Toward the genetic basis of adaptation: Arrays/Association Mapping

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

Widely Distributed

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

Olivier Loudet

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

17k SNPs 96 lines

Local Population Variation

Scott HodgesIvan Baxter

Seasonal Variation

Matt Horton

Megan Dunning

Light Affects the Entire Plant Life Cycle

de-etiolation

hypocotyl

}

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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Developmental Plasticity == BehaviorDevelopmental Plasticity == Behavior

Talk Outline• Arabidopsis Light Response

– PHYA, QTL mapping• Whole Genome Tiling Arrays

– Alternative splicing/Methylation – Single Feature Polymorphisms (SFPs)– Potential deletions/ Copy Number Variants– Genetic Mapping

• Resequencing/ Haplotypes– Variation Scanning

• Aquilegia for Genetics of Adaptive Radiations

• Arabidopsis Light Response– PHYA, QTL mapping

• Whole Genome Tiling Arrays– Alternative splicing/Methylation – Single Feature Polymorphisms (SFPs)– Potential deletions/ Copy Number Variants– Genetic Mapping

• Resequencing/ Haplotypes– Variation Scanning

• Aquilegia for Genetics of Adaptive Radiations

Quantitative Trait Loci

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

Which arrays should be used?

cDNA array

Long oligo array

Which 25mer arrays should be used?

Gene array

Exon array

Tiling array

Which 25mer arrays should be used?

Tiling/SNP array

SNP array

Ressequencing array

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

Alternative Splicing

V V V C C C

VanCol

Xu Zhang

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

Chip genotyping of a Recombinant Inbred Line

29kb interval

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)

Natural Variation on Tiling Arrays

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)

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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

SNP SFP MMMMM MSFP

SFP

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Chromosome (bp)

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start AAAAA

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Improved Genome Annotation

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

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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

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RgenesbHLH

Experimental Design of Association Study

• Sample > 3000 wild strains, ~100 SNPs

• Select 500 less structured reference fine mapping set for SFP resequencing

• Scan Genome for variation/selection

• Measure phenotype in Seasonal Chambers

• Haplotype map/ LD recombination blocks

• Associate Quantitative phenotypes with HapMap

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)• TIGR gene index 85,000 ESTs >16,00 SNPs• Complete BAC physical map Clemson• Nimblegen arrays

Aquilegia (Columbines)

Genetics of Speciationalong a Hybrid Zone

NSF Genome Complexity

• Microarray development – QTL candidates

• 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– VIGS

• Whole Genome Sequencing (JGI?)

Scott Hodges (UCSB)

Elena Kramer (Harvard)

Magnus Nordborg (USC)

Justin Borevitz (U Chicago)

Jeff Tompkins (Clemson)

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