1. Array Genotyping to Dissect Quantitative Trait Loci in Arabidopsis thaliana Justin Borevitz Ecology and Evolution University of Chicago http:// naturalvariation.org If you haven’t voted, leave now, I’ll/We’ll forgive you

3. Talk Outline QTL Intro Transcription based Cloning Single Feature Polymorphisms (SFPs) –Potential deletions Bulk Segregant Mapping –Extreme Array Mapping Haplotype analysis New Arrays, new models Aquilegia

4. Light Affects the Entire Plant Life Cycle de-etiolation hypocotyl }

7. Quantitative Trait Loci

8. QTL gene Confirmation Marker Identification Genotyping Genomics path Experimental Design Mapping population Phenotyping QTL Analysis Fine Mapping Candidate gene Polymorphisms gene expression loss of function QTL gene Confirmation Experimental Design Mapping population Phenotyping QTL Analysis Fine Mapping With the Aid of Genomics

9. Genomics to Clone QTL Recombination Fine Mapping Gene Expression Variation Hybridization Polymorphism Association Testing, LD mapping Direct Sequencing of Candidate Gene Quantitative Complementation Transgenic Complementation

11. Look for gene expression differences between genotypes Identify candidate genes that map to mutation Downstream targets that map elsewhere Transcription based cloning

15. differences may be due to expression or hybridization

16. PAG1 down regulated in Cvi PLALE GREEN1 knock out has long hypocotyl in red light

17. 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

18. Potential Deletions

19. Spatial Correction Spatial Artifacts Improved reproducibility Next: Quantile Normalization

21. 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

22. 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

23. SNP377 SM184 SM50 SM35 SM106 G2395 SNP65 SM40 SEQ8298 TH1 MSAT7964 MAT7787 CER45 5.50 5.87 6.34 7.01 7.30 7.44 7.60 7.79 7.96 8.13 8.29 8.65 9.32 MbMarker Near-Isogenic Lines for LIGHT1 Ler / Cvi #3 mm 81N-J17A-A/J114124189Ler 624333Plants Line RVE7 GI 194 3 5.0 5.8 5.15.95.7 5.8 Phenotype

24. LIGHT1 NIL

25. 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

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

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

28. Map bibb 100 bibb mutant plants 100 wt mutant plants

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

30. 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

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

32. 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

33. eXtreme Array Mapping BurC F2

34. XAM Lz x Col F2 QTL Lz x Ler F2

35. 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

36. 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

37. A star phylogeny 163 markers 73 accessions ~ 750kb/marker

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

39. 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

40. Transcriptome Viewer: http://signal.salk.edu

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

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

43. Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus Nordborg (USC) Justin Borevitz (U Chicago) Jeff Tompkins (Clemson) NSF Genomics of Adaptation to the Biotic and Abiotic Environment in Aquilegia

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

45. NSF Genomics of Adaptation to the Biotic and Abiotic Environment in Aquilegia 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 –and abiotic stress –QTL fine mapping/ LD mapping Develop transformation techniques

46. NaturalVariation.org Salk Jon Werner Sarah Liljegren Huaming Chen Joanne Chory Detlef Weigel Joseph Ecker UC San Diego Charles Berry Scripps Sam Hazen Steve Kay 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 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 Steve Kay Elizabeth Winzeler