1. Global analysis of genetic, epigenetic and transcriptional polymorphisms in Arabidopsis thaliana using whole genome tiling array

2. Linaria vulgaris flowers (Cubas et al., 1999) DNA methylation Tomato ripening mutant (Manning et al., 2006)  Genome defense against mobile elements  Regulation of gene activity

3.  Symmetric cytosine methylation: m CG m CNG  Asymmetric cytosine methylation: m CNN Plant DNA methylation

4.  Extent of CG methylation and methylation polymorphism among natural accessions  Inheritance of methylation polymorphisms  Any effect of methylation on gene expression What we want to know

5. 5’-C CGG- 3’-GGC C- Enzyme methylome approach 5’-CCGG- 3’-GGCC- 5’-CmCGG- 3’-GGCmC- 5’-mCmCGG- 3’-GGCmCm- 5’-mCCGG- 3’-GGCCm- HpaII cutting YNN Rare in plant MspI cutting YYN

6. CG-methylation and expression profiling  300ng genomic DNA  Digest with either mspI or hpaII  Label with biotin random primers  Hybridize to AtTILE1F Col♀ x Col♂Van ♀ x Van ♂Col ♀ x Van ♂Van ♀ x Col ♂  mRNA from 20ug totoal RNA  Double-stranded cDNA synthesis  Label with biotin random primers  Hybridize to AtTILE1F

7. HpaII digestion Random labeling MspI digestion * * * A) B) Constitutive CG methylation HpaII MspI HpaII MspI ColVan intensity

8. Methylation polymorphisms HpaII ColVan intensity MspI HpaII MspI HpaII digestion * * * A) B) Col genotype Van genotype MspI digestion HpaII digestion MspI digestion * * *

9. A) B) HpaII ColVan intensity MspI HpaII MspI Sequence polymorphisms * * Col genotype Van genotype

10. Simultaneous genetic and epigenetic profiling # of unique probes: 1,683,620 # of CCGG-containing probes: 54,519 model: Intensity ~ genotype + enzyme + genotype x enzyme

11. Summary of sequence polymorphisms FDRCalled a False b Sig- c Sig+ c 13.05% 211220 29007 58628 152592 6.22% 173611 11363 33227 140384 2.74% 153401 4431 23326 130075 1.16% 138552 1698 17742 120810 0.51% 126499 678 14131 112368 0.22% 116122 272 11448 104674 0.09% 106817 104 9347 97470 Called: significant features False: false positives based on permutation Sig-: Van greater signal Sig+: Col greater signal

12. Genome distribution of SFPs

13. CD c IntronUTRsPromoter d Downstream e Intergenic Total SFP a 23180198065130301903215850539161003 Feature b 5264073019471052604295854526815937572409637 Percentage4.40%6.56%4.87%7.03%7.10%8.51%6.68% Genic distribution of SFPs a The number of SFPs within each annotation category. b The number of features within each annotation category. c Coding sequences. d The sequences from transcriptional start to upstream 1kb. e The sequences from transcriptional stop to downstream 1kb.

14. EnzymeGenotype x enzyme p-valueHpaII > MspI a p-valueCol-specific b Van-specific c <0.012373<0.011062407 <0.054522<0.032389944 <0.16324<0.0537001515 Gene d 3628 (20%)Gene d 3498 (20%) Total gene e 17760Total gene e 17760 Promoter f 305 (6%)Promoter f 455 (9%) Total promoter g 5041Total promoter g 5041 Intergenic h 1298 (16%)Intergenic h 782 (9%) Total intergenic i 8264Total intergenic i 8264 Methylation polymorphisms are extensive a Features of constitutive CG methylation bc Features of Col- or Van-specific methylation df cDNAs or promoters with feature(s) of enzyme effect (p < 0.1) or genotype × enzyme interaction (p < 0.05) eg cDNAs or promoters containing CCGG feature(s) h Intergenic features (excluding cDNAs or promoters) of enzyme effect (p < 0.1) or genotype × enzyme interaction (p < 0.05) i Intergenic (excluding cDNAs or promoters) CCGG-containing features

15. Verification of methylation polymorphisms

17. bp Genome distribution of constitutive and polymorphic methylation sites

18. bp Co-methylation of pericentromere regions

19. Genic distribution of constitutive and polymorphic methylation sites

20. Correlation between gene size and constitutive CG methylation

21. Col Van Col♂ x Van♀ Van♂ x Col♀ CC*GG chromomethylase 2 (CMT2) exon19 epiTyper

22. Full model: Intensity ~ genotype + enzyme + genotype x enzyme Genotype: Additive (between parents) Dominant (between F1 and mid-parent) Maternal (between reciprocal F1s) Inheritance of CG methylation polymorphism

23. Additive effect describes intensity difference between parent strains across enzyme treatments. Additive effect HpaII MspI log intensity Col Van Col HpaII MspI log intensity Col Van Col Van SFP; Col has greater signal than Van. Van duplication or deletion in Col; Van has greater signal than Col F1c F1vF1c F1v F1c F1v F1c F1v Additive effect + Additive effect -

24. Dominant effect describes intensity difference between mid-parent (average of parents; dashed line) and average of F1 hybrids across enzyme treatments. Dominant effect Col Van Col F1c F1v F1c F1v HpaII MspI Increased F1 hybridization compared with expected from mid-parent log intensity Dominant effect + Col Van Col Dominant effect - F1c F1v F1cF1v HpaII MspI log intensity Reduced F1 hybridization compared with expected from mid-parent

25. Maternal effect describes intensity difference between reciprocal F1 hybrids across enzyme treatments. Maternal effect F1v F1c Maternal effect + HpaII MspI F1vF1c log intensity F1c HpaII MspI F1v F1c F1v Random variation; Col-mother F1 with greater signal than Van-mother F1 Random variation; Van-mother F1 with greater signal than Col- mother F1 log intensity Col Van Col Van Col Van Col Van Maternal effect -

26. Enzyme effect describes intensity difference between HpaII and MspI enzyme treatment across genotypes. Enzyme effect HpaII MspI Col Van Enzyme effect + Van F1c F1v F1c F1v log intensity HpaII MspI Col Van F1c F1v F1c F1v Constitutive CG methylation; HpaII samples have greater signal Normalization and/or preferential labeling of short fragment; MspI samples have greater signal log intensity Enzyme effect -

27. Additive x enzyme effect describes differential enzyme sensitivity between parent strains. Additive x enzyme interaction Additive x enzyme effect + log intensity Van Col Van HpaII MspI Col-specific methylation Van-specific methylation F1c F1v F1c F1v log intensity Van Col Van F1c F1v F1c F1v HpaII MspI Additive x enzyme effect -

28. Dominant x enzyme effect describes differential enzyme sensitivity between mid-parent (average of parents; dashed line) and average of F1 hybrids. Dominant x enzyme interaction Col Van Col F1c HpaII MspI Dominant x enzyme effect + F1v Van F1c F1v Col Van Col F1c HpaII MspI F1v Van F1c F1v log intensity Col-dominant methylation Van-dominant methylation log intensity Dominant x enzyme effect -

29. Maternal x enzyme effect describes differential enzyme sensitivity between reciprocal F1 hybrids Maternal x enzyme interaction Maternal x enzyme effect + Col-mother hybrid specific methylation Van-mother hybrid specific methylation log intensity Van Col Van HpaII MspI F1c F1v F1c F1v log intensity Van Col Van HpaII MspI F1c F1v F1c F1v Maternal x enzyme effect -

30. additivedominant maternal enzyme Significance of main effects

31. additive χ enzymedominant χ enzymematernal χ enzyme Significance of genotype x enyzme effects

32. Correlation of constitutive CG methylation and absolute gene expression

33. Correlation of polymorphic CG methylation and gene expresson variation

34. effectGOatermp-valueGOatermp-value addenz Col > VanVan > Col GO:0006457protein folding7.84E-05GO:0007242intracellular signaling cascade1.72E-03 GO:0009909regulation of flower development5.05E-03GO:0015979photosynthesis2.76E-03 GO:0007018microtubule-based movement8.56E-03GO:0006952defense response5.82E-03 GO:0006511ubiquitin-dependent protein catabolic process1.27E-02GO:0030001metal ion transport1.24E-02 GO:0007275multicellular organismal development1.50E-02GO:0009809lignin biosynthetic process2.49E-02 GO:0042254ribosome biogenesis and assembly2.03E-02GO:0006813potassium ion transport2.91E-02 GO:0019538protein metabolic process2.16E-02GO:0009739response to gibberellin stimulus4.85E-02 GO:0006470protein amino acid dephosphorylation2.73E-02 GO:0009567double fertilization forming a zygote and endosperm2.98E-02 GO:0045454cell redox homeostasis3.39E-02 GO:0007568aging4.67E-02 domenz F1 hybrids > parentsc parents > F1 hybridsc GO:0009965leaf morphogenesis2.13E-04GO:0042254ribosome biogenesis and assembly6.60E-03 GO:0009225nucleotide-sugar metabolic process4.21E-04GO:0009617response to bacterium1.36E-02 GO:0006869lipid transport2.96E-03GO:0009744response to sucrose stimulus2.31E-02 GO:0010119regulation of stomatal movement8.79E-03GO:0016192vesicle-mediated transport2.59E-02 GO:0000271polysaccharide biosynthetic process9.77E-03GO:0000074regulation of progression through cell cycle2.60E-02 GO:0015995chlorophyll biosynthetic process2.03E-02GO:0045449regulation of transcription3.60E-02 GO:0048364root development2.11E-02GO:0006810transport4.08E-02 GO:0009408response to heat2.33E-02 GO:0009908flower development4.08E-02 GO:0015979photosynthesis4.11E-02 GO:0045454cell redox homeostasis4.13E-02 GO:0019575sucrose catabolic process using beta-fructofuranosidase4.41E-02 GO:0009887organ morphogenesis4.49E-02 matenz Col-mother F1 > Van-mother F1dVan-mother F1 > Col-mother F1d GO:0015979photosynthesis1.17E-03GO:0015986ATP synthesis coupled proton transport1.09E-02 GO:0015995chlorophyll biosynthetic process1.22E-03GO:0006470protein amino acid dephosphorylation1.11E-02 GO:0009408response to heat1.76E-02GO:0009407toxin catabolic process1.14E-02 GO:0009416response to light stimulus2.87E-02GO:0006944membrane fusion2.60E-02 GO:0006520amino acid metabolic process3.38E-02GO:0009909regulation of flower development2.92E-02 GO:0042742defense response to bacterium3.50E-02GO:0009873ethylene mediated signaling pathway4.07E-02 GO:0006397mRNA processing4.44E-02 Gene set enrichment in genic CG methylation polymorphisms

35. Col methylation > Van methylation c Col-mother F1 expression > Van-mother F1 expression d GO e termp-valueGO e termp-value BP a GO:0006457protein folding*7.84E-05GO:0006412translation2.13E-32 GO:0009909regulation of flower development5.05E-03GO:0006457protein folding*2.09E-30 GO:0007018microtubule-based movement*8.56E-03GO:0042254ribosome biogenesis and assembly*2.82E-15 GO:0006511ubiquitin-dependent protein catabolic process1.27E-02GO:0007018microtubule-based movement*1.14E-11 GO:0007275multicellular organismal development1.50E-02GO:0006334nucleosome assembly1.88E-09 GO:0042254ribosome biogenesis and assembly*2.03E-02GO:0009408response to heat4.49E-09 MF b GO:0031072heat shock protein binding*1.67E-03GO:0003735structural constituent of ribosome6.21E-32 GO:0003777microtubule motor activity*7.56E-03GO:0003777microtubule motor activity*2.75E-13 GO:0051082unfolded protein binding*1.27E-02GO:0003723RNA binding1.34E-12 GO:0015035protein disulfide oxidoreductase activity1.90E-02GO:0051082unfolded protein binding*1.44E-12 GO:0005528FK506 binding*2.59E-02GO:0003755peptidyl-prolyl cis-trans isomerase activity*6.31E-10 GO:0003755peptidyl-prolyl cis-trans isomerase activity*3.19E-02GO:0005525GTP binding1.63E-08 GO:0031072heat shock protein binding*1.99E-08 GO:0005528FK506 binding*3.02E-06 Maternal methylome could be important for reciprocal F1 gene expression