Toward the genetic basis of adaptation using arrays

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

Arabidopsis thaliana• Genome Sequence 2000 (120Mb),• 20 strains by Perlegen, Weigel, Nordborg, Ecker• ~1% sequence variation, ~3000 collected lines• A. lyrata, Capsella rubella sister species JGI 2006• >5300 Research Labs (17th annual conference)• Fields study data to come, Annie Schmidt et al• 340k Sequence Indexed collection of KO lines• Gene Expression Atlas >300 tissues, time points• 15,000 full length cDNAs in recombination clones

Widely Distributed

http://www.inra.fr/qtlat/NaturalVar/NewCollection.htm

Olivier Loudet

Aranzana, et al PLOS genetics (2005), Sung Kim, Keyan Zhao

Local Population Variation

Scott HodgesIvan Baxter

Seasonal Variation

Matt Horton

Megan Dunning

Developmental Plasticity == Behavior

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

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Talk Outline

• Single Feature Polymorphisms (SFPs)– Methylation

– Potential deletions

– Genetic Mapping

• Resequencing/ Haplotypes

• Variation Scanning

• Single Feature Polymorphisms (SFPs)– Methylation

– Potential deletions

– Genetic Mapping

• Resequencing/ Haplotypes

• Variation Scanning

Tiling Arrays vs Resequencing Arrays

• AtTILE1, universal whole genome array

25mer every ~35bp, > 6.5 Million features

single array, many individuals.

• Re-sequencing array 120Mbp*8features

~1 Billion features, 8 wafers

20 Accessions available mid year

Perlegen, Max Planck (Weigel),

USC (Nordborg), Salk (Ecker)

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

Control for hybridization/genetic polymorphismsto understand true EXPRESSION polymorphismsTrue cis variation == Allele Specific Expression

SNP SFP MMMMM MSFP

SFP

MMMMM M

Chromosome (bp)

con

serv

atio

n

SNP

ORFa

start AAAAA

Tra

nsc

ripto

me

Atla

s

ORFb

deletion

Improved Genome Annotation

Potential Deletions

Delta p0 FALSE Called FDR

1.00 0.95 18865 160145 11.2%

1.25 0.95 10477 132390 7.5%

1.50 0.95 6545 115042 5.4%

1.75 0.95 4484 102385 4.2%

2.00 0.95 3298 92027 3.4%

SFP detection on tiling arrays

Intergenic Exon intron

SFPs 60770 23519 17216

total 685575 665524 301648

% 8.86% 3.53% 5.71%

SFPs/gene 0 >=1 >=2 >=3 >=4 >=5

genes 16322 9146 4304 2495 1687 1121

Methods for labeling

• Extract genomic 100ng DNA (single leaf)

• Digest with either msp1 or hpa2 CCGG

• Label with biotin random primers

• Hybridize to array

• Fit model

methylated features and mSFPs

>10,000 of 100,000 at 5% FDR

Enzyme effect, on CCGG features GxE

276 at 15% FDR

mQTL?

SFP Resequencing

• Advantages– Discovery and typing tool– Indels, rare variants, HMM tool– Quantitative score– Good for low polymorphism < 1%

• Caveats– No SNP knowledge, synonymous?– Bad for high polymorphism > 1%

• Rearrangements, Reference sequence

Natural Variation on Tiling Arrays

Potential Deletions

>500 potential deletions45 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

FLM

FLM natural deletion

(Werner et al PNAS 2005)

Chip genotyping of a Recombinant Inbred Line

29kb interval

Map bibb100 bibb mutant plants100 wt mutant plants

Array Mapping

Hazen et al Plant Physiology 2005

eXtreme Array Mapping

Histogram of Kas/Col RILs Red light

hypocotyl length (mm)

cou

nts

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02

46

81

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15 tallest RILs pooled vs15 shortest RILs pooled

LOD

eXtreme Array Mapping

Allele frequencies determined by SFP genotyping. Thresholds set by simulations

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Composite Interval Mapping

RED2 QTL

Chromosome 2

RED2 QTL 12cM

Red light QTL RED2 from 100 Kas/ Col RILs

Drosophila, Chao-Qiang Lai -Tufts University

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

(-4,-3.5] (-3,-2.5] (-2,-1.5] (-1,-0.5] (0,0.5] (1,1.5] (2,2.5] (3,3.5]

T statistic

fre

qu

en

cy

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00

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Distribution of T-stats

null (permutation)actual

Not Col ColNA NA duplications

32,427Calls

208,729

12,250 SFPs

Sequence confirmation of SFPs

SFP SNP Total FPR FDR Sensitivity

bay 44 61 1375 0.8% 25.0% 54.1%

bur 47 57 1320 1.1% 29.8% 57.9%

cvi 69 92 1325 1.2% 21.7% 58.7%

ler 41 51 1466 0.6% 22.0% 62.7%

lz 37 40 1441 0.5% 18.9% 75.0%

mr 67 87 1191 1.1% 17.9% 63.2%

mt 46 48 1413 0.9% 26.1% 70.8%

sorbo 37 53 1317 0.9% 29.7% 49.1%

ws 29 47 1369 0.3% 13.8% 53.2%

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

Experimental Design of Association Study

• Sample > 2000 wild strains, ~50-100 SNPs• Select 384 unstructured reference fine

mapping set• SFP resequencing of 384 lines• Haplotype map/ LD recombination blocks• Scan Genome for variation/selection• Measure phenotype in Seasonal Chambers• Associate Quantitative phenotypes with

HapMap

Review

• Single Feature Polymorphisms (SFPs) can be used to

• Methylation Variable sites• Potential deletions (candidate genes)• eXtreme Array Mapping

• Haplotyping• Diversity/Selection

• Association Mapping

NaturalVariation.orgNaturalVariation.orgUSC

Magnus NordborgPaul Marjoram

Max Planck

Detlef Weigel

Scripps

Sam Hazen

University of Michigan

Sebastian Zollner

University of Chicago

Xu ZhangEvadne SmithKen Okamoto

Michigan State

Shinhan Shui

PurdueIvan Baxter

University of Guelph, Canada

Dave Wolyn

Sainsbury Laboratory

Jonathan Jones

University of Chicago

Xu ZhangEvadne SmithKen Okamoto

Michigan State

Shinhan Shui

PurdueIvan Baxter

University of Guelph, Canada

Dave Wolyn

Sainsbury Laboratory

Jonathan Jones

USC

Magnus NordborgPaul Marjoram

Max Planck

Detlef Weigel

Scripps

Sam Hazen

University of Michigan

Sebastian Zollner