SNP/Tiling arrays for very high density marker based breeding and QTL candidate gene identification Justin Borevitz Ecology & Evolution University of Chicago

Major Issues in Breeding Complex Traits High throughput Phenotyping –Physiological dissection of 1000s correlated traits –Biological Variation Multiple genes under major QTL –High Density markers –High throughput seedling screens –Linkage Drag Environmental Interaction (GxE) –Good for optimizing local varieties Epistasis (GxG) –Magnify minor QTL in local backgrounds Multi species ecological interactions –“extended phenotype”

Genomic Breeding Path QTL gene Confirmation Marker Identification Genotyping Genomics path Experimental Design Mapping population Phenotyping QTL Analysis Fine Mapping Candidate gene Polymorphisms gene expression loss of function QTL gene Confirmation Experimental Design Mapping population Phenotyping QTL Analysis Fine Mapping Borevitz and Chory, COPB 2003

Talk Outline Phenotyping in multiple environments –Seasonal Variation in the Lab Germplasm Diversity –Population structure, Haplotype Mapping set SNP/Tiling microarrays –Very High Density Markers –Mapping Extreme Bulk Segregant –Expression, splicing, and allelic variation Ecological context –Arabidopsis and Aquilegia Phenotyping in multiple environments –Seasonal Variation in the Lab Germplasm Diversity –Population structure, Haplotype Mapping set SNP/Tiling microarrays –Very High Density Markers –Mapping Extreme Bulk Segregant –Expression, splicing, and allelic variation Ecological context –Arabidopsis and Aquilegia

Begin with regions spanning the Native Geographic range Nordborg et al PLoS Biology 2005 Li et al PLoS ONE 2007 Tossa Del Mar Spain Lund Sweden

Seasons in the Growth Chamber Changing Day length Cycle Light Intensity Cycle Light Colors Cycle Temperature Sweden Spain Seasons in the Growth Chamber Changing Day length Cycle Light Intensity Cycle Light Colors Cycle Temperature Geneva Scientific/ Percival

Kurt Spokas Version 2.0a June 2006 USDA-ARS Website Midwest Area (Morris,MN)

Flowering time QTL, Kas/Col RILs Sweden 1 Col-gl1 Kas1 Sweden 2 Col-gl1 Kas1 Spain 1 Col-gl1 Kas1 Spain 2 Col-gl1 Kas1 Number of RILs Flowering time QTL, Kas/Col RILs FRI FLM

Kas/Col flowering time QTL GxE Chr4 FRI Chr1 FLM Chr4 FRI

Global and Local Population Structure Olivier Loudet

144 Non singleton SNPs >2000 accessions Global, Midwest, and UK common haplotypes Local Population Structure Megan Dunning, Yan Li

80 Major Haplotypes Diversity within and between populations

RNA DNA Universal Whole Genome Array Transcriptome Atlas Expression levels Tissues specificity Transcriptome Atlas Expression levels Tissues specificity Gene/Exon Discovery Gene model correction Non-coding/ micro-RNA Gene/Exon Discovery Gene model correction Non-coding/ micro-RNA Alternative Splicing Comparative Genome Hybridization (CGH) Insertion/Deletions Copy Number Polymorphisms Comparative Genome Hybridization (CGH) Insertion/Deletions Copy Number Polymorphisms Methylation Chromatin Immunoprecipitation ChIP chip Chromatin Immunoprecipitation ChIP chip Polymorphism SFPs Discovery/Genotyping Polymorphism SFPs Discovery/Genotyping Control for hybridization/genetic polymorphisms to understand TRUE expression variation RNA Immunoprecipitation RIP chip RNA Immunoprecipitation RIP chip Antisense transcription Allele Specific Expression

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

Which arrays should be used? cDNA array Long oligo array BAC array

Which arrays should be used? Gene array Exon array Tiling array 35bp tile, 25mers 10bp gaps

Which arrays should be used? Tiling/SNP array k SNPs, 1.6M tiling probes SNP array Ressequencing array How about multiple species? Microbial communities? Pst,Psm,Psy,Psx, Agro, Xanthomonas, H parasitica, 15 virus,

Global Allele Specific Expression Zhang, X., Richards, E., Borevitz, J. Current Opinion in Plant Biology (2007) 65,000 SNPs Transcribed Accession Pairs 12,000 genes >= 1 SNP 6,000 >= 2 SNPs

Potential Deletions

Deltap0FALSECalledFDR % % % % % SFP detection on tiling arrays

Chip genotyping of a Recombinant Inbred Line 29kb interval

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

Array Mapping Hazen et al Plant Physiology (2005) chr1 chr2 chr3 chr4 chr5

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 (Wolyn et al Genetics 2004)

eXtreme Array Mapping BurC F2

XAM Lz x Col F2 QTL Lz x Ler F2 (Werner et al Genetics 2006)

X RED2 QTL mark1 mark2 Select recombinants by PCR >200 from >1250 plants High Low ~2Mb ~8cM >400 SFPs Col Kas Col het Col ~2 Kas hetCol het ~43 Kas Col Kashet Kas ~268 ~43~539 ~43 ~268~43 ~2 het ~539 Kas eXtreme Array Fine Mapping

Unite Genetic and Physical Map Shotgun genomic or 454 reads ESTs/ cDNAs/ BAC ends 1000s of contigs Genotype mapping population on arrays –Create very high density genetic map Known position of genes/contigs allow QTL candidatet gene identification –Control hybridization variation for gene expression

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 FLM MAF1 FLM natural deletion (Werner et al PNAS 2005)

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

Ecological and Evolutionary context Abiotic conditions –Light, temperature, humidity –Soil, water Biotic conditions –Pathogens and pollinators –Conspecifics, grasses, shrubs, trees Industrial Agriculture -> Sustainable EcoAgriculture Green, Super Hybrids!

Local adaptation under strong selection

Seasonal Variation Matt Horton Megan Dunning

Aquilegia (Columbines) Recent adaptive radiation, 350Mb genome

Genetics of Speciation along a Hybrid Zone

Aquilegia (Columbine) NSF Genome Complexity Microarray floral development –QTL candidates Physical Map (BAC tiling path) –Physical assignment of ESTs QTL for pollinator preference –~400 RILs, map abiotic stress –QTL fine mapping/ LD mapping Develop transformation techniques –VIGS Whole Genome Sequencing (JGI 2007) Scott Hodges (UCSB) Elena Kramer (Harvard) Magnus Nordborg (USC) Justin Borevitz (U Chicago) Jeff Tompkins (Clemson)

NaturalVariation.org USC Magnus Nordborg Paul Marjoram Max Planck Detlef Weigel Scripps Sam Hazen University of Michigan Sebastian Zoellner USC Magnus Nordborg Paul Marjoram Max Planck Detlef Weigel Scripps Sam Hazen University of Michigan Sebastian Zoellner University of Chicago Xu Zhang Yan Li Peter Roycewicz Evadne Smith Megan Dunning Joy Bergelson Michigan State Shinhan Shiu Purdue Ivan Baxter University of Chicago Xu Zhang Yan Li Peter Roycewicz Evadne Smith Megan Dunning Joy Bergelson Michigan State Shinhan Shiu Purdue Ivan Baxter