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
SUPPORTING ONLINE FIGURES Supplementary Figure 1 Intron: 1355bp 194 1839 197 1595 793 5941 496 559 114 124 112 130 213 640 315bp 207 89 199 59 108 46 114 69 66 101 180 213 267 57 162 97 389 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
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 99T748-26 SFB 99T748-30 SFB 99T748-49 SFB 99T748-61 SFB 99T748-66 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
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. 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
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
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 00T274-32 IL11-3 x LA2371 2.3 TA3113 1 3 3 3 3 3 3 3.1(0.4) 0.3030 3 3 3 3 3 3 3 2.8(0.2) 00T274-64 IL11-3 x LA2371 5.1 TA3114 3 1 1 1 1 1 1 12.2(2.0) 0.0014 3 3 3 3 3 1 1 5.3(1.6) 02T121-40 IL11-4 x LA2371 3.4 TA3116 1 1 1 1 1 1 1 5.1(1.1) 0.0474 1 3 3 3 3 3 3 3.3(0.1) 02T195-681 IL11-4 x LA2371 6.7 TA3120 3 1 1 1 1 1 1 15.7(4.1) 0.0268 3 3 3 3 3 3 3 7.4(1.1) 02T195-1693 IL11-4 x LA2371 11.8 TA3124 1 1 1 1 1 1 1 13.1(1.0) 0.1896 3 1 1 1 1 1 1 14.2(0.7) 02T195-1932 IL11-4 x LA2371 4.3 TA3132 1 1 1 1 1 1 1 11.8(02) <0.0001 1 3 3 3 3 3 3 4.3(0.8) 02T195-2146 IL11-4 x LA2371 4.7 TA3192 1 1 1 1 1 1 1 11.3(4.1) 0.001 1 3 3 3 3 3 3 5.6(2.2) 02T195-2649 IL11-4 x LA2371 5.2 TA3135 1 3 3 3 3 3 3 6.4(1.6) 0.2381 3 3 3 3 3 3 3 7.7(0.07) 02T195-3672 IL11-4 x LA2371 3.0 TA3194 3 1 1 1 1 1 1 4.8(0.4) 0.0021 3 3 3 3 3 3 3 3.1(0.2) 02T195-973 IL11-4 x LA2371 5.9 TA3122 1 1 3 3 3 3 3 14.0(1.4) 0.0007 3 3 3 3 3 3 3 4.7(1.0) 02T195-896 IL11-4 x LA2371 6.4 TA3121 3 3 3 1 1 1 1 8.4(1.9 0.6203 3 3 3 3 3 3 3 9.2(1.7) 00T275-76 IL11-4 x LA2371 2.9 TA3115 3 3 3 3 1 1 1 3.9(0.7) 0.0632 3 3 3 3 3 3 3 5.7(2.0) 02T195-1472 IL11-4 x LA2371 2.4 TA3123 3 3 3 3 3 1 1 2.9(0.3) 0.7059 3 3 3 3 3 3 3 2.8(0.0) 02T195-3223 IL11-4 x LA2371 4.1 TA3139 3 3 3 3 3 3 1 5.7(3.3) 0.4797 3 3 3 3 3 3 3 8.5(5.5) 02T195-921 IL11-4 x LA2371 3.2 TA3130 1 1 1 1 1 1 3 9.7(3.1) 0.0434 3 3 3 3 3 3 3 4.1(1.2) 02T195-2012 IL11-4 x LA2371 3.0 TA3133 1 1 1 1 1 1 3 7.9(2.6) 0.0231 3 3 3 3 3 3 3 2.6(0.2) 02T195-2112 IL11-4 x LA2371 4.7 TA3134 1 1 1 1 1 1 3 7.9(1.3) 0.0043 3 3 3 3 3 3 3 3.3(0.5) T1949 323E19S 53K9T 89M2T clet24j2 7
02T195-199 IL11-4 x LA2371 6.1 TA3117 1 1 1 1 1 1 3 13.5(1.3) 0.0092 3 3 3 3 3 3 3 5.8(2.5) 02T195-441 IL11-4 x LA2371 3.2 TA3128 3 3 3 3 3 3 1 6.8(2.2) 0.7427 3 3 3 3 3 3 3 6.3(1.6) 02T195-484 IL11-4 x LA2371 5.7 TA3119 1 1 1 1 1 1 1 9.7(3.9) 0.8242 1 1 1 1 1 1 3 10.4(3.7) 02T195-789 IL11-4 x LA2371 3.9 TA3129 1 1 1 1 1 1 1 10.1(1.7) 0.0068 3 3 3 3 3 3 1 4.5(0.8) 02T195-1453 IL11-4 x LA2371 3.2 TA3131 1 1 1 1 1 1 3 5.2(1.0) 0.0368 3 3 3 3 3 3 3 3.4(0.4) 02T195-2290 IL11-4 x LA2371 12.4 TA3125 1 1 1 1 1 1 1 15.5(3.4) 0.7995 1 1 1 1 1 1 3 14.7(4.4) 02T195-3087 IL11-4 x LA2371 2.5 TA3136 1 1 1 1 1 1 1 3.8(0.15) <0.0001 3 3 3 3 3 3 1 2.3(0.06) 02T195-3096 IL11-4 x LA2371 5.8 TA3137 3 3 3 3 3 3 1 4.2(0.5) 0.7458 3 3 3 3 3 3 3 4.5(1.3) 02T195-3132 IL11-4 x LA2371 5.0 TA3138 1 1 1 1 1 1 1 11.2(3.7) 0.0607 3 3 3 3 3 3 1 4.8(2.2) 02T195-3174 IL11-4 x LA2371 2.9 TA3126 1 1 1 1 1 1 1 8.8(4.4) 0.0978 3 3 3 3 3 3 1 3.3(0.6) 02T195-3616 IL11-4 x LA2371 3.7 TA3140 1 1 1 1 1 1 3 8.0(1.1) 0.0232 3 3 3 3 3 3 3 4.7(1.2) 02T195-3752 IL11-4 x LA2371 3.2 TA3141 3 3 3 3 3 3 1 4.3(1.3) 1 3 3 3 3 3 3 3 4.3(1.0) 02T195-3760 IL11-4 x LA2371 6.9 TA3127 1 1 1 1 1 1 1 14.9(0.3) 0.0005 3 3 3 3 3 3 1 6.6(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
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 12D23.1 2902 7360 Glycosyl hydrolases ABI49503 Solanum demissum 0.0 family 17 protein 2 12D23.2 9112 8780 Ribosomal protein ABI49504 Solanum demissum 1e-43 L7/L12 C-terminal domain containing protein 3 12D23.3 13842 18041 hypothetical protein AAK91899 Solanum demissum 0.0 4 12D23.4 22531 19108 KPHMT1/PANB1 NP_182135 Arabidopsis thaliana 2e-109 5 12D23.5 24537 27734 Amidase family protein AAK91890 Solanum demissum 0.0 6 12D23.6 29755 28596 Hsp20/alpha crystallin AAK91897 Solanum demissum 9e-126 family protein 7 12D23.7 30783 35041 tubulin family protein AAK91898 Solanum demissum 0.0 8 12D23.8 38208 39967 hypothetical protein AAT40511 Solanum demissum 0.0 9 12D23.9 44188 42967 hypothetical protein AAT40514 Solanum demissum 7e-48 10 12D23.10 45925 55440 aldehyde oxidase AAG22607 s.lycopersicum 0.0 11 12D23.11 58955 66392 aldehyde oxidase AAG22607 s.lycopersicum 0.0 12 12D23.12 72543 80446 aldehyde oxidase AAG22605 s.lycopersicum 0.0 13 12D23.13 88367 81629 Putative retrotransposon AAT38724 Solanum demissum 2e-07 protein 14 12D23.14 99442 105894 aldehyde oxidase AAG22605 s.lycopersicum 0.0 15 12D23.15 107659 107035 ARR22 NP_187078 Arabidopsis thaliana 3e-20 16 12D23.16 109500 110374 NB-ARC domain AAT39932 Solanum demissum 7e-88 containing protein 17 12D23.17 117696 120967 beta-d-glucan BAA33065 Nicotiana tabacum 0.0 exohydrolase 18 12D23.18 123262 127986 Exoglucanase precursor NP_001051275 Oryza sativa 0.0 19 12D23.19 132923 131319 hypothetical protein NP_001063358 Oryza sativa 3e-73 20 12D23.20 137185 138702 pentatricopeptide, NP_001049331 Oryza sativa 4e-143 putative 21 12D23.21 146583 141722 tousled-like kinase 2 NP_001051278 Oryza sativa 0.0 20 323E19.1 86 1235 pentatricopeptide, NP_001049331 Oryza sativa 2e-145 putative 21 323E19.2 16218 4381 TSL (TOUSLED) NP_568405 Arabidopsis thaliana 0.0 22 323E19.3 22832 18488 zinc knuckle (CCHCtype) NP_189945 Arabidopsis thaliana 1e-66 family protein 23 323E19.4 25090 30809 UBP12 NP_568171 Arabidopsis thaliana 0.0 24 323E19.5 36675 51362 putative ubiquitin AAF23207 Arabidopsis thaliana 9e-146 carboxyl-terminal hydrolase 25 323E19.6 52468 62739 kinesin motor proteinrelated NP_189991 Arabidopsis thaliana 0.0 26 323E19.7 65518 63267 regulator of gene AAT65969 Solanum 2e-72 silencing lycopersicum 27 323E19.8 67978 67382 regulator of gene AAT65969 Solanum 1e-108 silencing lycopersicum 28 323E19.9 74342 71268 elongation factor Tu family protein NP_188938 Arabidopsis thaliana 0.0 9
29 323E19.10 77554 76805 hypothetical protein ABD32418 Medicago truncatula 9e-10 30 323E19.11 82117 79081 unknown ABK92625 Populus trichocarpa 1e-48 31 323E19.12 86878 82862 strictosidine synthase NP_563818 Arabidopsis thaliana 3e-171 family 32 323E19.13 97661 91475 YABBY2-like AAS10179 Antirrhinum majus 4e-42 transcription factor 33 323E19.14 103668 106692 protein kinase family NP_680192 Arabidopsis thaliana 2e-63 34 323E19.15 110292 107285 putative DNAJ protein CAC12824 Nicotiana tabacum 0.0 35 323E19.16 116258 122259 calmodulin binding NP_188858 Arabidopsis thaliana 2e-82 36 323E19.17 125430 126500 unknown protein NP_190774 Arabidopsis thaliana 3e-109 37 323E19.18 134431 134835 zinc finger NP_190386 Arabidopsis thaliana 7e-18 38 323E19.19 137015 139648 ubiquitin carrier protein 4 AAF03236 Glycine max 2e-63 39 323E19.20 140447 141109 unnamed protein product CAO64369 Vitis vinifera 1e-50 a The BACs were annotated by FGENESH at http://linux1.softberry.com/berry.phtml b,c indicating start and stop codon d Homologs blasted in NCBI with accession number and species 10
Supplementary Table 3A. Summary of transgenic complementation tests in T0 T0 Transgenic Material Number of Fruit ALN a ± s.d. P-value b 06T689-01 LA2371 15 12.8667 ± 2.1995 0.0232 06T689-06 LA2371 13 10.3077 ± 2.7503 0.0001 06T689-07 LA2371 10 11.5000 ± 2.3214 0.0052 06T689-23 LA2371 16 11.8125 ± 2.8100 0.0020 06T689-33 LA2371 18 12.3889 ± 2.6377 0.0054 control LA2371 56 15.2143 ± 4.3179 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 06T814-02 T 18 11.44 ± 4.13 06T814-06 T 7 9.29 ± 3.35 06T814-08 T 18 11.33 ± 2.91 06T814-09 T 8 11.63 ± 1.41 06T814-10 T 14 11.43 ± 2.34 06T814-11 T 19 11.16 ± 1.77 06T814-12 T 21 11.24 ± 2.83 06T814-15 T 16 13.69 ± 3.82 06T814-24 T 10 11.7 ± 3.20 06T814-27 T 11 12.0 ± 2.72 06T814-28 T 16 11.56 ± 2.97 0.000 06T814-13 N 14 14.57 ± 2.71 06T814-19 N 23 12.7 ± 2.95 06T814-22 N 21 13.67 ± 2.37 06T814-25 N 17 13.88 ± 4.27 06T814-26 N 16 14.19 ± 2.90 06T814-35 N 19 13.89 ± 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
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 LA0517-3 7bp 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 LA2452-2 7bp 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
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
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 1706 53K9 T CATGGTTCCCACG TATTAAATG AAATGATCTTTTTC CAATGTCAA SNPs N/A BAC clone from Heinz 1706 89M2 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 www.sgn.cornell.edu 14
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 100805-101774 WPF2/F2 CTTTGGATCCAGATCCCGTA GAGGTGTGAACCGAAGTTAAAA 100014-100972 WPF3/R3 TAAGTTTCGAGTTCACAAAGAATTG AATGCGAGAAATAAGTGATCAAAA 99258-100107 prof/r GCTAACCGCATGTCAAACAC GCGTTCTGAGGAAGGTGAAG 97617-99313 WPF4/R4 TGTGTTGGATGAATTGTGACC TGTTGGTTTTGGAGATATCTGGT 98329-99128 prof2/r2 CTATCGCGTCATTGTTCTTCC TGGATCAAAGGGGAAGAGTG 97712-98526 FP1/RP1 TTCCCCTTTGATCCATGTTC CAAAATTCATGACACAAAGGACA 96373-97726 FP1/RP1a TTCCCCTTTGATCCATGTTC TGATCATACTCATCACACATGAGC 97426-97726 FP2/RP2 CAGGAGCACCAAAAGTAAAGG TGGAACAAATCGAAAAGGAAA 95276-96572 FP3/RP3 GTAAGATGTGGGCATTGTGC CCTGGTAGCCGGTTACACTT 94529-95623 FP4/RP4 TGTTGTTTGCAACATGTCCTT TTTGGCAGCAGTGCTAAAAG 93198-94601 FP5/RP5 CCTCTCTTGCCACCGAAAAT GGCCAAAAATAGACGCATGT 92165-93387 FP6/RP6 TGCGTCTATTTTTGGCCATT CATCAAATGGGACAAAGGAGTAT 91166-92181 EX4F/5R TCAAACTATGCAGCACCAGA GGACTTACATTTTTGGCAGCA 93187-93586 EX6F/6R AGTGGGCACATTTTCCACAT AAATCTTCGACGAAAACAACA 91396-91530 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
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, 413-22 (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, 669-79 (2004). 16