Genomics tools to identify the molecular basis of complex traits

Justin BorevitzSalk Institutenaturalvariation.org

Talk Outline

• Quick Natural Variation review

• Transcription profiling to identify QTL candidate genes

• Identification of Single Feature Polymorphisms

• Future Prospects

PHYB

Genes Expression for a candidate gene

Features (probes) for At1g22360

Spa

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ity

Gene Expression index that accounts for feature differences and polymorphisms

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

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

Effect of SNP position

340 CandidatePolymorphisms

False negative

True Positive

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

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

25 bp 25 bp

25 bp 25 bp16 bp

25 bp 25 bp8 bp

1st Set

2nd Set

3rd Set

Complete Genome Tilling 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

Future Projects

Design 2nd generation expression array $200

350,000 25mer features, expect 12,000 SFPs (2 accessions)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

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

SyngentaHur-Song ChangTong Zhu

SalkJon WernerHuaming ChenSarah LiljegrenJoanne ChoryDetlef WeigelJoseph Ecker

UC DavisJulin Maloof

UC San DiegoCharles Berry

ScrippsElizabeth Winzeler

SalkJon WernerHuaming ChenSarah LiljegrenJoanne ChoryDetlef WeigelJoseph Ecker

UC DavisJulin Maloof

UC San DiegoCharles Berry

ScrippsElizabeth Winzeler

NaturalVariation.orgNaturalVariation.org