High Resolution Patterns of Variation in the Arabidopsis Genome Justin Borevitz Ecology & Evolution University of Chicago naturalvariation.org

Talk Outline Natural Variation in Light Response Single Feature Polymorphisms (SFPs) –Potential deletions Haplotype analysis Patterns in gene Families Aquilegia Natural Variation in Light Response Single Feature Polymorphisms (SFPs) –Potential deletions Haplotype analysis Patterns in gene Families Aquilegia

Light Affects the Entire Plant Life Cycle Light response variation can be seen under constant conditions in the lab Natural Variation under selection? Test in field

Seasons in the Growth Chamber Changing Day length Changing Light Intensity/Color Changing Temperature

What is Array Genotyping? Affymetrix expression GeneChips contain 202,806 unique 25bp oligo nucleotides. 11 features per probset for genes New array’s have even more Genomic DNA is randomly labeled with biotin, product ~50bp. 3 independent biological replicates compared to the reference strain Col GeneChip

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

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

Distribution of T-stats null (permutation) actual Not ColColNANA duplications 32,427 Calls 208,729 12,250 SFPs

Sequence confirmation of SFPs

SFPs for reverse genetics 14 Accessions 30,950 SFPs`

Chromosome Wide Diversity

Diversity 50kb windows

Tajima’s D like 50kb windows RPS4 unknown

R genes vs bHLH Theta W RPS4

Rgenes vs bHLH Tajimas’ D RPS4

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

Aquilegia (Columbines) Recent adaptive radiation, 350Mb genome

> 20k dbEST 11/14/2003 Animal lineage: good coverage Plant lineage: crop plant coverage

NSF Genome Complexity 35,000 ESTs 5’ and 3’ 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)

NaturalVariation.org Salk Jon Werner Sarah Liljegren Huaming Chen Joanne Chory Detlef Weigel Joseph Ecker UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler Salk Jon Werner Sarah Liljegren Huaming Chen Joanne Chory Detlef Weigel Joseph Ecker UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler University of Chicago Xu Zhang Evadne Smith Syngenta Hur-Song Chang Tong Zhu UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones University of Chicago Xu Zhang Evadne Smith Syngenta Hur-Song Chang Tong Zhu UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones

Chip genotyping of a Recombinant Inbred Line 29kb interval Discovery 6 replicates X $500 12,000 SFPs = $0.25 Typing 1 replicate X $500 12,000 SFPs = $0.041

Map bibb 100 bibb mutant plants 100 wt mutant plants

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

eXtreme Array Mapping BurC F2

XAM Lz x Col F2 QTL Lz x Ler F2

X RED2 QTL mark1 mark2 Select recombinants by PCR >200 from >1250 plants High Low ~2Mb ~8cM >400 SFPs Col Kas Col het Col ~2 Kas hetCol het ~43 Kas Col Kashet Kas ~268 ~43~539 ~43 ~268~43 ~2 het ~539 Kas eXtreme Array Fine Mapping