Genomic Approaches to theGenetics of Adaptation

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

Widely Distributed

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

Olivier Loudet

Talk Outline• Wild Collections

– Local Population Sampling/ structure

• Seasonal Growth Chambers– KasC/ VanC RILs

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

• Aquilegia

• Wild Collections– Local Population Sampling/ structure

• Seasonal Growth Chambers– KasC/ VanC RILs

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

• Aquilegia

Local Population Variation

Local adaptation

under strong

selection

Seasonal Variation

Matt Horton

Megan Dunning

Collections• 807 Lines from 25 Midwest Populations

– (Diane Byers IL state) – growing!

• 1101 Lines from UK, 51 populations– (Eric Holub Warwick, UK) – growing!

• > 500 lines N and S Sweden (Nordborg)• > 400 Lines France and Midwest (Bergelson)• 400 lines Midwest (Borevitz)• 857 Accessions stock center (Randy Scholl)• Others welcome…

– Will be genotyped with Sequenom 149 SNPs $0.03 per SNP, 40plex > 400SNPs available

Variation within a field

Within and Between Variation

• BAKKER, E. G., STAHL, E. A., TOOMAJIAN, C., NORDBORG, M., KREITMAN, M. & BERGELSON, J.Distribution of genetic variation within and among local populations of Arabidopsis thaliana over its species range.Molecular Ecology 15 (5), 1405-1418.

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48 Non singleton SNPs of 87 tested Megan Dunning, poster #268

Local Population Structure in the Midwest

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120 SNPs of 149 tested including inbred lines Norman Warthmann`

Global Population Structure

Regional/Seasonal Variation

• What is Local Adaptation?

• Predictable Seasonal changes unique to each location.

Tossa Del MarSpain

LundSweden

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

Geneva Scientific/ Percival

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Developmental Plasticity == BehaviorDevelopmental Plasticity == Behavior

Kurt Spokas

Version 2.0a June 2006

USDA-ARS Website Midwest Area (Morris,MN)http://www.ars.usda.gov/mwa/ncscrl

May 1-3rd

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5Genetic map of the Kas-1 x Col-gl1 RIL population

55 markers from Wolyn et al. (2004) & 64 additional SNP markers.

Sweden 1

Col-gl1

Kas1

Sweden 2

Col-gl1

Kas1

Spain 1

Col-gl1

Kas1

Spain 2

Col-gl1

Kas1

Distribution of flowering time among 96 Kas-1/Col-gl1 RILs

Num

ber

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ILs

Num

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ILs

Kas/Col flowering time QTL

FRI

Chr3 Chr4 Chr5

Kas/Col flowering time QTL GxE

Chr4 FRI

Marker name Chr. position cM QTL QTL x Environment 2a* SE p-value 2a* SE p-value 21607030 chr1.27650179 99.3 -3.1 1.4 0.0353 0.0 2.0 0.9856 21607175 chr3.5140894 14.1 -2.5 1.3 0.0492 -0.7 1.8 0.6864 GL1 chr3.10361870 49.5 2.7 1.3 0.0435 -0.7 1.9 0.7145 MSAT4.39 chr4.89659 0 15.7 2.0 0.0000 -6.3 2.8 0.0289 44607955 chr4.5591486 28.8 3.8 1.3 0.0047 0.9 1.9 0.6497 44607234 chr5.1507224 7.7 2.2 1.4 0.1154 -1.8 1.9 0.3535 21607030 x MSAT4.39† chr1.27650179 x chr4.89659 - 6.6 2.9 0.0226 -1.7 4.0 0.6684 EnviSweden‡ - - 2.1 2.3 0.4586 - - -

1

Chr1 FLM Chr4 FRI

Environment and Epistasis

A B C

768 VanC AIL-RILs149 + 87 SNPsStock Center Release(Evadne Smith)

768 VanC AIL-RILs149 + 87 SNPsStock Center Release(Evadne Smith)

Van no mitochondrial insertion

FLC

Total Leaf Number

RNA DNA

Universal Whole Genome Array

Transcriptome AtlasExpression levelsTissues specificity

Transcriptome AtlasExpression levelsTissues specificity

Gene/Exon DiscoveryGene model correctionNon-coding/ micro-RNA

Gene/Exon DiscoveryGene model correctionNon-coding/ micro-RNA

Alternative SplicingAlternative Splicing

Comparative GenomeHybridization (CGH)

Insertion/DeletionsCopy Number Polymorphisms

Comparative GenomeHybridization (CGH)

Insertion/DeletionsCopy Number Polymorphisms

MethylationMethylation

ChromatinImmunoprecipitation

ChIP chip

ChromatinImmunoprecipitation

ChIP chip

Polymorphism SFPsDiscovery/Genotyping

Polymorphism SFPsDiscovery/Genotyping

Control for hybridization/genetic polymorphismsto understand TRUE expression variation

RNA ImmunoprecipitationRIP chip

RNA ImmunoprecipitationRIP chip

Antisense transcription

Allele Specific Expression

Which arrays should be used?

cDNA array

Long oligo array

BAC array

Which arrays should be used?

Gene array

Exon array

Tiling array35bp tile, 25mers 10bp gaps

Which arrays should be used?

Tiling/SNP array 2007 250k SNPs, 1.6M tiling probes

SNP array

Ressequencing array

How about multiple species? Microbial communities?

SNP SFP MMMMM MSFP

SFP

MMMMM M

Chromosome (bp)

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start AAAAA

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me

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Improved Genome Annotation

Transcription subUnits (TUs)

Exon1 Exon2Intron1

Tu1 Tu2 Tu3

?

cDNA1

cDNA2

cDNA3

X

v v v c c c v c RT-PCR gDNA PCR

Alternative spliced introns

ColVan

FDR for selection 3.5% 5%

Total introns tested 62,051 62,051

Total introns 43 228

Tested introns 32 72

Confirmed 20 36

percentage 71% 56%

FDR for selection 3%

Total exons tested

86,349

Total exons 69

Tested exons 5

confirmed 5

Alternative spliced exons

- verification

ColVan

v v v c c c v c RT-PCR gDNA PCR

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 Genotype effecton 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?

Copy Number Variation (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

Fast Neutron deletions

FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1

Het Sam Hazen (Scripps)

Natural Copy Variation on Tiling Arrays

Segregating self seed from wild ME isolate (Early – Late)

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)

Experimental Design of Association Study

• Sample > 3000 wild strains, 149 SNPs

• Select 3*384 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

Array Haplotyping

Inbred lines

Low effectiverecombinationdue to partialselfing

Extensive LDblocks

Col Ler Cvi Kas Bay Shah Lz Nd

Chr

omos

ome1

~50

0kb

Aquilegia (Columbines)

Recent adaptive radiation, 350Mb genome

Species with> 20k ESTs 11/14/2003

Animal lineage: good coverage

Plant lineage: crop plant coverage

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 Zoellner

USC

Magnus NordborgPaul Marjoram

Max Planck

Detlef Weigel

Scripps

Sam Hazen

University of Michigan

Sebastian Zoellner

University of Chicago

Xu ZhangYan Li

Peter RoycewiczEvadne Smith

Michigan State

Shinhan Shiu

PurdueIvan Baxter

University of Chicago

Xu ZhangYan Li

Peter RoycewiczEvadne Smith

Michigan State

Shinhan Shiu

PurdueIvan Baxter

http://www.plosone.org/

Aquilegia (Columbines)

Scott Hodges (UCSB)

Elena Kramer (Harvard)

Magnus Nordborg (USC)

Justin Borevitz (U Chicago)

Jeff Tompkins (Clemson)

• 300 F4 RILs growing (Evadne Smith)• TIGR gene index 85,000 ESTs >16,00 SNPs• Complete BAC physical map Clemson• Nimblegen arrays of 5 floral whorls

Whole Genome Shotgun Sequencing 2007 JGI