1. Fibre properties that affect paper quality Strength –Microfibril length/thickness –Hydrogen bonding between microfibrils and other cell wall constituents –Cell length –Cell wall thickness Length –Cell expansion (longitudinal vs. lateral) –Cell division (rate and/or extent) Lignin:cellulose content Compressibility Bondability Quantitative Traits trait lowhigh # of indiv.

2. Strategic Grant Goal: Investigate the genetic control of Arabidopsis fibre properties to improve poplar fibre properties Strategy: Identify fibre properties that vary across Arabidopsis ecotypes…optimize high-throughput means of quantification Choose extreme ecotypes for chosen traits…perform initial expression profiling and generate recombinant inbred lines (RILS) Identify suitable markers (50 for 500cM genome) for QTL analysis Perform ‘coarse’ QTL analysis to reveal large effect loci (LELs or QTLs)…support with expression profiling of most dissimilar RILs Identify candidate genes from ‘fine’ mapping and confirmation of QTLs (introgress QTL region into parentals) Investigate candidate genes through molecular genetics and knockout line characterization Identify orthologs in poplar through bioinformatics approaches Poplar studies including reverse genetics, marker identification

3. Aspects of PhD project defined in grant Strategic grant issuesApproach What expression differences exist between ecotypes that differ markedly for a specific fibre property? Expression profiling of >2 contrasting ecotypes for each fibre property Are transcriptional differences seen between extreme ecotypes more pronounced in the extreme RILs? 20 RILs from each extreme for each trait What additional genes emerge as trait-associated? Array tools: 30K cDNA 70mer oligomer cDNA arrays

4. Additional applications of arrays to the QTL approach Marker discovery Differential expressions can be used to detect polymorphisms Bulk segregant analysis Given known markers, arrays can be used to detect over-represented alleles of extreme traits Expression QTL (eQTL) analysis “gene expression is a complex trait” - eQTLs often map to QTLs Arraying tools: Arabidopsis Oligomer Arrays? Affymetrix?

5. Project Hurdles What interesting biological stories can be investigated in parallel with the strategic grant objectives? 1.What are relevant stages of development to investigate? 2.How can we compare expression profiles between ecotypes? 3.How do we relate the expression data to the QTL study? 4.What do these array experiments mean if no QTLs are identified? 5.How do we relate the Arabidopsis fibre story to poplar? “…seems likely that biophysical properties of fibres are closely linked to certain biological processes, such as cambial cell cycling, cell elongation, cell wall synthesis and lignification.”

6. Fibres within complex tissues Arabidopsis stems are composed of many cell types Cells and tissues are constantly changing (transcript profile included) Development is plastic Cell fate is largely dictated by external cues Adjacent cells Distal signals (hormones, transcription factors,…) Environmental cues (day length, temperature, H 2 O, nutrition) Larkin et al., 2003 Ehlting et al., 2005 What is the signaling story surrounding IFF development? …relating to the establishment of fibre phenotypes?

7. Gene expression varies widely between root zones C Auxin JA GA D Hormone-specific zones Plus, transcription factor families appear to be zone-specific Local organization centres in root development

8. Turning the Benfey approach up-side-down Scaled-down approach is reasonable given that… Precise identification of cell types is possible (growing libraries of developmental markers) Vast majority of Arabidopsis [pulped] fibers are intrafascicular (easy to identify), allowing a fibre-centric approach Growing number of developmental models for cell-cell interactions (Benfey, Larkin) Cell-specific expression profiling is becoming more common-place Fluorescence-assisted cell sorting (FACs) Laser capture microscopy (LCM) + RNA amplification Root Stem

9. Experimental approach Isolate mRNAs from IFFs and other cell types (FACs or LCM/RNA) at different developmental stages Extreme Ecotypes or RILs Fibre QTL analysis (Phenotyping +Genotyping of RILs) Expression profiling of developmentally-equivalent tissues between ecotypes and RILs

10. Challenges to this approach Tissue equivalence Window of equivalence Post hybridization alignment (tissue normalization) possible? Sample preparation mRNA levels for hybridization mRNA stability for given extraction method Array experimental design eQTL approaches feasible? (Affy vs. Oligomer arrays) Array analysis - directed approaches are required! Regulation common to all fibres (xylary, extraxylary) IFF transcription profile Localized centres of regulation of IFFs (ex. Endodermis?) Relating findings to poplar (apples and oranges?) Poplar pulp…xylary Arabidopsis pulp….extraxylary