Arrays as tools for Natural Variation studies:Mapping, Haplotyping, and gene expression

Justin BorevitzUniversity of Chicagonaturalvariation.org`

Talk Outline

• Single Feature Polymorphisms (SFPs)– Potential deletions

• Bulk Segregant Mapping– Extreme Array Mapping

• Haplotyping– Selection

• Transcriptional profiling– for QTL candidate genes

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

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

stamenstayLerSarah LiljegrenMapping confirmed

0 20 40 60 80 100

-0.5

0.0

0.5

stamenstaymut

cM Chromosome 1

alle

le fr

eque

ncy

0 20 40 60 80

-0.5

0.0

0.5

stamenstaymut

cM Chromosome 2

alle

le fr

eque

ncy

0 20 40 60 80

-0.5

0.0

0.5

stamenstaymut

cM Chromosome 3

alle

le fr

eque

ncy

0 20 40 60

-0.5

0.0

0.5

stamenstaymut

cM Chromosome 4

alle

le fr

eque

ncy

0 20 40 60 80 100

-0.5

0.0

0.5

stamenstaymut

cM Chromosome 5

alle

le fr

eque

ncy

0 20 40 60 80 100

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

ein6F2mut

cM Chromosome 1

alle

le fr

eque

ncy

0 20 40 60 80

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

ein6F2mut

cM Chromosome 2al

lele

freq

uenc

y

0 20 40 60 80

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

ein6F2mut

cM Chromosome 3

alle

le fr

eque

ncy

0 20 40 60

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

ein6F2mut

cM Chromosome 4

alle

le fr

eque

ncy

0 20 40 60 80 100

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

ein6F2mut

cM Chromosome 5

alle

le fr

eque

ncy ein6een

double mutantRamlah NehringMapping confirmed

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

Red light QTL RED2 from 100 Kas/ Col RILs

Allele frequencies determined by SFP genotyping. Thresholds set by simulations

15 tallest RILs pooled vs15 shortest RILs pooled

0

4

8

12

16

0 20 40 60 80 100cM

LO

D

Composite Interval Mapping

RED2 QTL

Chromosome 2

RED2 QTL 12cM

Fine Mapping with Arrays

0 100 200 300 400 500 600

-1.0

-0.5

0.0

0.5

1.0

Chromosome 1 (cM)

kb

geno

type

0 100 200 300 400 500 600

-1.0

-0.5

0.0

0.5

1.0

Chromosome 2 (cM)

kbge

noty

pe

0 100 200 300 400 500 600

-1.0

-0.5

0.0

0.5

1.0

Chromosome 3 (cM)

kb

geno

type

0 100 200 300 400 500 600

-1.0

-0.5

0.0

0.5

1.0

Chromosome 4 (cM)

kb

geno

type

0 100 200 300 400 500 600

-1.0

-0.5

0.0

0.5

1.0

Chromosome 5 (cM)

kb

geno

type

Single Additive Gene1000 F2sSelect recombinantsby PCR 1Mb region

Barley SFPs gDNA

• 9 arrays, random labeled genomic DNA

• 3 wild type, 3 parent 1, 3 parent 2

• Hope to verify some RNA SFPs

• Pairs plots, correlation matrix

• SFP table

Just better than permutations

delta ori.data perm.data difference FDR0.10 2866 2114.2 751.8 0.740.15 1870 578.4 1291.6 0.310.20 1274 269.3 1004.7 0.210.25 991 174.7 816.3 0.180.30 816 126.8 689.2 0.160.35 660 95.8 564.2 0.150.40 554 75.8 478.2 0.14

Increase specific activity with other labeling methodsPerform more replicates

• Single Feature Polymorphisms– Improve with replicates (easy)– Improved statistical models

• Genotyping– Precisely define recombination breakpoints– Fine mapping

• Potential Deletions– Candidate genes/ induced mutations

• Bulk segregant Mapping– eXtreme Array Mapping, F2s etc

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, Ler, Bay, Shah, Cvi, Kas, C24,

Est, Kin, Mt, Nd, Sorbo, Van, Ws2

C c c c C c C j j j j j j L L L B B B S S C C C k k c c E E E K K M M M N N N S S S v v V WWW

Cc

cc

Cc

Cj

jj

jj

jL

LL

BB

BS

SC

CC

kk

cc

EE

EK

KM

MM

NN

NS

SS

vv

VW

WW

o o o o o o o w w w w w w e e e a a a h h v v v a a 2 2 s s s e e t t t d d d o o o a a a s s s

oo

oo

oo

ow

ww

ww

we

ee

aa

ah

hv

vv

aa

22

ss

se

et

tt

dd

do

oo

aa

as

ss

l l l l l l l C C C L L L r r r y y y a a i i i s s 4 4 t t t n n 0 0 0 - - - r r r n n n - - -

ll

ll

ll

lC

CC

LL

Lr

rr

yy

ya

ai

ii

ss

44

tt

tn

n0

00

--

-r

rr

nn

n-

--

Pairwise Correlation between and within replicates

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

Accession FDR Sensitivity SNP Totalbay 0.0% 43% 51 563c24 0.2% 39% 64 580cvi 0.0% 38% 91 543est 0.0% 59% 39 548kas 1.9% 44% 66 577kendl 3.1% 33% 57 545ler 0.0% 49% 43 562lz 0.0% 53% 51 573mt 0.2% 61% 49 570nd 0.0% 47% 49 568shah 0.0% 24% 80 548sorbo 0.0% 45% 55 526van 0.2% 29% 92 571ws2 0.0% 49% 57 514

Sequence confirmation of SFPs

SFPs for reverse genetics

http://naturalvariation.org/sfp

14 Accessions 30,950 SFPs`

Chromosome Wide Diversity

Self Incompatibility-locus

Self Incompatibility-locus

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

Summery Haplotyping

• Patterns of variation across accessions

• Natural reverse genetics– Polymorphism database

• Increased polymorphism in centromere

• Selection on R/genes

• 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

SFPs from RNA

• Barley Affy array 22801 probe sets– Most probes sets 11 probes– Background correction “rma2”– Quantile normalization

• 36 arrays total– 3 replicates– 6 tissues, leaf, crown, root, radical, gem, col?– 2 genotypes (Golden Promise 7,459 ESTs)– (Morex 52,695 ESTs)

Look at some plots raw data

Remove probe effect

Remove Tissue + Genotype effect

Look at some plots raw data

Remove probe effect

Remove Tissue + Genotype effect

SAM False Discovery Rate

delta ori.data perm.data difference FDR0.1 13210 1210.34 11999.66 0.0916230130.2 7903 183.95 7719.05 0.0232759710.3 5462 49.18 5412.82 0.0090040280.4 4036 18.31 4017.69 0.0045366700.5 3024 8.49 3015.51 0.0028075400.6 2285 3.85 2281.15 0.001684902

Both + and – SFPs since no reference comparison

Need to compare with ESTs

Review• Single Feature

Polymorphisms (SFPs) can be used to identify recombination breakpoints, potential deletions, for eXtreme Array mapping, and haplotyping

• Expression analysis to identify QTL candidate genes and downstream responses that consider polymorphisms

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

~19 bp tile, eliminate repeat regionsboth strands “good” binding oligos

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

ChipViewer: Mapping of transcriptional units of ORFeome

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

2000 v Annotation (MIPS)

The latest AGI Annotation

NaturalVariation.org

SyngentaHur-Song ChangTong Zhu

SyngentaHur-Song ChangTong Zhu

University of Guelph, CanadaDave WolynUniversity of Guelph, CanadaDave Wolyn

Salk

Jon WernerTodd MocklerSarah LiljegrenRamlah NehringJoanne ChoryDetlef WeigelJoseph Ecker

UC Davis

Julin Maloof

UC San Diego

Charles Berry

Scripps

Sam HazenElizabeth Winzeler

NaturalVariation.orgSalk

Jon WernerTodd MocklerSarah LiljegrenRamlah NehringJoanne ChoryDetlef WeigelJoseph Ecker

UC Davis

Julin Maloof

UC San Diego

Charles Berry

Scripps

Sam HazenElizabeth Winzeler