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

2. Arabidopsis thaliana Genome Sequence 2000 (120Mb), 20 strains by Perlegen, Weigel, Nordborg, Ecker ~1% sequence variation, ~3000 collected lines A. lyrata, Capsella rubella sister species JGI 2006 >5300 Research Labs (17 th annual conference) Fields study data to come, Annie Schmidt et al 340k Sequence Indexed collection of KO lines Gene Expression Atlas >300 tissues, time points 15,000 full length cDNAs in recombination clones

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

4. Aranzana, et al PLOS genetics (2005), Sung Kim, Keyan Zhao

5. Local Population Variation Scott Hodges Ivan Baxter

6. Seasonal Variation Matt Horton Megan Dunning Developmental Plasticity == Behavior

7. 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

8. Talk Outline Single Feature Polymorphisms (SFPs) –Methylation –Potential deletions –Genetic Mapping Resequencing/ Haplotypes Variation Scanning Single Feature Polymorphisms (SFPs) –Methylation –Potential deletions –Genetic Mapping Resequencing/ Haplotypes Variation Scanning

9. Tiling Arrays vs Resequencing Arrays AtTILE1, universal whole genome array 25mer every ~35bp, > 6.5 Million features single array, many individuals. Re-sequencing array 120Mbp*8features ~1 Billion features, 8 wafers 20 Accessions available mid year Perlegen, Max Planck (Weigel), USC (Nordborg), Salk (Ecker) GeneChip

10. 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 Control for hybridization/genetic polymorphisms to understand true EXPRESSION polymorphisms True cis variation == Allele Specific Expression

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

12. Potential Deletions

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

14. 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

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

16. SFP Resequencing Advantages –Discovery and typing tool –Indels, rare variants, HMM tool –Quantitative score –Good for low polymorphism < 1% Caveats –No SNP knowledge, synonymous? –Bad for high polymorphism > 1% Rearrangements, Reference sequence

17. Natural Variation on Tiling Arrays

18. 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

19. 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)

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

21. Map bibb 100 bibb mutant plants 100 wt mutant plants

22. Array Mapping Hazen et al Plant Physiology 2005

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

24. 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 Drosophila, Chao-Qiang Lai -Tufts University

25. 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

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

27. Distribution of T-stats null (permutation) actual Not ColColNANA duplications 32,427 Calls 208,729 12,250 SFPs

28. Sequence confirmation of SFPs SFPSNPTotalFPRFDRSensitivity bay446113750.8%25.0%54.1% bur475713201.1%29.8%57.9% cvi699213251.2%21.7%58.7% ler415114660.6%22.0%62.7% lz374014410.5%18.9%75.0% mr678711911.1%17.9%63.2% mt464814130.9%26.1%70.8% sorbo375313170.9%29.7%49.1% ws294713690.3%13.8%53.2%

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

30. Chromosome Wide Diversity

31. Diversity 50kb windows

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

33. R genes vs bHLH

34. Experimental Design of Association Study Sample > 2000 wild strains, ~50-100 SNPs Select 384 unstructured reference fine mapping set SFP resequencing of 384 lines Haplotype map/ LD recombination blocks Scan Genome for variation/selection Measure phenotype in Seasonal Chambers Associate Quantitative phenotypes with HapMap

35. Review Single Feature Polymorphisms (SFPs) can be used to Methylation Variable sites Potential deletions (candidate genes) eXtreme Array Mapping Haplotyping Diversity/Selection Association Mapping

36. NaturalVariation.org USC Magnus Nordborg Paul Marjoram Max Planck Detlef Weigel Scripps Sam Hazen University of Michigan Sebastian Zollner University of Chicago Xu Zhang Evadne Smith Ken Okamoto Michigan State Shinhan Shui Purdue Ivan Baxter University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones University of Chicago Xu Zhang Evadne Smith Ken Okamoto Michigan State Shinhan Shui Purdue Ivan Baxter University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones USC Magnus Nordborg Paul Marjoram Max Planck Detlef Weigel Scripps Sam Hazen University of Michigan Sebastian Zollner