Genomic Systems underlying the genetics of adaptation in Arabidopsis thaliana

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

Light Affects the Entire Plant Life Cycle

de-etiolation

hypocotyl

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Talk OutlineTalk Outline• Predictable to Seasonal Variation

– Local Adaptation in the Lab

• Population Genetics– Population structure

– Extant diversity and new mutation

• Arrays– Genetic, epigenetic, expression,

splicing, and allelic variation

• Ecological context– Arabidopsis and Aquilegia

• Predictable to Seasonal Variation– Local Adaptation in the Lab

• Population Genetics– Population structure

– Extant diversity and new mutation

• Arrays– Genetic, epigenetic, expression,

splicing, and allelic variation

• Ecological context– Arabidopsis and Aquilegia

Begin with regions spanning the native geographic range

Nordborg et al PLoS Biology 2005

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

Day Length

0:00

2:00

4:00

6:00

8:00

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20:00

22:00

sep

oct

nov

dec

jan

feb

mar

apr

may jun jul

aug

month

hour

s

Sweden

Spain

standard

standard

Light Intensity

0

200

400

600

800

1000

1200

1400se

p

oct

nov

dec

jan

feb

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apr

may jun jul

aug

month

W/m

2

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Spain

standard

Temperature

-10

-5

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sep

oct

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Spain Low

Sweden High

Sweden Low

standard

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

Flowering time QTL, Kas/Col RILs

Sweden 1

Col-gl1

Kas1

Sweden 2

Col-gl1

Kas1

Spain 1

Col-gl1

Kas1

Spain 2

Col-gl1

Kas1

Num

ber

of R

ILs

Num

ber

of R

ILs

Flowering time QTL, Kas/Col RILs

FRI

FLM

Kas/Col flowering time QTL GxE

Chr4 FRIChr1 FLM Chr4 FRI

Environment and Epistasis

Globally Distributed

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

Olivier Loudet

Current collections• 807 Lines from 25 Midwest Populations

– (Diane Byers IL state) – pics published!

• 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)

– pics published

• Others welcome…Genotyped with Sequenom 149 SNPs $0.03 per

134 Non singleton SNPs of 1234 accessions

Global, Midwest, and UK

common haplotypes

Local Population Structure

Megan Dunning, Yan Li

17 Major Haplotypes

80 Major Haplotypes

Diversity within and between populations

Variation within a field http://naturalvariation.org/hapmapVariation within a field http://naturalvariation.org/hapmap

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

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

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?

Pst,Psm,Psy,Psx, Agro, Xanthomonas, H parasitica, 15 virus,

Col

Col

Van

Van

Col

Van

Van

Col

Genomic DNA RNA

No significant

allele specific expression

cis regulatory variation

(Van allele)

Paternal Imprinting Maternal Imprinting

cis regulatory variation

(Col allele)

RNA

RNA

RNA

RNA

GlobalAllele SpecificExpression

65,000 SNPsTranscribedAccession Pairs12,000 genes

>= 1 SNP6,000 >= 2 SNPs

Potential Deletions

SFPs and CC*GG Methylome

Extract genomic 100ng DNA (single leaf)

Digest with either msp1 or hpa2 CC*GG

Label with biotinRandom primers

Hybridize to array

HpaII digestion

Random labeling

Random labeling

MspI digestion

* * *

* * *

* * *

A)

B)

Hpa msp

Inte

nsit

y

*

*

Col

Col

Hpa msp

Col

Col

Hpa mspV

an

Van

mSFP

Hpa msp

Van

Van

SFP

Col♀ x Col♂

Van ♀ x Van ♂

Col ♀ x Van ♂

Van ♀ x Col ♂

Experimental design

Four genotypes, each with four biological replicates

4 day old seedlings, white light

Fit modelIntensity ~ additive + dominant + maternal +

additive:enzyme + dominant:enzyme + maternal:enzyme

Methylation Polymorphisms, mSFPs

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

methylated features and mSFPs

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

Enzyme effect, on CCGG features GxE

276 at 15% FDR

mQTL?

q-value enzyme addenz domenz matenz

0.001 2671 31 0 0

0.005 4000 141 0 481

0.01 4387 251 0 504

0.05 5950 725 0 507

0.1 6733 1263 43 507

Genomic Distribution of nonPolymorphic methylation sites

AT4G19020 tu19

AT1G27320 tu9

AT2G37080 tu1

AT4G27910 tu9

Col Van F1v F1c Col Van F1v F1c Col Van F1v F1c

Control HpaII MspI

AT1G51790 intron9

AT1G53240 intron4

AT2G01220 tu5

AT5G14600 intron3

AT1G49730 tu10

AT1G79990 intron9

AT1G20780 tu4

AT2G35350 tu2

AT2G45270 intron9

AT3G11460 tu1

AT4G04340 tu15

AT5G56370 tu3

AT2G18100 tu9

AT3G07740 tu2

AT3G48730 tu3

AT5G63190 tu6

AT5G13960 intron8

AT1G27900 intron7

AT2G45620 intron5

AT4G31140 tu2

Verification of additive x enzyme by genomic PCR

AT5G67130 tu7

AT1G19450 tu10

Col Van F1v F1c Col Van F1v F1c Col Van F1v F1c

Control HpaII MspI

AT2G32730 tu3

AT2G45670 tu11

AT3G04820 tu1

AT3G04610 intron5

AT3G53100 tu2

AT3G23590 intron4

AT1G10910 intron9

AT3G28880 tu8

AT4G23570 tu9

AT4G02500 tu1

AT4G17140 tu8

AT5G14950 tu1

AT5G04710 tu1

AT1G73730 tu2

AT1G19715 intron2

Verification of additive x enzyme by genomic PCR

ColColColVanVanVan

Col♂ x Van♀Col♂ x Van ♀

Van♂ x Col♀Van ♂ x Col ♀Van♂ x Col ♀

CC*GG

chromomethylase 2 (CMT2) exon19

0

1

2

3

4

5

6

hpaII mspI

log

inte

nsity

col van col♂xvan♀ van♂xcol♀

CC*GG

Verification of additive x enzyme by epiTyper

Next Questions

• What is the genetic architecture of methylation variation?

• How does it change with the environment and through development?

• Regional patterns, eg chromatin remodeling

• When does methylation effect expression?

Transcription subUnits (TUs)Intensity(gene/tu) ~ add + dom+mat + error

Exon1 Exon2Intron1

Tu1 Tu2 Tu3

?

cDNA1

cDNA2

cDNA3

X

Expression Analysis with Annotation

9.37%141150801508Maternal

1.2%22184901849Allele Additive

1.2%91741742cis Regulatory

1.1%40323323Imprinting

3.50%24561129690Dominant

3.50%5541411881602Additive

FDRExpected from perm

-Sig. Features

+ Sig. Features

Total Sig. Features

9.37%141150801508Maternal

1.2%22184901849Allele Additive

1.2%91741742cis Regulatory

1.1%40323323Imprinting

3.50%24561129690Dominant

3.50%5541411881602Additive

FDRExpected from perm

-Sig. Features

+ Sig. Features

Total Sig. Features

AT1G07350

AT1G29120

AT1G51350

AT1G53560

AT1G76170

D E

Additive, Dominant, Maternal, Genotype Variation

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

Ecological and Evolutionary context

• Abiotic conditions– Light, temperature, humidity– Soil, water

• Biotic conditions– Pathogens and polinators– Conspecifics, grasses trees

Local Population Variation

Local adaptation

under strong

selection

Seasonal Variation

Matt Horton

Megan Dunning

Aquilegia (Columbines)

Recent adaptive radiation, 350Mb genome

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)

http://www.plosone.org/

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

Megan DunningJoy Bergelson

Michigan State

Shinhan Shiu

PurdueIvan Baxter

University of Chicago

Xu ZhangYan Li

Peter RoycewiczEvadne Smith

Megan DunningJoy Bergelson

Michigan State

Shinhan Shiu

PurdueIvan Baxter