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

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)

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

My only collection

Quantitative Trait Loci

Epistasis scan Chr1 Chr2 Chr3 Chr4 Chr5 BQTL 43,956 pair-wise tests 163 markers and 133 intervals Permutation threshold p < 0.05 (5000 permutations)

replacement synonymous Between Species =Within Species = McDonald-Kreitman p = = 0.23 KaKsKaKs

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?

differences may be due to expression or hybridization

Potential Deletions

False Discovery and Sensitivity PM only SAM threshold 5% FDR GeneChip SFPs nonSFPs Cereon marker accuracy % Sequence Sensitivity Polymorphic % Non-polymorphic False Discovery rate: 3% Test for independence of all factors: Chisq = , df = 1, p- value = 1.845e -40 SAM threshold 18% FDR GeneChip SFPs nonSFPs Cereon marker accuracy % Sequence Sensitivity Polymorphic % Non-polymorphic False Discovery rate: 13% Test for independence of all factors: Chisq = , 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

Chip genotyping of a Recombinant Inbred Line 29kb interval

bibb mutant phenotypes Colbib-1det 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

Map bibb 100 bibb mutant plants 100 wt mutant plants

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

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

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

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 20% FDR

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

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

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

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)

ChipViewer: Mapping of transcriptional units of ORFeome From 2000v At1g09750 (MIPS) to the latest AGI At1g v Annotation (MIPS) The latest AGI Annotation

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