1. Microarrays for mapping and expression analysis: Toward the genetic determinants of light response adaptation in Arabidopsis and Aquilegia Justin Borevitz Ecology & Evolution University of Chicago naturalvariation.org

2. Light Affects the Entire Plant Life Cycle de-etiolation hypocotyl }

4. Light Affects the Entire Plant Life Cycle Light response variation can be seen under constant conditions in the lab

5. Seasons in the Growth Chamber Changing Day length Cycle Light Intensity Cycle Light Colors Cycle Temperature Sweden Spain Seasons in the Growth Chamber Changing Day length Cycle Light Intensity Cycle Light Colors Cycle Temperature

6. Local Population Variation

7. Talk Outline Single Feature Polymorphisms (SFPs) –Potential deletions –Bulk segregant/ eXtreme Mapping Haplotype analysis Tiling arrays Aquilegia Single Feature Polymorphisms (SFPs) –Potential deletions –Bulk segregant/ eXtreme Mapping Haplotype analysis Tiling arrays Aquilegia

8. What is Array Genotyping? Affymetrix tiling array GeneChips contain ~35bp spacing, 1.67million unique features Genomic DNA is randomly labeled with biotin dCTP, product ~50bp. 3 independent biological replicates compared to the reference strain Col GeneChip

9. Potential Deletions

11. 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

12. Chip genotyping of a Recombinant Inbred Line 29kb interval Discovery 6 replicates X $500 120,000 SFPs = $0.025 Typing 1 replicate X $500 120,000 SFPs = $0.0041

13. 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

14. Potential Deletions Suggest Candidate Genes FLOWERING1 QTL Chr1 (bp) Flowering Time QTL caused by a natural deletion in FLM (Werner et al, Genetics 2005) MAF1 FLM natural deletion

15. Fast Neutron deletions FKF1 80kb deletion CHR1cry2 10kb deletion CHR1 Het Hazen et al Plant Physiology 2005

16. Map bibb 100 bibb mutant plants 100 wt mutant plants

17. Array Mapping Hazen et al Plant Physiology 2005

18. LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms Hazen et al PNAS, 2005 Cloned with Array Mapping

19. eXtreme Array Mapping 15 tallest RILs pooled vs 15 shortest RILs pooled Wolyn et al Genetics 2004

20. 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

21. 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, Bur, Ler, Bay, Shah, Cvi, Kas, C24, Est, Kin, Mt, Nd, Sorbo, Van, Ws2 Fl-1, Ita-0, Mr-0, St-0, Sah-0

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

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

24. Chromosome Wide Diversity

25. Diversity 50kb windows

26. Tajima’s D like 50kb windows

27. R genes vs bHLH

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

29. 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

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

31. cDNA raw intensity 10% smoothed

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

33. 300 F3 RILs growing (Evadne Smith) >50,000 ESTs TIGR gene index and GenBank, arrays being designed by Nimblegen Aquilegia (Columbines)

34. Genetics of Speciation along a Hybrid Zone

35. Species with > 20k ESTs 11/14/2003 Animal lineage: good coverage Plant lineage: crop plant coverage

36. NSF Genome Complexity 52,000 ESTs 5’ and 3’ –>9k contigs, 4k singletons –>500 SNPs 350 arrays, RNA and genotyping –High density SFP Genetic Map Physical Map (BAC tiling path) –Physical assignment of ESTs QTL for pollinator preference –~400 RILs, map abiotic stress –QTL fine mapping/ LD mapping Develop transformation techniques http://www.AQgenome.org Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus Nordborg (USC) Justin Borevitz (U Chicago) Jeff Tompkins (Clemson)

37. University of Chicago Xu Zhang Evadne Smith University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones NaturalVariation.org Salk Jon Werner Joanne Chory Detlef Weigel Joseph Ecker UC Davis Julin Maloof UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler Salk Jon Werner Joanne Chory Detlef Weigel Joseph Ecker UC Davis Julin Maloof UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler University of Chicago Xu Zhang Evadne Smith University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones

39. differences may be due to expression or hybridization

40. PAG1 down regulated in Cvi PLALE GREEN1 knock out has long hypocotyl in red light