Regulatory Change in YABBY-like Transcription Factor Led to Evolution of Extreme Fruit Size during Tomato Domestication

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1 SUPPORTING ONLINE MATERIALS Regulatory Change in YABBY-like Transcription Factor Led to Evolution of Extreme Fruit Size during Tomato Domestication Bin Cong, Luz Barrero, & Steven Tanksley 1

2 SUPPORTING ONLINE FIGURES Supplementary Figure 1 Intron: 1355bp bp Exon UTR Intron cdna length: 2,722bp ORF: 2,019bp Exons: 16 Introns:15 Amino acids: 672aa Supplementary Figure 1. Depicts exons and introns of TOUSLED-like protein kinase found in the BAC. 2

3 Supplementary Figure 2 A tsl actin SFB MFB LFB carpel M W M W M W M W B tsl actin C Wild species Mutant Wild type Fasciated allele allele TA56 SFB TA56 MFB TA56 LFB LA925 SFB LA925 MFB LA925 LFB 99T748-9 SFB 99T SFB 99T SFB 99T SFB 99T SFB 99T SFB tsl actin Supplementary Figure 2. Expression analysis of the tousled gene using RT-PCR analysis. (A) tousled mrna was strongly expressed in all developing flower stages and carpels dissected from anthesis flowers in wild type TA209 and mutant LA2371. (B) Similar experiments done in mutant line LA0767 and wild type TA496. tousled expressed equally in wild type flowers buds and in mutant. W in (A) and (B) means wild type; M means mutant. (C) tousled mrna expression was examined in F2 individuals derived from S. lycopersicum (LA0925, high locule number) and S. pennellii (LA0716, wild species with low locule number) and their parental lines LA0925 and LA0716, respectively. tousled strongly expressed in lines containing wild type fas locus on chromosome 11 and lines containing mutant fas locus on chromosome 11. Actin was used as internal control. SFB: small flower buds; MFB: medium flower buds; and LFB: large flower buds. 3

4 Supplementary Figure 3 Supplementary Figure 3. A representative transformation result with the fasciated gene. Left fruit from high-locule number genotype T1 plants transformed with the fasciated wild type allele; Right: fruit from non-transformed, high-locule number genotype T1 plants derived from the same hemizygous T0 individual. Note: The extremely large tomato fruit found in modern cultivars (including LA2371 which was used in the transformation experiments) is due, not only to the fasciated QTL, but also other QTLs that increase carpel/locule number (e.g. the locule-number QTL) 1,2. Transformation/ complementation experiments using the fasciated gene were thus not expected to reduce carpel/locule number to the extent found in fruit produced by primitive or wild tomato varieties (e.g. 2-4 carpels/locules). Rather, it was only expected to significantly decrease carpel/locule number as observed. 4

Supplementary Figure 4 Marker EcoRI EcoRV HaeIII ScaI HindIII 1 2 1 2 1 2 1 2 1 2 Supplementary Figure 4.

5 Supplementary Figure 4 Marker EcoRI EcoRV HaeIII ScaI HindIII Supplementary Figure 4. Southern blot showing a 6-8kb insertion in the first intron of the fasciated gene in mutant. Lane labeled with 1 indicating DNA extracted from wild type TA209; 2 indicating DNA from mutant LA2371. DNAs were digested with EcoRI, EcoRV, HaeIII, ScaI and HindIII. 5

6 Supplementary Figure 5 fasciated_1 fasciated 1 ATGTCATTCGATATGACTTTTTCTTCTTCACCTTCCTCAGAACGCGTTTGTTACTTGCAA 1 ATGTCATTCGATATGACTTTTTCTTCTTCACCTTCCTCAGAACGCGTTTGTTACTTGCAA fasciated_1 fasciated 61 TGTAATTTTTGCAACACCATTCTTGCGGTTAGTGTTCCATGCAGCAACATGTTAACCTTA 61 TGTAATTTTTGCAACACCATTCTTGCGGTTAGTGTTCCATGCAGCAACATGTTAACCTTA fasciated_1 121 GTGACAGTAAGATGTGGGCATTGTGCAAATATGCTTTCTGTTAATATTGGATCTTTAATT fasciated 121 GTGACAGTAAGATGTGGGCATTGTGCAAATATGCTTTCTGTTAATATTGGATCTTTAATT fasciated_1 181 CAAGCTCTACCCCTTCAAGATGTTCAAAAGCTGCAAAGGCAACAGTACACAAATGTTGAA fasciated 181 CAAGCTCTACCCCTTCAAGATGTTCAAAAGCTGCAAAGGCAACAGTACACAAATGTTGAA fasciated_1 241 AATAATTCTAGTAATTATAAAGCTTATGGTTCATCATCATCATCCTCTTCAAAGTTCAAC fasciated 241 AATAATTCTAGTAATTATAAAGCTTATGGTTCATCATCATCATCCTCTTCAAAGTTCAAC fasciated_1 301 AGATATTCTTCCATTGTTTCTCCTCAAATTGAACCTAAAATCCCTTCGATTCGTTCACCA fasciated 301 AGATATTCTTCCATTGTTTCTCCTCAAATTGAACCTAAAATCCCTTCGATTCGTCCACCA fasciated_1 361 GAGAAAAAGACAACGTGTTCCTTCTGCGTACAACCGATTCATCAAGGAAGAGATTCAAAG fasciated 361 GAGAAAA-GACAACGTGTTCCTTCTGCGTACAACCGATTCATCAAGGAAGAGATTCAAAG fasciated_1 421 GATCAAGGCCAGTAATCCTGATATTAGCCATCGTGAAGCTTTTAGCACTGCTGCCAAAAA fasciated 420 GATCAAGGCCAGTAATCCTGATATTAGCCATCGTGAAGCTTTTAGCACTGCTGCCAAAAA fasciated_1 481 TTGGGCACATTTTCCACATATTCACTTTGGACTCAAGCTGGAGGGCAACAAATAG fasciated 480 TTGGGCACATTTTCCACATATTCACTTTGGACTCAAGCTGGAGGGCAACAAATAG Supplementary Figure 5. Sequence comparison between a new allele of fasciated gene found in S.lycopersicum cv. cerasiforme with high-locule nubmer and cultivated wild type tomato showing a nucleotide substitution and followed by one nucleotide insertion which results a truncated protein in the new allele. 6

7 SUPPORTING ONLINE TABLES Supplementary Table 1. F3 progeny analysis of the selected recombinants in the fasciated region between makers T302 and clet24j2a on Chromosome 11 F2 family F3 family Markers b Parental pedigree ALN a F3 plant T302 T1948 TG105A F2 plant ALN(SD) P-value c 00T IL11-3 x LA TA (0.4) (0.2) 00T IL11-3 x LA TA (2.0) (1.6) 02T IL11-4 x LA TA (1.1) (0.1) 02T IL11-4 x LA TA (4.1) (1.1) 02T IL11-4 x LA TA (1.0) (0.7) 02T IL11-4 x LA TA (02) < (0.8) 02T IL11-4 x LA TA (4.1) (2.2) 02T IL11-4 x LA TA (1.6) (0.07) 02T IL11-4 x LA TA (0.4) (0.2) 02T IL11-4 x LA TA (1.4) (1.0) 02T IL11-4 x LA TA ( (1.7) 00T IL11-4 x LA TA (0.7) (2.0) 02T IL11-4 x LA TA (0.3) (0.0) 02T IL11-4 x LA TA (3.3) (5.5) 02T IL11-4 x LA TA (3.1) (1.2) 02T IL11-4 x LA TA (2.6) (0.2) 02T IL11-4 x LA TA (1.3) (0.5) T E19S 53K9T 89M2T clet24j2 7

8 02T IL11-4 x LA TA (1.3) (2.5) 02T IL11-4 x LA TA (2.2) (1.6) 02T IL11-4 x LA TA (3.9) (3.7) 02T IL11-4 x LA TA (1.7) (0.8) 02T IL11-4 x LA TA (1.0) (0.4) 02T IL11-4 x LA TA (3.4) (4.4) 02T IL11-4 x LA TA (0.15) < (0.06) 02T IL11-4 x LA TA (0.5) (1.3) 02T IL11-4 x LA TA (3.7) (2.2) 02T IL11-4 x LA TA (4.4) (0.6) 02T IL11-4 x LA TA (1.1) (1.2) 02T IL11-4 x LA TA (1.3) (1.0) 02T IL11-4 x LA TA (0.3) (1.4) a ALN- average locule number b Marker score: 1 homozygous for S.lycopersicum alleles; 3 homozygous for S.pennellii alleles c P-values of t-tests for comparisons within each F3 family 8

9 Supplementary Table 2. Summary of gene content for assembly 12D23 and 323E19 Start b End c Putative Function Accession/Species d E ID BAC Gene ID a Value 1 12D Glycosyl hydrolases ABI49503 Solanum demissum 0.0 family 17 protein 2 12D Ribosomal protein ABI49504 Solanum demissum 1e-43 L7/L12 C-terminal domain containing protein 3 12D hypothetical protein AAK91899 Solanum demissum D KPHMT1/PANB1 NP_ Arabidopsis thaliana 2e D Amidase family protein AAK91890 Solanum demissum D Hsp20/alpha crystallin AAK91897 Solanum demissum 9e-126 family protein 7 12D tubulin family protein AAK91898 Solanum demissum D hypothetical protein AAT40511 Solanum demissum D hypothetical protein AAT40514 Solanum demissum 7e D aldehyde oxidase AAG22607 s.lycopersicum D aldehyde oxidase AAG22607 s.lycopersicum D aldehyde oxidase AAG22605 s.lycopersicum D Putative retrotransposon AAT38724 Solanum demissum 2e-07 protein 14 12D aldehyde oxidase AAG22605 s.lycopersicum D ARR22 NP_ Arabidopsis thaliana 3e D NB-ARC domain AAT39932 Solanum demissum 7e-88 containing protein 17 12D beta-d-glucan BAA33065 Nicotiana tabacum 0.0 exohydrolase 18 12D Exoglucanase precursor NP_ Oryza sativa D hypothetical protein NP_ Oryza sativa 3e D pentatricopeptide, NP_ Oryza sativa 4e-143 putative 21 12D tousled-like kinase 2 NP_ Oryza sativa E pentatricopeptide, NP_ Oryza sativa 2e-145 putative E TSL (TOUSLED) NP_ Arabidopsis thaliana E zinc knuckle (CCHCtype) NP_ Arabidopsis thaliana 1e-66 family protein E UBP12 NP_ Arabidopsis thaliana E putative ubiquitin AAF23207 Arabidopsis thaliana 9e-146 carboxyl-terminal hydrolase E kinesin motor proteinrelated NP_ Arabidopsis thaliana E regulator of gene AAT65969 Solanum 2e-72 silencing lycopersicum E regulator of gene AAT65969 Solanum 1e-108 silencing lycopersicum E elongation factor Tu family protein NP_ Arabidopsis thaliana 0.0 9

10 29 323E hypothetical protein ABD32418 Medicago truncatula 9e E unknown ABK92625 Populus trichocarpa 1e E strictosidine synthase NP_ Arabidopsis thaliana 3e-171 family E YABBY2-like AAS10179 Antirrhinum majus 4e-42 transcription factor E protein kinase family NP_ Arabidopsis thaliana 2e E putative DNAJ protein CAC12824 Nicotiana tabacum E calmodulin binding NP_ Arabidopsis thaliana 2e E unknown protein NP_ Arabidopsis thaliana 3e E zinc finger NP_ Arabidopsis thaliana 7e E ubiquitin carrier protein 4 AAF03236 Glycine max 2e E unnamed protein product CAO64369 Vitis vinifera 1e-50 a The BACs were annotated by FGENESH at b,c indicating start and stop codon d Homologs blasted in NCBI with accession number and species 10

11 Supplementary Table 3A. Summary of transgenic complementation tests in T0 T0 Transgenic Material Number of Fruit ALN a ± s.d. P-value b 06T LA ± T LA ± T LA ± T LA ± T LA ± control LA ± a ALN average locule number b P-Values are results from one-tailed student s t-test between transgenic plants vs. nontransgenic plants Supplementary Table 3B. Summary of transgenic plants vs. non-transgenic plants segregated int1 from T0 complementation line (06T689-06) T1 Transgene a Number of fruit ALN ± s.d. P-value b 06T T ± T T ± T T ± T T ± T T ± T T ± T T ± T T ± T T ± T T ± T T ± T N ± T N ± T N ± T N ± T N ± T N ± 3.31 a Transgenic plants (T) and Non-transgenic plants (N) segregated in T1 family b P-Value is result from one-tailed student s t-test between transgenic plants vs. nontransgenic plants segregated from the same T0 line 11

12 Supplementary Table 4. Summary of indels among fasciated stocks and wild types Accessions Indel at about 330bp to 5' ATG 1 st intron insertion Locule Number RT- PCR Species 1 LA0014 7bp deletion 6-8kb fas S.lycopersicum 2 LA0020 7bp deletion 6-8kb fas S.lycopersicum 3 LA bp deletion 6-8kb fas S.lycopersicum 4 LA0767 7bp deletion 6-8kb fas No S.lycopersicum 5 LA0925 7bp deletion 6-8kb fas No S.lycopersicum 6 LA1113 7bp deletion 6-8kb fas No S.lycopersicum 7 LA1786 7bp deletion 6-8kb fas S.lycopersicum 8 LA2367 7bp deletion 6-8kb fas S.lycopersicum 9 LA2371 7bp deletion 6-8kb fas No S.lycopersicum 10 LA bp deletion 6-8kb fas S.lycopersicum 11 LA2595 7bp deletion 6-8kb fas S.lycopersicum 12 LA2799 7bp deletion 6-8kb fas S.lycopersicum 13 TA1486 7bp deletion 6-8kb fas S.lycopersicum 14 TA3757 7bp deletion 6-8kb fas S.lycopersicum 15 TA3762 7bp deletion 6-8kb fas S.lycopersicum 16 TA3765 7bp deletion 6-8kb fas S.lycopersicum 17 TA3766 7bp deletion 6-8kb fas S.lycopersicum 18 TA3767 7bp deletion 6-8kb fas S.lycopersicum 19 TA3768 7bp deletion 6-8kb fas S.lycopersicum 20 TA3769 7bp deletion 6-8kb fas S.lycopersicum 21 TA3770 7bp deletion 6-8kb fas S.lycopersicum 22 TA3771 7bp deletion 6-8kb fas S.lycopersicum 23 TA3772 7bp deletion 6-8kb fas No S.lycopersicum 24 TA3773 7bp deletion 6-8kb fas S.lycopersicum 25 TA3774 7bp deletion 6-8kb fas S.lycopersicum 26 TA209 No No Normal Yes S.lycopersicum 27 M82 No No Normal Yes S.lycopersicum 28 TA3752 7bp insertion No Normal S.lycopersicum 29 TA3753 7bp insertion No Normal S.lycopersicum 30 TA3754 7bp deletion No Normal Yes S.lycopersicum 31 TA3755 7bp insertion No Normal S.lycopersicum 32 TA3756 7bp insertion No Normal S.lycopersicum 33 TA3758 7bp insertion No Normal S.lycopersicum 34 TA3759 No No Normal S.lycopersicum 35 TA3760 7bp insertion No Normal S.lycopersicum 36 TA3761 7bp insertion No Normal S.lycopersicum 37 TA3763 7bp insertion No Normal S.lycopersicum 12

13 38 TA3764 7bp insertion No Normal S.lycopersicum 39 LA1654 No No 5-8 Yes cerasiforme 40 LA1654 No No 5-8 Yes cerasiforme 41 LA2121 7bp insertion No 5-8 Yes cerasiforme 42 LA2126 No No 5-8 Yes cerasiforme 43 LA2136 7bp insertion No 4-6 Yes cerasiforme 44 LA2095 7bp insertion No 4-6 weak cerasiforme 45 LA2845 No No 4-8 Yes cerasiforme 46 LA1247 No No Normal cerasiforme 47 LA1338 No No Normal Yes cerasiforme 48 LA1386 No No Normal cerasiforme 49 LA1423 No No Normal cerasiforme 50 LA1701 No No Normal cerasiforme 51 LA2135 No No Normal cerasiforme 52 LA2137 4bp deletion No Normal cerasiforme 53 LA2177 7bp insertion No Normal cerasiforme 54 LA2312 No No Normal cerasiforme 55 LA2844 7bp insertion No Normal cerasiforme 56 LA3623 7bp deletion No Normal Yes cerasiforme 57 LA3623 No No Normal Yes cerasiforme 58 LA3623 7bp deletion No Normal cerasiforme 59 LA1226 4bp deletion No Normal Yes cerasiforme 60 LA1228 4bp deletion No Normal cerasiforme 61 LA1231 Normal No Normal cerasiforme 62 LA1420 7bp insertion No Normal Yes cerasiforme 63 LA1429 No No Normal cerasiforme 64 LA1549 No No Normal Yes cerasiforme 64 LA2131 7bp insertion No 5-8 Yes cerasiforme 66 LA2308 No No Normal cerasiforme 67 LA2616 7bp insertion No Normal cerasiforme 68 LA1320 7bp deletion No Normal Yes cerasiforme 69 LA483 Normal S.cheesmanii 70 LA1589 No Normal pimpinellifolium 71 LA1708 No Normal S.peruvianum 72 LA1777 No Normal S.hirsutum 73 LA2133 No No Normal S.parviflorum 75 LA0716 No Normal Yes S.pennellii 13

14 Supplementary Table 5. Summary of tomato markers used for the fasciated region high-resolution map Marker a Forward Primer Reverse primer Marker Type Restrict Enzyme Source b T302 TGGCTCATCCTGA AGCTGATAGCGC AGTGTACATCCTTG CCATTGACT INDEL/ RFLP XbaI Clone cled-24- J21 323E19 T AGATAAGCGAAA GGGGTTTTACC TTATCACATCAGGA GTAATATCCTTC CAPs MspI BAC clone from Heinz 1706 T1948 TTCCCAATTTCAC CAATTTTTC ATCCAGAACCAGC ACATTTCTT CAPs/ RFLP DraI/ EcoRI, V Clone clet-45- E9 TG105A N/A N/A RFLP DraI Genomic clone T1949 AAAGATCACCCG GGAATTCCACGATG CAPs/ RsaI/ Clone cleg13c3 AAGTTCAAGA AAATGAAT RFLP HaeIII, DraI 323E19 S GAGCCACACAAC AGAGGATTC TGAAGAAAAATCA TTCTTGATGGT CAPs DpnII, ApoI BAC clone from Heinz K9 T CATGGTTCCCACG TATTAAATG AAATGATCTTTTTC CAATGTCAA SNPs N/A BAC clone from Heinz M2 T AGGGACTGACAA GTTTTGGTTTT TCCTAAAGGTTTAA TGAGAGTTATCCA CAPs RsaI BAC clone from Heinz 1706 clet24j2a CAACCATCCTAG CAATGAAATCT GAGGCATTCACTCT CTTCGATAC CAPs/ RFLP KpnI/ ScaI cdna clone a markers were developed from cdna, or BAC clones. If markers developed from BAC clones, with S indicating SP6 end; T indicating T7 end b detailed information may be found in 14

15 Supplementary Table 6. The primer pairs used to amplify wide type TA209 and mutant LA2371 and LA0767 Primer name Forward primer Reverse primer Corresponding region to 323E19 (bp) WPF1/R1 TGACCCATTGTTGCTTCTGA TGAAACGTAGTCCTCCGAAAA WPF2/F2 CTTTGGATCCAGATCCCGTA GAGGTGTGAACCGAAGTTAAAA WPF3/R3 TAAGTTTCGAGTTCACAAAGAATTG AATGCGAGAAATAAGTGATCAAAA prof/r GCTAACCGCATGTCAAACAC GCGTTCTGAGGAAGGTGAAG WPF4/R4 TGTGTTGGATGAATTGTGACC TGTTGGTTTTGGAGATATCTGGT prof2/r2 CTATCGCGTCATTGTTCTTCC TGGATCAAAGGGGAAGAGTG FP1/RP1 TTCCCCTTTGATCCATGTTC CAAAATTCATGACACAAAGGACA FP1/RP1a TTCCCCTTTGATCCATGTTC TGATCATACTCATCACACATGAGC FP2/RP2 CAGGAGCACCAAAAGTAAAGG TGGAACAAATCGAAAAGGAAA FP3/RP3 GTAAGATGTGGGCATTGTGC CCTGGTAGCCGGTTACACTT FP4/RP4 TGTTGTTTGCAACATGTCCTT TTTGGCAGCAGTGCTAAAAG FP5/RP5 CCTCTCTTGCCACCGAAAAT GGCCAAAAATAGACGCATGT FP6/RP6 TGCGTCTATTTTTGGCCATT CATCAAATGGGACAAAGGAGTAT EX4F/5R TCAAACTATGCAGCACCAGA GGACTTACATTTTTGGCAGCA EX6F/6R AGTGGGCACATTTTCCACAT AAATCTTCGACGAAAACAACA Supplementary Table 7. Primers used in the methods Primer name sequence BB25 GACTCGAGTCGACATCGA(dT) 17 BB26 GACTCGAGTCGACATCGA Actin (TOM52 F) CCAAAAGCCAATCGAGAGAA Actin (TOM52 R) GGTACCACCACTGAGGACGA 3 UTR F GCAACAAATAGCGAGGGAAA 3 UTR R CACAACTCTGAAAATAATATTAAGACGA 15

16 SUPPORTING ONLINE REFERENCES 1. Lippman, Z. & Tanksley, S.D. Dissecting the genetic pathway to extreme fruit size in tomato using a cross between the small-fruited wild species Lycopersicon pimpinellifolium and L. esculentum var. Giant Heirloom. Genetics 158, (2001). 2. Barrero, L.S. & Tanksley, S.D. Evaluating the genetic basis of multiple-locule fruit in a broad cross section of tomato cultivars. Theor Appl Genet 109, (2004). 16

Nature Genetics: doi: /ng Supplementary Figure 1. The phenotypes of PI , BR121, and Harosoy under short-day conditions.

Nature Genetics: doi: /ng Supplementary Figure 1. The phenotypes of PI , BR121, and Harosoy under short-day conditions. Supplementary Figure 1 The phenotypes of PI 159925, BR121, and Harosoy under short-day conditions. (a) Plant height. (b) Number of branches. (c) Average internode length. (d) Number of nodes. (e) Pods