toward the genomics of adaptation to seasonal environments in arabidopsis thaliana justin borevitz...

Toward the genomics of Adaptation to seasonal environments in Arabidopsis thaliana

Justin BorevitzEcology & EvolutionUniversity of Chicagohttp://naturalvariation.org

Talk Outline

• Natural Variation/ QTL mapping• Single Feature Polymorphisms (SFPs)• eXtreme Array Mapping• Potential deletions• Haplotype analysis• Patterns in gene Families• Aquilegia

Light Affects the Entire Plant Life Cycle

de-etiolation

hypocotyl

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Light Affects the Entire Plant Life Cycle

Light response variation can be seen under constant conditions in the labNatural Variation != Natural Selection

Seasons in the Growth Chamber

• Changing Day length• Cycle Light Intensity• Cycle Light Colors• Cycle Temperature

Day Length

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2:00

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sep

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may jun jul

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hour

s

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standard

Light Intensity

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1400se

p

oct

nov

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may jun jul

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W/m

2

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Temperature

-10

-5

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35

sep

oct

nov

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feb

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may jun jul

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monthde

gree

s C

Spain High

Spain Low

Sweden High

Sweden Low

standard

Quantitative Trait Loci

QTL geneConfirmation

MarkerIdentificationGenotyping

Genomics path

Experimental DesignMapping population PhenotypingQTL AnalysisFine Mapping

Candidate genePolymorphismsgene expressionloss of function

QTL gene

Confirmation

Experimental Design

Mapping population

Phenotyping

QTL Analysis

Fine Mapping

With the Aid of Genomics

Which arrays should be used?

• Spotted arrays Arizona 29,000 - 70mers• 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

RNA DNA

Universal Whole Genome Array

Transcriptome AtlasExpression levelsTissues specificity

Transcriptome AtlasExpression levelsTissues specificity

Gene DiscoveryGene model correctionNon-coding/ micro-RNAAntisense transcription

Gene DiscoveryGene model correctionNon-coding/ micro-RNAAntisense transcription

Alternative SplicingAlternative Splicing Comparative GenomeHybridization (CGH)

Insertion/Deletions

Comparative GenomeHybridization (CGH)

Insertion/Deletions

MethylationMethylation

ChromatinImmunoprecipitation

ChIP chip

ChromatinImmunoprecipitation

ChIP chip

Polymorphism SFPsDiscovery/Genotyping

Polymorphism SFPsDiscovery/Genotyping

~35 bp tile, non-repetitive regions, “good” binding oligos, evenly spaced

Potential Deletions

False Discovery and Sensitivity

PM only

SAM threshold

5% FDR

GeneChip SFPs nonSFPs Cereon marker accuracy 3806 89118 100% Sequence 817 121 696 Sensitivity

Polymorphic 340 117 223 34% Non-polymorphic 477 4 473

False Discovery rate: 3% Test for independence of all factors: Chisq = 177.34, df = 1, p-value = 1.845e-40 SAM threshold 18% FDR

GeneChip SFPs nonSFPs Cereon marker accuracy 10627 82297 100% Sequence 817 223 594 Sensitivity

Polymorphic 340 195 145 57% Non-polymorphic 477 28 449

False Discovery rate: 13% Test for independence of all factors: Chisq = 265.13, 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%

Cereonmay be asequencingError

TIGRmatch isa match

Chip genotyping of a Recombinant Inbred Line

29kb interval

Discovery 6 replicates X $500 12,000 SFPs = $0.25Typing 1 replicate X $500 12,000 SFPs = $0.041

Map bibb100 bibb mutant plants100 wt mutant plants

bibb mapping

ChipMapAS1

Bulk segregantMapping usingChip hybridization

bibb maps toChromosome2 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

bibb as1-101

MYB

bib-1W49*

as-101Q107*

as1bibb

AS1 (ASYMMETRIC LEAVES1) =MYB closely related toPHANTASTICA located at 64cM

Array Mapping

Hazen et al Plant Physiology (in press)

chr1 chr2 chr3 chr4 chr5

eXtreme Array Mapping

Histogram of Kas/Col RILs Red light

hypocotyl length (mm)

cou

nts

6 8 10 12 14

02

46

81

01

2

15 tallest RILs pooled vs15 shortest RILs pooled

LOD

eXtreme Array Mapping

Allele frequencies determined by SFP genotyping. Thresholds set by simulations

0

4

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16

0 20 40 60 80 100cM

LO

D

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 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 CHR1 cry2 10kb deletion CHR1

Het

Array Haplotyping

• What about Diversity/selection across the genome?

• A genome wide estimate of population genetics parameters, θw, 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, Ws2Fl-1, Ita-0, Mr-0, St-0, Sah-0

Array Haplotyping

Inbred lines

Low effectiverecombinationdue to partialselfing

Extensive LDblocks

Col Ler Cvi Kas Bay Shah Lz Nd

Chr

omos

ome1

~50

0kb

SFPs for reverse genetics

http://naturalvariation.org/sfp

14 Accessions 30,950 SFPs`

Chromosome Wide Diversity

Diversity 50kb windows

Tajima’s D like 50kb windows

RPS4 unknown

R genes vs bHLH

(-1,-0.8] (-0.6,-0.4] (-0.2,0] (0.2,0.4] (0.6,0.8]

Selection

Tajima's D like statistic

freq

uen

cy

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RgenesbHLH

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

> 20k dbEST 11/14/2003

Animal lineage: good coverage

Plant lineage: crop plant coverage

NSF Genome Complexity

• 45,000 ESTs 5’ and 3’ ends• 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.orgNaturalVariation.orgSalk

Jon WernerJoanne ChoryJoseph Ecker

Max Planck

Detlef Weigel

UC San Diego

Charles Berry

Scripps

Sam HazenElizabeth Winzeler

Salk

Jon WernerJoanne ChoryJoseph Ecker

Max Planck

Detlef Weigel

UC San Diego

Charles Berry

Scripps

Sam HazenElizabeth Winzeler

University of Chicago

Xu ZhangEvadne Smith

UC Davis

Julin Maloof

University of Guelph, Canada

Dave Wolyn

Sainsbury Laboratory

Jonathan Jones

University of Chicago

Xu ZhangEvadne Smith

UC Davis

Julin Maloof

University of Guelph, Canada

Dave Wolyn

Sainsbury Laboratory

Jonathan Jones