1. Genomic Approaches to the Genetics of Adaptation Justin Borevitz Ecology & Evolution University of Chicago http://naturalvariation.org/

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

3. Talk Outline Wild Collections –Local Population Sampling/ structure Seasonal Growth Chambers –KasC/ VanC RILs Whole Genome Tiling Arrays –Alternative splicing –Single Feature Polymorphisms (SFPs) –Potential deletions/ Copy Number Variants Aquilegia Wild Collections –Local Population Sampling/ structure Seasonal Growth Chambers –KasC/ VanC RILs Whole Genome Tiling Arrays –Alternative splicing –Single Feature Polymorphisms (SFPs) –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 SNP, 40plex > 400SNPs available

8. Variation within a field

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 Ecology 15 (5), 1405-1418.

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

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

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 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 Geneva Scientific/ Percival Developmental Plasticity == Behavior

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

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

18. Kas/Col flowering time QTL FRI Chr3 Chr4Chr5

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

20. Environment and Epistasis ABC

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

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

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

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

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

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

27. Transcription subUnits (TUs) Exon1Exon2 Intron1 Tu1Tu2Tu3 ? cDNA1 cDNA2 cDNA3 X

28. v v v c c c v c RT-PCR gDNA PCR Alternative spliced introns Col Van FDR for selection3.5%5% Total introns tested62,051 Total introns43228 Tested introns3272 Confirmed2036 percentage71%56%

29. FDR for selection3% Total exons tested 86,349 Total exons69 Tested exons5 confirmed5 Alternative spliced exons - verification Col Van v v v c c c v c RT-PCR gDNA PCR

30. Potential Deletions

31. Deltap0FALSECalledFDR 1.000.951886516014511.2% 1.250.95104771323907.5% 1.500.9565451150425.4% 1.750.9544841023854.2% 2.000.953298920273.4% SFP detection Genotype effect on tiling arrays IntergenicExonintron SFPs607702351917216 total685575665524301648 %8.86%3.53%5.71% SFPs/gene0>=1>=2>=3>=4>=5 genes1632291464304249516871121

32. 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

33. methylated features and mSFPs >10,000 of 100,000 at 5% FDR Enzyme effect, on CCGG featuresGxE 276 at 15% FDR mQTL?

34. 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

35. Fast Neutron deletions FKF1 80kb deletion CHR1cry2 10kb deletion CHR1 Het Sam Hazen (Scripps)

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

37. 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)

38. 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

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

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

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

42. 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)

43. 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

44. http://www.plosone.org/

45. Aquilegia (Columbines)

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

47. 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