Arabidopsis thaliana

1. Toward the genetic basis of adaptation using arraysJustin BorevitzEcology & EvolutionUniversity of Chicagohttp://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 (17th 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 Olivier Loudet http://www.inra.fr/qtlat/NaturalVar/NewCollection.htm

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

5. Local Population Variation Ivan Baxter Scott Hodges

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

7. Seasons in the Growth Chamber Seasons in the Growth Chamber Sweden Spain • Changing Day length • Cycle Light Intensity • Cycle Light Colors • Cycle Temperature • 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. 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 Control for hybridization/genetic polymorphisms to understand true EXPRESSION polymorphisms True cis variation == Allele Specific Expression

11. 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)

12. Potential Deletions

13. SFP detection 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%

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 Enzyme effect, on CCGG features GxE mQTL? >10,000 of 100,000 at 5% FDR 276 at 15% FDR

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. 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)

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

21. 100 bibb mutant plants Map bibb 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. 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 Drosophila, Chao-Qiang Lai -Tufts University Allele frequencies determined by SFP genotyping. Thresholds set by simulations Red light QTL RED2 from 100 Kas/ Col RILs

25. 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

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

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

28. Sequence confirmation of SFPs

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

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 NaturalVariation.org 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 USC Magnus Nordborg Paul Marjoram Max Planck Detlef Weigel Scripps Sam Hazen University of Michigan Sebastian Zollner