eXtreme Array Mapping and Haplotype analysis Using Arrays Justin Borevitz Salk Institute naturalvariation.org

Talk Outline Bulk Segregant Mapping of –Mendelian mutations eXtreme Array Mapping of QTL –Kas x Col RILs and Simulations Haplotype analysis –Patterns Global Variation Selection

Potential Deletions

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

Potential Deletions 111 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

Deletions between Accessions

Fast Neutron deletions FKF1 80kb deletion CHR1cry2 10kb deletion CHR1 Het

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

Other Mendelian mutations aar21 arhythmicein6 ethylene insensitive (no een?) Also aar90, aar60 and stamenstay

Short pool – Tall pool Kas x Col RILs all Features RED2 QTL

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

Simulation Genotypes 15 eXtreme RILs of QTL chr2 37%var chr5 13%var

Simulation Genotypes 100 eXtreme RILs of QTL

Simulation Genotypes 50 eXtreme F2s of QTL

Simulation Chip Noise 50 eXtreme F2s of QTL

Simulation Likelihood 50 eXtreme F2s of QTL

Array Haplotyping Hybridize 48 arrays with 15 accessions ~300ng DNAeasy MiniPrep leaf tissue Overnight Bioprime Klenow labeling 25C "col", "lz", "ler", "bay", "shah", "cvi", "kas", "c24", "est", "kendl", "mt", "nd", "sorbo", "van", "ws2"

Linkage Disequilibrium explained 1 SNP 2 haplotypes Mutation 2 SNPs 3 haplotypes 2 SNPs 4 haplotypes recombination

Pairwise Correlation between and within replicates

Feature Density chr1

Diversity measure

LIGHT1 tstat and raw data

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

Quantitative Trait Loci

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

Review Single Feature Polymorphisms (SFPs) can be used to identify recombination breakpoints, potential deletions Bulk segregant mapping, and –eXtreme Array Mapping of QTL Haplotyping Diversity scans

NaturalVariation.org Salk Jon Werner Sam Hazen Sarah Liljegren Ramlah Nehring Joanne Chory Joseph Ecker UC San Diego Charles Berry Scripps Elizabeth Winzeler Syngenta Hur-Song Chang Tong Zhu Salk Jon Werner Sam Hazen Sarah Liljegren Ramlah Nehring Joanne Chory Joseph Ecker UC San Diego Charles Berry Scripps Elizabeth Winzeler NaturalVariation.org