Array Genotyping to Dissect Quantitative Trait Loci in Arabidopsis thaliana

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

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Talk Outline

• QTL Intro

• Transcription based Cloning

• Single Feature Polymorphisms (SFPs)– Potential deletions

• Bulk Segregant Mapping– Extreme Array Mapping

• Haplotype analysis

• New Arrays, new models Aquilegia

Light Affects the Entire Plant Life Cycle

de-etiolation

hypocotyl

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Quantitative Trait Loci

QTL geneConfirmation

MarkerIdentificationGenotyping

Genomics path

Experimental DesignMapping population PhenotypingQTL AnalysisFine Mapping

Candidate genePolymorphismsgene expressionloss of function

QTL gene

Confirmation

Experimental Design

Mapping population

Phenotyping

QTL Analysis

Fine Mapping

With the Aid of Genomics

Genomics to Clone QTL

• Recombination Fine Mapping

• Gene Expression Variation

• Hybridization Polymorphism

• Association Testing, LD mapping

• Direct Sequencing of Candidate Gene

• Quantitative Complementation

• Transgenic Complementation

• Look for gene expression differences between genotypes

• Identify candidate genes that map to mutation

• Downstream targets that map elsewhere

Transcription based cloning

differences may be due to expression or hybridization

PAG1 down regulated in Cvi

PLALE GREEN1 knock out has long hypocotyl in red light

What is Array Genotyping?

• Affymetrix expression GeneChips contain 202,806 unique 25bp oligo nucleotides.

• 11 features per probset for 21546 genes• New array’s have even more• Genomic DNA is randomly labeled with

biotin, product ~50bp.• 3 independent biological replicates

compared to the reference strain Col

GeneChip

Potential Deletions

Spatial Correction

Spatial Artifacts

Improved reproducibilityNext: Quantile Normalization

False Discovery and Sensitivity

PM only

SAM threshold

5% FDR

GeneChip SFPs nonSFPs Cereon marker accuracy 3806 89118 100% Sequence 817 121 696 Sensitivity

Polymorphic 340 117 223 34% Non-polymorphic 477 4 473

False Discovery rate: 3% Test for independence of all factors: Chisq = 177.34, df = 1, p-value = 1.845e-40 SAM threshold 18% FDR

GeneChip SFPs nonSFPs Cereon marker accuracy 10627 82297 100% Sequence 817 223 594 Sensitivity

Polymorphic 340 195 145 57% Non-polymorphic 477 28 449

False Discovery rate: 13% Test for independence of all factors: Chisq = 265.13, df = 1, p-value = 1.309e-59

3/4 Cvi markers were also confirmed in PHYB

90% 80% 70%

41% 53% 85%

90% 80% 70%

67% 85% 100%

Cereonmay be asequencingError

TIGRmatch isa match

Chip genotyping of a Recombinant Inbred Line

29kb interval

Discovery 6 replicates X $500 12,000 SFPs = $0.25Typing 1 replicate X $500 12,000 SFPs = $0.041

SNP377

SM184

SM50

SM35

SM106

G2395

SNP65

SM40

SEQ8298

TH1

MSAT7964

MAT7787

CER45

5.50

5.87

6.34

7.01

7.30

7.44

7.60

7.79

7.96

8.13

8.29

8.65

9.32

MbMarker

Near-Isogenic Lines for LIGHT1 Ler / Cvi #3

mm

81N-J 17A-A/J 114 124 189Ler

6 2 4 3 3 3 Plants

Line

RVE7

GI

194

3

5.0 5.8 5.8 5.1 5.9 5.7 5.8 Phenotype

LIGHT1 NIL

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 MAF1

MAF1

MAF1 natural deletion

Fast Neutron deletions

FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1

Het

Map bibb100 bibb mutant plants100 wt mutant plants

bibb mapping

ChipMapAS1

Bulk segregantMapping usingChip hybridization

bibb maps toChromosome2 near ASYMETRIC LEAVES1

BIBB = ASYMETRIC LEAVES1

Sequenced AS1 coding region from bib-1 …found g -> a change that would introduce a stop codon in the MYB domain

bibb as1-101

MYB

bib-1W49*

as-101Q107*

as1bibb

AS1 (ASYMMETRIC LEAVES1) =MYB closely related toPHANTASTICA located at 64cM

eXtreme Array Mapping

Histogram of Kas/Col RILs Red light

hypocotyl length (mm)

cou

nts

6 8 10 12 14

02

46

81

01

2

15 tallest RILs pooled vs15 shortest RILs pooled

LOD

eXtreme Array Mapping

Allele frequencies determined by SFP genotyping. Thresholds set by simulations

0

4

8

12

16

0 20 40 60 80 100cM

LO

D

Composite Interval Mapping

RED2 QTL

Chromosome 2

RED2 QTL 12cM

Red light QTL RED2 from 100 Kas/ Col RILs

eXtreme Array Mapping BurC F2

XAMLz x Col

F2

QTLLz x Ler

F2

XRED2 QTL

mark1 mark2

Select recombinants by PCR >200 from >1250 plants

HighLow~2Mb ~8cM

>400 SFPsCol

Kas

Col Col

Col het

Col

~2

Kas

het Col

het het

het

~43

Kas

Kas Col

Kas het

Kas

~268

~43 ~539 ~43

~268 ~43 ~2

Kas

eXtreme Array Fine Mapping

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

A star phylogeny

163 markers 73 accessions ~ 750kb/marker

Array Haplotyping

Inbred lines

Low effectiverecombinationdue to partialselfing

Extensive LDblocks

Col Ler Cvi Kas Bay Shah Lz Nd

Chr

omos

ome1

~50

0kb

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

~35 bp tile, non-repetitive regions, “good” binding oligos, evenly spaced

Transcriptome Viewer: http://signal.salk.edu

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

Review

• Transcription Based Cloning• Single Feature Polymorphisms

(SFPs) can be used to• Potential deletions (candidate genes)• Identify recombination breakpoints• eXtreme Array Mapping

• Haplotyping• Diversity/Selection• Association Mapping

Scott Hodges (UCSB)

Elena Kramer (Harvard)

Magnus Nordborg (USC)

Justin Borevitz (U Chicago)

Jeff Tompkins (Clemson)

NSF Genomics of Adaptation to the Biotic and Abiotic Environment in Aquilegia

Aquilegia (Columbines)

Recent adaptive radiation, 350Mb genome

NSF Genomics of Adaptation to the Biotic and Abiotic Environment in Aquilegia

• 35,000 ESTs 5’ and 3’

• 350 arrays, RNA and genotyping– High density SFP Genetic Map

• Physical Map (BAC tiling path)– Physical assignment of ESTs

• QTL for pollinator preference – and abiotic stress– QTL fine mapping/ LD mapping

• Develop transformation techniques

NaturalVariation.orgNaturalVariation.orgSalk

Jon WernerSarah LiljegrenHuaming ChenJoanne ChoryDetlef WeigelJoseph Ecker

UC San Diego

Charles Berry

Scripps

Sam HazenSteve KayElizabeth Winzeler

University of Chicago

Xu ZhangEvadne Smith

Syngenta

Hur-Song ChangTong Zhu

UC Davis

Julin Maloof

University of Guelph, Canada

Dave Wolyn

Sainsbury Laboratory

Jonathan Jones

University of Chicago

Xu ZhangEvadne Smith

Syngenta

Hur-Song ChangTong Zhu

UC Davis

Julin Maloof

University of Guelph, Canada

Dave Wolyn

Sainsbury Laboratory

Jonathan Jones

Salk

Jon WernerSarah LiljegrenHuaming ChenJoanne ChoryDetlef WeigelJoseph Ecker

UC San Diego

Charles Berry

Scripps

Sam HazenSteve KayElizabeth Winzeler