Supplemental Figure 1 s2

SUPPLEMENTAL FIGURE 1

TaSEP-2A AGACATCGCTCTGCTGGCTGCGGAAAATAAAAGGAGACTTAGAGAGAGAGAAAAGAAAGA 60

TaSEP-2B AGACATCGCTCTGCTGGCTGCGGAAAATAAAAGGAGACTTAGAGAGAGAGAAAAGAAAGA 60

TaSEP-2A GAGGAGGAGGAGGAGATGGGTCGGGGGAAGGTGGAGATGAGGCGGATCGAGAACAAGATA 120

TaSEP-2B GAGGAGGAGGAGGAGATGGGTCGGGGGAAGGTGGAGATGAGGCGGATCGAGAACAAGATA 120

TaSEP-2A AGCCGGCAGGTGACGTTCGCCAAGCGCCGGAATGGGCTGCTCAAGAAGGCCTACGAGCTC 180

TaSEP-2B AGCCGGCAGGTGACGTTCGCCAAGCGCCGGAATGGGCTGCTCAAGAAGGCCTACGAGCTC 180

TaSEP-2A TCGCTGCTCTGCGACGCCGAGGTCGCCCTCATCATCTTCTCCGGCCGCGGCCGCCTCTTC 240

TaSEP-2B TCGCTGCTCTGCGACGCCGAGGTCGCCCTCATCATCTTCTCCGGCCGCGGCCGCCTCTTC 240

TaSEP-2A GAGTTCTCAAGCTCCTCATGCATGTACAGAACACTTGAGAGATACCGTACCTGCAACTCC 300

TaSEP-2B GAGTTCTCAAGCTCCTCATGCATGTACAGAACACTTGAGAGATACCGTACCTGCAACTCC 300

TaSEP-2A AACTCACAGGAAGCAACACCTCCGCTAGAAAATGAAATTAATTACCAGGAATATTTGAAG 360

TaSEP-2B AACTCACAGGAAGCAACACCTCCGCTAGAAAATGAAATTAATTACCAGGAATATTTGAAG 360

TaSEP-2A CTCAAGACCAGAGTTGAATTTCTTCAAAGTTCACAAAGAAATATTCTCGGTGAGGATCTG 420

TaSEP-2B CTCAAGACCAGAGTTGAATTTCTTCAAAGTTCACAAAGAAATATTCTCGGTGAGGATCTG 420

TaSEP-2A GGCCCACTTAGCATGAAGGAGCTTGACCAGATAGAGAACCAAATAGATGCATCCCTCAAG 480

TaSEP-2B GGCCCACTTAGCATGAAGGAGCTTGACCAGATAGAGAACCAAATAGATGCATCCCTCAAG 480

TaSEP-2A CATATCAGGTCAAAGAAGAATCAAGTATTACTCGATCAGCTGTTTGAACTGAAAAGTAAG 540

TaSEP-2B CATATCAGGTCAAAaAAGAATCAAGTATTACTCGATCAGCTaTTTGAACTGAAAAGTAAG 540

TaSEP-2A GAGCAAGAATTGCAGGATGAAAACAAAGACTTGAGGAAGAAGTTGCGAGATACCACCAGC 600

TaSEP-2B GAGCAAGAATTGCAGGATGAAAACAAtGACTTGAGGAAGAAGTTGCaAGATACCACCAGt 600

TaSEP-2A AGCTGCGGAGAGAATGCGGTCCATATGTCCTGGCAAGACGGAGGGCAGTCTAGCTCCAGA 660

TaSEP-2B tGCTGCGGAGAcAATGCGGTCCATATGTCCTGGCAAGACGGAGGGCAGTgTAGCTCCAGA 660

TaSEP-2A GTACTCCAACACCCGGAGCATGATACCTCCATGCAAATTGGGTATCCTCAGGCCTACATG 720

TaSEP-2B GTACT...ACACCCGGAGCATGATACCTCCATGCAAATTGGGTATCCTCAGGCCTACATG 717

TaSEP-2A GACCAGCTGAACAG.CAGAGATCACGTGGCTTCTGAACGCCCTGGTGGAGGATCGTCTGC 779

TaSEP-2B GACCAGCTGAACAaaCAGAGATCACGTGGCTTCTGAgCGCCCTGGTGGAGGATCGTCTGC 777

TaSEP-2A AGGGTGGATATGATTAACTGATGTGGCGCTGCGTCGTGTGTACTGTAGTTAATGTATCTG 839

TaSEP-2B AGGtTGGATATGATTAACTGATGTGGCGCTGCGTCaTGTGTACTGTAGcTAATGTATtTG 837

TaSEP-2A TACTTCGGTGACAATTGTTATA..TTTTTTGTGTACTGGAGTTGTGGTGATGAGCACCAC 897

TaSEP-2B TACTTCGGTGACAATTGTTATAttTTTTTTGTGTACTGGAGTTGTGGTGATGAGCACCAC 897

TaSEP-2A ATGTATCTACTGAGATTACGTGTGTGCTAGGCTGCGTGGTACGCTGACGGCCCAAAAGTT 957

TaSEP-2B ATGTATCTACTGAaATTACGTGTGTGCTAaGCTGCGTGGTACGCaGAaGGCCCAAAAGTT 957

TaSEP-2A TGACAGCTTGTGCGGTGTG 976

TaSEP-2B TGACAGCTTGTGCGGTGTG 976

SUPPLEMENTAL FIGURE 2

TaAG-4A TGCAAGGAGGTCAAGCTGATTCTTCCTTGTGTTAGAATTGCTTGCTACCATGGGGAGGGG 60

TaAG-4B TGCAAGGAGGTCAAGCTGATTCTTCCTTGTGTTAGAgTTGCTTGCTACCATGGGGAGGGG 60

TaAG-4A GAAGATCGAGATCAAGAGGATCGAGAACACGACGAGCCGCCAGGTGACCTTCTGCAAGCG 120

TaAG-4B GAAGATCGAGATCAAGAGGATCGAGAACACGACGAGCCaCCAGGTGACCTTCTGCAAGCG 120

TaAG-4A CAGGAACGGGCTGCTCAAGAAGGCCTATGAGCTCTCCGTCCTCTGCGAAGCCGAGATCGC 180

TaAG-4B CAGGAACGGGCTGCTCAAGAAGGCCTATGAGCTCTCCGTCCTCTGCGAAGCCGAGATCGC 180

TaAG-4A CTTGATCGTCTTCTCCGCCCGCGGCCGCCTCTACGAATATGCTAGTAACAGCACGAGGAC 240

TaAG-4B CTTGATCGTCTTCTCCGCCCGCGGCCGCCTCTACGAgTATGCTAGTAACAGCACGAGGAC 240

TaAG-4A GACGATCGACAGGTACAAGAAGGCTTCCGCAAGCGCTTCTGGCTCTGCTCCAGCTATAGA 300

TaAG-4B GACGATCGACAGGTACAAGAAGGCTTCtGCAAGCGCTTCTGGCTCTGCTCCAGCTATAGA 300

TaAG-4A CGTCAATTCTCAGCAATACTTTCAGCAAGAATCAGCGAAACTGCGCCATCAGATACAGTC 360

TaAG-4B CGTCAATTCTCAGCAATACTTTCAGCAAGAATCAGCGAAACTGCGCCATCAGATACAGTC 360

TaAG-4A CCTGCAAAATGCAAACAGGAACCTGATGGGCGAATCCGTCGGCAACTTGACACTGAAGGA 420

TaAG-4B CCTGCAAAATGCAAACAGGAACCTGATGGGCGAATCCGTCGGCAACTTGACACTGAAGGA 420

TaAG-4A GCTCAAGAGCCTGGAGAACAGGCTCGACAAGGGCATCGGCCGGATCAGAGCAAAGAAGCA 480

TaAG-4B GCTCAAGAGCCTGGAGAACAGGCTCGACAAGGGCATCGGCCGGATCAGAGCAAAGAAGCA 480

TaAG-4A CGAACTGCTGTTCGCGGAGATCGAATACATGCAGAAGCTGGAGGTGGATCTGCAGAGCGA 540

TaAG-4B CGAgCTGCTGTTtGCGGAGATCGAATACATGCAGAAGCTGGAGGcGGATCTGCAGAGtGA 540

TaAG-4A GAACATGTATCTCCGAGCCAAGGTGGCGGACGCGGAGCGGCTAGCCCTGGCGGCGCCGCC 600

TaAG-4B GAACATGTATCTCCGAGCCAAGGTGGCGGACGCGGAGCGGCTgGCCCTGGCGGCGCCGCC 600

TaAG-4A GCCGGCGCCGGGCGGGGCGGAGCTGGAGGTGCTCCCGACGTTCGACGCGAGGAGCTACTA 660

TaAG-4B GCCGtCGtCGGGCGGGGCGGAGCTGGAGGTGCTCCCGACGTTCGACGCGAGGAcCTACTA 660

TaAG-4A CCACCACCAGGCAGTGAACATGCTGCAGGACGCCGCGGCGGCCTCCTCCTCCTCGCGCTA 720

TaAG-4B CCACCACCAGGCAGTGAgCATGCTGCtGGACGCgGCGGCGGCCTCCTCCTCCTCGCGCTA 720

TaAG-4A CTCCCAGTCGTCCCAGGCGGCGGCGGCG...... ACCACCGCTCTTCACCTCGGCTACCA 774

TaAG-4B CTCCCAGTCGTCCCAGGCGGCGGCGGCGgcggcgACCACCGCTCTTCACCTCGGCTACCA 780

TaAG-4A GATTAAGGGGTCCCAAGTCCCAACTGAACTGATCGACCGCTAGCTC.CTCCACTCGGAGG 833

TaAG-4B GATTAAGGGGgg...... CCAACTGAACTGATCGACCGCTAGCTagCTCCACTCGGcGG 833

TaAG-4A CTCGGGACGCGCGCGCGTCACGGCAGGAGAAAGCACCCGCGCCGTCGATCGATCCATGTC 893

TaAG-4B CTCGGGACGCGCGCGCGTCACGGCgGGAGAtAGCACCCGCGCtGTCGAcCGATCCATGTC 893

TaAG-4A CTCTGCTAGTTCGATGGATTTGCTGAATGGCCTACGTG....TACTTAATTACCACAGTA 949

TaAG-4B CTCTaCTAGTTCGATaGgTTTGCTGgATaaCCTACGTacgtcTACTTtATTACCACAGTA 953

TaAG-4A CTGTATTGCGGTATGCTACCTAAATAACTGCGTGATGTGTGAGAAACAAGGAAGCGTATA 1009

TaAG-4B CTGTATTGCGGTATGCTACCTAAATAAtTGCGTGATGTGTGcGAAgCgAGGAAGtGTATg 1013

TaAG-4A CTGGTACTATGGGACCTGGCTAGTGAGTACGAG....CGTGCATGTTGTGAAGAATGACC 1065

TaAG-4B taGtacgTAcGGGACgTGGCTAGTGAGTACGAGagcgtGTGCgTaTTGTGAAGAATGACC 1073

TaAG-4A TGGCCTTT 1073

TaAG-4B TGGCCTTT 1081

Supplemental Fig.3

Figure legends

Supplemental Figure 1 - Alignment of nucleotide sequences of TaSEP-2A and TaSEP-2B. The translation start (ATG) and stop (TGA) codons are boxed. Nucleotide substitutions and insertions/deletions are showed in bold. The arrow indicate the insertion of a single nucleotide in the C-terminal region of TaSEP-2B, which determined a frameshift mutation with the introduction of a premature stop codon 18 bp downstream.

Supplemental Figure 2 - Alignment of nucleotide sequences of TaAG-4A and TaAG-4B. The translation start (ATG) and stop (TGA/TAG) codons are boxed. Nucleotide substitutions and insertions/deletions are showed in bold. The arrow indicate the deletion of seven bp in the C-terminal region of TaAG-4B which introduced a premature stop codon (TGA) 10 bp downstream.

Supplemental Figure 3 - Southern analysis of CS genomic DNA digested with EcoRI (EI), BamHI (B), HindIII (H), EcoRV (EV) and SacI (S) and hybridised with specific probes of 23 MIKC-type sequences of wheat cloned in this study. The sizes of the molecular-weight marker (kb) are shown on the right side.

Supplemental Figure 4 - Phylogenetic tree based on amino acid sequences of 125 MIKC-type MADS box genes: 33 from Arabidopsis (At), 31 from rice (Os), 32 from maize (Zm) and 29 cloned wheat sequences (Ta). The five Arabidopsis sequences of the FLC subfamily were used as outgroups. Numbers on major branches indicate bootstrap percentage for 1,000 replicates. The wheat genes are indicated by arrows. Subfamilies of the plant MIKC-type genes are enclosed by square brackets at the right margin.

Supplemental Table 1 - List of primers used for the isolation of the full-lenght cDNA sequences of wheat MADS-box genes

Supplemental Tab 2 - List of primers used for probe labelling and expression analyses by RT-PCR and real time RT-PCR (*)

Supplemental Table 3 - Characteristics of nucleotide and deduced amino acid sequences of 45 full-length cDNAs of wheat MIKC-type genes.

Clone Sequence length (nt) Protein Domains (aa)

Tot. 5’UTR 3’UTR ORF (aa) 5’ext. M I K C

TaSOC1-1A 1000 84 226 690 230 - 60 12 101 57

TaSOC1-1B 999 84 228 687 229 - 60 12 100 57

TaAG-1 1101 18 276 807 269 36 60 13 100 60

TaAG-3A 1071 148 167 756 252 - 60 14 101 77

TaAG-3B 1077 148 167 762 254 - 60 14 101 79

TaSEP-1 1076 15 317 744 248 - 60 14 97 77

TaSEP-2A 976 75 187 714 238 - 60 14 98 66

TaSEP-2B 976 75 226 675 225 - 60 14 98 53

TaAP1-1 1163 144 287 732 244 - 60 14 100 70

TaAP1-2 1111 116 194 801 267 - 60 15 100 92

TaAP1-3 1255 140 293 822 274 - 60 14 100 100

TaAGL6-A 1144 126 241 777 259 - 60 13 99 87

TaAGL6-B 1141 126 241 774 258 - 60 13 99 86

TaAGL6-C 1158 134 250 774 258 - 60 13 99 86

TaSEP-3A 1226 113 357 756 252 - 60 14 102 76

TaSEP-3B 1233 113 364 756 252 - 60 14 102 76

TaSEP-4 1033 67 228 738 246 - 60 14 102 70

TaAGL12 890 124 94 672 224 - 60 9 105 50

TaAP3 970 104 179 687 229 - 60 10 100 59

TaPI-1 1035 217 194 624 208 - 60 10 100 38

TaPI-2 953 24 302 627 209 - 60 10 100 39

TaWM16 986 118 280 588 196 - 60 11 100 25

TaSOC1-2 967 32 158 777 259 33 60 17 100 49

TaSEP-5A 1202 214 280 708 236 - 60 14 100 62

TaSEP-5B 1186 197 281 708 236 - 60 14 100 62

TaSEP-6 1028 91 256 681 227 - 60 14 98 55

TaSOC1-3A 937 144 127 666 222 - 60 13 99 50

TaSOC1-3B 931 138 127 666 222 - 60 10 102 50

TaSVP-1A 1282 183 415 684 228 - 60 9 103 56

TaSVP-1B 1297 198 415 684 228 - 60 9 103 56

TaSVP-2A 1041 86 277 678 226 - 60 18 95 53

TaSVP-2B 1043 89 276 678 226 - 60 18 95 53

TaGGM13 1155 134 265 756 252 - 60 10 101 81

TaAG-4A 1073 49 259 765 255 - 60 12 102 81

TaAG-4B 1081 49 279 753 251 - 60 12 102 77

TaSVP-3A 1163 187 298 678 226 - 60 20 94 52

TaSVP-3B 1171 191 302 678 226 - 60 20 94 52

TaAG-2A 1141 107 215 819 273 37 60 13 101 62

TaAG-2B 1135 107 200 828 276 37 60 13 101 65

TaAGL17-1 1119 123 276 720 240 - 60 12 100 68

TaAGL17-2A 1155 117 342 696 232 - 60 13 100 59

TaAGL17-2B 1164 126 342 696 232 - 60 13 100 59

TaAGL17-2C 1122 91 341 690 230 - 60 13 100 57

TaAGL17-3A 1012 40 249 723 241 - 60 12 99 70

TaAGL17-3B 1013 40 250 723 241 - 60 12 99 70

APPENDIX I

The present paper describes the cloning and characterization of 45 wheat cDNA sequences of MICK-type MADS-box genes. Several of them are the same studied by Zhao et al. (2006, Mol. Gen. Genomics, 276:334-350). This gave us the opportunity of comparing their and our results in terms of nucleotide sequences, expression and phylogenetic analyses and relationships among homoeologous genes.

A careful comparison and analysis of our cDNA sequences cloned from Chinese Spring and of those cloned by Zhao et al. (2006) from Nogda 3338 showed that several dissimilarities can not be ascribed to genetic polymorphism between the two bread wheat genotypes and can only be explained by technical problems, as we will demonstrate here. For the sake of brevity we are reporting only the most evident and noteworthy discrepancies, but many additional differences have been detected.

1) The deduced proteins encoded by TaSEP2-A (this paper) and TaAGL24 (Zhao et al., 2006) differ for nine amino acids within the K domain (App. I, Fig. 1). This change is due to a frameshift mutation caused by the insertion of a seventh adenine in the ORF of TaAGL24, while the correct reading frame is restored after 27 base pairs by the deletion of a thymine (App. I, Fig. 2, the two frameshift mutations are indicated by arrows). However, none of the ESTs found in the databases corresponding to these sequences shows the two frameshift mutations observed in TaAGL24. Moreover, the translation of TaAGL24 is incorrect, in fact the start of the coding region should be located at nucleotide number 53 and not 74 (App. I, Fig 2), this would cause an increase of 7 amino acids of the translated protein (App. I, Fig. 1).

2) The nucleotide sequences of our clones TaSVP-3A and TaSVP-3B, presumably corresponding to homoeologous genes, show a high identity with the sequences TaAGL13 (97.0% and 98.8%, respectively) and TaAGL19 (95.5% and 96.7%, respectively) cloned by Zhao et al., (2006). TaAGL19 diverges from TaSVP-3A/B for a deletion of 102 bp located within the sequence encoding the MADS domain (App. I, Figs. 3 and 4); however, none of the ESTs in the databases corresponding to these sequences shows such deletion. Since the region including the deleted sequence is very rich of G/C (65/102 bp), one could hypothesize the occurrence of a PCR artefact, due to the formation of a loop during the amplification.

3) Our sequence TaAG-3A is different from TaAGL9 and TaAGL31 of Zhao et al. (2006) for two nucleotide substitutions, one of them, in the coding region, causing the change of an amino acid. However, the only difference between the sequences cloned using two different primer pairs by Zhao et al. (2006) rests witj the length of the 5’UTR and the deletion of a thymine in the 5’UTR of TaAGL31 (App. I, Fig. 5); most probably this deletion is a sequencing artifact, because the deleted thymine was contained in the forward primer used to amplify this sequence.

4) Our sequence TaSEP-6 is closely related to five sequences (TaAGL5, TaAGL8, TaAGL3, TaAGL34 and TaAGL40) cloned by Zhao et al. (2006), with nucleotide and amino acid identities higher than 90%. However, the Southern hybridisation of Chinese Spring DNA with a specific probe for the TaSEP-6 sequence showed the presence, as expected in an allohexaploids, of three copies of the corresponding gene, which were located in the three group 7 homoeologous chromosomes. Then the additional homologous sequences found by Zhao et al. (2006) can only be explained by mistakes introduced during the amplification and/or sequencing of the cloned sequences, as can be deduced by comparison of TaAGL3 with TaAGL34 (e.g. App. I, Fig. 6) and of TaAGL5 with TaAGL8.

5) In the first 64 bp the sequence TaAGL29 (Acc. n. DQ512346) by Zhao et al. (2006) contains a stretch of plasmid sequence, most probably of the vector used for cloning (App. I, Fig. 7).

6) It is difficult to understand how the sequences TaAGL40 (Acc. n. DQ512370) and TaAGL2 (Acc. n. DQ512337) were amplified and cloned, because the primers forward for TaAGL40 and reverse for TaAGL2, reported in Tab. 1 of Zhao et al. (2006), were not present in the corresponding sequences (App. I, Fig. 8).

7) Several primers show short (1-3 nt) nucleotide substitutions or deletions at their 5’ or 3’ ends in comparison to the corresponding sequences in the clones. Moreover, it is not explained the meaning of the small and capital letters used in the list of primers in Tab. 1 of Zhao et al. (2006).

8) Zhao et al. (2006) do not give any information on the primers used for expression analysis, we guess that the same primers employed for cloning and listed in Tab. 1 were also exploited for RT-PCR. If this deduction is correct, however, it would be difficult to explain why and how the RT-PCR by non-specific primers, designed in conserved regions of highly homologous sequences (identity >90%, likely deriving from homoeologous genes), produces diversified expression patterns.

9) As an example, the sequence TaAGL31 by Zhao et al. (2006) is 100 bp shorter than TaAGL9, but the remaining sequences are identical, they differ just for the deletion of a thymine in TaAGL31, most probably due to the sequencing (App. I, Fig. 5). Thus, if our assumption that the same primer pairs were used for cDNA cloning and for RT-PCR is correct, the expression patterns of the two sequences should have been identical or, anyhow, the primer pair amplifying the shorter sequence (TaAGL31) should have detected also the transcripts of the longer one (TaAGL9), producing amplification in the same tissues and development stages. Surprisingly, Zhao et al. (2006) report the expression with the primers corresponding to the longer sequence (TaAGL9) in 28 h embryos of imbibed seeds, but not with those of the shorter sequence (TaAGL31) (Fig. 4 of Zhao et al. 2006).