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e X treme A rray M apping and Haplotype analysis Using Arrays Justin Borevitz Salk Institute naturalvariation.org

e X treme A rray M apping 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

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e X treme A rray M apping and Haplotype analysis Using Arrays Justin Borevitz Salk Institute naturalvariation.org

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  1. eXtreme Array Mappingand Haplotype analysisUsing ArraysJustin BorevitzSalk Institutenaturalvariation.org

  2. 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

  3. Potential Deletions

  4. False Discovery and Sensitivity PM only GeneChip SAM threshold SFPs nonSFPs Cereon marker accuracy 5% FDR 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: value = 1.845e Chisq = 177.34, df = 1, p - - 40 real data Observed t statistics vs Null (permuted) t statistics 5% FDR Permuted data

  5. Chip genotyping of a Recombinant Inbred Line 29kb interval

  6. 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

  7. FLOWERING1 QTL deletion of MAF1 MAF1 Flowering Time QTL caused by a natural deletion in MAF1 Potential Deletions Suggest Candidate Genes Chr1 (bp)

  8. Deletions between Accessions

  9. Het Fast Neutron deletions FKF1 80kb deletion CHR1 cry2 10kb deletion CHR1

  10. 100 bibb mutant plants Map bibb 100 wt mutant plants

  11. bibb mapping Bulk segregant Mapping using Chip hybridization bibb maps to Chromosome2 near ASYMETRIC LEAVES1 AS1 ChipMap

  12. BIBB = ASYMETRIC LEAVES1 AS1 (ASYMMETRIC LEAVES1) = MYB closely related to PHANTASTICA located at 64cM as1 bibb Sequenced AS1 coding region from bib-1 …found g -> a change that would introduce a stop codon in the MYB domain bib-1 W49* as-101 Q107* bibb as1-101 MYB

  13. Other Mendelian mutations aar21 arhythmic ein6 ethylene insensitive (no een?) Also aar90, aar60 and stamenstay

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

  15. eXtreme Array Mapping Chromosome 2 LOD 16 12 RED2 QTL LOD 8 4 0 0 20 40 60 80 100 cM RED2 QTL Composite Interval Mapping 15 tallest RILs pooled vs 15 shortest RILs pooled QTL likelihood model using bulk segregant analysis with SFP genotyping Red light QTL RED2 from 100 Kas/ Col RILs

  16. Simulation Genotypes 15 eXtreme RILs of 100 2 QTL chr2 37%var chr5 13%var

  17. Simulation Genotypes 100 eXtreme RILs of 700 2 QTL

  18. Simulation Genotypes 50 eXtreme F2s of 500 2 QTL

  19. Simulation Chip Noise 50 eXtreme F2s of 500 2 QTL

  20. Simulation Likelihood 50 eXtreme F2s of 500 2 QTL

  21. 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"

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

  23. S e q u e n c e V a r i a t i o n a t a C a n d i d a t e L o c u s , L I G H T 2 l o c u s ( 6 . 5 k b ) P H Y B L e r P H Y B p r o t e i n ( 1 1 7 2 a a ) S I H L L e r O I L R C o l T I L E U k - 4 R L V S o r b o L V T s u - 1 L V W e i - 0 T L V A V a n - 0 L L V L E m a - 1 E L V C v i - 0 R L V T s - 1 L V S f - 0 L V S e - 0 T h e s e p o l y m o r p h i s m s a r e i n c o m p l e t e L D I 1 4 3 / L 1 0 7 2 x = 8 . 4 ± 0 . 8 m m a s s o c i a t i o n t e s t i n g L 1 4 3 / V 1 0 7 2 x = 1 0 . 2 ± 0 . 4 m m p < 0 . 0 1 ( p e r m u t a t i o n t e s t i n g )

  24. Pairwise Correlation between and within replicates

  25. Feature Density chr1

  26. Diversity measure

  27. LIGHT1 tstat and raw data

  28. LIGHT1 tstat and raw data

  29. Chromosome1 ~500kb Col Ler Cvi Kas Bay Shah Lz Nd Array Haplotyping Inbred lines Low effective recombination due to partial selfing Extensive LD blocks

  30. Quantitative Trait Loci

  31. Feature level model Gene Expression index that accounts for feature effect and polymorphisms FLC controls flowering time Difference detected it 3 day old seedlings

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

  33. 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

  34. NaturalVariation.org NaturalVariation.org Salk Jon Werner Sam Hazen Sarah Liljegren Ramlah Nehring Joanne Chory Joseph Ecker UC San Diego Charles Berry Scripps Elizabeth Winzeler 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

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