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Talk Outline

High Resolution Patterns of Variation in the Arabidopsis Genome Justin Borevitz Ecology & Evolution University of Chicago naturalvariation.org. Talk Outline. Natural Variation in Light Response Single Feature Polymorphisms (SFPs) Potential deletions Haplotype analysis

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Talk Outline

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  1. High Resolution Patterns of Variationin the Arabidopsis GenomeJustin BorevitzEcology & EvolutionUniversity of Chicagonaturalvariation.org

  2. Talk Outline • Natural Variation in Light Response • Single Feature Polymorphisms (SFPs) • Potential deletions • Haplotype analysis • Patterns in gene Families • Aquilegia • Natural Variation in Light Response • Single Feature Polymorphisms (SFPs) • Potential deletions • Haplotype analysis • Patterns in gene Families • Aquilegia

  3. Light Affects the Entire Plant Life Cycle Light response variation can be seen under constant conditions in the lab Natural Variation under selection? Test in field

  4. Seasons in the Growth Chamber • Changing Day length • Changing Light Intensity/Color • Changing Temperature

  5. What is Array Genotyping? • Affymetrix expression GeneChips contain 202,806 unique 25bp oligo nucleotides. • 11 features per probset for 21546 genes • New array’s have even more • Genomic DNA is randomly labeled with biotin, product ~50bp. • 3 independent biological replicates compared to the reference strain Col GeneChip

  6. Potential Deletions

  7. 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

  8. FLM natural deletion MAF1 Flowering Time QTL caused by a natural deletion in FLM Potential Deletions Suggest Candidate Genes FLOWERING1 QTL Chr1 (bp)

  9. 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

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

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

  12. Sequence confirmation of SFPs

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

  14. Chromosome Wide Diversity

  15. Diversity 50kb windows

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

  17. RPS4 R genes vs bHLH Theta W

  18. RPS4 Rgenes vs bHLH Tajimas’ D

  19. R genes vs bHLH

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

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

  22. Plant lineage: crop plant coverage > 20k dbEST 11/14/2003 Animal lineage: good coverage

  23. NSF Genome Complexity • 35,000 ESTs 5’ and 3’ • 350 arrays, RNA and genotyping • High density SFP Genetic Map • 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 Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus Nordborg (USC) Justin Borevitz (U Chicago) Jeff Tompkins (Clemson)

  24. NaturalVariation.org NaturalVariation.org Salk Jon Werner Sarah Liljegren Huaming Chen Joanne Chory Detlef Weigel Joseph Ecker UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler University of Chicago Xu Zhang Evadne Smith Syngenta Hur-Song Chang Tong Zhu UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones Salk Jon Werner Sarah Liljegren Huaming Chen Joanne Chory Detlef Weigel Joseph Ecker UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler University of Chicago Xu Zhang Evadne Smith Syngenta Hur-Song Chang Tong Zhu UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones

  25. Chip genotyping of a Recombinant Inbred Line 29kb interval Discovery 6 replicates X $500 12,000 SFPs = $0.25 Typing 1 replicate X $500 12,000 SFPs = $0.041

  26. 100 bibb mutant plants Map bibb 100 wt mutant plants

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

  28. 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 Allele frequencies determined by SFP genotyping. Thresholds set by simulations Red light QTL RED2 from 100 Kas/ Col RILs

  29. eXtreme Array Mapping BurC F2

  30. QTLLz x Ler F2 XAMLz x Col F2

  31. eXtreme Array Fine Mapping ~2Mb ~8cM Col Low RED2 QTL >400 SFPs High Kas X mark2 mark1 ~2 ~268 ~43 Kas Col Col Col het Col ~43 ~43 ~539 ~539 Kas het Col het het het het het ~268 ~2 ~43 Kas Kas Kas Kas Col het Select recombinants by PCR >200 from >1250 plants

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