1. Genetics and Genomics of Light Response adaptation in Arabidopsis thalianaJustin BorevitzEcology & EvolutionUniversity of Chicagohttp://naturalvariation.org/

2. Widely Distributed Olivier Loudet http://www.inra.fr/qtlat/NaturalVar/NewCollection.htm

3. Talk Outline Talk Outline • Wild Collections • Local Population Sampling/ structure • Seasonal Growth Chambers • KasC/ VanC RILs • Whole Genome SNP/Tiling Arrays • Single Feature Polymorphisms (SFPs) • 5-Methylcytosine, the 5th base • Potential deletions/ Copy Number Variants • Aquilegia • Wild Collections • Local Population Sampling/ structure • Seasonal Growth Chambers • KasC/ VanC RILs • Whole Genome SNP/Tiling Arrays • Single Feature Polymorphisms (SFPs) • 5-Methylcytosine, the 5th base • Potential deletions/ Copy Number Variants • Aquilegia

4. Local Population Variation

5. Local adaptation under strong selection

6. Seasonal Variation Matt Horton Megan Dunning

7. Collections • 807 Lines from 25 Midwest Populations • (Diane Byers IL state) – growing! • 1101 Lines from UK, 51 populations • (Eric Holub Warwick, UK) – growing! • > 500 lines N and S Sweden (Nordborg) • > 400 Lines France and Midwest (Bergelson) • 400 lines Midwest (Borevitz) • 857 Accessions stock center (Randy Scholl) • Others welcome… Will be genotyped with Sequenom 149 SNPs $0.03 per

8. Variation within a field (web link)

9. Within and Between Variation • BAKKER, E. G., STAHL, E. A., TOOMAJIAN, C., NORDBORG, M., KREITMAN, M. & BERGELSON, J.Distribution of genetic variation within and among local populations of Arabidopsis thaliana over its species range.Molecular Ecology15 (5), 1405-1418.

10. Local Population Structure in the Midwest 48 Non singleton SNPs of 87 tested Megan Dunning, poster #268

11. Global Population Structure 120 SNPs of 149 tested including inbred lines Norman Warthmann`

12. Regional/Seasonal Variation • What is Local Adaptation? • Predictable Seasonal changes unique to each location. Tossa Del Mar Spain Lund Sweden

13. Seasons in the Growth Chamber Seasons in the Growth Chamber Developmental Plasticity == Behavior Developmental Plasticity == Behavior Sweden Spain • Changing Day length • Cycle Light Intensity • Cycle Light Colors • Cycle Temperature • Changing Day length • Cycle Light Intensity • Cycle Light Colors • Cycle Temperature Geneva Scientific/ Percival

14. Solar Calc II • Kurt Spokas • Version 2.0a June 2006 • USDA-ARS Website Midwest Area (Morris,MN) • http://www.ars.usda.gov/mwa/ncscrl

15. May 1-3rd

16. Genetic map of the Kas-1 x Col-gl1 RIL population I II III IV V 55 markers from Wolyn et al. (2004) & 64 additional SNP markers.

17. Distribution of flowering time among 96 Kas-1/Col-gl1 RILs Col-gl1 Col-gl1 Number of RILs Sweden 2 Sweden 1 Kas1 Kas1 Col-gl1 Col-gl1 Spain 1 Spain 2 Number of RILs Kas1 Kas1

18. Kas/Col flowering time QTL FRI Chr4 Chr5 Chr3

19. Kas/Col flowering time QTL GxE Chr4 FRI Chr1 FLM Chr4 FRI

20. A B C Environment and Epistasis

21. FLC Total Leaf Number Van no mitochondrial insertion 768 VanC AIL-RILs 149 + 87 SNPs Stock Center Release (Evadne Smith) 768 VanC AIL-RILs 149 + 87 SNPs Stock Center Release (Evadne Smith)

22. Universal Whole Genome Array DNA RNA Gene/Exon Discovery Gene model correction Non-coding/ micro-RNA Chromatin Immunoprecipitation ChIP chip Alternative Splicing Methylation Antisense transcription Polymorphism SFPs Discovery/Genotyping Transcriptome Atlas Expression levels Tissues specificity Comparative Genome Hybridization (CGH) Insertion/Deletions Copy Number Polymorphisms RNA Immunoprecipitation RIP chip Allele Specific Expression Control for hybridization/genetic polymorphisms to understand TRUE expression variation

23. Which arrays should be used? BAC array cDNA array Long oligo array

24. Which arrays should be used? Gene array Exon array Tiling array 35bp tile, 25mers 10bp gaps

25. Which arrays should be used? SNP array How about multiple species? Microbial communities? Ressequencing array Tiling/SNP array 2007 250k SNPs, 1.6M tiling probes

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

27. Potential Deletions

28. SFP detectionGenotypeeffect on tiling arrays Delta p0 FALSE Called FDR 1.00 0.95 18865 160145 11.2% 1.25 0.95 10477 132390 7.5% 1.50 0.95 6545 115042 5.4% 1.75 0.95 4484 102385 4.2% 2.00 0.95 3298 92027 3.4%

29. Chip genotyping of a Recombinant Inbred Line 29kb interval

30. Methods for labeling • Extract genomic 100ng DNA (single leaf) • Digest with either msp1 or hpa2 CCGG • Label with biotin random primers • Hybridize to array • Fit model Intensity(feature) ~ additive + dominance + maternal + enzyme + add:enz + dom:enz + mat:enz

31. SFPs and mSFPs

32. Genic and Intergenic Composition

33. EpiTyper CmG Col Col Col Van Van Van Col♂ x Van♀ Col♂ x Van♀ Van ♂ x Col ♀ Van ♂ x Col ♀ Van ♂ x Col ♀ CC*GG CC*GG CC*GG histidine kinase (AHK3) exon9

34. EpiTyper CmG Col Col Col Van Van Van Col♂ x Van♀ Col♂ x Van ♀ Van♂ x Col♀ Van ♂ x Col ♀ Van♂ x Col ♀ CC*GG chromomethylase 2 (CMT2) exon19 CC*GG mQTL?

35. Copy Number Variation (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

36. Het Fast Neutron deletions FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1 Sam Hazen (Scripps)

37. Natural Copy Variation on Tiling Arrays Segregating self seed from wild ME isolate (Early – Late)

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

39. Experimental Design of Association Study • Sample > 3000 wild strains, 149 SNPs • Select 3*384 less structured reference fine mapping set for SFP resequencing • Scan Genome for variation/selection • Measure phenotype in Seasonal Chambers • Haplotype map/ LD recombination blocks • Associate Quantitative phenotypes with HapMap

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

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

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

43. Genetics of Speciationalong a Hybrid Zone

44. NSF Genome Complexity • Microarray development • QTL candidates • 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 • VIGS • Whole Genome Sequencing (JGI?) Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus Nordborg (USC) Justin Borevitz (U Chicago) Jeff Tompkins (Clemson)

45. NaturalVariation.org NaturalVariation.org USC Magnus Nordborg Paul Marjoram Max Planck Detlef Weigel Scripps Sam Hazen University of Michigan Sebastian Zoellner USC Magnus Nordborg Paul Marjoram Max Planck Detlef Weigel Scripps Sam Hazen University of Michigan Sebastian Zoellner University of Chicago Xu Zhang Yan Li Peter Roycewicz Evadne Smith Michigan State Shinhan Shiu Purdue Ivan Baxter University of Chicago Xu Zhang Yan Li Peter Roycewicz Evadne Smith Michigan State Shinhan Shiu Purdue Ivan Baxter

46. http://www.plosone.org/

47. Aquilegia (Columbines)

48. Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus Nordborg (USC) Justin Borevitz (U Chicago) Jeff Tompkins (Clemson)

49. 300 F4 RILs growing (Evadne Smith) • TIGR gene index 85,000 ESTs >16,00 SNPs • Complete BAC physical map Clemson • Nimblegen arrays of 5 floral whorls Whole Genome Shotgun Sequencing 2007 JGI