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Talk Outline

Methods for the identification of Quantitative Trait Loci Justin Borevitz Salk Institute naturalvariation.org. Talk Outline. Intro Natural Variation/ QTL Transcription profiling to identify QTL candidate genes Identification of S ingle F eature P olymorphisms Future Prospects. PHYB.

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Talk Outline

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  1. Methods for the identification of Quantitative Trait LociJustin BorevitzSalk Institutenaturalvariation.org

  2. Talk Outline • Intro Natural Variation/ QTL • Transcription profiling to identify QTL candidate genes • Identification of Single Feature Polymorphisms • Future Prospects

  3. PHYB

  4. differences may be due to expression or hybridization

  5. Deletions

  6. False Discovery and Sensitivity • Cereon • may be a • sequencing • Error • TIGR • match is • a match 90% 80% 70% 41% 53% 85% 90% 80% 70% 67% 85% 100% 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 GeneChip SAM threshold SFPs nonSFPs Cereon marker accuracy 18% FDR 10627 82297 100% Sequence 817 223 594 Sensitivity Polymorphic 340 195 145 57% Non - polymorphic 477 28 449 False Discovery rate: 13% Test for indep endence of all factors: Chisq = 265.13, df = 1, p - value = 1.309e - 59

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

  8. bibb mutant phenotypes • three independent recessive alleles • medial sepals remain attached • “cabbage-like” rosette leaves • flowers open prematurely • fruit appear more slender 23 days Col bib-1 det2-1 Ler bib-3 bib-3 bib-3

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

  10. Future Bulk segregant Mapping 20% variance Extend to Quantitative Traits Map multiple genes (mutation modifiers) Fine mapping after PCR identification of recombinants Multiple models (epistasis) Changes in allele F2, F3 frequency (selection) 20 simulations

  11. 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

  12. Gene expression revisited • Now that we know what features are polymorphic we can determine which gene expression differences are real and which are due to polymorphism. • New RNA analysis algorithm— account for spatial correction, polymorphisms, and feature differences

  13. Genes Expression for a candidate gene Gene Expression index that accounts for feature differences and polymorphisms Spatially corrected log PM intensity Features (probes) for At1g22360

  14. Gene expression revisited SAM True False Difference FDR threshold Positive Positive 0.02 1464 2913.6 -1449.6 1.990164 0.04 831 1067.5 -236.5 1.284597 0.06 409 390.8 18.2 0.9555012 0.08 242 160.2 81.8 0.6619835 0.1 171 73.6 97.4 0.4304094 0.12 131 35.4 95.6 0.270229 0.14 96 20.6 75.4 0.2145833 0.16 60 12.2 47.8 0.2033333 0.18 44 8.2 35.8 0.1863636 0.2 41 6.2 34.8 0.1512195

  15. Gene expression revisited FLC controls flowering time Difference detected at 3 day old seedling stage

  16. Gene expression revisited PAG1 down regulated in Cvi pag1 Knock Out is pale

  17. pag1 KO is light insensitive pag1 KO has long hypocotyl in red light

  18. pag1 KO is early flowering

  19. Salk Jon Werner Todd Mockler Sarah Liljegren Joanne Chory Detlef Weigel Joseph Ecker UC Davis Julin Maloof UC San Diego Charles Berry Scripps Elizabeth Winzeler Salk Jon Werner Todd Mockler Sarah Liljegren Joanne Chory Detlef Weigel Joseph Ecker UC Davis Julin Maloof UC San Diego Charles Berry Scripps Elizabeth Winzeler NaturalVariation.org NaturalVariation.org Syngenta Hur-Song Chang Tong Zhu

  20. Complete Genome Tiling Chip 1st Set 25 bp 25 bp 8 bp 25 bp 25 bp 2nd Set 16 bp 25 bp 25 bp 3rd Set Extra Chips: 9 Whole-Genome Expression Chips 2 Splicing Chips 2 5’ Mapping Chips New Gene Discovery Improve Annotation Alternative Splicing Micro RNAs ChIP – Chip (DNA binding sites) Polymorphisms (re-sequencing) Global methylation (Methylome) Comparative Genomics (Brassica) Validate features

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

  22. Future Projects Design 2nd generation expression array $200 350,000 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

  23. 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

  24. 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

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