Haplotype mapping withSingle Feature Polymorphismsin Arabidopsis

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

Talk Outline

• Natural Variation in Light Response

• Single Feature Polymorphisms (SFPs)– Potential deletions

• Haplotype analysis

• Patterns in gene Families

• Aquilegia

• Natural Variation in Light Response

• Single Feature Polymorphisms (SFPs)– Potential deletions

• Haplotype analysis

• Patterns in gene Families

• Aquilegia

Light Affects the Entire Plant Life Cycle

Light response variation can be seen under constant conditions in the labNatural Variation under selection? Test in field

Seasons in the Growth Chamber

• Changing Day length• Cycle Light Intensity• Cycle Light Colors• Cycle Temperature

Day Length

0:00

2:00

4:00

6:00

8:00

10:00

12:00

14:00

16:00

18:00

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

mar

apr

may jun jul

aug

month

W/m

2

Sweden

Spain

standard

Temperature

-10

-5

0

5

10

15

20

25

30

35

sep

oct

nov

dec

jan

feb

mar

apr

may jun jul

aug

monthde

gree

s C

Spain High

Spain Low

Sweden High

Sweden Low

standard

Which arrays should be used?

• Spotted arrays Arizona 29,000 - 70mers• ATH1, Affymetrix expression GeneChip

202,806 unique 25bp oligo nucleotides features• AtTILE1, universal whole genome array

every ~35bp, > 3Million PM features• Re-sequencing array 120M*8bp

– 20 Accessions, Perlegen,

– Max Planck (Weigel), USC (Nordborg)

GeneChip

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

ChipViewer: Mapping of transcriptional units of ORFeome

From 2000v At1g09750 (MIPS) to the latest AGI At1g09750

2000 v Annotation (MIPS)

The latest AGI Annotation

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

Potential Deletions

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

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

Array Mapping

Hazen et al Plant Physiology (submitted)

chr1 chr2 chr3 chr4 chr5

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 (Wolyn et al Genetics 2004)

eXtreme Array Mapping BurC F2

XAMLz x Col

F2

QTLLz x Ler

F2

(Werner et al Genetics in press)

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

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

MAF1

FLM natural deletion

(Werner et al PNAS 2005)

Fast Neutron deletions

FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1

Het

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

~50

0kb

(-4,-3.5] (-3,-2.5] (-2,-1.5] (-1,-0.5] (0,0.5] (1,1.5] (2,2.5] (3,3.5]

T statistic

fre

qu

en

cy

0

e+

00

4

e+

04

8

e+

04

Distribution of T-stats

null (permutation)actual

Not Col ColNA NA duplications

32,427Calls

208,729

12,250 SFPs

Sequence confirmation of SFPs

SFP SNP Total FPR FDR Sensitivity

bay 44 61 1375 0.8% 25.0% 54.1%

bur 47 57 1320 1.1% 29.8% 57.9%

cvi 69 92 1325 1.2% 21.7% 58.7%

ler 41 51 1466 0.6% 22.0% 62.7%

lz 37 40 1441 0.5% 18.9% 75.0%

mr 67 87 1191 1.1% 17.9% 63.2%

mt 46 48 1413 0.9% 26.1% 70.8%

sorbo 37 53 1317 0.9% 29.7% 49.1%

ws 29 47 1369 0.3% 13.8% 53.2%

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 Theta W

RPS4

Rgenes vs bHLH Tajimas’ D

RPS4

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

freq

uen

cy

01

02

03

04

05

06

07

0

RgenesbHLH

Review

• Single Feature Polymorphisms (SFPs) can be used to

• Identify recombination breakpoints• eXtreme Array Mapping• Potential deletions (candidate genes)

• Haplotyping• Diversity/Selection

• Association Mapping

Aquilegia (Columbines)

Recent adaptive radiation, 350Mb genome

> 20k dbEST 11/14/2003

Animal lineage: good coverage

Plant lineage: crop plant coverage

NSF Genome Complexity

• 45,000 ESTs 5’ and 3’ ends• 350 arrays, RNA and genotyping

– High density SFP Genetic Map

• 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

Scott Hodges (UCSB)

Elena Kramer (Harvard)

Magnus Nordborg (USC)

Justin Borevitz (U Chicago)

Jeff Tompkins (Clemson)

NaturalVariation.orgNaturalVariation.orgSalk

Jon WernerJoanne ChoryJoseph Ecker

Max Planck

Detlef Weigel

UC San Diego

Charles Berry

Scripps

Sam HazenElizabeth Winzeler

Salk

Jon WernerJoanne ChoryJoseph Ecker

Max Planck

Detlef Weigel

UC San Diego

Charles Berry

Scripps

Sam HazenElizabeth Winzeler

University of Chicago

Xu ZhangEvadne Smith

UC Davis

Julin Maloof

University of Guelph, Canada

Dave Wolyn

Sainsbury Laboratory

Jonathan Jones

University of Chicago

Xu ZhangEvadne Smith

UC Davis

Julin Maloof

University of Guelph, Canada

Dave Wolyn

Sainsbury Laboratory

Jonathan Jones