1. Toward the genetic basis of adaptation using arrays Justin Borevitz Ecology & Evolution University of Chicago http://naturalvariation.org

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

4. Local Population Variation Scott Hodges Ivan Baxter

5. Seasonal Variation Matt Horton Megan Dunning

6. 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

7. Talk Outline Natural Variation in Light Response Single Feature Polymorphisms (SFPs) –Potential deletions –Bulk segregant/ eXtreme Mapping Barley RNA SFPs Aquilegia Natural Variation in Light Response Single Feature Polymorphisms (SFPs) –Potential deletions –Bulk segregant/ eXtreme Mapping Barley RNA SFPs Aquilegia

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

13. Quantitative Trait Loci

15. Which arrays should be used? Spotted oligo 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 120Mbp*8features –20 Accessions, Perlegen, –Max Planck (Weigel), USC (Nordborg) GeneChip

16. Which arrays should be used? cDNA array Long oligo array

17. Which 25mer arrays should be used? Gene array Exon array Tiling array

18. 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

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

20. Potential Deletions

21. Deltap0FALSECalledFDR 1.000.951886516014511.2% 1.250.95104771323907.5% 1.500.9565451150425.4% 1.750.9544841023854.2% 2.000.953298920273.4%

22. 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

23. Chip genotyping of a Recombinant Inbred Line 29kb interval Discovery 8 replicates X $500 80,000 SFPs = $0.05 Typing 1 replicate X $500 80,000 SFPs = $0.00625

24. Map bibb 100 bibb mutant plants 100 wt mutant plants

25. Array Mapping Hazen et al Plant Physiology 2005

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

27. 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 (Wolyn et al Genetics 2004)

28. 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

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

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

31. Natural Variation on Tiling Arrays

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

33. Complex, Large Genomes? Signal to Noise with Large Genomes RNA, less complex, but differential expression Barley SFPs

34. RNA 2 genotypes, 18 replicates

35. False Discovery Rate RNA RNA hybridization 17 Golden Promise 19 Morex, 6 tissues SAM Analysis for the Two-Class Unpaired Case Assuming Unequal Variances s0 = 0.0342 (The 5 % quantile of the s values.) Number of permutations: 500 MEAN number of falsely called genes is computed. Deltap0CalledFALSEFDR 0.50.952715958840.206 1.00.95177445940.032 1.50.9513285650.005 2.00.951050470.001 2.50.95858300.000

36. Sequence Verification of SFPs RNAGeneChip mxSFPnonSFPgpSFP Sequence 53012403075203 MX1781154518 Non- polymorphic2200272045128 GP223761155 Chisq = 2049.2, df = 4, p-value = 0

37. Position of SNP

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

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

40. 300 F3 RILs growing (Evadne Smith) 85,000 5’ 3’ ESTs -- 51,000 clones, >16,00 SNPs TIGR gene index and GenBank arrays being designed by Nimblegen Aquilegia (Columbines)

41. Genetics of Speciation along a Hybrid Zone

42. NSF Genome Complexity 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)

43. 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 Ken Okamoto Purdue Ivan Baxter UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones University of Chicago Xu Zhang Evadne Smith Ken Okamoto Purdue Ivan Baxter UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones

44. Barley SFPs Genomic DNA 3 genotypes 3 replicates

45. False Discovery Rate DNA Genomic DNA hybridizaiton 3 replicates 3 genotypes SAM Analysis for the Multi-Class Case with 3 Classes s0 = 0.0123 (The 25 % quantile of the s values.) Number of permutations: 100 MEAN number of falsely called genes is computed. Deltap0CalledFALSEFDR 10.95401720730.47 20.9517285830.31 30.9510902580.22 40.957891390.16 50.95631860.13