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Hardy Hall PhD Prospect July 6, 2006 Ph.D. Candidacy Examination

Investigation of the Genetic Control of Fibre Length in Arabidopsis thaliana through Gene Expression Profiling of the Intrusive Growth Phase of Interfascicular Fibres. Hardy Hall PhD Prospect July 6, 2006 Ph.D. Candidacy Examination. Outline. Background Why study fibres?

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Hardy Hall PhD Prospect July 6, 2006 Ph.D. Candidacy Examination

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  1. Investigation of the Genetic Control of Fibre Length in Arabidopsis thaliana through Gene Expression Profiling of the Intrusive Growth Phase of Interfascicular Fibres Hardy Hall PhD Prospect July 6, 2006 Ph.D. Candidacy Examination

  2. Outline • Background • Why study fibres? • What regulates fibre growth? • How can we study complex traits? • Why study genetic regulation of fibre length in Arabidopsis? • Thesis objectives • Work plan • Timeline • Conclusion

  3. Hemp Poplar Arabidopsis Bromeliadleaf High-rise pith fibres ≈ rebar W. Barthloltt, ‘83. Queens Univ. ifl1 Fibres provide strength and elasticity • Long • Tapered • 2o cellwall (thick, lignified) • Dead at maturity Mutations affecting fibre properties affect stem architecture Burk, ‘02. Burk, ‘02 Fibre cells are key structural cells in vascular plants Fibres occur in many seed plants phloem xylem interfascicular South Dakota State Univ. Zhong, ‘97

  4.       • Programmed cell death Mature fibre property determinants • Cell expansion • Intrusive growth • Cessation of elongation • Cell wall fortification • Developmental gradients

  5. Systematic genetics approaches to fibre properties • Classical genetics (phenotype  gene) • Mutant libraries + complex phenotyping • Reverse genetics (gene phenotype) • NOT PRACTICAL • Quantitative genetics (phenotype  gene) • Natural variation + inbreeding (RILs) + genetic maps • Expression profiling (gene - phenotype) • Natural variation + microarrays • eQTL : Genetic maps + RILs + microarrays

  6. 18 Lehle Ecotypes 150 ABRC accessions 30 20 Frequency 10 0 0.4 0.6 0.8 1.0 1.2 Length (mm) 0.3 0.5 0.7 0.9 1.1 Length (mm) Model systems for studying fibre length • Poplar and eucalyptus are model woody species • Poplar exhibits natural variation in fibre length • Poplar is a challenging genetic model • Arabidopsis exhibits natural variation in fibre length

  7. Size Generation time Genetic and physical maps Ploidy Zhang, ‘03 Shimuzu, ‘00 Gunning (web) Oppenheimer (web) Functional annotation Compatibility Knowledge base Genome size Arabidopsis is a model for studying fibre length • Arabidopsis is a genetic model for complex traits • Arabidopsis is a model for cell expansion

  8. What genes affect fibre development? Ifl1/rev fra1,2,3 Problem statement Thesis objectives • Identify milestone and gradients of fibre morphogenesis • Characterize growth mode of fibres (intrusive?) • Identify genes that regulate fibre length • Characterize function of fibre length genes What are the genetic determinants of fibre length?

  9. Ontogenesis • Prophase-specific cyclin activity, mitotic figures, DNA replication B. Programmed cell death • Mitochondrial PT, vacuolar collapse, DNA fragmentation C. Correlate ‘A’ and ‘B’ with stem morphometrics • Diffuse stem elongation rates, internode number, silique emergence Work plan 1. Determination of fibre developmental gradients and milestones Dan, ‘03 Gunurwardena, ‘04

  10. Work plan 2. Examination of mode of fibre cell expansion bamboofibres • Identify intrusive growth events • Symplastic disruption and isolation, degradation of middle lamella Symplastic continuity by plasmodesmata B. Investigate cell wall and cytoskeletal ultrastructure (evidence for diffuse or tip growth) • Vesicle distribution, microtubule/microfilament dynamics, microfibril orientations C. Less-destructive indicators of diffuse growth • Epidermal cells as diffuse/intrusive expansion indicators? • Live-cell imaging (non-transgenic approaches) Gritsch, ‘05 Ageeva, ‘05 Suh, ‘05

  11. Sampling strategies 3. Cellular ontogeny dividing mature 1. Internode # from SAM 2. Distance from SAM Establishing developmental equivalence • Variable morphologies • Bolt timing • Growth rate • Branch number • Internode spacing • Sources • Genotype • Microclimate • Stochastic development Hertzberg, ‘04

  12. sampling point observation period LCM sampling Epidermis (E) Vascular bundle (VB) Pith (P) Segment sectioning Interfascicular fibres (IFF) Epidermal cell length Cortex (C) Fibre length Biochemical analysis Chemical Fixation LCM RNA amplification Cryostat Expression profiling Live cell imaging Pooling microarray qPCR Epidermal cell length Sampling schematic Segment selection Expansion rate (mm hr -1) 5 mm increments 1 2 3 4 5 6 7 8 Time (days) 3 mm h-1

  13. D. Expression QTL • Correlate 30K oligo expression profiles with mapped markers Work plan3. Gene expression profiling • qPCR of known developmental markers • Correlate with fibre development milestones • Direct expression profile sampling B. Global expression profiling (multi-factor) • Cell type • Developmental stage (expanding vs. fortifying) • Genotype (short- vs. long-fibred) Ehlting, ‘05 C. Follow-up qPCR • Validate interesting array expressions • Investigate candidate genes over wider factor range

  14. Work plan4. Functional characterization of candidate genes B. Identify candidate genes • Bioinformatics (Genevestigator, AtGenExpress) • Consensus QTL and expression profiling (eQTL) A. Functional genomics • Clustering, PCA, gene ontologies, pathway-mapping C. Localization • Gene expression (promoter::GUS/xFP) • Protein expression (ORF::xFP)

  15. Determine fibre developmental gradients/milestones Monitor stem expansion Fibre origin - DAPI Fibre cell wall status (microfibril angle, lignification) Fibre death - TUNEL Determine intrusive growth timing/localization Locate intrusive growth events Epidermal cell study Find ultrastructure correlatives Stem prep for confocal work Tissue-specific expression profiling qPCR marker survey qPCR follow-up RNA amplification trials Conventional microarrays (20 genotypes/2 stages) LCM trials eQTL (100 RILs, 1 stage) Conventional QTL of fibre length RILs Candidate gene selection RIL population generation RILs Available Fine-mapping Functional characterization of candidate genes UPSC collaboration? Reverse genetics of candidate genes - intrusive events Identification of poplar homologs Timeline 2006 2007 2008 2009 Main Tasks Key Standardizations

  16. Conclusions • Fibre length varies amongst natural accessions of Arabidopsis • Understanding the genetic regulation of fibre development requires systematic approach (QTL + expression profiling) • Description of fibre morphogenesis in Arabidopsis is novel and will help expression profiling • This project offers many opportunities to take advantage of new developments

  17. Acknowledgements • Collaborators • Rodger Beatson (BCIT/Forestry,UBC) • Thomas Berleth (Botany, UofT) • Richard Chandra (Forestry,UBC) • Marcus Shi (Botany, UofT) • Harry Chang (Forestry,UBC) • George Soong (Forestry,UBC) • Brian Poole (BCIT) • Paul Bicho (Paprican) • Committee • Brian Ellis (Supervisor, UBC) • Carl Douglas (Co-supervisor, UBC) • Lacey Samuels (Botany,UBC) • Geoff Wasteneys (Botany,UBC) • Shawn Mansfield (Forestry,UBC) • Substitute advisor • Ljerka Kunst (Botany,UBC) • Personal support team • - Noriko Tanaka (Home) • Botany technical consultants • Eiko Kawamura (Botany, UBC) • Minako Kaneda (Botany,UBC) • David Johnston (Botany,UBC) • Michael Friedman (Forestry, UBC)

  18. Khumbu ice falls, Mount Everest

  19. Supplements • List of contingencies • Fibre length variation along mature Col-0 stems • Fibre length does not correlate with plant height

  20. Contingencies List Problems Solutions Fibre growth is diffuse Consider more general process (other cell types) Fibre growth already characterized Good! On to the arrays! LCM is not possible Hand cut sectioning RNA can’t be amplified reliably Pool tissue from replicates Array lists are unintelligible Be pre-emptive, Do more bioinformatics! Conventional QTL yields no candidates Utilize published QTL data, UPSC? Fine! Lots of great fibre biology left to explore! Arabidopsis fibre length genes already found

  21. N=5 N=5 N=4 N=7 Fibre length variation along mature Col-0 stems FQA analysis of 3-5mg samples 31% fines 28% fines 28% fines 32% fines

  22. Fibre length does not correlate with plant height

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