Fibre properties that affect paper quality
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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)

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Fibre properties that affect paper quality

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Fibre properties that affect paper quality

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

# of

indiv.

low

high

trait


Strategic grant

  • Goal:

    • Investigate the genetic control of Arabidopsis fibre properties to improve poplar fibre properties

Strategic Grant

  • 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 poplarthrough bioinformatics approaches

    • Poplar studies including reverse genetics, markeridentification


Aspects of phd project defined in grant

Aspects of PhD project defined in grant

Array tools:

30K cDNA 70mer oligomer cDNA arrays

What additional genes emerge as trait-associated?


Additional applications of arrays to the qtl approach

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?


Project hurdles

Project Hurdles

What are relevant stages of development to investigate?

How can we compare expression profiles between ecotypes?

How do we relate the expression data to the QTL study?

What do these array experiments mean if no QTLs are identified?

How do we relate the Arabidopsis fibre story to poplar?

What interesting biological stories can be investigated in parallel

with the strategic grant objectives?

“…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.”


Fibres within complex tissues

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, H2O, nutrition)

Ehlting et al., 2005

Larkin et al., 2003

What is the signaling story surrounding IFF development?

…relating to the establishment of fibre phenotypes?


Gene expression varies widely between root zones

Gene expression varies widely between root zones

D

Hormone-specific zones

Auxin JA GA

C

  • Plus, transcription factor families appear to be zone-specific

Local organization centres in root development


Turning the benfey approach up side down

Turning the Benfey approach up-side-down

Root

Stem

  • 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


Experimental approach

Experimental approach

Extreme Ecotypes or RILs

Isolate mRNAs from IFFs and other cell types (FACs or LCM/RNA) at different developmental stages

Expression profiling of developmentally-equivalent tissues between ecotypes and RILs

Fibre QTL analysis (Phenotyping +Genotyping of RILs)


Challenges to this approach

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


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