Markers, mapping, and expression using arrays

Justin BorevitzSalk Institutenaturalvariation.org

Talk Outline

• Intro Natural Variation/ QTL

• Transcription profiling to identify QTL candidate genes

• Identification of Single Feature Polymorphisms

• Future Prospects

PHYB

differences may be due to expression or hybridization

Deletions

Spatial Correction

Spatial Artifacts

Improved reproducibility

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

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

Future Bulk segregant Mapping

Extend to Quantitative Traits

Map multiple genes (mutation modifiers)

Fine mapping after PCRidentification of recombinants

Multiple models(epistasis)

Changes in allele F2, F3frequency (selection)

20% variance

20 simulations

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

• Now that we know what features are polymorphic we can determine which gene expression differences are real and which are due to polymorphism.

• New RNA analysis algorithm—

account for spatial correction, polymorphisms, and feature differences

Genes Expression for a candidate gene

Features (probes) for At1g22360

Spa

tially

cor

rect

ed lo

g P

M in

tens

ity

Gene Expression index that accounts for feature differences and polymorphisms

Gene expression revisited

0.02 1464 2913.6 -1449.6 1.990164 0.04 831 1067.5 -236.5 1.284597 0.06 409 390.8 18.2 0.9555012 0.08 242 160.2 81.8 0.6619835 0.1 171 73.6 97.4 0.4304094 0.12 131 35.4 95.6 0.270229 0.14 96 20.6 75.4 0.2145833 0.16 60 12.2 47.8 0.2033333 0.18 44 8.2 35.8 0.1863636 0.2 41 6.2 34.8 0.1512195

SAM True False Difference FDR threshold Positive Positive

Gene expression revisited

FLC controls flowering time

Difference detected at 3 day old seedling stage

Gene expression revisited

PAG1 down regulated in Cvi pag1 Knock Out is pale

pag1 KO is light insensitive

pag1 KO has long hypocotyl in red light

pag1 KO is early flowering

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

SyngentaHur-Song ChangTong Zhu

NaturalVariation.orgNaturalVariation.orgSalkJon 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

Future Projects

Design 2nd generation expression array $200

350,000 25mer features, expect 12,000 SFPs (2accessions)Validated gene models (exon intron)2 features per exon, alternative splicing5’ 3’ Untranslated region for gene family/ polymorphismMicro RNAsValidated “good” hybridization intensities

Haplotype Map –20 Accessions 3 replicates, SFP discovery

estimated 85,000 SFPs 1.4kb resolution

Association Studies –

120 Accessions 1 replicate, Genotyping

Bulk Segregant Mapping –Confirm Associations in specific crosses

Future Projects DNA

Future work with Natural Variation

• VanC advanced intercross RIL population• Backcross collections

True natural variation in gene expression20 Accessions 3 replicates, (polymorphism accounted for)

Cis regulatory variation/ Imprintingreciprocal F1s 3 replicates

Transcriptome QTL Map –100 best VanC Advanced Intercross Lines

How many loci control the variation in gene transcription? Candidate TF and binding sites?

Future Projects RNA