Nature Genetics: doi: /ng Supplementary Figure 1. ssp mutant phenotypes in a functional SP background.

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1 Supplementary Figure 1 ssp mutant phenotypes in a functional SP background. (a,b) Statistical comparisons of primary and sympodial shoot flowering times as determined by mean values for leaf number on the primary shoot (a) and mean number of leaves in the first four sympodial shoots (b) in WT (black bar) and ssp-2129 SP and ssp-1906 SP (gray bars) plants. Alternating white and gray boxes indicate sympodial shoot flowering time on the main shoot. Note that the ssp mutation has no effect on sympodial shoot flowering time in an SP background, and the ssp-1906 mutation causes only slight delays, resulting in an average of four leaves per sympodial shoot. Mean values (± s.e.m.) were compared to WT values using Student s t test: **P <

2 Supplementary Figure 2 Cloning of ssp-1906 (renamed sft-1906). (a) The ssp-1906 mapping interval on chromosome 3 defined by flanking insertion/deletion (indel) markers (red font) (Online Methods). Bottom, detailed gene structure showing the exons (black boxes) and introns of the primary candidate gene SFT. The ssp-1906 allele carried a nonsynonymous mutation (GTG to ATG; red bar; red A ) in exon 4. (b) Multiple-sequence alignment (ClustalW2) of the 2

3 florigen proteins from rice (Hd3a), Arabidopsis (FT) and tomato (SFT), and florigen antagonist from tomato (SP). The yellow box indicates the external loop domain, which is identical among florigen proteins but diverged in SP. Putative binding sites are conserved (red asterisks), and red boxes indicate residues that are conserved in the binding surface. Blue boxes indicate putative ligand-binding sites, and red and green characters therein indicate different conserved residues within the SFT subfamily and the SP subfamily. An arrow indicates the sft-1906 mutation. (c) Complementation tests confirming that sft-1906 is a weak allele of sft. Two known sft loss-of-function alleles, sft-4537 and sft-7187, were crossed with sft Bar graphs indicate leaf numbers produced on the primary shoot (green bar), alternating sympodial shoots (white and gray bars) and axillary shoot resulting from a failure to initiate sympodial growth (yellow bar). Mean values (± s.d.) are shown. Note that ssp-1906 sp failed to complement sft-4537 sp and sft7187 sp, as indicated by late flowering and the promotion of axillary meristem growth after generating a few late-flowering sympodial shoots. AxM, axillary shoot growth; ID, indeterminate shoot growth; D, determinate shoot growth. AxM growth is distinguished from indeterminate growth in that more than three leaves are produced on axillary shoots compared to typical sympodial shoots. AxM/ID indicates a mixed growth pattern of AxM and ID. 3

4 Supplementary Figure 3 Cloning of ssp-2129 and ssp-610. (a) The ssp-2129 mapping interval on chromosome 2 defined by flanking indel markers (red font). Five nonsynonymous mutations (arrows) in four genes were identified from ssp-2129 RNA sequencing data (Online Methods and Supplementary Table 2). Bottom, detailed gene structure of Solyc02g showing the exons (black boxes) and introns. Red bars indicate nonsynonymous mutations (ssp-2129, CCA to CTA; ssp-610, ACT to ATT; red T ) in exon 3. (b) Complementation tests confirming that ssp-2129 and ssp-610 are allelic. ssp-2129 sp and ssp-610 sp were crossed with each other. Both parents were backcrossed two times to sp plants to eliminate unlinked background mutations. Flowering times as determined by leaf number from the primary shoot (green bar) and successive sympodial shoots (alternating white and gray bars) are shown. Note that ssp-610 sp failed to complement ssp-2129 sp. ID, indeterminate shoot growth; D, determinate shoot growth. Error bars, s.d. 4

5 Supplementary Figure 4 A paralog of SSP and molecular evidence supporting functional redundancy. (a) Phylogenetic tree of the basic leucine-zipper (bzip) transcription factor family in tomato and Arabidopsis. Full-length bzip proteins from A. thaliana and tomato were aligned with ClustalW2 ( and a 5

6 phylogenetic tree was constructed (neighbor joining) using the FigTree v1.3 program. The blue wedge indicates FD paralogs and orthologs in Arabidopsis and tomato (red fonts). bzip family genes were retrieved from AGRIS ( and from the Solanaceae Genomics Network (SGN) (ftp://ftp.solgenomics.net/tomato_genome/). Scale bar, 0.05 substitutions per site. (b) Multiple-sequence alignment of the C termini from FD homologs using ClustalW2. The bzip domain (red bar and background) and the SAP motif required for protein interactions (green bar and background) are highly conserved. Note that we found a misannotation on the C terminus of the Solyc02g (SPGB2) protein and corrected it for this analysis. The red asterisk on the green bar indicates a putative phosphorylation site. Blue fonts, binding motif. (c) Yeast two-hybrid assays showing interaction between the SPGB2 and proteins. Tomato proteins binding with SFT interact with SPGB2 but not an spgb2 mutant having a deletion of the SAP motif. 3AT, 3 aminotriazole; L, lysine; T, tryptophan; H, histidine. (d) Normalized read counts for SPGB2 across five primary shoot meristem (PSM) stages and two sympodial meristems from our tomato meristem maturation atlas. A dashed line separates the PSM stages from the sympodial meristems. EVM, MVM and LVM, early (fifth leaf initiated), middle (sixth leaf initiated) and late (seventh leaf initiated) vegetative meristems, respectively; TM, transition meristem (eighth (final) leaf initiation before termination); FM, floral meristem; SIM, sympodial inflorescence meristem; SYM, sympodial shoot meristem. 6

7 Supplementary Figure 5 Functional conservation among proteins in rice and tomato. (a) Multiple-sequence alignment (ClustalW2) of three tomato homologs of proteins with rice proteins involved in florigen activation complex formation. The three tomato homologs have residues identical to the Hd3a-binding sites and OsFD1-interacting sites of the rice proteins (GF14b, GF14c and GF14e). Shown are putative SFT-binding sites (red asterisks) and putative SSP 7

8 interaction sites (blue boxes). The green bar indicates a highly conserved motif for a putative nuclear export signal (NES). Black and gray backgrounds indicate identity and high similarity. (b,c) Expression dynamics of genes from our tomato meristem maturation atlas generated by RNA sequencing of five primary shoot meristem (PSM) stages and two sympodial meristems (SIM and SYM) (c). Normalized read counts (RPKM) indicate expression values. EVM, MVM and LVM, early (fifth leaf initiated), middle (sixth leaf initiated) and late (seventh leaf initiated) vegetative meristems, respectively; TM, transition meristem (eighth (final) leaf initiated before termination); FM, floral meristem; SIM, sympodial inflorescence meristem; SYM, sympodial shoot meristem. (d,e) Protein interaction tests between proteins using yeast two-hybrid assays and BiFC assays in tobacco (N. benthamiana) leaf cells proteins that interact with SSP form homo- and heterodimers in yeast (d). Confocal images showing that GFP /74 localizes to the cytosol and nucleus (e, top). Confocal images detecting EYFP signals indicate BiFC interactions between N-YFP /74 and C-YFP /74, which are also localized to the cytosol and nucleus (e, bottom). A nuclear expression marker (NLS-RFP) is coexpressed in the epidermal cells of leaves from 3-week-old plants (e). 3AT, 3-aminotriazole; L, lysine; T, tryptophan; H, histidine; Scale bar, 50 µm. 8

9 Supplementary Figure 6 Confocal images showing the effects of coexpressing SSP and the ssp-2129 mutant protein on the subcellular localization of the SFT /74 interaction. EYFP signals are visualizing SFT /74 interaction via BiFC assay. Coexpression of CFP-SSP strongly increases nuclear localization of the interaction signals (bottom left), but coexpression of CFP ssp-2129 slightly affects the movement of the SFT /74 interaction into the nucleus (top left). CFP-SSP and CFP ssp-2129 are colocalized in the nuclei of N. benthamiana leaf cells. To quantify the SFT /74 interaction signals visualized by EYFP (Fig, 2g), the whole-cell (white dashed line) and nuclear (red dashed line) areas were drawn, on the basis of DIC (differential interference contrast) images, nuclear marker (NLS-RFP) expression and the expression of the SSP proteins (middle). YFP florescence signals from confocal images were captured by z-stack scanning and were then quantified for the whole-cell and nuclear areas. Scale bar, 50 µm. 9

10 Supplementary Figure 7 mrna in situ hybridization showing the spatial expression of SSP. (a c) SSP expression and dynamics during PSM maturation (a), in the SIM and SYM (b), and in canonical axillary meristems (c). SSP is expressed in all meristem types and in all cell layers but is relatively weakly expressed in the SIM and FM. SSP is also weakly expressed in vasculature cells (arrowheads). SSP, antisense probe; AxM, axillary meristem; FM3, third floral meristem (the second floral meristem is not in this plane); L4, fourth leaf, etc. Scale bar, 100 µm. 10

11 Supplementary Figure 8 Combining sft and ssp mutations enhances suppression of sp. (a) Representative images of the main shoots from sft ssp sp triple-mutant plants (at 3 months) showing that sft-1906 ssp-2129 sp triple mutants produce more leaves in successive sympodial shoots and enhance bract/leaf and branch production in inflorescences. (b) The sft-4537 ssp-2129 sp triple mutant shows stronger vegetative reversions of inflorescences, having less flower production compared to sft-4537 sp and ssp-2129 sp plants. Note that the enhanced sp suppression from combining ssp and sft alleles is likely because the ssp and sft mutations affect different components of the same complex. L, leaf; BR, bract; B, branched inflorescence; R, reversion to vegetative shoot; ID, indeterminate growth; D, determinate growth; AxM, axillary shoot growth. AxM/ID indicates a mixed growth pattern of alternating AxM and ID growth. Scale bar, 5 cm. 11

12 Supplementary Figure 9 Flowering times of the side (axillary) shoots and sympodial shoots derived from axillary shoots. (a) Average leaf number produced from the basal axillary shoots originating from the axil of the first leaf on the primary shoot. (b) Average leaf number produced from proximal axillary shoots from the axil of the last leaf on the primary shoot. (c) Average leaf number from four successive sympodial shoots derived from the basal axillary shoot. (d) Average leaf number from four successive sympodial shoots derived from the proximal axillary shoot. All single- and double-heterozygous genotypes are shown along with controls. The vertical dashed line indicates a switch to indeterminate growth (see also Supplementary Fig. 10). Mean values (± s.e.m.) are shown, and statistically significant differences (Student s t test) compared to WT and sp are represented by black and red asterisks, respectively: *P < 0.05, **P <

13 Supplementary Figure 10 Conversion of sp determinate growth to indeterminate growth using sft and ssp mutations. (a d) Quantification and comparison of shoot determinacy from primary shoots (a,b), basal axillary shoots (c) and proximal axillary shoots (d). Shown are data from 16 field-grown plants after 3 months (a,c,d) and from 10 greenhouse-grown plants after 3 months (b). Shoots were qualitatively scored as determinate (D, red bar) or indeterminate (ID, light-green bar) and then quantified from replicates. Note that, in some cases, determinacy was ambiguous at the time of data collection (D/ID, orange bar). AxM, axillary shoot (dark-green bar). 13

14 Supplementary Figure 11 Breakdown of component traits for two yield trials in Israel and New York. Statistical comparisons of mean values (± s.e.m.) for red-fruit yield (a,e), plant weight (b,f), fruit weight (c,g) and Brix (d,h) for M82 sp control (white bar), ssp-2129 (indeterminate control; black bar), single heterozygotes (light-gray bar) and double heterozygotes (darkgray bar). The vertical dashed line indicates a switch to indeterminate growth (see also Supplementary Fig. 10). Asterisks indicate significant differences from the M82 sp control plants according to the Dunnett s compare with control test: *P < 0.05, **P < Results were also obtained using multiple-range comparison analysis (Tukey-Kramer test; P < 0.05) (Supplementary Table 4). 14

15 Supplementary Figure 12 Double heterozygosity of the ssp mutations with sft-1906 produces the highest yields in multiple genetic backgrounds. Hybrids with three distinct determinate inbred lines ( SAR (a), REB (b) and LEA (c)) were generated by crossing with M82 determinate plants (as controls) and with all single and double mutants of ssp and sft (in the sp background). Double heterozygotes (black bars) were generated by crossing homozygous double mutants for ssp and sft with each inbred. Each genotype was evaluated for at least 15 replicates (Online Methods). Asterisks indicate significantly different yields from control hybrids (light-gray bar) calculated on the basis of Dunnett s compare with control test (*P < 0.05, **P < 0.01). Results were also obtained using a multiple-range comparison test (Tukey-Kramer test; P < 0.05) for total fruit yield and Brix-yield (Supplementary Table 5). Dark-gray bar, single heterozygote. Error bars, s.e.m. 15

16 Supplementary Figure 13 The ssp mutations improve shoot architecture and inflorescence production in different tomato types. (a) Representative main shoot of the cherry tomato cv. Sweet100, cv. M82 and the large-fruited cv. M99, each with introgressions of the ssp-2129 and sp mutations. All genotypes are indeterminate (ID) and produce less than three leaves in each sympodial shoot 16

17 compared to WT parental lines. Three-month-old plants are shown. Gray arrows indicate representative fruits from each cultivar. Red arrowheads point to inflorescences. L, leaf. Scale bar, 5 cm. (b,c) Quantification and comparison of average leaf number per sympodial shoot (b) and flowering times from five successive sympodial shoots (c; stacked gray and white bars) of the ssp-2129 and ssp-610 mutations in the three different genotypes shown above. The ssp and sp mutations were introgressed into each genotype by backcrossing and genotyping three times followed by self-fertilization to identify double-mutant progeny by genotyping. M99 was already in the background of sp. Mean values (± s.e.m.) from ten replicates were compared to WT and sp values using Student s t test. Black and red asterisks represent significant differences from WT and sp, respectively: **P <

18 Supplementary Table 1. Quantification of flowering time measured by leaf number in successive sympodial shoots derived from the primary shoots of sp, WT, and ssp plants. The first five sympodial shoots were examined from main shoots in 3-month old plants. STDEV, standard deviation; N, number of replicates. 1

19 Supplementary Table 2. List of nonsynonymous mutations within the SSP mapping interval on chromosome 2 identified from the mrna-sequencing of meristems from ssp-2129 mutants. Tomato gene identifiers are shown along with the position of the SNP and quality of the RNA-seq data and read depth supporting the SNP. SNP quality was calculated using SAMtools and bcftools. Position reflects the nucleotide in the coding sequence. The C to T change at position 647 in Soly02g (highlighted in red) was verified by Sanger sequencing and further confirmed by PCR genotyping. 2

20 Supplementary Table 3. Quantification of flowering time measured by leaf number in successive sympodial shoots derived from the primary shoots. The first five sympodial shoots were examined from the main shoot in 3-month old plants. STDEV, standard deviation; N, number of replicates. 3

21 Supplementary Table 4. Statistical comparisons of tomato yield components from two yield trials in two different environments. Shown are Red fruit (Red-kg), green fruit (Green-kg) and total yields (TY-kg) for each indicated genotype. Plant weight (PW-kg) is the 2 vegetative biomass after removing fruits. Comparisons of Fruit weight (FW-g), Brix (BX-%) and Brix-yield (BY-g/m ) are also shown. The percentage increase for each trait in each genotype over sp is shown (red font). Mean values were compared using a multiple range comparison test (Tukey-Kramer test; P < 0.05). Different letters represent significant differences. 4

22 Supplementary Table 5. Statistical comparisons of yield under optimal field conditions involving ssp and sft single and double mutant heterozygosity in the hybrid state with three distinct genetic backgrounds. Shown are total yields (kg) and Brix-yield (BY-g/m 2 ) for each indicated genotype. The percentage increase for each trait in each genotype over corresponding control hybrids (M82 x SAR, M82 x REB, and M82 x LEA) is shown. The Average effect change columns show average percentage changes for total yield and Brix-yield across four distinct genetic backgrounds (M82, and three hybrids with SAR, REB, and LEA). Mean values were compared using a multiple range comparison test (Tukey-Kramer test; P < 0.05). Different letters represent significant differences. 5

23 Supplementary Table 6. Primer sequences used in this study. 6