Flowering time Rosette leaf number 50 45 40 35 30 25 20 15 10 5 0 Col C24 Cvi C24xCol C24xCvi ColxCvi Figure S1. Flowering time in three F 1 hybrids and their parental lines as measured by leaf number at bolting. Data represent mean values ± standard error obtained from more than twenty plants.
Figure S2. Phenotype of the pol IV mutant hybrid. Seedlings at 20 DAS (Upper Left), 24 DAS (Upper middle), and 29 DAS plants (Upper Right). Pol IV mutant hybrid showed late flowering time and greater biomass than its parents (Bottom).
Relative expression level (vs. C24 x Col) 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 U1 U2 U3 U4 U5 U6 U7 U8 U9 U10 U11 U12 U13 U14 U15 U16 C24 x Col ** sde4-2 x nrpd1-3 Relative expression level (vs. C24 x Col) 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 ** D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 C24 x Col sde4-2 x nrpd1-3 Relative expression level (vs. Col) 4.0 3.0 2.0 1.0 0.0 ** ** U9 U10 D2 D9 D10 Relative expression level (vs. C24) 4.0 3.0 2.0 1.0 0.0 U9 U10 D2 D9 D10 Col nrpd1a-3 C24 sde4-2 Figure S3. Expression analysis of non-additively expressed genes between C24 x Col hybrids and parental lines in pol IV mutant hybrids. Expression levels of up- (U1-U16, Upper left) and down-regulated (D1-D11, Upper right) genes in C24 x Col hybrids were compared between C24 x Col and pol IV mutant hybrids. Expression levels of U9, U10, D2, D9, and D10 genes were compared between Col-0 and nrpd1a-3 (Bottom left) or between C24 and sde4-2 (Bottom right). Data represent mean values ± standard error obtained from three biological and technical replications. **, p<0.01
A Relative expression level of MET1 (vs. WT) 1.0 0.8 0.6 0.4 0.2 0.0 C Rosette diameter (cm) at 40 DAS 12 11 10 9 8 7 6 5 B Rosette diameter (cm) at 18 DAS 4.0 3.5 3.0 2.5 2.0 Figure S4. Rosette diameter in the hybrids between met1-rnai plants and met1-1. (A) MET1 expression levels in leaves of T 1 plants analyzed by RT-qPCR. Relative expression levels of met1-rnai plants were calculated with a control of wild type plants C24 being given a value of 1.0. The actin gene was used as a standard of expression level. The rosette diameter at 18 DAS (B) and 40 DAS (C). Data represent mean values ± standard error obtained from more than twenty plants.
A (%) 100 FIS2 promoter 50 CG CHG CHH 0 met1 / met1 met1-rnai / met1 MET1/MET1 BC 2 F 1 (C24) met1-rnai (C24) C24 x Col x met1-1 (Col) x met1-1 (%) 100 At3g19360 (%) 100 At5g58410 50 50 CG CHG CHH 0 met1 / met1 MET1 / met1 0 met1 / met1 MET1 / met1 BC 2 F 1 (C24) BC 2 F 1 (C24) BC 2 F 1 (C24) BC 2 F 1 (C24) x met1-1 (Col) x met1-1 (Col) x met1-1 (Col) x met1-1 (Col) B At3g19360 C24 x Col BC 2 F 1 (C24) x met1-1 (Col) Figure S5. Validation of decrease in DNA methylation in met1-rnai (C24) x met1-1 or BC 2 F 1 (C24) x met1-1 hybrid by bisulfite-sequencing. (A) The percentage of DNA methylation in FIS2 (Upper), At3g19630 (Bottom left), and At5g58410 (Bottom right) is shown in the bar graph. Ten clones from bisulfite-treated templates were sequenced. (B) DNA methylation pattern visualized by CyMATE (http://www.cymate.org/) in exon regions of At3g19630. Closed circles, triangles and squares represent methylated cytosine for CG, CHG, and CHH context, respectively. Open circles, triangles and squares represent non-methylated cytosine for CG, CHG, and CHH context, respectively.
Ratio of DNA methylation levels (d/d vs. d/d) 18 16 14 12 10 8 6 4 2 0 Methylated region Non-methylated region Figure S6. Ratio of DNA methylation levels in the heterozygous ddm1 hybrids compared with the homozygous ddm1 hybrids. At1g51172, At1g52010, At1g58561, At4g08720, At4g10990, At5g19165 and EDV are methylated in Col, and PORC and SAHH1 have no DNA methylation.
C24 ddm1 (Cvi) x C24 Rosette diameter (cm) 10.00 Cvi 8.00 6.00 4.00 2.00 0.00 14 DAS 18 DAS ddm1-1 (Cvi) ddm1-1 x ddm1-9 ddm1 (C24) x Cvi ddm1 (Cvi) x ddm1 (C24) ddm1 (C24) x ddm1 (Cvi) ddm1 (Cvi) C24xCol 32 DAS ddm1-9 (C24) ddm1-1 x C24 10.00 Rosette diameter (cm) ddm1 (C24) 25 DAS 8.00 6.00 4.00 2.00 0.00 14 DAS 16 DAS ddm1-1 (Cvi) ddm1-9 x ddm1-1 23 DAS 30 DAS ddm1-9 (C24) ddm1-9 x Cvi Figure S7. Phenotype of the ddm1 mutant hybrid between C24 and Cvi Fig. S7 background lines. Time course of rosette diameter is shown in right panel. Data represent mean values ± standard error obtained from more than twenty plants.
Figure S8. Phenotype of the ddm1 mutant hybrid between Cvi and Col background lines at 21 DAS (Upper left) and 36 DAS (Bottom left). Time course of rosette diameter is shown in right panel. Data represent mean values ± standard error obtained from more than twenty plants.
Number of plants 12 10 8 6 4 2 0 6-7 7-8 8-9 9-10 10-11 11-12 12-13 13-14 Rosette Diameter (cm) C24xCol (DDM1/DDM1) Rosette duaneter (cm) 14 12 10 8 6 4 2 0 unknown ddm1/ddm1 ddm1/ddm1 Figure S9. Rosette diameter (RD) in the hybrid between C24 (ddm1/ddm1) x Col (DDM1/ddm1) at 28 DAS. Frequency distribution of the RD for C24 (ddm1/ddm1) x Col (DDM1/ddm1) hybrids (Left panel). Rosette diameter of heterozygous and homozygous ddm1 hybrids and C24 x Col wild type hybrid (DDM1/DDM1) at 28 DAS (Right panel). Unknown represents plants genotypes has not been determined.
Figure S10. Phenotype of the hybrids between ddm1-9 (C24) and met1-1 (Col) mutant. Seedling of ddm1-9 (C24) x met1-1 (Col) hybrids at 21 (A) and 34 (B) DAS. (C) Time course of rosette diameter. Data represent mean values ± standard error obtained from more than twenty plants.
Table S1 Primer sequences for genotyping of mutants Target Primer sequences (5'-3') ddm1-1 ATTTGCTGATGACCAGGTCCT CATAAACCAATCTCATGAGGC CAPS: Nsi I ddm1-9 AGATTTCAGTGATGAGAAGAGCAGC GACCCTTCACTGGAAAAGCTTCAGC Direct sequencing met1-1 ACTTTGGCTTCCCTTCTCGA CAGCCCCAGGCCTAGTTGGG CAPS: Hae III
Table S2 Primer sequences for expression analysis Target Primer sequences (5'-3') RT-PCR MET1 TGCCAATGATTATCCTCTCCCATCG TGTTTCATCGGTAGAAGTTCCAGTG sololtr ATCAATTATTATGTCATGTTAAAACCGATTG TGTTTCGAGTTTTATTCTCTCTAGTCTTCATT AtSN1 AACGTGCTGTTGGCCCAGT CTGGAAGTTCAAGCCCAAAG GAP CACTTGAAGGGTGGTGCCAAG CCTGTTGTCGCCAACGAAGTC RT-qPCR U1 At1g27730 TTCTCGCTCGCGACAACCGTCAGCC CCCGCACCTTCGGAGTTGGACACGC U2 At1g54040 AACTGTATCCTGCTTAGGCGTGCGC ATTGCACAGCATTAGACTCATAGTC U3 At4g03060 TGAATCATAATAATCTCTCCGAGAG ATTATCATTAACACCGGTATCAGCG U4 At4g29200 GCTCTTTGGACAGATTCGTGCTCGG TCTCTATCAAATGGTGTCCATGGCC U5 At4g16860 AAGCTTCAGTGGGGTAGATGTTCGC TTCTGATCCTCTGGTTTGTCCTCGC U6 At1g61500 CTCGTATTTATCGTTCATAATCGTG ATGACCTTAAGATTTCCGCTATCTG U7 At3g14210 ATGACCTTAAGATTTCCGCTATCTG GGTATTAACCATCTTCCATAATCCG U8 At5g56030 AGATCTTCCTCCGTGAACTCATCAG AGACTCCCACACGTACTGCTCATCG U9 At2g30790 GAGAAGGATTCAAAATCCAGATCCC CCTAGCTCCTTTGAACCACCTCTTG U11 At1g28370 ATGGCACCGA CAGTTAAAAC GGCGG AGTTGGTTTT GGCTTTAGCT CCACG U12 At2g26530 ATGGAAGTGA TGAGCTTGAC TGCCC GTGATCTTCC TAGGCGTGCC TGGAG U13 At4g20480 TGATGAGGGT GATGGAGCCT CCGCC TTAAGGATGT CACGAGGGAC CTCTG U14 At5g26030 GCAGGCAACG GCTTTATCAT CTGGG GTTTCATCAT AAGAACAGTC TCCAG U15 At1g31580 TGGCATCTTC TATAGTCTCT TCCAT AACAGAAAGG GCACCTTCTG GGACG U16 At5g15360 ATGATCAGTT GGAATTTTGA AGTGA CAGATGCTGT CCACGAGCAA TCTAG U17 At5g48540 TGGCTCTTGT ATGTAGCTGC AGAGC TGCTGATATC TCCTCTACAT TGAGC D1 At2g43570 CGCCTCTCAAAACTGTGGCTGCGCC CCCTGTTTCTTGGGCTACGTGAGCG D2 At3g57260 ACGGGATGCTAGGCGATACCTTGCC ACCTTGACTTCAAGCCCTGCTCCAG D3 At5g10760 CCATTCAAGTCAGCTCTCTGTTCCC TCACTTCCGGTGTCGAAGACCAGCG D4 At5g54610 TATTCTCTTATTCAAGCGGCTCCCG CTCGTATTTATCGTTCATAATCGTG D5 At2g24850 CCTCCGCCCATTCCAACTTCAGGAC TCTCGGGGAGAAGATCGTATTTGCG D6 At4g20110 GTCGAAGCACGACGGCTCGATAGCC CCAAATGATTTATCGATTAACACCG D7 At1g02340 CCAAATGATTTATCGATTAACACCG ACTTGCTGTGAAAGATCAGGGCATG D8 At2g14610 GTCTTTGTAGCTCTTGTAGGTGCTC ACTTTGGCACATCCGAGTCTCACTG D9 At2g45660 AGAATGCAACAAGCAGACAAGTGAC TTGCTCAATCTGTTGCAGCTCCTCG D10 At1g45145 ATGGCCGGTG AAGGAGAAGT GATTG TTTGAATTCC TGAGCAACAG CTTGC D11 At3g50770 GGCAACTCAA AAAGAGAAAC CTTCC GATCTTACCG TCGCCGTCGC TGTCG
Table S3 Sequences of DNA oligo used for Northern blotting. Target Sequence (5'-3') sirna02 ATC GGC TGG CGG ACT GGT CAA C sirna1003 ATG CCA AGT TTG GCC TCA CGG TCT mir171 GAT ATT GGC GCG GCT CAA TCA
Table S4 Primer sequences for DNA methylation analysis Target Primer sequences (5'-3') Chop-PCR AtSN1 AGGATTTATTTCAATCCACGAACCT CGACTCCCATAAGTAACGAGTTG actin CTCATGAAGATTCTCACTGAG ACAACAGATAGTTCAATTCCCA Bisulfite sequencing FIS2 AYAATAATTAAGGTYYAATYGYATA AAAAARAAATCATRRAAACAACAAR At3g19630 GAGGTTGTGYATGAAGTTTGA TCRATCCARTCTCCATATATTC At5g58410 GTTGGTYTTGATATATAGAGGG TTCTAACACTTCRRTCATCCTT MeDIP-qPCR At1g51172 GAATCTGAAGCTCGCAGGTC GCGAAGCGAAGGTAGTTGTC At1g52010 ATGGTGAGACCGATGCTTCT GAGAGGTTTCGCTCAAGGTG At1g58561 ATCCAACAAAGGCAGAGGTG GCAGCTAAGGCAAGTGGAAG At4g08720 TCAAACCGATACCGTCTTCC ATTGGACGGACAAGGAACAG At4g10990 AGAAGGTGGCTCAAGTTGGA GTTCCATGGCGAAATGAGTT At5g19165 CGAAGCCATTTAAGCATATATCTGG CGTGAGATAATTGGGAGGTTATTGT EDV TGGCATGCTAGGCTAGGYCATCCTC GTGTATTCTCCTCCATTRTCGGTCC PORC GGCTTAGCGTCAGGATTGAATGGGC TTTGCCAGCTTCCTCTTCAGATACG SAHH1 GCTACTCTTTTGATTCATGAGGGTG AGGGAACAAAAGAGTTCCATTTTGC
Table S5 Primer sequences for RNAi construct Target Primer sequences (5'-3') MET1 TCTGAACACCTGCCTCACAG CCCATGGACGACCAAATATC