MicroArray Evolution: expression to mapping and back again Justin Borevitz Salk Institute naturalvariation.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 Expression Revisited

Natural Mutation in PHYA photoreceptor

Quantitative Trait Loci EPI1 EPI2

differences may be due to expression or hybridization

What is Array Genotyping? Affymetrix expression GeneChips contain 202,806 unique 25bp oligo nucleotides. 11 features per probset for 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 reproducibility Next: Quantile Normalization

False Discovery and Sensitivity Permuted data real data 5% FDR PM only SAM threshold 5% FDR GeneChip SFPs nonSFPs Cereon marker accuracy % Sequence Sensitivity Polymorphic % Non-polymorphic False Discovery rate: 3% Test for independence of all factors: Chisq = , df = 1, p- value = 1.845e -40 Observed t statistics vs Null (permuted) t statistics

Chip genotyping of a Recombinant Inbred Line 29kb interval Discovery 6 replicates X $500 12,000 SFPs = $0.25 Typing 1 replicate X $500 12,000 SFPs = $0.041

Potential Deletions >500 potential deletions 45 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 CHR1cry2 10kb deletion CHR1

Map bibb 100 bibb mutant plants 100 wt mutant plants

bibb mapping ChipMap AS1 Bulk segregant Mapping using Chip hybridization bibb maps to Chromosome2 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 bibbas1-101 MYB bib-1 W49* as-101 Q107* as1 bibb AS1 (ASYMMETRIC LEAVES1) = MYB closely related to PHANTASTICA 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 cM LOD Composite Interval Mapping RED2 QTL Chromosome 2 15 tallest RILs pooled vs 15 shortest RILs pooled RED2 QTL 12cM

Simulation Likelihood 50 eXtreme of 500 F2s chr2 QTL 37% var chr5 QTL 13% var Can detect additive or epistatic QTL but hard to determine which

Fine Mapping with Arrays Single Additive Gene 1000 F2s Select recombinants by PCR 1Mb region

Array Haplotyping What about Diversity/selection across the genome? A genome wide estimate of population genetics parameters, θ w, 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

Array Haplotyping Hybridize 48 arrays with 15 accessions 3 replicates Analyze 1/10 Genome 12Mb 24,507 features x 14 = 343,098 T-tests Spatial Correction, quantile normalization, SAM like T-tests, permutation null distribution.

Array Haplotyping Inbred lines Low effective recombination due to partial selfing Extensive LD blocks ColLerCviKasBayShahLzNd Chromosome1 ~500kb

Distribution of T-stats null (permutation) actual Not ColColNANA duplications 32,427 Calls 208,729 12,250 SFPs

Diversity

Tajima’s D

25 bp 16 bp 25 bp 8 bp 1st Set 2nd Set 3rd Set Complete Genome Tiling Chip Polymorphisms (re-sequencing) Global methylation (Methylome) Comparative Genomics (Brassica) New Gene Discovery Improve Annotation Alternative Splicing Micro RNAs 9 Whole-Genome Expression Chips 2 Splicing Chips 2 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 At1g v Annotation (MIPS) The latest AGI Annotation

Expression Analysis Revisited FLC controls flowering time Difference detected it 3 day old seedlings Feature level model 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

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 eXtreme Array mapping, and haplotyping

NaturalVariation.org Syngenta Hur-Song Chang Tong Zhu Syngenta Hur-Song Chang Tong Zhu Salk Jon Werner Todd Mockler Sarah Liljegren Joanne Chory Detlef Weigel Joseph Ecker UC Davis Julin Maloof UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler University of Guelph, Canada Dave Wolyn University of Guelph, Canada Dave Wolyn Salk Jon Werner Todd Mockler Sarah Liljegren Joanne Chory Detlef Weigel Joseph Ecker UC Davis Julin Maloof UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler NaturalVariation.org

Effect of SNP position 340 Candidate Polymorphisms False negative True Positive

Fine Mapping LIGHT1 500kb 2 nd North QTL

Future Projects Design 2 nd generation expression array $ ,000 25mer features, expect 12,000 SFPs (2 accessions) Validated gene models (exon intron) 2 features per exon, alternative splicing 5’ 3’ Untranslated region for gene family/ polymorphism Micro RNAs Validated “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 expression 20 Accessions 3 replicates, (polymorphism accounted for) Cis regulatory variation/ Imprinting reciprocal 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

Pairwise Correlation between and within replicates

LIGHT1 tstat and raw data