Genomics of Natural Variation in Arabidopsis thaliana Justin Borevitz Salk Institute naturalvariation.org

Talk Outline Natural Variation in Light Response –PHYTOCHROME A/QTL mapping –Fine Mapping/ Gene Expression candidates Single Feature Polymorphisms –Deletion/ Candidate genes –Bulk Segregant/ eXtreme Mapping Haplotype analysis

Light Affects the Entire Plant Life Cycle de-etiolation hypocotyl }

Quantitative Trait Loci EPI1 EPI2

Epistasis scan Chr1 Chr2 Chr3 Chr4 Chr5 BQTL 43,956 pair-wise tests 163 markers and 133 intervals Permutation threshold p < 0.05 (5000 permutations)

SNP377 SM184 SM50 SM35 SM106 G2395 SNP65 SM40 SEQ8298 TH1 MSAT7964 MAT7787 CER MbMarker Near-Isogenic Lines for LIGHT1 Ler / Cvi #3 mm 81N-J17A-A/J Ler Plants Line RVE7 GI Phenotype

differences may be due to expression or hybridization Downstream players`

PAG1 down regulated in Cvi PLALE GREEN1 knock out has long hypocotyl in red light

Abundant Genetic Variation in Light Response –Quickly map in new crosses (XAM still to come!) QTL map to novel loci and candidate genes –New crosses find major loci and new loci Gene Expression –NILs, pools of extreme RILs or F2s, –Identify candidate genes at QTL (linked) – or downstream effects of QTL (unlinked)

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

physical location known Genetic Markers in genes

False Discovery and Sensitivity 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 SAM threshold 18% FDR GeneChip SFPs nonSFPs Cereon marker accuracy % Sequence Sensitivity Polymorphic % Non-polymorphic False Discovery rate: 13% Test for independence of all factors: Chisq = , 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% Cereon may be a sequencing Error TIGR match is a match

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 FLOWERING1 QTL Chr1 (bp) Flowering Time QTL caused by a natural deletion in MAF1 MAF1 MAF1 natural deletion

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

eXtreme Array Mapping 15 tallest RILs pooled vs 15 shortest RILs pooled

LOD eXtreme Array Mapping Allele frequencies determined by SFP genotyping. Thresholds set by simulations cM LOD Composite Interval Mapping RED2 QTL Chromosome 2 RED2 QTL 12cM Red light QTL RED2 from 100 Kas/ Col RILs

Single Feature Polymorphisms –Improve with replicates (easy) –Improved statistical models Genotyping –Precisely define recombination breakpoints –Fine mapping –Gene conversion Potential Deletions –Candidate genes/ induced mutations Bulk segregant Mapping –eXtreme Array Mapping, F2s etc

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 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

Sequence confirmation of SFPs

SFPs for reverse genetics 14 Accessions 30,950 SFPs`

Chromosome Wide Diversity

Diversity 50kb windows

Tajima’s D like 50kb windows

Remember to think about hybridization polymorphism in RNA analysis (affy or cDNA) Keep in mind that DNA can be used on many arrays Example for mapping ESTs Haplotyping Diversity/Selection Association Mapping –Population Genomics (hybrid zones)

RNADNA Universal Whole Genome Array Transcriptome Atlas Expression levels Tissues specificity Transcriptome Atlas Expression levels Tissues specificity Gene Discovery Gene model correction Non-coding/ micro-RNA Antisense transcription Gene Discovery Gene model correction Non-coding/ micro-RNA Antisense transcription Alternative Splicing Comparative Genome Hybridization (CGH) Insertion/Deletions Comparative Genome Hybridization (CGH) Insertion/Deletions Methylation Chromatin Immunoprecipitation ChIP chip Chromatin Immunoprecipitation ChIP chip Polymorphism SFPs Discovery/Genotyping Polymorphism SFPs Discovery/Genotyping ~35 bp tile,non-repetitive regions, “good” binding oligos,evenly spaced

ChipViewer: Mapping of transcriptional units of ORFeome From 2000v At1g09750 (MIPS) to the latest AGI At1g v Annotation (MIPS) The latest AGI Annotation

SNP SFP MMMMMM MMMMMM Chromosome (bp) conservation SNP ORFa start AAAAA Transcriptome Atlas ORFb deletion Improved Genome Annotation

Haplotype Map – Linkage Disequilibrium, Gene Family (R genes) Association Studies – Whole Genome Arrays 192 Accessions, > 200,000 SFPs (~600bp resolution ) Confirm Associations in specific crosses with eXtreme Array Mapping Future Projects DNA

True natural variation in gene expression polymorphism accounted for, alternative splicing Cis regulatory variation/ Imprinting reciprocal F1s 3 replicates Transcriptome QTL Map – VanC Advanced Intercross Recombinant Inbred Lines How many loci control the variation in gene transcription? Candidate TF and binding sites? Future Projects RNA

Future work with Natural Variation VanC advanced intercross RIL population Backcross collections

Salk Jon Werner Todd Mockler Sarah Liljegren Olivier Loudet Huaming Chen Joanne Chory Detlef Weigel Joseph Ecker UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones USC Magnus Nordborg Tina Hu Syngenta Hur-Song Chang Tong Zhu NaturalVariation.org Salk Jon Werner Todd Mockler Sarah Liljegren Olivier Loudet Huaming Chen Joanne Chory Detlef Weigel Joseph Ecker UC San Diego Charles Berry Scripps Sam Hazen Elizabeth Winzeler UC Davis Julin Maloof University of Guelph, Canada Dave Wolyn Sainsbury Laboratory Jonathan Jones USC Magnus Nordborg Tina Hu Syngenta Hur-Song Chang Tong Zhu Helen Hay Whitney Foundation