Microarrays for mapping and expression analysis: Toward the genetic determinants of light response adaptation in Arabidopsis and Aquilegia Justin Borevitz Ecology & Evolution University of Chicago naturalvariation.org

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

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

Local Population Variation

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

What is Array Genotyping? Affymetrix tiling array GeneChips contain ~35bp spacing, 1.67million unique features Genomic DNA is randomly labeled with biotin dCTP, product ~50bp. 3 independent biological replicates compared to the reference strain Col GeneChip

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 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

Chip genotyping of a Recombinant Inbred Line 29kb interval Discovery 6 replicates X $ ,000 SFPs = $0.025 Typing 1 replicate X $ ,000 SFPs = $0.0041

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 (Werner et al, Genetics 2005) MAF1 FLM natural deletion

Fast Neutron deletions FKF1 80kb deletion CHR1cry2 10kb deletion CHR1 Het Hazen et al Plant Physiology 2005

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 Wolyn et al Genetics 2004

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

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

SFPs for reverse genetics 14 Accessions 30,950 SFPs`

Chromosome Wide Diversity

Diversity 50kb windows

Tajima’s D like 50kb windows

R genes vs bHLH

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

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

cDNA raw intensity 10% smoothed

Aquilegia (Columbines) Recent adaptive radiation, 350Mb genome

300 F3 RILs growing (Evadne Smith) >50,000 ESTs TIGR gene index and GenBank, arrays being designed by Nimblegen Aquilegia (Columbines)

Genetics of Speciation along a Hybrid Zone

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

NSF Genome Complexity 52,000 ESTs 5’ and 3’ –>9k contigs, 4k singletons –>500 SNPs 350 arrays, RNA and genotyping –High density SFP Genetic Map 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)

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

differences may be due to expression or hybridization

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