1. Whole genome transcriptome variation in Arabidopsis thaliana Xu Zhang Borevitz Lab Whole genome transcriptome variation in Arabidopsis thaliana Xu Zhang Borevitz Lab

2. Arabidopsis thaliana have been adapted to highly variable environments

3. Transcription and splicing Chromosomal DNA Transcription Nuclear RNA Exon 1 Exon 2 Exon 3 Intron 1Intron 2 RNA splicing Messenger RNA Exon 1Exon 2Exon 3Exon 1Exon 3

4. Whole genome tiling array Genetic hybridization polymorphisms could affect the estimation of gene expression  High density and resolution: 1.6M unique probes at 35bp spacing  Without bias toward known transcripts

5. Col♀ x Col♂Van ♀ x Van ♂Col ♀ x Van ♂Van ♀ x Col ♂  parental strains and reciprocal F1 hybrids  mRNA from total RNA; genomic DNA The experiment

6. Double-stranded random labeling Random reverse transcription Double-stranded cDNA Random priming AAAAA

7.  Sequence polymorphisms  Gene expression variation  Splicing variation  A functional network of differentially spliced genes  HMM for a de novo transcription profiling Outlines

8.  Sequence polymorphisms  Gene expression variation  Splicing variation  A functional network of differentially spliced genes  HMM for a de novo transcription profiling Outlines

9. SFP deletion or duplication in Van Single Feature Polymorphisms and indels SFPs SFP

10. Sequence polymorphisms SPFs and indels (>200bp) were removed before gene expression analysis SFPs a FDRCol > Van c Van > Col c Total 11.82%13576914934150703 7.66%1264439479135922 5.22%1183816662125043 3.88%1108614979115840 3.15%1041153820107935 Indels b Model selectiondeletionduplicationTotal BIC d 51822540 AIC e 16451361781

11. Deletions vs duplications

12. Distribution of indels along chromosomes

13.  Sequence polymorphisms  Gene expression variation  Splicing variation  A functional network of differentially spliced genes  HMM for a de novo transcription profiling Outlines

14. Additive, dominant and maternal effects of gene expression

15. The linear model Gene probe Intensity ~ additive + dominant + maternal + ε intensity Col Van F1c F1v additive maternal dominant genotypes

16. Gene expression variation between genotypes Delta a Sig+ b Sig- c TotalFalse d FDR additive 0.549113967887890110.15% 12674173644102154.88% 1.516269232549702.76% 1.812496761925392.03% 2.56903341024131.24% dominant 0.515113190470176716.31% 1405152119261869.65% 1.5157811968676.93% 1.892575667405.99% 2.541270311144.65% maternal 0.5599895609373512.06% 12046820541517.37% 1.54800 4910.29% 1.81630 2817.33% 2.5410 922.84%

17. Mean gene intensity Van dominant Col dominant over dominant F1v dominantF1c dominant Maternal paternal The pattern of gene expression inheritance Col Van F1v F1c

18. The pattern of gene expression inheritance

19. Enrichment in GO functional categories GO enrichment for additive dominant maternal effect genes Defense response genes are highly expressed in F1 hybrid lines, while many growth related pathway are down-regulated

20.  Sequence polymorphisms  Gene expression variation  Splicing variation  A functional network of differentially spliced genes  HMM for a de novo transcription profiling Outlines

21. Default expression status of exon and intron  Exons: correction for gene expression corrected by gene mean corrected by a gene median splicing index (Mean exon /Mean gene )  Introns: direct comparison Exon/intron probe Intensity ~ additive + dominant + maternal + ε

22. Differential exon splicing Exon probe Intensity ~ additive + dominant + maternal + ε Delta a Sig+ b Sig- c TotalFalse d FDR corrected by gene mean 0.3287190477559117% 0.417712930620567.0% 0.51271092369741.0% 0.692861785530.8% 0.777691463423.4% Corrected by gene median 0.352328080355669.2% 0.432817250020340.6% 0.52231203439628.0% 0.6154762305423.5% 0.7123521753419.3% Splicing index 0.340723564242566.0% 0.429217546713228.0% 0.52301433735013.0% 0.6178104282217.50% 0.714886234104.30%

23. Differential intron splicing Intron probe Intensity ~ additive + dominant + maternal + ε Delta a Sig+ b Sig- c TotalFalse d FDR 0.35611034159533220.8% 0.4405523928859.17% 0.5316352668284.26% 0.6239220459122.61% 0.720215535771.91% 0.817612029651.53%

24. Differential exon splicing is predominantly additive in F1 hybrids

25. Some dominant effect in differential intron splicing in F1 hybrids

26. Comparison for enrichment in known alternatively spliced exons Threshold 1Threshold 2 CalledNot calledCalledNot called Corrected by gene mean Known 2899171012 Not known 397551459055452 Fold enrichment 3.924.26 p-value 5.97E-091.90E-03 Corrected by gene median polish Known 2499561013 Not known 430551128555457 Fold enrichment 3.093.86 p-value 3.60E-066.14E-03 Splicing index Known 24109351112 Not known 537723288872777 Fold enrichment 2.963.72 p-value 6.84E-061.36E-02

27. AT1G21350 AT1G34180 AT1G76170 AT1G29120 AT1G51350 AT1G80960 AT1G07350 Experimental determined FDR for differential splicing # of significant calls estimated FDR # of tested # of confirmed experimental FDR Exon (corrected by mean) 477117%452251.1% 11120.8%181044.4% Exon (corrected by median) 50040.6%402147.5% 10315.60%171041.2% Exon (splicing index) 64266.0%502354.0% 1021.00%201050.0% intron 4592.61%653841.5% 1951.15%583343.1%

28.  Sequence polymorphisms  Gene expression variation  Splicing variation  A functional network of differentially spliced genes  HMM for a de novo transcription profiling Outlines

29. Enrichment of differentially spliced genes in chloroplast thylakoid enrichment of differentially spliced genes

30. Chloroplast thylakoid

31. Differrentially spliced genes which are located in chloroplast thylakoid Photosynthesis related genes AT5G38660 APE1 (Acclimation of Photosynthesis to Environment) mutant has altered acclimation responses

32. AT1G07350transformer serine/arginine-richribonucleoprotein putative AT1G55310SC35-like splicing factor 33 kD(SCL33) AT2G29210splicing factor PWIdomain-containing protein AT5G04430KH domain-containing proteinNOVA putative Splicing regulator tend to be differentially spliced

33.  Sequence polymorphisms  Gene expression variation  Splicing variation  A functional network of differentially spliced genes  HMM for a de novo transcription profiling Outlines

34. Generalized tiling array HMM  3-state HMM  Discrete distribution for emission probability  Transition probability counts for probe spacing  Baum-Welch parameter estimation (by Jake Byrnes)

35. An example of HMM detected segments

36. A nice model also needs better array  Array density is not enough to distinguish exon/intron boundaries  Probe quality

37. Differential segments >=3 continuous probes with posterior probability >0.99. Differentially expressed genes annotated genes for which ≥33% of their probes reside within the observed differential segments. Differentially spliced genes annotated genes for which <33% of probes resided within the differential segment, or annotated genes containing ≥2 differential segments with different states. Novel gene boundaries differential segments with >= 5 probes extending beyond annotated gene boundary Novel transcripts differential segments with >= 5 probes and outside any annotated gene boundary.

38. Length distribution of segments called by HMM

39. Comparison of annotation-based analysis and HMM Col > VanVan > ColTotal Annotation differential expression a 16269232549 differential exonic splicing b 287190477 differential intronic splicing c 202155357 HMM differential expression d 16549622616 differential splicing e 8745301404 un-annotated transcript f 344276 un-annotated 5' g 301949 un-annotated 3' g 28836

40. Comparison of annotation-based analysis and HMM Annotation Expression (Col>Van) Expression (Van>Col) Splicing (Col>Van) Splicing (Van>Col) HMM 1654962921550 Expression (Col>Van) 16261270 225 Expression (Van>Col) 923 727132 Splicing (Col>Van) 44118147 Splicing (Van>Col) 300 9038

41. Acknowledgements Justin Borevitz Yan Li Christos Noutsos Geoff Morris Andy Cal Jake Byrnes Josh Rest