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Toward the genetic basis of adaptation: Arrays/Association Mapping Justin Borevitz Ecology & Evolution University of Chicago http://naturalvariation.org/. Arabidopsis thaliana. Genome Sequence 2000 (120Mb), 20 strains by Perlegen, Weigel, Nordborg, Ecker

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Toward the genetic basis of adaptation: Arrays/Association MappingJustin BorevitzEcology & EvolutionUniversity of Chicagohttp://naturalvariation.org/


Arabidopsis thaliana
Arabidopsis thaliana

  • Genome Sequence 2000 (120Mb),

  • 20 strains by Perlegen, Weigel, Nordborg, Ecker

  • ~1% sequence variation, LD extends 50-250kb

  • ~3000 collected “inbred” lines, >50 RIL sets

  • 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

Olivier Loudet

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



Local Population Variation

Ivan Baxter

Scott Hodges


Seasonal Variation

Matt Horton

Megan Dunning



Sweden spain

Seasons in the Growth Chamber

Seasons in the Growth Chamber

Developmental Plasticity == Behavior

Developmental Plasticity == Behavior

Sweden Spain

  • Changing Day length

  • Cycle Light Intensity

  • Cycle Light Colors

  • Cycle Temperature

  • Changing Day length

  • Cycle Light Intensity

  • Cycle Light Colors

  • Cycle Temperature


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



Tiling arrays vs resequencing arrays
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
Which arrays should be used?

cDNA array

Long oligo array


Which 25mer arrays should be used
Which 25mer arrays should be used?

Gene array

Exon array

Tiling array


Which 25mer arrays should be used1
Which 25mer arrays should be used?

SNP array

Ressequencing array

Tiling/SNP array


Universal Whole Genome Array

RNA

DNA

Chromatin

Immunoprecipitation

ChIP chip

Gene Discovery

Gene model correction

Non-coding/ micro-RNA

Antisense transcription

Methylation

Transcriptome Atlas

Expression levels

Tissues specificity

Polymorphism SFPs

Discovery/Genotyping

Comparative Genome

Hybridization (CGH)

Insertion/Deletions

Alternative Splicing

Control for hybridization/genetic polymorphisms

to understand true EXPRESSION polymorphisms

True cis variation == Allele Specific Expression


Alternative splicing
Alternative Splicing

Van

Col

V V V C C C

Xu Zhang



SFP detection on tiling arrays

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%


Methods for labeling
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
methylated features and mSFPs

Enzyme effect, on CCGG features

GxE

mQTL?

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

276 at 15% FDR


Sfp resequencing
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



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


FLM natural deletion

FLM

Potential Deletions Suggest Candidate Genes

FLOWERING1 QTL

Chr1 (bp)

Flowering Time QTL caused by a natural deletion in FLM

(Werner et al PNAS 2005)



100 bibb mutant plants

Map bibb

100 wt mutant plants


Array Mapping

Hazen et al Plant Physiology 2005


eXtreme Array Mapping

15 tallest RILs pooled vs

15 shortest RILs pooled


Extreme array mapping

Chromosome 2

16

12

RED2 QTL

LOD

8

4

0

0

20

40

60

80

100

cM

RED2 QTL 12cM

Composite Interval Mapping

eXtreme Array Mapping

LOD

Drosophila, Chao-Qiang Lai -Tufts University

Allele frequencies determined by SFP genotyping. Thresholds set by simulations

Red light QTL RED2 from 100 Kas/ Col RILs


Improved Genome Annotation

ORFa

Transcriptome Atlas

ORFb

start

AAAAA

deletion

M

M

M

M

M

M

M

M

M

M

M

M

SFP

SNP

SNP

SFP

SFP

conservation

Chromosome (bp)


Array haplotyping
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, Ws2

    Fl-1, Ita-0, Mr-0, St-0, Sah-0


Array haplotyping1

Chromosome1 ~500kb

Col

Ler

Cvi

Kas

Bay

Shah

Lz

Nd

Array Haplotyping

Inbred lines

Low effective

recombination

due to partial

selfing

Extensive LD

blocks


SFPs for reverse genetics

14 Accessions 30,950 SFPs`

http://naturalvariation.org/sfp






Experimental design of association study
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
Aquilegia (Columbines)

Recent adaptive radiation, 350Mb genome


Species with 20k ests 11 14 2003

Plant lineage:

crop plant coverage

Species with> 20k ESTs 11/14/2003

Animal lineage:

good coverage


Aquilegia columbines1
Aquilegia (Columbines)

  • 300 F3 RILs growing (Evadne Smith)

  • TIGR gene index 85,000 ESTs >16,00 SNPs

  • Complete BAC physical map Clemson

  • Nimblegen arrays


Genetics of speciation along a hybrid zone
Genetics of Speciationalong a Hybrid Zone


Nsf genome complexity
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.org

NaturalVariation.org

University of Chicago

Xu Zhang

Evadne Smith

Ken Okamoto

Michigan State

Shinhan Shui

Purdue

Ivan Baxter

University of Guelph, Canada

Dave Wolyn

Sainsbury Laboratory

Jonathan Jones

University of Chicago

Xu Zhang

Evadne Smith

Ken Okamoto

Michigan State

Shinhan Shui

Purdue

Ivan Baxter

University of Guelph, Canada

Dave Wolyn

Sainsbury Laboratory

Jonathan Jones

USC

Magnus Nordborg

Paul Marjoram

Max Planck

Detlef Weigel

Scripps

Sam Hazen

University of Michigan

Sebastian Zollner

USC

Magnus Nordborg

Paul Marjoram

Max Planck

Detlef Weigel

Scripps

Sam Hazen

University of Michigan

Sebastian Zollner


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