Natural Variation in Light Response using Whole Genome Tiling Arrays Justin Borevitz Ecology & Evolution University of Chicago

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

Light Affects the Entire Plant Life Cycle Light response variation can be seen under constant conditions in the lab

Quantitative Trait Loci

differences may be due to expression or hybridization

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

Local Population Variation

Seasons in the Growth Chamber Changing Day length Cycle Light Intensity Cycle Light Colors Cycle Temperature Sweden Spain Seasons in the Growth Chamber Changing Day length Cycle Light Intensity Cycle Light Colors Cycle Temperature

Talk Outline Single Feature Polymorphisms (SFPs) –Potential deletions –Bulk segregant/ eXtreme Mapping Haplotype analysis Aquilegia Single Feature Polymorphisms (SFPs) –Potential deletions –Bulk segregant/ eXtreme Mapping Haplotype analysis Aquilegia

Which arrays should be used? Spotted arrays Arizona 29, mers ATH1, Affymetrix expression GeneChip 202,806 unique 25bp oligo nucleotides features AtTILE1, universal whole genome array every ~35bp, > 3Million PM features Re-sequencing array 120M*8bp –20 Accessions, Perlegen, –Max Planck (Weigel), USC (Nordborg) GeneChip

RNADNA Universal Whole Genome Array Transcriptome Atlas Expression levels Tissues specificity Transcriptome Atlas Expression levels Tissues specificity Gene Discovery Gene model correction Non-coding/ micro-RNA Antisense transcription Gene Discovery Gene model correction Non-coding/ micro-RNA Antisense transcription Alternative Splicing Comparative Genome Hybridization (CGH) Insertion/Deletions Comparative Genome Hybridization (CGH) Insertion/Deletions Methylation Chromatin Immunoprecipitation ChIP chip Chromatin Immunoprecipitation ChIP chip Polymorphism SFPs Discovery/Genotyping Polymorphism SFPs Discovery/Genotyping ~35 bp tile,non-repetitive regions, “good” binding oligos,evenly spaced

SNP SFP MMMMMM MMMMMM Chromosome (bp) conservation SNP ORFa start AAAAA Transcriptome Atlas ORFb deletion Improved Genome Annotation

Potential Deletions

Deltap0FALSECalledFDR % % % % % SFP detection on tiling arrays

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

Map bibb 100 bibb mutant plants 100 wt mutant plants

Array Mapping Hazen et al Plant Physiology 2005

LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms Hazen et al PNAS, 2005 Cloned with Array Mapping

eXtreme Array Mapping 15 tallest RILs pooled vs 15 shortest RILs pooled

LOD eXtreme Array Mapping Allele frequencies determined by SFP genotyping. Thresholds set by simulations cM LOD Composite Interval Mapping RED2 QTL Chromosome 2 RED2 QTL 12cM Red light QTL RED2 from 100 Kas/ Col RILs (Wolyn et al Genetics 2004)

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

Potential Deletions Suggest Candidate Genes FLOWERING1 QTL Chr1 (bp) Flowering Time QTL caused by a natural deletion in FLM MAF1 FLM natural deletion (Werner et al PNAS 2005)

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

Natural Variation on Tiling Arrays

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, Bur, Ler, Bay, Shah, Cvi, Kas, C24, Est, Kin, Mt, Nd, Sorbo, Van, Ws2 Fl-1, Ita-0, Mr-0, St-0, Sah-0

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

Chromosome Wide Diversity

Diversity 50kb windows

Tajima’s D like 50kb windows RPS4 unknown

R genes vs bHLH

Review Single Feature Polymorphisms (SFPs) can be used to Identify recombination breakpoints eXtreme Array Mapping Potential deletions (candidate genes) Haplotyping Diversity/Selection Association Mapping

Aquilegia (Columbines) Recent adaptive radiation, 350Mb genome

Species with > 20k ESTs 11/14/2003 Animal lineage: good coverage Plant lineage: crop plant coverage

300 F3 RILs growing (Evadne Smith) 85,000 5’ 3’ ESTs -- 51,000 clones, >3500 SNPs TIGR gene index and GenBank arrays being designed by Nimblegen Aquilegia (Columbines)

Genetics of Speciation along a Hybrid Zone

NSF Genome Complexity Physical Map (BAC tiling path) –Physical assignment of ESTs QTL for pollinator preference –~400 RILs, map abiotic stress –QTL fine mapping/ LD mapping Develop transformation techniques Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus Nordborg (USC) Justin Borevitz (U Chicago) Jeff Tompkins (Clemson)

NaturalVariation.org Salk Jon Werner Joanne Chory Joseph Ecker Max Planck Detlef Weigel UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler Salk Jon Werner Joanne Chory Joseph Ecker Max Planck Detlef Weigel UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler University of Chicago Xu Zhang Evadne Smith Ken Okamoto Purdue Ivan Baxter UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones University of Chicago Xu Zhang Evadne Smith Ken Okamoto Purdue Ivan Baxter UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones