1. Natural Variation in Arabidopsis thaliana Light Response: Genomic Approaches Justin Borevitz Salk Institute naturalvariation.org

2. Talk Outline Survey of light response in Arabidopsis thaliana accessions QTL mapping of light response in Ler/Cvi Recombinant Inbred Lines Transcriptional profiling for QTL candidate genes Analysis of Single Feature Polymorphisms (SFPs)

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

7. My only collection

13. Quantitative Trait Loci

15. Epistasis scan Chr1 Chr2 Chr3 Chr4 Chr5 BQTL http://hacuna.ucsd.edu/bqtl 43,956 pair-wise tests 163 markers and 133 intervals Permutation threshold p < 0.05 (5000 permutations)

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

20. Review Natural Variation Light response variation is correlated with latitude Functional variation in Photoreceptors PHYA, PHYB, CRY2 (El-Din El-Assal et al 2001) New QTL identified in common and unique photoreceptor pathways QTL may be under selection?

24. differences may be due to expression or hybridization

26. Potential Deletions

28. 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 90%80%70% 41%53%85% 90%80%70% 67%85%100% Cereon may be a sequencing Error TIGR match is a match

29. Chip genotyping of a Recombinant Inbred Line 29kb interval

30. bibb mutant phenotypes Colbib-1det2-1 23 days bib-3 three independent recessive alleles medial sepals remain attached “cabbage-like” rosette leaves flowers open prematurely fruit appear more slender bib-3 Ler bib-3

31. Map bibb 100 bibb mutant plants 100 wt mutant plants

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

33. BIBB=AS1 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

34. Potential Deletions 111 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

37. Gene expression revisited FLC controls flowering time Difference detected at 3 day old seedling stage Gene Expression index that accounts for feature effect and polymorphisms ~100 genes @ 20% FDR

38. Gene expression revisited PAG1 down regulated in Cvi PLALE GREEN1 knock out

39. pag1 KO is light insensitive pag1 KO has long hypocotyl in red light

40. Review Transcriptional profiling to identify candidate genes and downstream responses Single Feature Polymorphisms (SFPs) can be used for identifying recombination breakpoints, bulk segregant mapping, and haplotyping Consider polymorphisms for expression analysis

41. 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)

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

43. NaturalVariation.org Syngenta Hur-Song Chang Tong Zhu Syngenta Hur-Song Chang Tong Zhu Salk Jon Werner Todd Mockler Sarah Liljegren Joanne Chory Detlef Weigel Joseph Ecker UC Davis Julin Maloof UC San Diego Charles Berry Scripps Elizabeth Winzeler Salk Jon Werner Todd Mockler Sarah Liljegren Joanne Chory Detlef Weigel Joseph Ecker UC Davis Julin Maloof UC San Diego Charles Berry Scripps Elizabeth Winzeler