Studies of Genome Wide Molecular Variation in Arabidopsis thaliana using Arrays

Justin BorevitzSalk Institutenaturalvariation.org

Talk Outline

• Natural Variation in Light Response

• Transcriptional profiling– for QTL candidate genes

• Single Feature Polymorphisms (SFPs)– Potential deletions

• Bulk Segregant Mapping– Extreme Array Mapping

• Haplotype analysis

Natural Mutation in PHYA photoreceptorNatural Mutation in PHYA photoreceptor

Quantitative Trait Loci

differences may be due to expression or hybridization

Feature level model

FLC controls flowering time Difference detected it 3 day old seedlings

Gene Expression index that accounts for feature effect and polymorphisms

PAG1 down regulated in Cvi

PLALE GREEN1 knock out has long hypocotyl in red light

Potential Deletions

False Discovery and Sensitivity

Permuted data

real data

5% FDR

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

Observed t statistics vsNull (permuted) t statistics

Chip genotyping of a Recombinant Inbred Line

29kb interval

Potential Deletions

111 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

deletion of MAF1

FLOWERING1 QTL

Chr1 (bp)

Flowering Time QTL caused by a natural deletion in MAF1

MAF1

Fast Neutron deletions

FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1

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

LOD

eXtreme Array Mapping

Red light QTL RED2 from 100 Kas/ Col RILs

QTL likelihood model using bulk segregant analysis with SFP genotyping

0

4

8

12

16

0 20 40 60 80 100cM

LO

D

Composite Interval MappingRED2 QTL

RED2 QTL

Chromosome 2Chromosome 2Chromosome 1

Chromosome 4

15 tallest RILs pooled vs15 shortest RILs pooled

Array Haplotyping

Inbred lines

Low effectiverecombinationdue to partialselfing

Extensive LDblocks

Col Ler Cvi Kas Bay Shah Lz Nd

Chr

omos

ome1

~50

0kb

Review

• Transcriptional profiling to identify candidate genes and downstream responses that consider polymorphisms

• Single Feature Polymorphisms (SFPs) can be used to identify recombination breakpoints, potential deletions, for bulk segregant mapping, and haplotyping

NaturalVariation.org

SyngentaHur-Song ChangTong Zhu

SyngentaHur-Song ChangTong Zhu

SalkJon WernerTodd MocklerSarah LiljegrenJoanne ChoryDetlef WeigelJoseph Ecker

UC DavisJulin Maloof

UC San DiegoCharles Berry

ScrippsElizabeth Winzeler

SalkJon WernerTodd MocklerSarah LiljegrenJoanne ChoryDetlef WeigelJoseph Ecker

UC DavisJulin Maloof

UC San DiegoCharles Berry

ScrippsElizabeth Winzeler

University of Guelph, CanadaDave WolynUniversity of Guelph, CanadaDave Wolyn