGGGAAGAGGAAA
NdAP3-1ΔC_BamHI AATGGGATCCCTTACTAAATTCTTTCTTCATATT
NdAP3-2_NdeI CAGACATATGGGAAGAGGAAAG
NdAP3-2_BamHI ATCAGGATCCATTATTAACCTAAACGGAG
NdAP3-3_NcoI GGAGTACCATGGGTAGAGGG
NdAP3-3_EcoRI ATCTAAGAATTCCTATTACTTAACCC
NdPI1_NdeI AGGAACATATGGGGAGAGGAAAG
NdPI1_EcoRI CTTTGAATTCTTACTATTTATTCTCCTGC
NdPI2_NdeI GAAACATATGGGGAGAGGTAAGATTG
NdPI2_EcoRI TGAAGAATTCCTATTAATGCTCTTG
NdAG1_NcoI CAAAGAACCATGGGAAGAGGAAAG
NdAG1_EcoRI ATAGAATTCTCACCCAAGTTGAAGTGCCG
NdAG2_NcoI CCACACAAACCATGGGAAGAG
NdAG2_EcoRI GCTGAATTCTCAACTAATTAATTGGAG
NdSEP1_NdeI GAACATATGGGAAGAGGGAAAG
NdSEP1_EcoRI TGCTGAATTCTCAGAGCATCCACCCAGGA
NdSEP2_NdeI AGAACATATGGGAAGAGGGAAAG
NdSEP2_BamHI AATAGGATCCCTCAGAGCATCCATCTAGG
NdSEP2ΔC_NdeI AGAACATATGGGAAGAGGGAAAG
NdSEP2ΔC_BamHI TCTGGGATCCCCTAATCCTGGCTTTTTAATAATC
NdSEP3_NcoI TCAATCGCCATGGGTAGAGGAAG
NdSEP3_EcoRI TGAGGAATTCCTATCAAGCTAACCATC
NdAGL6_NdeI TTAGCATATGGGTAGAGGAAG
NdAGL6_EcoRI CATAGAATTCTCAGAGAACCCATCG
AD_r AGATGGTGCACGATGCACAG
BD_r TAAGAGTCACTTTAAAATTTGTAT
T7 TAATACGACTCACTATAGGGC
Bold font indicates introduced restriction enzyme recognition site.
18 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
S1
S2S3
S4
S5
S1
S2S3
S4
S5
d e1st
2nd
3rd
1st/2nd
a b c
# of
tota
l flo
ral o
rgan
s
0 2 4 640
50
60
70
80 ρ = 0.8291
P1
S1
S2
S3
S4
S5
P2
P4
P5
P6
P7
P8
St1
St2St3
St4
St5
St6
P3
h
S4
P2P1
S1
S2S3
S4
S5f g
3
4th
5th
6th
) 3
Height of receptacle (mm)
# of
tota
l flo
ral o
rgan
s
0 1 2 340
50
60
70
80
Width of receptacle (mm)0 2 4 6
0 9018
ρ = 0.7499
Supplementary Figure 1 Correlation between the number of floral organs per flower
C1-5
C2
C4C3
C5
-13-21
-8 -3
-16 -24
-11-19
-2-10
-18
-7-15-23
-4-12
-20
-1
-6
-9-17
-25
-14-22S4
S5
S3S1
P1P6
St6 P4St1St3
h i
Heig
ht o
f rec
epta
cle
(mm
Width of receptacle (mm)0 2 4 6
0
1
2
3 ρ = 0.9018
Supplementary Figure 1. Correlation between the number of floral organs per flower and the size of floral meristem in Nigella damascena. a, An individual plant at anthesis. b, A diagram showing the structure of the plant, in which the 1st, 2nd and 3rd bloomed flowers are indicated. c, Correlation between the total number of floral organs and the height and width of the floral receptacle in mature flowers (stage 16). A total of 20 flowers were investigated. The Spearman correlation coefficient (ρ) is shown for each chart. The regression equation for the bottom chart is height = -0.0254 + 0.4852*width. d-e Scanning electron microscopy (SEM) images of flower buds at stage 4 showing thee, Scanning electron microscopy (SEM) images of flower buds at stage 4, showing the difference in the size of floral meristem. f-i, SEM images of flower buds at stages 4, 5, 6 and 8, respectively. Br, bract; S, sepal; P, petal; St, stamen; C, carpel. The numbers indicate the forward (in f-h) or reverse (in i) order of the organ initiated. Scale bar = 100 µm.
NATURE PLANTS | www.nature.com/natureplants 19
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188
a b Aquilegia AqvuAP3-1Aquilegia AqcoAP3-1
Delphinium DeexAP3-1Cimicifuga CiraAP3-1
Anemone AnneAP3-1Nigella NdAP3-1
Menispermum MedaAP3-3_1Cocculus CotrAP3-1
Epimedium EpgrAP3-1
100
100
100
AP3-
1
Arabido
p pgSinofranchetia SichAP3-1
Akebia AktrAP3-3Akebia AktrAP3-2Akebia AkquAP3-1_1
Eschscholzia EscaDEF1Sanguinaria SacaAP3
Dicentra DiexAP3
100100
9595
Aquilegia AqcoAP3-2Aquilegia AqvuAP3-2Anemone AnneAP3-2
Delphinium DeexAP3-2Nigella NisaAP3-2
Nigella NdAP3-2Cimicifuga CiraAP3-2
Berberis BegiAP3 2 1
100
100
51100
AAP
3-2
0.1
opsis SEP3
B2 (PI)
Aquilegia AqcoAP3-3bAquilegia AqvuAP3-3Aquilegia AqcoAP3-3
Anemone AnneAP3-3_1Delphinium DeexAP3-3
Nigella NdAP3-3Nigella NisaAP3-3
Cimicifuga CiraAP3-3E i di E AP3 3
10098
1005591
98
Berberis BegiAP3-2_1Cocculus CotrAP3-2
Menispermum MedaAP3-2Sinofranchetia SichAP3-2
Akebia AktrAP3-1Akebia AkquAP3-2_1
100100
97
AAP
3-3
Epimedium EpgrAP3-3Berberis BegiAP3-1
Menispermum MedaAP3-1Cocculus CotrAP3-3
Sinofranchetia SichAP3-3Eschscholzia EscaDEF2
Euptelea EuplAP3Trochodendron TrarAP3
Pachysandra PaprAP3-1Meliosma MediAP3-1
Magnolia MaprMADS7Chloranthus ChspAP3
100
87
96
100
64
93
0.1
A
RanunculaceaeBerberidaceaeMenispermaceaeLardizabalaceaePapaveraceaeEupteleaceaeOthers
Supplementary Figure 2. Classification and phylogenetic analysis of floral MADS-box genes. a, An unrooted tree showing the classification of the N. damascena genes into lineages. b-f, Phylogenetic trees of each gene lineage. Bootstrap values higher than 50% are indicated for each node. Yellow stars indicate inferred gene duplication events.
20 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
Delphinium DeajPI1Delphinium DeexPI
Aconitum AcsiPI1Aconitum AcsiPI2
Nigella NisaPI2Nigella NdPI2
Nigella NdPI1Nigella NisaPI1
Cimicifuga CiraPI2Cimicifuga CiraPI3
90
100
97
100
100100
100
100
dc Nigella NdFL2Nigella NisaFL2
Helleborus HehyFL2Ranunculus RabuFL1
Ranunculus RabuFL2Ranunculus RascFL3
Aquilegia AqcoFL2Hydrastis HycaFL2Sanguinaria SacaFL2
Eschscholzia EscaFL3Papaver PasoFL1
Lamprocapnos LaspFL2E ptelea E plFL2
5962
95
10088
100100
78100
FL2
0.1 Cimicifuga CiraPI1Euptelea EuplFL2Eranthis ErhyFL2Eranthis ErhyFL1
Clematis CljoFL1Helleborus HehyFL1
Ranunculus RascFL2Ranunculus RascFL1Ranunculus RabuFL3
Ranunculus RabuFL4Anemone AnsyFL1
Nigella NisaFL1Nigella NdFL1
Aquilegia AqcoFL1BAquilegia AqcoFL1A
Hydrastis HycaFL1Berberis BethFL174
100
100
97
5796
100
100
96100
10055
FL1
Clematis ClinAG1Ranunculus RafiAG1Aquilegia AqcoAG1Aquilegia AqcoAG1-2
Thalictrum ThthAG1Helleborus HeorAG1b
Helleborus HeorAG1a100 100
67
100100
AG1
e
Berberis BethFL1Berberis BegiFL1Berberis BebeFL
Berberis BegiFL2Berberis BegiFL3Berberis BegiFL4
Lamprocapnos LaspFL1Papaver PasoFL2
Eschscholzia EscaFL1Eschscholzia EscaFL2
Sinofranchetia SichFL1Akebia AktrFL1
Akebia AktrFL2Sinofranchetia SichFL2
Euptelea EuplFL1Chloranthus ChspAP1
M li M AP1
91
7492
99
100
80
54
10099
74100
52
F Helleborus HeorAG1aNigella NdAG1
Akebia AktrAG1Euptelea EuplAG2
Clematis ClinAG2Ranunculus RafiAG2
Aquilegia AqcoAG2Thalictrum ThthAG2
Nigella NdAG2Berberis BegiAG
Euptelea EuplAG1Eschscholzia EscaAGL2
Eschscholzia EscaAGL1Chloranthus ChspAG
100
9571
100
100
94
6360
53
AG2
Aquilegia AqcoSEP2Nigella NdSEP2
Epimedium EpsaAGL2-2Akebia AktrSEP1-2
Aquilegia AqcoSEP1Nigella NdSEP1
SEP2
191
5962
9889
f
0.1
Magnolia MagrAP1Michelia MifiFL
Amborella AmtrAP1Nuphar NuadAP1
100
10054
0.1Magnolia MaprMADS2
Amborella AmtrAG52
RanunculaceaeBerberidaceaeM i
Supplementary Figure 2 (Continued)
0.1
Nigella NdSEP1Epimedium EpsaAGL2-1
Akebia AktrSEP1-1Euptelea EuplSEP1Magnolia MagrAGL2
Amborella AmtrAGL2Nuphar NuadSEP2
SEP1
5290
MenispermaceaeLardizabalaceaePapaveraceaeEupteleaceaeOthers
NATURE PLANTS | www.nature.com/natureplants 21
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.188
0
0.5
1
1.5
2
Rela
tive
expr
essio
n
0
0.5
1
1.5
2NdAP3-1 NdAP3-2 NdAP3-3
B F S P St C B F S P St C0
0.5
1
1.5
2
B F S P St C
0
0.5
1
1.5
2
Rela
tive
expr
essio
n NdPI1 NdPI2
B F S P St C0
0.5
1
1.5
2
B F S P St C
2
ion 2
Rela
tive
expr
essio
n
0.5
1
1.5
2
0
NdAG1 NdAG2
NdSEP1 NdSEP2 NdSEP3
B F S P St C0
0.5
1
1.5
2
B F S P St C
2
0
1
B F S P St C
0.5
1.5
Rela
tive
expr
essi
0
0.5
1
1.5
1.5
2
essio
n NdAGL6 NdFL11.5
2
B F S P St C0
0.5
1
1.5
B F S P St C
0
0.5
1
Rela
tive
expr
0.5
1
0B F S P St CB F S P St C
Supplementary Figure 3. Expression patterns of 12 Nigella damascena genes as revealed by qRT-PCR analyses. Flowers at stage 12 were assayed, and the relative
i l l f th li d t th t f NdAP3 3 i t l E bexpression levels of other genes were normalized to that of NdAP3-3 in petals. Error bars indicate the collective standard deviations of three biological replicates and three technical replicates. B, bract; F, flower bud; S, sepal; P, petal; St, stamen; C, carpel.
22 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
a1 a2 a3 a4 a5S3 S5 S6 S9 S11
sepe
br se pepe
st
pe
sest
ca
stca
se
sepest
brse
br
stpe
cast
pe
se
cast
pese
br
br
b1 b2 b3 b4 b5S5 S6 S9 S10
se
S3
se
br
st
NdFL1
NdFL2
sepe
st
sepe
st
br br
sepest
ca
se
st
pe
ca
c1 c2 c3
d1 d2 d3
c4
d4
c5
d5
S4 S6
S4 S6
S6 S8
S9S6
S10
S10
sepe
NdFL2
NdAP3-1
stpe
se sepe
st
pese
st
ca
sepe
stca
pese
ca st
sepe
stca
se pe
ca
st
sepe
br
br
e1 e2 e3 e4 e5S2 S5 S7 S9 S10
sepe
st
NdAP3-2
NdPI1
pe
se sepe
st
pest
ca
se se
stpe
ca
br
f1 f2 f3 f4 f5S3 S4 S9S6 S10
NdPI1
NdPI2
ca
g1 g2 g3 g4 g5S5S3 S6 S8 S10
st
sepe
sepe
st
sepe
st castpesebr
br
sest
NdAG1
Supplementary Figure 4. Expression patterns of 12 Nigella damascena genes as revealed by RNA in situ hybridization. a, NdFL1. b, NdFL2. c, NdAP3-1. d, NdAP3-2. e,NdPI1. f, NdPI2. g, NdAG1. h, NdAG2. i, NdSEP1. j, NdSEP2. k, NdSEP3. l, NdAGL6. The stages of the flowers are shown in the top left corners of the images br bract; se sepal;stages of the flowers are shown in the top left corners of the images. br, bract; se, sepal; pe, petal; st, stamen; ca, carpel. Scale bar = 100 µm.
NATURE PLANTS | www.nature.com/natureplants 23
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S3 S4
st ca
h1 h2 h3 h4 h5S6 S8 S10
sebr
se
st
se
ca
st
sepe
st cape
pe
br
S2 S6 S9 S10
pe
i1 i2 i3 i4 i5S4
br
se se sepe
st
pese
stca
stpese
pe
br
NdAG2
NdSEP1
br
se
se pest
ca
sepe
cast
sepe
st
S2 S3 S8S6 S10
br
j1 j2 j3 j4 j5
cast
k1 k2 k3 k4 k5S5S3 S6 S9 S10
st
NdSEP2
cast
pesesepe
stpe
se
st
sepe
sepe
sepe pe
sebr
stst
ca
brse
stca
pe
l1 l2 l3 l4 l5
se
br
br
S2 S4 S6 S9 S10
NdSEP3
NdAGL6
Supplementary Figure 4 (Continued)
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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
1.5 NdAP3-1
on 1.5 NdAP3-2 1.5 NdAP3-3
0
0.5
1
Rela
tive
expr
essi
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C 0
0.5
1
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C
1.5NdPI1
1.5
NdPI2
ssio
n
0
0.5
1
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C
0
0.5
1
0
0.5
1
Rela
tive
expr
e
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C
1
1.5 NdAG1
1
1.5 NdAG2
ress
ion
0
0.5
1
0
0.5
1
Rela
tive
exp
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C
1
1.5
1
1.5NdSEP1 NdSEP2
1
1.5NdSEP3
xpre
ssio
n
0 5
1
1.5 NdFL1
0 5
1
1.5 NdFL2NdAGL6
0 5
1
1.5
e ex
pres
sion
0
0.5
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C 0
0.5
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C 0
0.5
Rela
tive
ex
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C
0
0.5
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C 0
0.5
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C0
0.5
Rela
tive
S6~7 S9 S12 S16S P St C S P St C S P St C S P St C
Supplementary Figure 5. Expression patterns of 13 Nigella damascena genes as revealed by digital gene expression profiling (DGE). Flowers at stages 6-7, 9, 12 and 16 were used, and the relative expression levels of floral MADS-box genes were normalized to those of NdAP3-3 in petals at stage 12. Error bars indicate the collective standard deviations of three biological replicates. S, sepal; P, petal; St, stamen; C, carpel.
NATURE PLANTS | www.nature.com/natureplants 25
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NdFL1 M I K C
NdFL2 M I K C
100bp EcoRI XbaI BamHIKpnIXhoI
NdFL1 NdANS
EcoRI XbaI BamHI KpnI XhoI
NdFL2NdANS
TRV2–NdFL1–NdANS
TRV2–NdFL2–NdANS
ca
NdAP3-1 M I K C
NdAP3-2 M I K C
NdAP3-3 M I K C
NdPI1 M I K C
EcoRI XbaI BamHIKpnI XhoI
EcoRI XbaI BamHI KpnIXhoI
NdAP3-3
EcoRI XbaI BamHI KpnI XhoI
NdAP3-1NdANS
EcoRI XbaI BamHI KpnI XhoI
NdAP3-2NdANS
EcoRI XbaI BamHIKpnI
XhoI
NdPI1NdANS
TRV2–NdAP3-3
TRV2–NdAP3-1–NdANS
TRV2–NdAP3-2–NdANS
TRV2–NdPI1–NdANS
NdPI2 M I K C
NdAG1 M I K C
NdAG2 M I K C
NdSEP2 M I K C
NdSEP1 M I K C
EcoRI XbaI BamHI KpnI XhoI
NdANS NdAG1
EcoRI XbaI BamHI KpnI XhoI
NdANS NdSEP1
EcoRI XbaI BamHIKpnI XhoI
NdANSNdSEP2
EcoRI XbaIBamHI
KpnI XhoI
NdANS NdAG2
NdANSNdPI2
TRV2–NdAG1–NdANS
TRV2–NdSEP1–NdANS
TRV2–NdSEP2–NdANS
TRV2–NdAG2–NdANS
TRV2–NdPI2–NdANS
NdSEP3 M I K C
NdAGL6 M I K C
LB2×35S
CPEcoRIXbaI
b EcoRI XbaI BamHI KpnI XhoI
NdAP3-2 NdAP3-1NdAP3-3
EcoRIXbaI
BamHIKpnI
XhoI
EcoRI XbaIBamHI
KpnI XhoI
NdAP3-2 NdAP3-1
EcoRI XbaIBamHI KpnI XhoI
NdAGL6
EcoRI XbaI BamHI KpnI XhoI
NdSEP3NdANS
TRV2–NdAP3-1–NdAP3-2–NdAP3-3
TRV2–NdAP3-1–NdAP3-2
TRV2–NdAGL6
TRV2–NdSEP3–NdANS
Kan BamHIKpnI
XhoIRzNOSt
RBpYL1569663bp
XbaI KpnINdPI1NdPI2
EcoRI XbaIBamHI KpnI XhoI
NdAG1 NdAG2
EcoRI XbaI BamHI KpnI XhoI
NdFL2NdFL1 NdANS
EcoRI XbaI BamHI KpnI XhoI
NdFL2NdFL1 NdAGL6-2
EcoRI XbaI BamHI KpnI XhoI
NdSEP3NdSEP2 NdSEP1
TRV2–NdPI1–NdPI2
TRV2–NdAG1–NdAG2
TRV2–NdFL1–NdFL2–NdANS
TRV2–NdFL1–NdFL2–NdAGL6
TRV2–NdSEP1–NdSEP2 NdSEP3NdSEP2–NdSEP3
Supplementary Figure 6. VIGS construction. a, Schematics showing the positions of the primers used to amplify fragments for VIGS construction. Rectangles and lines indicate the coding regions and untranslated regions (UTRs), respectively. M, I, K and C stand for the sequences that encode the MADS, I, K and C regions of the proteins, respectively. Wavy lines indicate the regions that were used for the VIGS assays. The relative positions of the
i d f RT PCR d i it h b idi ti l i di t d b fill d dprimers used for qRT-PCR and in situ hybridization are also indicated by filled and open triangles, respectively. b, A simplified diagram of the tobacco rattle virus PYL156 vector. Red fonts indicate the restriction enzyme recognition sites used in this study. c, Schematics of VIGS constructs.
26 NATURE PLANTS | www.nature.com/natureplants
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
ba1.5
1
0.5
0
NdAP3-3 NdAP3-1 NdAP3-2
0
1
0.75
0.5
0.25
Rela
tive
expr
essio
n
NdPI1 NdPI21
0.75
0.5
0.25
dcNdAP3-1 NdAP3-2 NdAP3-3
0.5
1
0.75
0.25ativ
e ex
pres
sion
0
NdAG1 NdAG2
1.5
2fe
0
1
0.75
NdFL1 NdANS
Rela
xpre
ssio
n
Supplementary Figure 7. Relative expression levels of genes in wild-type, mock-treated,
0.5
1
0
0.5
0.25
0
Rela
tive
ex
and gene-specific construct-treated flowers with strong phenotypic changes. a, TRV2–NdAP3-3. b, TRV2–NdAP3-1–NdAP3-2. c, TRV2–NdAP3-1–NdAP3-2–NdAP3-3. d, TRV2–NdPI1–NdPI2. e, TRV2–NdAG1–NdAG2. f, TRV2–NdFL1–NdFL2–NdANS. g, TRV2–NdFL1–NdFL2–NdAGL6. h, TRV2–NdAGL6. i, TRV2–NdSEP1–NdSEP2–NdSEP3. Light grey, wild-type flowers; black, mock-treated flowers; colors, gene-specific construct-treated flowers with strong phenotypic changes. Error bars indicate the collective standard deviations of three technical replicates.
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NdAGL61
0.75
0.5
0.25
0
hgNdFL1 NdAGL6
0
0.25
0.5
0.75
1
Rela
tive
expr
essio
n
0
i
0
1
0.75
0.5
NdSEP1 NdSEP2 NdSEP3
e ex
pres
sion
0.25
0
Rela
tive
Supplementary Figure 7 (Continued)
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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
a b c d
ndap3-3 TRV2–NdAP3-3, strong
e f
TRV2–NdAP3-3, moderate
g
h i j k
TRV2–NdAP3-2–NdANS, strongTRV2–NdAP3-1–NdANS, strong
h i j k
l m n o
TRV2–NdAP3-1–NdAP3-2, moderateTRV2–NdAP3-1–NdAP3-2, strong
p r s tq
ndap3-3 TRV2–NdAP3-1–NdAP3-2, strong
TRV2–NdAP3-1–NdAP3-2–NdAP3-3, strong
TRV2–NdPI1–NdPI2, strong
u v w x
Supplementary Figure 8. Phenotypes of the flowers in which AP3- and PI-lineage members were knocked down. a-x, Flowers and floral organs. For each flower, both side and top views are shown, and “strong” and “moderate” indicate the flowers with strong and moderate phenotypic changes, respectively. Note that: 1) five organs, which display different degree of petal-to-sepal transformation in TRV2–NdAP3-3–treated flowers with moderate phenotypic changes, are shown in (g); 2) a petal of TRV2–NdAP3-1–NdANS–treated flower with strong phenotypic changes is shown in (p); 3) a petal of TRV2–NdAP3-treated flower with strong phenotypic changes is shown in (p); 3) a petal of TRV2 NdAP32–NdANS–treated flower with strong phenotypic changes is shown in (q); and 4) two petals of TRV2–NdAP3-1–NdAP3-2–treated flowers with moderate phenotypic changes are shown in (r). y, Numbers of floral organs in the aforementioned flowers. 1st, 2nd and 3rd represent the first, second and third flowers, respectively.
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40
60
Sepal
10
Petaly
75
Stamen 60 Carpel
0
20
1st 2nd 3rd
0
5
1st 2nd 3rd
0
25
50
75
0
20
40
20
40
60Sepal+Petal
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75
100 Stamen+Carpel
1st 2nd 3rd 1st 2nd 3rd
Petal+Stamen100 90
Total
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20
1st 2nd 3rd
0
25
1st 2nd 3rd
0
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50
75
1st 2nd 3rd
0
30
60
1st 2nd 3rd
ndap3 3 TRV2 NdAP3 3MockWt ndap3-3
TRV2–NdAP3-1–NdAP3-2–NdAP3-3
TRV2–NdAP3-3
TRV2–NdPI1–NdPI2
MockWt
TRV2–NdAP3-1–NdAP3-2
ndap3-3 TRV2–NdAP3-1–NdAP3-2
Supplementary Figure 8 (Continued)
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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
a b c d
TRV2–NdANS, strong
e fTRV2–NdAG1–NdANS, strong
TRV2 NdAG1 NdAG2 strongTRV2–NdAG2–NdANS, strong TRV2–NdAG1–NdAG2, strong
g h
TRV2–NdAG1–NdAG2, strongTRV2 NdAG2 NdANS, strong
TRV2–NdAG1–NdAG2, moderate TRV2–NdAG1–NdAG2, moderate
TRV2 NdAG1 NdAG2, strong
i j k l
m n o p
ndap3-3 TRV2–NdAG1–NdAG2, strong
q Sepal Petal Stamen Carpel Total
7.5
10
100
150
40
60
6
8
100
***
******100
150Petal+Stamen
TRV2–NdAG1–NdAG2Mock
0
2.5
5
1st 2nd 3rd0
50
1st 2nd 3rd0
20
1st 2nd 3rd0
2
4
1st 2nd 3rd0
50
1st 2nd 3rd0
50
100
1st 2nd 3rd
Supplementary Figure 9. Phenotypes of the flowers in which AG-lineage members were knocked down. a-p, Flowers and floral organs. For each flower, both side and top views are shown, and “strong” and “moderate” indicate the flowers with strong and moderate phenotypic changes, respectively. Note that: 1) three organs, which display different degree of stamen-to-petal transformation in TRV2–NdAG1–NdAG2–treated flowers with moderate phenotypic changes, are shown in (m); and 2) two organs showing different degree of carpel-to-sepal transformation in TRV2–NdAG1–NdAG2–treated flowers with strong and moderate phenotypes are shown in (n). q, Numbers of floral organs in the aforementioned flowers. 1st, 2nd and 3rd represent the first, second and third flowers, respectively, and stars (*) indicate the P values obtained with a Mann-Whitney U test. *, P<0.05; **, P< 0.01; ***, P< 0.001.
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a b c d
e f g hTRV2–NdFL1–NdFL2–NdANS, strong TRV2–NdAGL6, strong
TRV2–NdFL1–NdFL2–NdAGL6, strong TRV2–NdAGL6, moderate
i j k l
TRV2–NdSEP1–NdSEP2–NdSEP3, moderate
q r s t
ndap3-3 TRV2–NdAGL6, strong
TRV2–NdSEP1–NdSEP2–NdSEP3, strong
m n o p
TRV2–NdSEP1–NdSEP2–NdSEP3, moderate TRV2–NdSEP1–NdSEP2–NdSEP3, moderate
Supplementary Figure 10. Phenotypes of the flowers in which AP1-, AGL6- and SEP-lineage members were knocked down a t Flowers and floral organs For each flowerlineage members were knocked down. a-t, Flowers and floral organs. For each flower, both side and top views are shown, and “strong” and “moderate” indicate the flowers with strong and moderate phenotypic changes, respectively. Two sepals of TRV2–NdAGL6–treated flowers with moderate (left) and weak (right) phenotypic changes are shown in (i). Three petals of TRV2–NdAGL6–treated flowers with nearly strong (left), moderate (middle) and weak (right) phenotypic changes are shown in (j). u, Numbers of floral organs in the aforementioned flowers. 1st, 2nd and 3rd represent the first, second and third flowers respectively and stars (*) indicate the P values obtained with a Mann-third flowers, respectively, and stars ( ) indicate the P values obtained with a MannWhitney U test. *, P<0.05; **, P< 0.01; ***, P< 0.001.
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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
u10
Sepal20
Petal50
Stamen
2.5
5
7.5
10
20
10
20
30
40
0 0 0
5
7.5Carpel
20
Sepal+Petal
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60 Stamen+Carpel
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
0
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Petal+Stamen Total
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
Total
25
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75
20
40
60eta Sta e
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60
ota
****
ota
TRV2–NdAGL6
TRV2–NdFL1–NdFL2–NdANS
TRV2–NdFL1–NdFL2–NdAGL6
Mock
00 0
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
TRV2–NdSEP1–NdSEP2–NdSEP3
Supplementary Figure 10 (Continued)
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Supplementary Figure 11. Interactions among proteins of floral MADS-box genes as revealed by using Y2H assays a Test for auto activation b Test for interaction Left panelrevealed by using Y2H assays. a, Test for auto-activation. b, Test for interaction. Left panel, colonies grown on the SD-LW medium. Middle panel, colonies grown on the selective medium (SD-LWHA + 5 mM 3-AT). Right panel, lacZ test results of the positive colonies on the selective medium. Positive colonies in left panel indicate the co-existence of two vectors in the cells; positive colonies in middle panel and blue coloration in right panel indicate the capability of auto-activation in (a) and the interaction of the paired proteins in (b). ΔC indicates the truncation of the C-terminal regions in NdAP3-1 and NdSEP2, and the C-terminal truncated versions of NdAP3-1 and NdSEP2 were inserted into the pGBKT7the C terminal truncated versions of NdAP3 1 and NdSEP2 were inserted into the pGBKT7 vectors to avoid auto-activation in (b). L, leucine; W, tryptophan; H, histidine; A, adenine; 3-AT, 3-amino-1,2,4-triazole.
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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
NdAG1 expression in wild-type andndap3 3 mutant floral organs
a dNdAG2 expression in mock and
NdAP3 1/2 silenced floral organsndap3-3 mutant floral organs
0
0.2
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0.6
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NdAP3-1/2-silenced floral organs
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lativ
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NdAP3-1 expression in wild-type and ndap3-3 mutant floral organs
NdAP3-3 expression in mock and NdAG1/2-silenced floral organs
200
300
b e
stamenstamen-derived
carpel
petalpetal-derived
sepal
outer stamenstamen-derived
sepal
inner stamenstamen
e ex
pres
sion
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NdAP3-2 expression in wild-type and ndap3-3 mutant floral organs
NdAGL6 expression in mock and NdAG1/2-silenced floral organs
stamenstamen-derived
petal
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c f
petalpetal-derived
sepal
outer stamenstamen-derived
sepal
inner stamenstamen
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tive
ndap3-3 mutant floral organs NdAG1/2-silenced floral organs
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tive
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essio
n
stamenstamen-derived
petal
petalpetal-derived
sepal
outer stamenstamen-derived
sepal
inner stamenstamen
Supplementary Figure 12. qRT-PCR assessment of gene expression levels in floral organs of wild-type, “Double Sepals” mutant (ndap3-3), mock and VIGS-treated flowers. a-c, “Double Sepals”. d-e, TRV2–NdAG1–NdAG2. f, TRV2–NdAP3-1–NdAP3-2. Light grey, wild-type floral organs; black, mock-treated floral organs; colors, “Double Sepals” or gene-specific construct treated floral organs with strong phenotypic changes Error barsspecific construct-treated floral organs with strong phenotypic changes. Error bars indicate the collective standard deviations of three technical replicates.