ddm1a (PFG_3A-51065) ATG ddm1b (PFG_2B-60109) ATG osdrm2 (PFG_3A-04110) osdrm2 osdrm2 osdrm2
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1 Relative expression OsDDM1a OsDDM1b OsDRM2 TG TG TG ddm1a (PFG_3-5165) P1 P3 ddm1b (PFG_2-619) P2 531bp 5233bp P1 P4 P3 P2 F1 R1 F1 TG TG TG F1 R1 C ddm1a -/- -/- ddm1b -/- +/- +/- -/- D (PFG_3-411) 3827bp 2.5cm P1 P2 R1 1cm P1+P2 P3+P2 F1+R2 ctin1 E ddm1a ddm1b ddm 1a F ddm1b Figure S1. Related to Figure 1. Characterization of expression and T-DN insertion mutants of OsDDM1a, OsDDM1b, and OsDRM2.. Transcript levels of OsDDM1a, OsDDM1b, and OsDRM2 relative to that of ctin 1 in difference organs, detected by qrt-pcr. ars = means +/-SD from 3 repeats.. Diagrams of OsDDM1a, OsDDM1b, and OsDRM2 genes. Open triangles indicate the T-DN insertion sites. Primers used for genotyping are indicated by arrows. C. Genotyping of T-DN insertion plants. D. osddm1a and osddm1b single mutants displayed no obvious phenotype at seedling and mature stages. E. Phenotypes of osddm double mutants in comparison with wild type. The double mutant is severely dwarfed at mature stage. The panicle is much smaller and no viable pollens were produced in the double mutant. Empty and dark colored seeds produced in the double mutant are indicated by arrows. Close-up shows ectopic tillers produced in the double mutant compared to wild type. F. Phenotype of mutant in comparison with wild type. The mutant has pleiotropicgrowth phenotypes at both vegetative and reproductive stages, including growth defects, abnormal panicle and spikelet morphology, and complete sterility.
2 ddm TEG TEGs Figure S2. Related to Figure 2. Cytosine methylation average levels at all sequence contexts in TE-related genes (TEG).. Cytosine methylation average levels at all sequence contexts plotted against TEG size. Note that high levels of wild type CG methylation (>8) in TEG were reduced to about 4% in average in osddm, and wild type CHG (8% in average) in TEG were reduced to low levels (below 2%) in osddm, while remained unchanged in.. Patterns and genome-wide average levels of cytosine methylation (CG, CHG, and CHH) of TEGs with their contiguous 2 kb upstream and downstream flanks in wild type and mutant rice plants.
3 ddm C ddm 1g553 (MX1) 5g142 (OsCYP51G3) 1g3685 2g453 (OsMSP) 7g134 (OsG2ox5) 3g3156 3g243 (SDG713) 8g1942 4g31984 D 4g4759 (Kala4) 8g429 (FIE1) McrC ddm triple 8g429 E 2g453 (OsMSP) HaeIII triple control CG CHG CHH MITE 5g317 Genes 3g243 1g553 4g4795 control 5g124 McrC control 5g124 1g3685 ddm triple TEGs 3g3156 4g g317 2g453 Figure S3. Related to Figure 2. Genome browser and validation of DN methylationloss in the mutants.. Genome browser display of methylation of protein coding genes in ddm.. Genome browser display of methylation of MITE-associated protein-coding genes in. C. Genome browser display of methylation of TE-related genes (TEGs) in osddm. D. McrC-digestion and PCR tests of methylationof genes and TEGs (shown in, C) in osddm double and osddm triple mutants compared to wild type. E. McrC- or Hae III-digestion and PCR tests of methylationof genes (shown in ) in, osddm, and the triple mutants compared to wild type.
4 Mutator LINE y1-copia Ty LTR non-ltr Retrotransposon Figure S4. Related to Figure 3. Patterns of cytosine methylation (CG, CHG, and CHH) of additional TE and repeats in wild type and osddm1a, osddm1b, osddm, and mutants. TEs were aligned at the 5 and the 3 ends, and average methylation for all cytosines within each 1 bp interval is plotted.
5 Chr 4 Centromere CCT Ty3-gypsy H3K9me2 Chr 1 Centromere CCT Ty3-gypsy H3K9me2 ddm CG CHG CHH Centromere 3g3889 2g2199 CCT 11g47 4g2192 Ty3-gypsy 3g4567 3g3345 Figure S5. Related to Figure 3. Heterochromatin localization of hyper-chh methylated TEs in osddm plants.. Distribution of hyper-chh methylated CCT, Ty3-gypsy, and centromere repeats in chromosome 1 and chromosome 4.. Genome browser of methylation levels at CG, CHG and CHH sites of 2 CCT, 2 Ty3- gypsy, and 2 centromere repeats in osddm compared to wild type.
6 osddm Figure S6. Related to Figure 6.. Transcriptomic analysis.. Reproducibility of RN-seq repeats of Cr_, osddm, and seedling leaves.. Overlap of differentially expressed genes (> 2 fold changes) in osddm and mutants.
7 1g3685 3g3156 4g g317 7g Fold change 1g553 2g453 ddm up TEGs 3g243 4g4759 8g Fold change ddm up Genes C 9 2g g g g1942 Fold change F up Genes D Fold change 1,4 DMT71 1,4 DMT73 2,5 DMT74 1,8 JMJ719 1,2 SDG714 1,2 DNG71 Figure S7. Related to Figure 6. Relative transcripts levels (to ctin 1) tested by qrt-pcr.. Relative transcript levels of 5 TE-related genes shown in Fig S3 in osddm compared Callus-regenerated wild type (Cr-).. Relative transcript levels of 5 protein-coding genes shown in Fig S3 in osddm compared wild type. C. Relative transcript levels of 4 protein-coding genes shown in Fig S3 in compared wild type. D. Transcripts levels of H3K9me2 methyltransferaseand demethylase genes in osddm compared wild type. DMT71 and DMT74 (OsCMT3); DMT73 (OsCMT2); JMJ719 (OsIM1); SDG714 (OsSUVH4).
8 Table S1. Cytosine methylation levels in wild type and mutant rice leaves measured by mass spectrometry. Genotype n 5-meC content (% of total cytosine) Percentage of wild-type levels Percentage decrease ±.51 ddm1a ± ddm1b ± ** ddm ± ** Table S2. Related to Figure 1; S-seq data of wild type and mutants. Sample Raw Reads Raw ases Clean Reads Clean ases unique mapping unique reads ases Coverage (X) all mapping reads all mapping smap Cr R G G G % x Cr R G G G % _R G G G % x _R G G G % ddm_r G G 65.2% G % x ddm_r G G 63.3% G % ddm1a_r G G 67.9% G % x ddm1a_r G G 67.1% G % ddm1b_r G G 67.1% G % x ddm1b_r G G 65.9% G % Table S3. Related to Figure 1; H3K27me3 and H3K9me2 ChIP-seq data Clean paired aligned concordantly aligned reads exactly 1 time concordantly >1 times Fail to align nti-h3k27me (75.42%) (22.36%) (2.22%) nti-h3k9me (35.26%) (63.%) (1.74%)
9 Table S4. Related to Figure 6; RN-seq data (two repeats per sample). pair mapping Sample raw reads ases read count clean data rate Cr_1_1_R G G 96.4% Cr_1_1_R G G Cr_1_2_R G G 96.3% Cr_1_2_R G G _1_R G G 95.5% _1_R G G _2_R G G 95.1% _2_R G G Cr_2_1_R G G 92.3% Cr_2_1_R G G Cr_2_2_R G G 92.% Cr_2_2_R G G ddm_1_r G G 91.1% ddm_1_r G G ddm_2_r G G 91.% ddm_2_r G G ddm_1_r G G 91.1% ddm_1_r G G ddm_2_r G G 91.% ddm_2_r G G
10 Table S5. Related to Figure 5. Numbers of hypo or hyper DMC, DMR and DMGs in CG, CHG, and CHH contexts in the mutants. DMCs CG CHG CHH Hypo Hyper Hypo Hyper Hypo Hyper ddm ddm1a ddm1b DMRs CG CHG CHH Hypo Hyper Hypo Hyper Hypo Hyper ddm ddm1a ddm1b DMGs ddm CG hypo CHG hypo CHH hypo CG hypo CHG hypo CHH hypo non-te TE CG hyper CHG hyper CHH hyper CG hyper CHG hyper CHH hyper non-te TE
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