1. QTL mapping using Single Feature Polymorphisms Justin Borevitz Salk Institute naturalvariation.org

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

4. 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

5. Potential Deletions

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

10. 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

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

13. 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

14. 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

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

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

17. Map bibb 100 bibb mutant plants 100 wt mutant plants

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

19. 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

20. arythmic11 Mapping confirmed Sam Hazen

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

22. stamenstay Ler Sarah Liljegren Mapping confirmed

23. ein6een double mutant Ramlah Nehring Mapping confirmed

24. 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

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

26. 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, Ler, Bay, Shah, Cvi, Kas, C24, Est, Kin, Mt, Nd, Sorbo, Van, Ws2

27. Pairwise Correlation between and within replicates

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

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

30. Diversity 100kb windows 3 pollen allergen like proteins

31. Tajima’s D 100kb windows

32. Tajima’s D distribution

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

36. differences may be due to expression or hybridization

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

38. 25 bp 16 bp 25 bp 8 bp 1st Set 2nd Set 3rd Set Complete Genome Tiling Chip Polymorphisms (re-sequencing) Global methylation (Methylome) Comparative Genomics (Brassica) New Gene Discovery Improve Annotation Alternative Splicing Micro RNAs 9 Whole-Genome Expression Chips 2 Splicing Chips 2 5’ Mapping Chips Validate features Extra Chips: ChIP – Chip (DNA binding sites)

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

40. True natural variation in gene expression 20 Accessions 3 replicates, (polymorphism accounted for) Cis regulatory variation/ Imprinting reciprocal F1s 3 replicates Transcriptome QTL Map – 100 best VanC Advanced Intercross Lines How many loci control the variation in gene transcription? Candidate TF and binding sites? Future Projects RNA

41. Future Projects Arabidopsis single chip tiling array $300 6.3M unique 25mer features ~17 bp tile both strands. Model based prediction of “good” hybridization intensities Discover/Validate gene models (exon intron) Antisense transcription, Alternative Splicing, Micro RNAs Expect 252,000 SFPs, High Density LD map > 200 Accessions

42. 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

43. 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