1. Genomic Methods for Cloning QTL Justin Borevitz University of Chicago http:// naturalvariation.org

2. Talk Outline Survey of light response in Arabidopsis thaliana accessions QTL mapping of light response in Ler/Cvi Recombinant Inbred Lines Sequence variation at a PHYTOCHROME B Extent of Linkage Disequilibrium and Association Mapping

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

6. Light Affects the Entire Plant Life Cycle Light response variation can be seen under constant conditions in the lab Natural Variation != Natural Selection

7. My only collection

13. Future work with Natural Variation VanC advanced intercross RIL population Backcross collections

16. Quantitative Trait Loci

17. Epistasis scan Chr1 Chr2 Chr3 Chr4 Chr5

18. 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

19. Cloning QTL Recombination Fine Mapping Gene Expression Variation Hybridization Polymorphism Association Testing, LD mapping Direct Sequencing of Candidate Gene Quantitative Complementation Transgenic Complementation

21. 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

22. LIGHT1 NIL

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

28. differences may be due to expression or hybridization

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

31. replacement synonymous Between Species =Within Species = McDonald-Kreitman p = 0.027 23 62 8 58 = 0.23 KaKsKaKs

34. Review Light response variation is correlated with latitude Functional variation in Photoreceptors PHYA, PHYB, CRY2 New loci identified in common and unique photoreceptor pathways QTL may be under selection?

35. 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

36. Potential Deletions

38. >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

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

40. Review Single Feature Polymorphisms (SFPs) can be used to identify recombination breakpoints, potential deletions, for eXtreme Array mapping, and haplotyping Expression analysis to identify QTL candidate genes and downstream responses that consider polymorphisms

41. NaturalVariation.org Syngenta Hur-Song Chang Tong Zhu Syngenta Hur-Song Chang Tong Zhu University of Guelph, Canada Dave Wolyn University of Guelph, Canada Dave Wolyn Salk Jon Werner Todd Mockler Sarah Liljegren Ramlah Nehring Joanne Chory Detlef Weigel Joseph Ecker UC Davis Julin Maloof UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler NaturalVariation.org Salk Jon Werner Todd Mockler Sarah Liljegren Ramlah Nehring Joanne Chory Detlef Weigel Joseph Ecker UC Davis Julin Maloof UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler