Talk Outline

1. Microarray Based Methods to Determine the Causes of Natural Phenotypic Variation in Arabidopsis thalianaJustin BorevitzEcology & EvolutionUniversity of Chicagohttp://naturalvariation.org

2. Talk Outline • Natural Variation/ QTL mapping • Single Feature Polymorphisms (SFPs) • eXtreme Array Mapping • Potential deletions • Haplotype analysis • Patterns in gene Families • Aquilegia

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

6. Light Affects the Entire Plant Life Cycle Light response variation can be seen under constant conditions in the lab Natural Variation != Natural Selection

7. Seasons in the Growth Chamber • Changing Day length • Cycle Light Intensity • Cycle Light Colors • Cycle Temperature

13. Quantitative Trait Loci

16. With the Aid of Genomics Experimental Design Mapping population Marker Identification Genotyping Phenotyping QTL Analysis Fine Mapping Candidate gene Polymorphisms gene expression loss of function QTL gene Confirmation QTL gene Confirmation Phenotyping QTL Analysis Fine Mapping Genomics path Experimental Design Mapping population

17. Which arrays should be used? • Spotted arrays Arizona 29,000 - 70mers • ATH1, Affymetrix expression GeneChip 202,806 unique 25bp oligo nucleotides features • AtTILE1, universal whole genome array every ~35bp, > 3Million PM features • Re-sequencing array 120M*8bp • 20 Accessions, Perlegen, • Max Planck (Weigel), USC (Nordborg) GeneChip

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

19. Potential Deletions

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

22. 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

23. 100 bibb mutant plants Map bibb 100 wt mutant plants

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

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

26. Array Mapping chr1 chr2 chr3 chr4 chr5 Hazen et al Plant Physiology (in press)

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

28. Chromosome 2 16 12 RED2 QTL LOD 8 4 0 0 20 40 60 80 100 cM RED2 QTL 12cM Composite Interval Mapping eXtreme Array Mapping LOD Allele frequencies determined by SFP genotyping. Thresholds set by simulations Red light QTL RED2 from 100 Kas/ Col RILs (Wolyn et al Genetics 2004)

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

30. Het Fast Neutron deletions FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1

31. Array Haplotyping • What about Diversity/selection across the genome? • A genome wide estimate of population genetics parameters, θw, 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

32. Chromosome1 ~500kb Col Ler Cvi Kas Bay Shah Lz Nd Array Haplotyping Inbred lines Low effective recombination due to partial selfing Extensive LD blocks

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

34. Chromosome Wide Diversity

35. Diversity 50kb windows

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

37. R genes vs bHLH

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

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

40. Plant lineage: crop plant coverage > 20k dbEST 11/14/2003 Animal lineage: good coverage

41. NSF Genome Complexity • 45,000 ESTs 5’ and 3’ ends • 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 Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus Nordborg (USC) Justin Borevitz (U Chicago) Jeff Tompkins (Clemson)

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