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
Clone / Forward primer / Reverse primerTaSOC1-1 / 5’-CCTCGCTTAGCCATTTCTGTTG-3’ / 5’-CAGCCAGGCCAAGAAACAA-3’
TaAG-1 / 5’-CGCCCACGAAACACAAAC-3’ / 5’-TCGCCACCAGTACTATTGCAT-3’
TaAG-3 / 5’-TTTCTGCCTTCGGCTTGG-3’ / 5’-TCTACACCAGCGGCAAATTTA-3’
TaSEP-1 / 5’-GGAGGGAGAAAGGAGATGGG-3’ / 5’-TTGGTCCTGCATATGGTTCGT-3’
TaSEP-2 / 5’-AGACATCGCTCTGCTGGCT-3’ / 5’-CACACCGCACAAGCTGTCA-3’
TaAP1-1 / 5’-TCTCTTCCACCTCACGTCCT-3’ / 5’-TCCCACTAGAGACGGGTATCA-3’
TaAP1-2 / 5’-CACCGCATTTCTCATTCTTCC-3’ / 5’-CCGCAGGTCCATTAAAGCTTA-3’
TaAP1-3 / 5’-TCCTCTTCCTCTCCCATCTTT-3’ / 5’-ACACACGTCATCACACAACCTAGCTT-3’
TaAGL6 / 5’-TCCCAAGCCCTATGCGCTA-3’ / 5’-TGCATGGACAGCTTGGAACTT-3’
TaSEP-3 / 5’-TGAGCTGCTTTGGTGGTG-3’ / 5’-AGGCACACTCAGTTGATAACATC-3’
TaSEP-4 / 5’-TGGTGTGTGTATGGTTGCTG-3’ / 5’-TCACATAGTCACTCTAGTAA-3’
TaAGL12 / 5’-AACCGCAGCAACCTCGAA-3’ / 5’-AGACCGGAGCCAATCATACG-3’
TaAP3 / 5’-TTCTTCTCCACCCGTCGC-3’ / 5’-CAGTCGAGCACTACGGCGTTA-3’
TaPI-1 / 5’-GGCAGCCACCCTCCTTTACT-3’ / 5’-GCATCTTGACAGGGACAGGAA-3’
TaPI-2 / 5’-CGGAGGAAGAAGAAGGAGGC-3’ / 5’-TTCACCGTCCAGCAATTGTG-3’
TaWM16 / 5’-GCGGGCAATCCAAACCTT-3’ / 5’-CACGAATTGTCCCATTGACG-3’
TaSOC1-2 / 5’-CGTCTCTCCCAGATCCGCCCGT-3’ / 5’-ATGCTGCAGCCCGTCAGTT-3’
TaSEP-5 / 5’-GGTTGAGAGCGAGGAATCG-3’ / 5’-GAGATGCAGGACACATAGGCA-3’
TaSEP-6 / 5’-TCCTCCGGCTAGCTAGTGGT-3’ / 5’-TGTAGCTATGCACTGACACGGTG-3’
TaSOC1-3 / 5’-AGCTACGGCCGAACCCTACA-3’ / 5’-TTCCGCTGCACAGGGTTT-3’
TaSVP-1 / 5’-TCCTCTCCTTTCGCATCCC-3’ / 5’-CATGCCCTTCAACTTCTGAGC-3’
TaSVP-2 / 5’-TTGTTCGTTCGTGCGGCT-3’ / 5’-CCGTGGCAGGCACATACATA-3’
TaGGM13 / 5’-CGTCCGCACATCACAAGT-3’ / 5’-CGTACGTACCAACATTTGACCA-3’
TaAG-4 / 5’-TGCAAGGAGGTCAAGCTGATT-3’ / 5’-AAAGGCCAGGTCATTCTTCACA-3’
TaSVP-3 / 5’CGCAGATCTATTGCAACACCTG-3’ / 5’-TGTGCCAGTGGTTGATTTACTTG-3’
TaAG2 / 5’-TCCTGCCTATCCCACACTACA-3’ / 5’-CGATCGACTAACCAGCACCTA-3’
TaAGL17-1 / 5’-CCTGGGAACTTTCTCCTTC-3’ / 5’-TGTCAGTAAGCTAGCTGCTCAT-3’
TaAGL17-2 / 5’-AGCAGCCTAGGTAGCACCA-3’ / 5’-TTAAGTATGGGAAACATGTCCG-3’
TaAGL17-3 / 5’-TTCTTCTCAGTCCGCCTGC-3’ / 5’-AATGTCATGTGGGCATAAGCA-3’
Supplemental Tab 2 - List of primers used for probe labelling and expression analyses by RT-PCR and real time RT-PCR (*)
Clone / Forward primer / Reverse primerTaSOC1-1 / 5’-AAGCGCGCAACCTTGAGG-3’ / 5’-CAATTGGTGTGCTGGGCAT-3’
TaAG-1 / 5’-TTCATGCAGCAGCAGCCT-3’ / 5’-TCGCCACCAGTACTATTGCAT-3’
TaAG3 / 5’-CGGTTGCAGCAGGTGACTAT-3’ / 5’-TCTACACCAGCGGCAAATTTA-3’
TaSEP-1 / 5’-TGGATCATCTGCAGGTTGGA-3’ / 5’-TTGGTCCTGCATATGGTTCGT-3’
TaSEP-1* / 5’-GCGGGCATGTCATTGATACTA-3’ / 5’-TCTTTCTCGTCGCCATAA-3’
TaSEP-2 / 5’-CATATGTCCTGGCAAGACG-3’ / 5’-CACACCGCACAAGCTGTCA-3’
TaSEP-2* / 5’-GTTGCTGCGGAGACAATG-3’ / 5’-GCCACATCAGTTAATCATATCC-3’
TaAP1-1 / 5’-GCAGCTACCAGCATTCATCC-3’ / 5’-TCCCACTAGAGACGGGTATCA-3’
TaAP1-2 / 5’-AGCGACAGCCCAAGATTC-3’ / 5’-CCGCAGGTCCATTAAAGCTTA-3’
TaAP1-3 / 5’-CCCAGAACCCATACCCAA-3’ / 5’-ACACACGTCATCACACAACCTAGCTT-3’
TaAGL6 / 5’-AGCAGCAGCAGCACCCTAA-3’ / 5’-TGCATGGACAGCTTGGAACTT-3’
TaSEP-3 / 5’-GCCCTGAAGATCCACGAT-3’ / 5’-AGGCACACTCAGTTGATAACATC-3’
TaSEP-3* / 5’-CCAACTTGCTCGGCTACG-3’ / 5’-TAGAATGCGACGGCTGTG-3’
TaSEP-4 / 5’-CAAGTTCATGGGCAGCAGC-3’ / 5’-TCACATAGTCACTCTAGTAA-3’
TaSEP-4* / 5’-TCTGGGAGCACAACAACAATG-3’ / 5’-CCATCTTCACTCAAGGCAACC-3’
TaAGL12 / 5’-TGAAAGCTGCCAACGAAATTC-3’ / 5’-AGACCGGAGCCAATCATACG-3’
TaAP3 / 5’-CGGCTAATCGATCACTTCG-3’ / 5’-CAGTCGAGCACTACGGCGTTA-3’
TaPI-1 / 5’-CTGTGTCAACTGAAGCTCCTCTA-3’ / 5’-GCATCTTGACAGGGACAGGAA-3’
TaPI-2 / 5’-ACTCTCCACGCCACTCCAT-3’ / 5’-TTCACCGTCCAGCAATTGTG-3’
TaWM16 / 5’-AAGCTGAGCGCTACGGCCTA-3’ / 5’-CACGAATTGTCCCATTGACG-3’
TaSOC1-2 / 5’-AAAGCTGTTGACGGAGAACG-3’ / 5’-ATGCTGCAGCCCGTCAGTT-3’
TaSEP-5* / 5’-GAAATGTGCGACCTGAAG-3’ / 5’-CTGGAAGAAGTGCTCTGG-3’
TaSEP-6 / 5’-GGGTATCAACGAAATTTCCTGGA-3’ / 5’-TGTAGCTATGCACTGACACGGTG-3’
TaSEP-6* / 5’-AAGAGCCAGCAGTCACTTGATC-3’ / 5’-CCTATACGCAGGGAAGGGTCAC-3’
TaSOC1-3 / 5’-GCATCAGGGCAATGAAGACTC-3’ / 5’-TTCCGCTGCACAGGGTTT-3’
TaSVP-1 / 5’-CAGTTGTCGTCCGTTCGGTT-3’ / 5’-CATGCCCTTCAACTTCTGAGC-3’
TaSVP-2 / 5’-GGGAAGCTCACAGGACAATG-3’ / 5’-CCGTGGCAGGCACATACATA-3’
TaGGM13 / 5’-TACTACACGGGCGAGGAGTC-3’ / 5’-CGTACGTACCAACATTTGACCA-3’
TaAG-4 / 5’-CTACCACCACCAGGCAGTGA-3’ / 5’-AAAGGCCAGGTCATTCTTCAC-3’
TaSVP-3 / 5’-CCGATACGTCTCTCAGGCTCG-3’ / 5’-TGTGCCAGTGGTTGATTTACTTG-3’
TaAG-2 / 5’-CAGCGAATACGATCACATGG-3’ / 5’-CGATCGACTAACCAGCACCTA-3’
TaAGL17-1 / 5’-TTGCAGTTGTTGAAGATGCCA-3’ / 5’-TGTCAGTAAGCTAGCTGCTCAT-3’
TaAGL17-2 / 5’-TTTATTGACCTTGAGTTGCGGC-3’ / 5’-TTAAGTATGGGAAACATGTCCG-3’
TaAGL17-3 / 5’-TGAACTGAGCCAGGCACA-3’ / 5’-AATGTCATGTGGGCATAAGCA-3’
ACTIN* / 5’-TGGTCAGGTCATCACGATTGG-3’ / 5’-ATCTCCTTGCTCATACGGTCAG-3’
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).