1. Identification and Genotyping of Single Feature Polymorphisms in Complex Genomes Justin Borevitz University of Chicago naturalvariation.org

2. Talk Outline Intro/QTL mapping Single Feature Polymorphisms (SFPs) –Potential deletions Bulk Segregant Mapping –Extreme Array Mapping Transcriptional profiling – for QTL candidate genes

5. Quantitative Trait Loci EPI1 EPI2

7. 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

8. 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

9. Potential Deletions

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

14. 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

15. Effect of SNP position 340 Candidate Polymorphisms False negative True Positive

17. 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

18. LIGHT1 NIL

19. 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

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

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

22. Map bibb 100 bibb mutant plants 100 wt mutant plants

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

24. 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

25. arythmic11 Mapping confirmed Sam Hazen

26. arythmic90 Gene cloned Sam Hazen arythmic21 Allelic to arr90 Sam Hazen

27. stamenstay Ler Sarah Liljegren Mapping confirmed

28. ein6een double mutant Ramlah Nehring Mapping confirmed

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

30. LOD eXtreme Array Mapping Red light QTL RED2 from 100 Kas/ Col RILs Allele frequencies determined by SFP genotyping. Thresholds set by simulations 15 tallest RILs pooled vs 15 shortest RILs pooled 0 4 8 12 16 020406080100 cM LOD Composite Interval Mapping RED2 QTL Chromosome 2 RED2 QTL 12cM

31. Fine Mapping with Arrays Single Additive Gene 1000 F2s Select recombinants by PCR 1Mb region

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

33. Barley SFPs gDNA 9 arrays, random labeled genomic DNA 3 wild type, 3 parent 1, 3 parent 2 Hope to verify some RNA SFPs Pairs plots, correlation matrix SFP table

34. Just better than permutations delta ori.data perm.data difference FDR 0.10 2866 2114.2 751.8 0.74 0.15 1870 578.4 1291.6 0.31 0.20 1274 269.3 1004.7 0.21 0.25 991 174.7 816.3 0.18 0.30 816 126.8 689.2 0.16 0.35 660 95.8 564.2 0.15 0.40 554 75.8 478.2 0.14 Increase specific activity with other labeling methods Perform more replicates

35. Single Feature Polymorphisms –Improve with replicates (easy) –Improved statistical models Genotyping –Precisely define recombination breakpoints –Fine mapping Potential Deletions –Candidate genes/ induced mutations Bulk segregant Mapping –eXtreme Array Mapping, F2s etc

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

39. differences may be due to expression or hybridization

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

41. SFPs from RNA Barley Affy array 22801 probe sets –Most probes sets 11 probes –Background correction “rma2” –Quantile normalization 36 arrays total –3 replicates –6 tissues, leaf, crown, root, radical, gem, col? –2 genotypes (Golden Promise 7,459 ESTs) – (Morex 52,695 ESTs)

42. Look at some plots raw data

43. Remove probe effect

44. Remove tissue effect

45. Remove Genotype effect

46. SAM False Discovery Rate delta ori.data perm.data difference FDR 0.1 13210 1210.34 11999.66 0.091623013 0.2 7903 183.95 7719.05 0.023275971 0.3 5462 49.18 5412.82 0.009004028 0.4 4036 18.31 4017.69 0.004536670 0.5 3024 8.49 3015.51 0.002807540 0.6 2285 3.85 2281.15 0.001684902 Both + and – SFPs since no reference comparison Need to compare with ESTs

47. 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

48. 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

49. 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