1. High Resolution Patterns of Variation in the Arabidopsis Genome Justin Borevitz University of Chicago naturalvariation.org

3. Talk Outline Single Feature Polymorphisms (SFPs) –Potential deletions Bulk Segregant Mapping –Extreme Array Mapping Haplotype analysis Patterns in gene Families New Arrays Aquilegia

4. What is Array Genotyping? Affymetrix expression GeneChips contain 202,806 unique 25bp oligo nucleotides. 11 features per probset for 21546 genes New array’s have even more Genomic DNA is randomly labeled with biotin, product ~50bp. 3 independent biological replicates compared to the reference strain Col GeneChip

5. Potential Deletions

6. Spatial Correction Spatial Artifacts Improved reproducibility Next: Quantile Normalization

8. False Discovery and Sensitivity PM only SAM threshold 5% FDR GeneChip SFPs nonSFPs Cereon marker accuracy 3806 89118 100% Sequence 817 121 696 Sensitivity Polymorphic 340 117 223 34% Non-polymorphic 477 4 473 False Discovery rate: 3% Test for independence of all factors: Chisq = 177.34, df = 1, p-value = 1.845e-40 SAM threshold 18% FDR GeneChip SFPs nonSFPs Cereon marker accuracy 10627 82297 100% Sequence 817 223 594 Sensitivity Polymorphic 340 195 145 57% Non-polymorphic 477 28 449 False Discovery rate: 13% Test for independence of all factors: Chisq = 265.13, df = 1, p-value = 1.309e-59 3/4 Cvi markers were also confirmed in PHYB 90%80%70% 41%53%85% 90%80%70% 67%85%100% Cereon may be a sequencing Error TIGR match is a match

9. Chip genotyping of a Recombinant Inbred Line 29kb interval Discovery 6 replicates X $500 12,000 SFPs = $0.25 Typing 1 replicate X $500 12,000 SFPs = $0.041

10. Potential Deletions >500 potential deletions 45 confirmed by Ler sequence 23 (of 114) transposons Disease Resistance (R) gene clusters Single R gene deletions Genes involved in Secondary metabolism Unknown genes

11. Potential Deletions Suggest Candidate Genes FLOWERING1 QTL Chr1 (bp) Flowering Time QTL caused by a natural deletion in MAF1 MAF1 MAF1 natural deletion

12. Fast Neutron deletions FKF1 80kb deletion CHR1cry2 10kb deletion CHR1 Het

13. Map bibb 100 bibb mutant plants 100 wt mutant plants

14. bibb mapping ChipMap AS1 Bulk segregant Mapping using Chip hybridization bibb maps to Chromosome2 near ASYMETRIC LEAVES1

15. BIBB = ASYMETRIC LEAVES1 Sequenced AS1 coding region from bib-1 …found g -> a change that would introduce a stop codon in the MYB domain bibbas1-101 MYB bib-1 W49* as-101 Q107* as1 bibb AS1 (ASYMMETRIC LEAVES1) = MYB closely related to PHANTASTICA located at 64cM

16. arythmic90 Gene cloned Sam Hazen arythmic21 Allelic to arr90 Sam Hazen

17. stamenstay Ler Sarah Liljegren Mapping confirmed

18. eXtreme Array Mapping 15 tallest RILs pooled vs 15 shortest RILs pooled

19. LOD eXtreme Array Mapping Allele frequencies determined by SFP genotyping. Thresholds set by simulations 0 4 8 12 16 020406080100 cM LOD Composite Interval Mapping RED2 QTL Chromosome 2 RED2 QTL 12cM Red light QTL RED2 from 100 Kas/ Col RILs

20. Array Haplotyping What about Diversity/selection across the genome? A genome wide estimate of population genetics parameters, θ w, π, Tajima’D, ρ LD decay, Haplotype block size Deep population structure? Col, Lz, Ler, Bay, Shah, Cvi, Kas, C24, Est, Kin, Mt, Nd, Sorbo, Van, Ws2

21. Pairwise Correlation between and within replicates

22. Array Haplotyping Inbred lines Low effective recombination due to partial selfing Extensive LD blocks ColLerCviKasBayShahLzNd Chromosome1 ~500kb

23. Distribution of T-stats null (permutation) actual Not ColColNANA duplications 32,427 Calls 208,729 12,250 SFPs

24. Sequence confirmation of SFPs

25. SFPs for reverse genetics http://naturalvariation.org/sfp 14 Accessions 30,950 SFPs`

26. Chromosome Wide Diversity

27. Diversity 50kb windows

28. Tajima’s D like 50kb windows RPS4 unknown

29. R genes vs bHLH Theta W RPS4

30. Rgenes vs bHLH Tajimas’ D RPS4

31. R genes vs bHLH

32. RNADNA Universal Whole Genome Array Transcriptome Atlas Expression levels Tissues specificity Transcriptome Atlas Expression levels Tissues specificity Gene Discovery Gene model correction Non-coding/ micro-RNA Antisense transcription Gene Discovery Gene model correction Non-coding/ micro-RNA Antisense transcription Alternative Splicing Comparative Genome Hybridization (CGH) Insertion/Deletions Comparative Genome Hybridization (CGH) Insertion/Deletions Methylation Chromatin Immunoprecipitation ChIP chip Chromatin Immunoprecipitation ChIP chip Polymorphism SFPs Discovery/Genotyping Polymorphism SFPs Discovery/Genotyping ~35 bp tile,non-repetitive regions, “good” binding oligos,evenly spaced

33. ChipViewer: Mapping of transcriptional units of ORFeome From 2000v At1g09750 (MIPS) to the latest AGI At1g09750 2000 v Annotation (MIPS) The latest AGI Annotation

34. SNP SFP MMMMMM MMMMMM Chromosome (bp) conservation SNP ORFa start AAAAA Transcriptome Atlas ORFb deletion Improved Genome Annotation

35. Review Single Feature Polymorphisms (SFPs) can be used to Potential deletions (candidate genes) Identify recombination breakpoints eXtreme Array Mapping Haplotyping Diversity/Selection Association Mapping

36. Aquilegia (Columbines) Recent adaptive radiation, 350Mb genome

37. NSF Genome Complexity 35,000 ESTs 5’ and 3’ 350 arrays, RNA and genotyping –High density SFP Genetic Map Physical Map (BAC tiling path) –Physical assignment of ESTs QTL for pollinator preference –and abiotic stress –QTL fine mapping/ LD mapping Develop transformation techniques

38. NSF Genome Complexity Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus Nordborg (USC) Justin Borevitz (U Chicago) Jeff Tompkins (Clemson)

39. NaturalVariation.org Salk Jon Werner Sarah Liljegren Huaming Chen Joanne Chory Detlef Weigel Joseph Ecker UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler Salk Jon Werner Sarah Liljegren Huaming Chen Joanne Chory Detlef Weigel Joseph Ecker UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler University of Chicago Xu Zhang Evadne Smith Syngenta Hur-Song Chang Tong Zhu UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones University of Chicago Xu Zhang Evadne Smith Syngenta Hur-Song Chang Tong Zhu UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones