Natural Variation in Arabidopsis thalianaLight Response:Genomic Approaches

Justin BorevitzSalk Institutenaturalvariation.org

Talk Outline

• Survey of light response in

Arabidopsis thaliana accessions

• QTL mapping of light response in

Ler/Cvi Recombinant Inbred Lines

• Transcriptional profiling for QTL candidate genes

• Analysis of Single Feature Polymorphisms

(SFPs)

Light Affects the Entire Plant Life Cycle

de-etiolation

hypocotyl

}

My only collection

Quantitative Trait Loci

Epistasis scan

Chr1 Chr2 Chr3 Chr4 Chr5

Chr

1

C

hr2

Chr

3

Chr

4

Chr

5

BQTLhttp://hacuna.ucsd.edu/bqtl43,956 pair-wise tests 163 markers and 133 intervalsPermutation threshold p < 0.05 (5000 permutations)

replacementsynonymous Between Species =Within Species =

McDonald-Kreitmanp = 0.027

2362

858

= 0.23Ka

Ks

Review Natural Variation

• Light response variation is correlated with latitude

• Functional variation in Photoreceptors PHYA, PHYB, CRY2 (El-Din El-Assal et al 2001)

• New QTL identified in common and unique photoreceptor pathways

• QTL may be under selection?

differences may be due to expression or hybridization

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

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

bibb mutant phenotypes

Col bib-1 det2-1

23 days

bib-3

• three independent recessive alleles • medial sepals remain attached• “cabbage-like” rosette leaves • flowers open prematurely• fruit appear more slender

bib-3

Ler

bib-3

Map bibb100 bibb mutant plants100 wt mutant plants

bibb mapping

ChipMapAS1

Bulk segregantMapping usingChip hybridization

bibb maps toChromosome2 near ASYMETRIC LEAVES1

BIBB=AS1

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 to PHANTASTICAlocated at 64cM

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

Gene expression revisited

FLC controls flowering time Difference detected at 3 day old seedling stage

Gene Expression index that accounts for feature effect and polymorphisms

~100 genes @ 20% FDR

Gene expression revisited

PAG1 down regulated in CviPLALE GREEN1 knock out

pag1 KO is light insensitive

pag1 KO has long hypocotyl in red light

Review

• Transcriptional profiling to identify candidate genes and downstream responses

• Single Feature Polymorphisms (SFPs) can be used for identifying recombination breakpoints, bulk segregant mapping, and haplotyping

• Consider polymorphisms for expression analysis

25 bp 25 bp

25 bp 25 bp16 bp

25 bp 25 bp8 bp

1st Set

2nd Set

3rd Set

Complete Genome Tiling Chip

Polymorphisms (re-sequencing)Global methylation (Methylome)Comparative Genomics (Brassica)

New Gene DiscoveryImprove AnnotationAlternative SplicingMicro RNAs

9 Whole-Genome Expression Chips2 Splicing Chips2 5’ Mapping Chips

Validate features

Extra Chips:

ChIP – Chip (DNA binding sites)

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

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