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Linaria vulgaris flowers (Cubas et al., 1999)

Global analysis of genetic, epigenetic and transcriptional polymorphisms in Arabidopsis thaliana using whole genome tiling array. DNA methylation. Genome defense against mobile elements Regulation of gene activity. Linaria vulgaris flowers (Cubas et al., 1999).

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Linaria vulgaris flowers (Cubas et al., 1999)

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

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

  3. Plant DNA methylation • Symmetric cytosine methylation: mCG mCNG • Asymmetric cytosine methylation: mCNN

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

  5. 5’-C CGG- 3’-GGC C- Enzyme methylome approach

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

  7. Constitutive CG methylation * * * A) HpaIIdigestion * * * Random labeling B) * * * MspI digestion Random labeling HpaII HpaII intensity MspI MspI Col Van

  8. Methylation polymorphisms * * * A) Col genotype * * * HpaIIdigestion * * * MspIdigestion B) Van genotype HpaIIdigestion HpaII intensity HpaII MspI MspI MspIdigestion Col Van

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

  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 Called: significant features False: false positives based on permutation Sig-: Van greater signal Sig+: Col greater signal

  12. Genome distribution of SFPs

  13. Genic distribution of SFPs aThe number of SFPs within each annotation category.  bThe number of features within each annotation category. cCoding sequences. dThe sequences from transcriptional start to upstream 1kb. eThe sequences from transcriptional stop to downstream 1kb.

  14. 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

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

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

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

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

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

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

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

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

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

  30. additive dominant enzyme maternal Significance of main effects

  31. Significance of genotype x enyzme effects additive χ enzyme dominant χ enzyme maternal χ enzyme

  32. Correlation of constitutive CG methylation and absolute gene expression

  33. Correlation of polymorphic CG methylation and gene expresson variation

  34. Gene set enrichment in genic CG methylation polymorphisms

  35. Maternal methylome could be important for reciprocal F1 gene expression

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