Figure S1 Expression of AHL gene family members in diploid (Ler Col) and triploid (Ler
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1 Supplemental material Supplemental figure legends Figure S Expression of AHL gene family members in diploid (Ler ) and triploid (Ler osd) seeds. AHLs from clade B are labelled with (I), and AHLs from clade A are labelled with (II). Figure S2 Localization and expression of ADM and SUVH2/9. Fusion constructs with GFP (A C) or N or C terminal domains of YFP (nyfp or cyfp) (D G) were expressed in N. benthamiana leaves. Water mounted sections of leaf tissue were examined by confocal microscopy. Scale bars, 5µm. (A) SUVH9 GFP, (B) ADM GFP, (C) AHL0 GFP, (D) ADMcEYFP and neyfp SUVH9, (E) ADM ceyfp and AHL0 neyfp, (F) AHL0 ceyfp and neyfp SUVH9, (G) AHL0 neyfp and AHL0 ceyfp. Scale bars, 0 µm. (H) Quantitative RT PCR analysis of SUVH2 and SUVH9 expression in seeds derived from indicated crosses at 6 DAP. Data were normalized to PP2A. Figure S3 Bar chart representing z scored normalized H3K27me3 values on maternal and paternal alleles of PHE, ADM, SUVH9, and AHL0. Figure S4 Triploid seed abortion is rescued by suvh9 and ahl0. (A) Percentage of noncollapsed (gray bars) and germinated seeds (black bars) of indicated genotypes. Numbers on top of bars correspond to number of analyzed seeds. (B and C) Pictures of seedlings at 7 days after germination of indicated genotypes. Scale bars, cm. (D) Percentage of non collapsed seeds of indicated genotypes. Numbers on top of bars correspond to number of analyzed seeds. (E and F) Complementation of osd ahl0 and
2 osd suvh9 using constructs expressing AHL0 and SUVH9 under control of the UBIQUITIN (UBQ) and (PHE) promoters. plants were pollinated with pollen of osd ahl0 or osd suvh9 with or without the indicated complementation construct. plants pollinated with osd pollen served as a control. Independent transgenic lines are numbered. Numbers on top of bars correspond to number of analyzed seeds. Figure S5 (A) Metagene plots showing z score normalized H3K9me2 for all five chromosomes in the endosperm of seeds derived from crosses (), osd (), 2x adm (2x adm), osd adm (3x adm), 2x suvh2 suvh9 (2x suvh2 suvh9), 4x suvh2 suvh9 (3x suvh2 suvh9), 2x ahl0 (2x ahl0), 4x ahl0 (3x ahl0). (B) Metagene plots showing z score normalized H3K9me2 at TEs in the genotypes specified in A. (C) Metagene plots showing CHH DNA methylation (#C/(#C+#T) at TEs in the endosperm of,, and 3x adm. Figure * S6 (A) Box plots showing median values of z score normalized H3K9me2 in predicted matrix attachment regions (MAR), regions excluding MARs (Not MAR), predicted MARs overlapping transposable elements (MAR (TE)), and transposable elements (TE) in the endosperm of seeds derived from crosses (), osd (), osd adm (3x adm), 4x suvh2 suvh9 (3x suvh2 suvh9), 4x ahl0 (3x ahl0). The difference between and, and 3x adm, and 3x suvh2 suvh9, and 3x ahl0 in MARs, MAR(TE)s, and TEs are significant (Kolmogorov Smirnov test, p<0e 5 ). (B) Boxplot showing z score normalized H3K9me2 in predicted matrix attachment regions (MAR), regions excluding MARs (Not MAR), predicted MARs overlapping transposable elements (MAR (TE)), and transposable
3 elements (TE) in the indicated genotypes. Boxes show medians and the interquartile range, error bars show the full range excluding outliers. Figure S7 (A) Log2 fold expression change of transposable elements gaining H3K9me2 in (p<0.05) with detectable transcript levels in 2x,, or 3x adm seeds. (B) Log2 fold expression change of genes flanking TEs significantly gaining H3K9me2 in 3x (p<0.05) in 2x and seeds. All genes were used as control. The difference is significant (Kolmogorov Smirnov testp<0.0). (C) Log2 fold expression change of genes with increased expression in and genes flanking TEs significantly gaining H3K9me2 in (p<0.05) in and 3x adm seeds. The difference is significant (Kolmogorov Smirnov test, p <0.05). Figure S8 H3K9me2 methylation profiles of HDG0, ATG3583, PEG4, PEG7, and PEG9 in the endosperm. The profiles are derived from endosperm of the following genotypes: (brown), (cyan), 3x adm (blue), 3x suvh2;9 (red), 3x ahl0 (green). Regions with changes in H3K9me2 are marked by red dashed rectangles. Black horizontal bar: coding region of genes; blue horizontal bar: transposable element. Figure S9 (A) Metagene plots showing z score normalized in 4 DAP endosperm derived from crosses osd (), osd adm (3x adm), and (2x ) seeds. TEs belonging to RC/helitron, DNA MuDR, and DNA/HAT families that gained H3K9me2 in triploid seeds are shown. (B) Metagene plots showing DNA methylation (#C/(#C+#T) in each sequence context in samples specified in A. (C)
4 Metagene plots showing CHH methylation of TEs defined above in 6 DAP endosperm of and 2x seeds. Figure S0 Log2 fold change of expression of MORCs in versus.
5 Jiang_Fig S RNAseq (rpkm) 7.00E E E E E E+03.00E E+00 Lerx x Lerxosd x osd ()
6 Jiang_Fig S2 A B C D D SUVH9-GFP E ADM-GFP F AHL0-GFP G ADM-cEYFP neyfp-suvh9 ADM-cEYFP AHL0-nEYFP AHL0-cEYFP neyfp-suvh9 AHL0-nEYFP AHL0-cEYFP H Relative expression values x x 2x 2x x 4x 0 SUVH9 SUVH2
7 Jiang_Fig S3 2.5 H3K27me3 levels x xler_mat maternal xler_pat paternal Lerx xmaternal _mat Ler x_pat paternal 0.0 PHE ADM SUVH9 AHL0
8 Jiang_Fig S4 A 00% 382 B C Non-collapsed/ germinated seeds 80% 60% 40% 20% 0% x x osd suvh9 2x x osd ahl0 D Non-collapsed seeds 00% 80% 60% 40% 20% 0% E 80% 60% 40% 20% 0% Non-collapsed seeds00% x x osd ahl0 3 B F 80% 60% 40% 20% 0% Non-collapsed seeds00% x x osd suvh9
9 Jiang_Fig S5 A 2X 2X adm 2x suvh2 suvh9 2x ahl0 Chr Chr Chr Chr 3X 3X adm 3x suvh2 suvh9 3x ahl0 Chr Chr Chr Chr B 0.8 3x adm 2x adm 0.8 3x suvh2 suvh9 2x suvh2 suvh kb 0% 00% +2 kb kb 0% 00% +2 kb C x adm mchh 3x ahl0 2x ahl kb 0% 00% +2 kb kb 0% 00% +2 kb
10 Jiang_Fig S6 A 2 0 3x adm 3x suvh2 suvh9 3x ahl0 - MAR Not MAR MAR(TE) TE B 2 0 (2x) adm (2x) suvh2 suvh9 (2x) ahl0 (2x) - MAR Not MAR MAR(TE) TE
11 Jiang_Fig S7 A 5 Log 2 expression fold change B Log 2 expression fold change ( / 2x ) C Log 2 expression fold change (3x adm / 2x )
12 Jiang_Fig S8 2x 3x adm 3x suvh9 3x ahl0-2x 3x adm- 3x suvh9-3x ahl0- TE gene ATG34650, HDG0 500 bp 2x 3x adm 3x suvh9 3x ahl0-2x 3x adm- 3x suvh9-3x ahl0- TE gene AT3G50720, PEG7 500 bp 2x 3x adm 3x suvh9 3x ahl0-2x 3x adm- 3x suvh9-3x ahl0- TE gene ATG49290, PEG2 250 bp 2x 3x adm 3x suvh9 3x ahl0-2x 3x adm- 3x suvh9-3x ahl0- TE gene ATG bp AT5G540, PEG9 2x 3x adm 3x suvh9 3x ahl0-2x 3x adm- 3x suvh9-3x ahl0- TE gene 500 bp
13 Jiang_Fig S9 A 3x adm 0-2kb 0% 50% 00% +2kb B x adm C 0.2 mchh mcg mchg mchh kb 0% 50% 00% +2kb -2kb 0% 50% 00% +2kb -2kb 0% 50% 00% +2 kb kb 0% 50% 00% +2kb
14 Jiang_Fig S0.5 Log 2 expression fold change ( /2x ) 0 MORC MORC2 MORC6.5
ddm1a (PFG_3A-51065) ATG ddm1b (PFG_2B-60109) ATG osdrm2 (PFG_3A-04110) osdrm2 osdrm2 osdrm2
Relative expression.6.5.4.3.2.1 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)
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