Genomics tools to identify the molecular basis of complex traits Justin Borevitz Salk Institute naturalvariation.org

Talk Outline Quick Natural Variation review Transcription profiling to identify QTL candidate genes Identification of Single Feature Polymorphisms Future Prospects

PHYB

Genes Expression for a candidate gene Features (probes) for At1g22360 Spatially corrected log PM intensity Gene Expression index that accounts for feature differences and polymorphisms

Deletions

Spatial Correction Spatial Artifacts Improved reproducibility

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 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

Effect of SNP position 340 Candidate Polymorphisms False negative True Positive

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

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

Future Bulk segregant Mapping Extend to Quantitative Traits Map multiple genes (mutation modifiers) Fine mapping after PCR identification of recombinants Multiple models (epistasis) Changes in allele F2, F3 frequency (selection) 20% variance 20 simulations

25 bp 16 bp 25 bp 8 bp 1st Set 2nd Set 3rd Set Complete Genome Tilling 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

Future Projects Design 2 nd generation expression array $ ,000 25mer features, expect 12,000 SFPs (2 accessions) Validated gene models (exon intron) 2 features per exon, alternative splicing 5’ 3’ Untranslated region for gene family/ polymorphism Micro RNAs Validated “good” hybridization intensities

Haplotype Map – 20 Accessions 3 replicates, SFP discovery estimated 85,000 SFPs 1.4kb resolution Association Studies – 120 Accessions 1 replicate, Genotyping Bulk Segregant Mapping – Confirm Associations in specific crosses Future Projects DNA

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

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