1. Haplotype mapping with Single Feature Polymorphisms in Arabidopsis Justin Borevitz Ecology & Evolution University of Chicago http://naturalvariation.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 Cycle Light Intensity Cycle Light Colors Cycle Temperature

5. Which arrays should be used? Spotted arrays Arizona 29,000 - 70mers ATH1, Affymetrix expression GeneChip 202,806 unique 25bp oligo nucleotides features AtTILE1, universal whole genome array every ~35bp, > 3Million PM features Re-sequencing array 120M*8bp –20 Accessions, Perlegen, –Max Planck (Weigel), USC (Nordborg) GeneChip

6. 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 ~35 bp tile,non-repetitive regions, “good” binding oligos,evenly spaced

7. ChipViewer: Mapping of transcriptional units of ORFeome From 2000v At1g09750 (MIPS) to the latest AGI At1g09750 2000 v Annotation (MIPS) The latest AGI Annotation

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

9. Potential Deletions

11. False Discovery and Sensitivity PM only SAM threshold 5% FDR GeneChip SFPs nonSFPs Cereon marker accuracy 3806 89118 100% Sequence 817 121 696 Sensitivity Polymorphic 340 117 223 34% Non-polymorphic 477 4 473 False Discovery rate: 3% Test for independence of all factors: Chisq = 177.34, df = 1, p-value = 1.845e-40 SAM threshold 18% FDR GeneChip SFPs nonSFPs Cereon marker accuracy 10627 82297 100% Sequence 817 223 594 Sensitivity Polymorphic 340 195 145 57% Non-polymorphic 477 28 449 False Discovery rate: 13% Test for independence of all factors: Chisq = 265.13, df = 1, p-value = 1.309e-59 3/4 Cvi markers were also confirmed in PHYB 90%80%70% 41%53%85% 90%80%70% 67%85%100% Cereon may be a sequencing Error TIGR match is a match

12. 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

13. Map bibb 100 bibb mutant plants 100 wt mutant plants

14. bibb mapping ChipMap AS1 Bulk segregant Mapping using Chip hybridization bibb maps to Chromosome2 near ASYMETRIC LEAVES1

15. BIBB = ASYMETRIC LEAVES1 Sequenced AS1 coding region from bib-1 …found g -> a change that would introduce a stop codon in the MYB domain bibbas1-101 MYB bib-1 W49* as-101 Q107* as1 bibb AS1 (ASYMMETRIC LEAVES1) = MYB closely related to PHANTASTICA located at 64cM

16. Array Mapping Hazen et al Plant Physiology (submitted) chr1 chr2 chr3 chr4 chr5

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

18. 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 (Wolyn et al Genetics 2004)

19. eXtreme Array Mapping BurC F2

20. XAM Lz x Col F2 QTL Lz x Ler F2 (Werner et al Genetics in press)

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

22. 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

23. Potential Deletions Suggest Candidate Genes FLOWERING1 QTL Chr1 (bp) Flowering Time QTL caused by a natural deletion in FLM MAF1 FLM natural deletion (Werner et al PNAS 2005)

24. Fast Neutron deletions FKF1 80kb deletion CHR1cry2 10kb deletion CHR1 Het

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 Theta W RPS4

34. Rgenes vs bHLH Tajimas’ D RPS4

35. R genes vs bHLH

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

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

38. > 20k dbEST 11/14/2003 Animal lineage: good coverage Plant lineage: crop plant coverage

39. NSF Genome Complexity 45,000 ESTs 5’ and 3’ ends 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)

40. NaturalVariation.org Salk Jon Werner Joanne Chory Joseph Ecker Max Planck Detlef Weigel UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler Salk Jon Werner Joanne Chory Joseph Ecker Max Planck Detlef Weigel UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler University of Chicago Xu Zhang Evadne Smith UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones University of Chicago Xu Zhang Evadne Smith UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones