Studies of Genome Wide Molecular Variation in Arabidopsis thaliana using Arrays Justin Borevitz Salk Institute naturalvariation.org

Talk Outline Natural Variation in Light Response Transcriptional profiling – for QTL candidate genes Single Feature Polymorphisms (SFPs) –Potential deletions Bulk Segregant Mapping –Extreme Array Mapping Haplotype analysis

Natural Mutation in PHYA photoreceptor

Quantitative Trait Loci

differences may be due to expression or hybridization

Feature level model FLC controls flowering time Difference detected it 3 day old seedlings Gene Expression index that accounts for feature effect and polymorphisms

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

Potential Deletions

False Discovery and Sensitivity Permuted data real data 5% FDR 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 Observed t statistics vs Null (permuted) t statistics

Chip genotyping of a Recombinant Inbred Line 29kb interval

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

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

Fast Neutron deletions FKF1 80kb deletion CHR1cry2 10kb deletion CHR1

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 = 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

LOD eXtreme Array Mapping Red light QTL RED2 from 100 Kas/ Col RILs QTL likelihood model using bulk segregant analysis with SFP genotyping cM LOD Composite Interval Mapping RED2 QTL Chromosome 2 Chromosome 1 Chromosome 4 15 tallest RILs pooled vs 15 shortest RILs pooled

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

Review Transcriptional profiling to identify candidate genes and downstream responses that consider polymorphisms Single Feature Polymorphisms (SFPs) can be used to identify recombination breakpoints, potential deletions, for bulk segregant mapping, and haplotyping

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 University of Guelph, Canada Dave Wolyn University of Guelph, Canada Dave Wolyn