Supplemental Data. Jeong et al. (2012). Plant Cell /tpc

Transcription

1 Suppmemental Figure 1. Alignment of amino acid sequences of Glycine max JAG1 and its homeolog JAG2, At-JAG and NUBBIN from Arabidopsis thaliana, LYRATE from Solanum lycopersicum, and Zm- JAG from Zea mays. Boxed amino acids denote conserved residues. The EAR motif, putative nuclear localization signal (NLS) sequence, single C2H2-type zinc finger motif, and proline-rich motif are indicated. 1

2 Supplemental Figure 2. Silique phenotypes observed in Arabidopsis wild-type Landsberg erecta (Ler), jag- 3 mutant, three jag-3 mutant lines transformed with transgene PAtJAG:gGmJAG1, and three jag-3 mutant lines transformed with transgene PAtJAG:gGmjag1. (A) Comparison of number of seeds per silique. (B) Comparison of silique length. (C) Comparison of number of seeds per unit silique length. Statistically significant differences are indicated with different letters (one-way ANOVA, Fisher s Protected LSD test, P < 0.05). The number of siliques in each line ranged from 24 to 45. 2

3 Supplemental Figure 3. Comparison of phenotypes of 35S:GmJAG1, 35S:Gmjag1, PGmJAG1:gGmJAG1, PGmjag1:gGmjag1, PAtJAG:gGmJAG1 EAR, and PAtJAG:gGmJAG2 gene constructs in the Arabidopsis 3

4 jag-3 mutant. (A) Ler flower. (B) jag-3 flower. (C) 35S:GmJAG1 flower. (D) 35S:Gmjag1 flower. (E) PGmJAG1:gGmJAG1 flower. (F) PGmjag1:gGmjag1 flower. (G) PAtJAG:gGmJAG1 EAR flower. (H) PAtJAG:gGmJAG2 flower. (I) Ler rosette leaves. (J) jag-3 rosette leaves. (K) 35S:GmJAG1 rosette leaves. (L) 35S:Gmjag1 rosette leaves. (M) PAtJAG:gGmJAG1 EAR rosette leaves. (N) PAtJAG:gGmJAG2 rosette leaves. Leaves were excised from 4-week-old plants in (I-N). The first leaves are seen on the left. (O) Dehiscent silique of Ler. (P) Dehiscent silique of jag-3. (Q) Dehiscent silique of 35S:GmJAG1. (R) Dehiscent silique of 35S:Gmjag1. (S) Dehiscent silique of PAtJAG:gGmJAG1 EAR. (T) Dehiscent silique of PAtJAG:gGmJAG2. A valve was removed from each dehiscent silique to display the seeding pattern (O-T). Scale bar: 1 mm (A-H, O- T); 10 mm (I-N). 4

5 Supplemental Figure 4. Copy number analysis of Gm-JAG1 and Gm-JAG2. (A) Schematic of the Gm-JAG1 and Gm-JAG2 chromosomal regions probed with a GmJAG1 probe. Genic regions of Gm-JAG1 and Gm-JAG2 from start to stop codons are indicated by gray boxes. Arrows indicate the gene orientation. Sites of restriction enzyme BglII (B) and HindIII (H) used for Southern blot analysis are shown above the map. A BglII site present in V (Ln) and absent in Sowon (ln) is highlighted by. (B) Southern blot analysis of Gm-JAG1 and Gm-JAG2 in soybean. Southern blot analysis of BglII (B) and HindIII (H) digests of DNA isolated from V (Ln) and Sowon (ln) cultivars. DNA size standards are indicated as kilobase pairs. The probe that was generated from a Gm-JAG1 genic region hybridized with three HindIII-digested DNA fragments of different sizes, which were expected from the Williams 82 sequence in both V and Sowon: 12.2 kb from the Gm- JAG2 region and Gm-JAG1-derived 1.5 kb and 1.1 kb hybridized with 236-bp and 952-bp regions, respectively, of the probe (1188 bp) that contains a HindIII site. The probe hybridized with a 3.4-kb region of the BglIIdigested genomic DNA fragment from V and Sowon from the Gm-JAG2 region and hybridized with 11.2-kb (Sowon) and 6.2-kb (V ) fragments from the Gm-JAG1 region, which was caused by a single nucleotide substitution at the BglII recognition site at the N-terminal region of Gm-JAG1. 5

6 Supplemental Figure 5. Variation in leaflet shape among cultivated and wild soybean accessions. Shown are trifoliolate leaves from six wild soybean accessions with broad leaflet (IT182932, PI423991, PI518282, PI549046, PI407290, and PI378691), three cultivated soybean accessions with narrow leaflet (Myeongjunamul, Pungsannamul, Sowon), two cultivated soybean accessions with broad leaflet (Sowon and Williams 82), and three putative hybrids between cultivated and wild soybeans with narrow leaflet (IT178535, IT183014, and IT191201). The representative leaf of each accession is a fully expanded trifoliolate leaf at the third or fourth node on the main stem from the top at the early flowering stage plant. 6

7 Supplemental Figure 6. Polymorphic sites identified from comparison of Gm-JAG1-containing sequences from 14 soybean lines: V , Williams 82, Sowon, Pungsannamul, Myeongjunamul, IT178535, IT191201, IT183014, IT182932, PI423991, PI518282, PI549046, PI407290, and PI Nine haplotypes, designated by Roman numerals, were grouped by comparison of all the identified polymorphic sites but the C- repeat site. Each of the polymorphic sites is separated by a space. Nucleotide positions of the V sequence at single nucleotide polymorphic sites, 5 -nucleotide positions of the V sequence at insertion sites, and 3 -nucleotide positions of the V sequence at deletion sites are identified above the sequence line with an exception of the C-repeat polymorphic site, where 5 - and 3 -nucleotide positions are identified. Nucleotide missing positions are indicated by. The A nucleotide of the ATG translation initiation codon is assigned as position 1 (not shown), the nucleotide positions upstream of position 1 are given negative numbers, and those downstream of the A nucleotide (position 1866, not shown) of the TAA stop codon are given positive numbers, starting from +1. Two polymorphic sites of the predicted protein coding sequence are in red. 7

8 Supplemental Table 1. Attributes of single-nucleotide polymorphism markers for fine mapping (Figure 1B) Marker Range of template sequence on chromosome 20 Type of polymorphism * Primer specificity Sequence (5 3 ) Ln_43k A(V)/C(S) F V-sp CCCTTTAGTTCTAGCCACTGA F S-sp CCCTTTAGTTCTAGCCACTCC Com R TTGGAAAATATCTGTTTCTT Ln_44k A(V)/G(S) Com F TCTAAAATATCGAACGCTAC R V-sp GGTTTTTAGAAGACACATACAA R S-sp GGTTTTTAGAAGACACATACAG Ln_AH G(V)/C(S) F V-sp CCAGAACGAAACCCCTTCG F S-sp CCAGAACGAAACCCCTTCC Com R GGCAGTGTTCAACTTTGTTT Ln_54k T(V)/G(S) F V-sp CTTGTGGGATTTGTTTCTT F S-sp CTTGTGGGATTTGTTTCTG Com R TTAGGGGATAAAAAGCATGA Ln_57k G(V)/T(S) F V-sp CATTACTTTcTTTAATTGTGG F S-sp CATTACTTTcTTTAATTGTGT Com R GTGGATCAGTATAGTGGAGA * V, V ; S, Sowon. F, forward; R, reverse; Com, common; sp, specific. 8

9 Supplemental Table 2. Primers for sequencing, RT-PCR, and probe generation used in this study. Use Target Name Orientation Sequence (5-3 ) Sequencing of Gm JAG1 and Gm JAG2 cjag-5 Forward CAGTCTCAAACTGAAAGTCTAAAT transcripts GAACAAAATCTACACTCGTAGACC Gm JAG1 and Gm JAG2 cjag-5-1 Forward AAAAGCTTTAGTTTTATCCCTACC GAACAAAATCTACACTCGTAGACC Gm JAG1 and Gm JAG2 cjag-m-2 Forward AAGGTCTACGAGTGTAGATTTTGT AGAAGTATCTTGGGAATCTTAGAG Gm JAG1 cjag-m-3 Forward GTCAACTGGTCTTTCGTTGT CTCTCTCCTCCAACATAGTGTC Gm JAG2 cjag-mh-3 Forward GTCAACTGGTaTTTCGTaGc CTCTCTCCTCCAACATAGTGTC Gm JAG1 and Gm JAG2 cjag-4 Forward ATGCAGTGAACGATTACTATGTG TCTCTCTCTCTCTGGATTGC Gm JAG1 and Gm JAG2 cjag-5 Forward GAAGGGGCTATTCAGGTGCAC CGTGTCTTTATGATGATACCAC Expression analysis Gm JAG1 cjag-sp Forward CCTCGAAGACCATACCTCTTCAT GTGCAGCAATGTTATGATCACA Gm JAG2 cjag-hsp Forward CCTCGAAGACCATACCTCTTCAc GTGgAGCAATGaTATGATCgCt Actin 11 Forward CGGTGGTTCTATCTTGGCATC GTCTTTCGCTTCAATAACCCTA UBQ10 Forward GATCTTTGCCGGAAAACAATTGGAGGATGGT 9

10 Sequencing of genomic DNA Gm-JAG1-containing region CGACTTGTCATTAGAAAGAAAGAGATAAC Ln-44k Forward TAAAAGTAGGGCATCTTGGA TCGAAAAAGGTGAAACTGAT Ln-45k Forward TGGAGAGAATATTACCGAAAA ACAGCATAATCCGAAGAGAA Ln-46k Forward CCCCTTCTCTTTGGTTTTAT TTTTGAATCTTCTGCTCCAT Ln-47k Forward TGCTGTCCGTATCTGTGTAA CAAACAAAGCCAAAGATTGT Ln-48k Forward CCCCACGTTTTTATCAATAG CTTTGAGCATTAAAGGTTGC SoyJAG-1 Forward AGTCTAAGTCCCCTGGGTAG ATCATGAAGTTTACCGGATG SoyJAG-1a Forward CTTGCTTTCTTTCTGGTATAAATG CTGTTTCATGGTCAAGGAAT SoyJAG-2 Forward ACACCCTTCTTTTCTTCCTC GTAATGGGTGCATACCCTAA SoyJAG-3 Forward GATTGCTGAAATTCGATCAT AGGAAAAGGAGTTTGGAAAG SoyJAG-4 Forward CTACAGTAACCAGGGACAGC GTCACTAATCATCGCCTTTC Ln-53k Forward ACGCACAGTAAGTGAGGATT 10

11 AATTAAAGCGAGATGGATCA Ln-54k Forward GCTTGGCTGGTAAAAGTAAA AAGTCAAATAAACCCGCATA Ln-55k Forward TGCCCCCATAATATAAATGT TACGTGTTTTTGGATGTTCA Ln-56k Forward CACTTGGCATTCAACTGTTA TCTAATTGGAATGGCCTAAA Ln-57k Forward TTTCAACAGTTTTGTCCTGA TTTGTCCAGTTTGCCTATTT Gm-JAG2-containing SoyJAG-1a Forward CTTGCTTTCTTTCTGGTATAAATG region CTGTTTCATGGTCAAGGAAT SoyJAG-h-p Forward TACCAACCAGCTAGTCAAGG CAGTCAAATGCATTGAGAAA SoyJAG-h Forward AAGCCTTTCACTTTTCACAG ATGTGAGTTGAGGATGAACC SoyJAG-h-1 Forward GCATTTGTTAACGTTGTTTTT CAAAACTTCTCTGCGTCATT Vector construction JAG promoter patjag Forward CACCGGAATAGAGCTGATGTAGTAGCCGTG XhoI-pAtJAG TACTCGAGTAAAGAAGAGAGGTTCGAAAGTTTTCTATC Gm JAG1 for XhoI-soyJAG- Forward CACCCTCGAGTCAACCAGCCTCTATATTGCAGTCTC PAtJAG::gGmJAG1 1 soyjag TTAGAAACCATCTTGAAACCGATTG Gm JAG1 for p35s-so Forward CACCATGAGACCAGAACGAAACCCCTTACA 35S::gGmJAG1 SoyJAG TTAGAAACCATCTTGAAACCGATTG 11

12 Gm JAG1 and Gm jag1 SoyJAG-p Forward CACCCTGGTTTGTAAGTTTGCCTTGCTAGAGT promoters SoyJAG-B GAGGTATGGTCTTCGAGGACTTGTTTGC Coding region of Gm SoyJAG Forward CACCATGAGACCAGAACGAAACCCCTTAGA JAG1 SoyJAG TTAGAAACCATCTTGAAACCGATTG EAR-motif-deleted Gm- XhoIdeltaEAR Forward CACCCTCGAGATGGAGTACTCTAGAGATGGCAAACAAG JAG1 soyjag TTAGAAACCATCTTGAAACCGATTG Gm JAG2 Xho1-hmsoyJAG Forward CACCCTCGAGAGTCTCACAAATTTTGTAGAAAACTG hm-soyjag TTAGAAACCATCATG AAACCGATTG Southern probe Gm JAG1 SoyJAG-2 Forward ACACCCTTCTTTTCTTCCTC SoyJAG-2 GTAATGGGTGCATACCCTAA 12

13 Supplemental Table 3. Leaflet shapes of cultivated and wild soybean accessions and their genotype at marker Ln-AH locus Accession or cultivar Leaflet shape Nucleotide base at Ln- AH locus Species Collection site of Glycine soja and G. max x G. soja Pungsannamul Narrow C Glycine max Sowon Narrow C G. max Myeongjunamul Narrow C G. max Bokwang Narrow C G. max Saeal Narrow C G. max Jangyeob Narrow C G. max Eunha Narrow C G. max Someyng Narrow C G. max T136 Narrow C G. max IT Narrow C G. max x G. soja Kyunggi, Korea IT Narrow C G. max x G. soja Chungnam, Korea IT Narrow C G. max x G. soja Kyunggi, Korea V Broad G G. max Williams 82 Broad G G. max Daewon Broad G G. max Jangmi Broad G G. max Jinpum2 Broad G G. max Ilpumgeomjeong Broad G G. max Nokchae Broad G G. max Sodam Broad G G. max Jinpum Broad G G. max Daewon Broad G G. max Taekwang Broad G G. max Saeol Broad G G. max Dangkyung Broad G G. max Cheongja Broad G G. max T243 Broad G G. max T210 Broad G G. max Essex Broad G G. max Hwangkeum Broad G G. max SS2-2 Broad G G. max T202 Broad G G. max L29 Broad G G. max Jack Broad G G. max 13

14 Pureun Broad G G. max Wye Broad G G. max Namhae Broad G G. max Hutcheson Broad G G. max PI96983 Broad G G. max Williams Broad G G. max Muhan Broad G G. max T181 Broad G G. max T117 Broad G G. max Ogden Broad G G. max T201 Broad G G. max Kwangan Broad G G. max York Broad G G. max Evans Broad G G. max Geomjeong2 Broad G G. max Peking Broad G G. max Marshall Broad G G. max Samnam Broad G G. max Lee68 Broad G G. max T244 Broad G G. max T176 Broad G G. max T54 Broad G G. max Jinyul Broad G G. max T245 Broad G G. max T245 Broad G G. max IT Broad G G. soja Kyunggi, Korea PI Broad G G. soja Miyazaki, Japan PI Broad G G. soja Jilin, China PI Broad G G. soja Amur, Russia PI Broad G G. soja Taiwan PI Broad G G. soja Shaanxi, China IT Broad G G. soja IT Broad G G. soja IT Broad G G. soja IT Broad G G. soja IT Broad G G. soja IT Broad G G. soja 14