**The *Populus* Genome Science Plan**

**High-Throughput Phenotyping Panel**

(Panel Members: Mark Davis, Stephen Kelly, Bob Kodrzycki, Laigeng Li,
Gary Peter, Simon Potter, Lacey Samuels, Steve Strauss, Bjorn Sundberg,
Gail Taylor, and Stan Wullschleger)

**Background**

Considerable progress has been made in the development of techniques for
generating populations of transgenic plants. Activation tagging,
gene/promoter trapping, fast-neutron deletion, and RNA silencing have
all been shown effective in creating loss- and gain-of-function mutants
in higher plants. Application of these techniques has led to the
development of large populations of organisms with altered patterns of
gene expression. These populations are proving to be extremely useful in
a wide variety of studies aimed at better understanding processes that
control gene expression and in characterizing the role of specific
genes, gene families, and gene products (i.e., proteins) in plant
biochemistry, physiology, and growth and development.

**Goals and Objectives**

Considering the size of current transgenic populations, and recognizing
that capabilities for generating even larger numbers of transgenic lines
will undoubtedly be enhanced in a post-genomics world, scientists are
finding it necessary to also develop rapid and reproducible methods to
identify changes in plant characteristics (i.e., phenotype). Such
information will be critical if we are to associate altered phenotypes
with the presence or absence of a particular gene, and therein to
determine gene function for the thousands of genes present within the
poplar genome.

With this as one of the principle goals of the International *Populus*
Science Plan, here we identify the tools and technologies required for
the high-throughput characterization of altered plant phenotypes in
trees. Our aim is to identify potential approaches that while still in
their infancy might ultimately serve as high-throughput tools for
assessing gene function in trees. Our emphasis lies in the development
of new and rapid approaches for quantifying variation in wood and/or
tree-specific anatomical, morphological or physiological traits as part
of a future functional genomics effort.

**Scientific Objective #1**

Develop phenotyping tools for traits of interest to the tree biologist.

**1) Wood quality**

Science Question:

(Short term) What is the range of variation in wood quality phenotypes
for selected genotypes, clonal material and control transformed Populus
trees? What are the appropriate ages for wood quality determinations?

(Mid term) What genes affect wood phenotypes?

(Long term) What are the molecular mechanisms that underlay the
variation in wood quality phenotypes? What controls the developmental
mechanisms that affect wood quality phenotypes?

Short-term Goals (1-3 yr):

-   With existing methods, (Molecular Beam MS, Fiber Quality Analyzer,
    Silviscan II, Microcat Scan) determine a baseline and variation in
    wood quality phenotypes (tip to base: pith to bark) for pedigrees in
    breeding populations, clones and Nisqually 1 tissue culture
    regenerated lines for multiple ages

-   Determine appropriate age(s) of trees for reliable comparisons of
    wood quality phenotypes useful for detection of altered phenotypes

-   Develop reliable tests for multiple wood quality property
    determinations from young single seedlings

-   Develop in forest methods to accurately determine wood quality
    phenotypes for standing trees, e.g., via Near Infrared
    Spectroscopy...

Mid-term Goals: (3-10 yr)

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Long-term Goals: (\>10 yr)

-   Isolate and obtain proof of genes that underlay wood quality QTLs

-   Understand the role of allelic interactions in controlling wood
    quality traits

-   Understand interactions between growth rate and juvenility changes
    with wood quality phenotypes

**2) Wood chemistry**

Science Question:

(Short term) What is the range of variation in wood chemistry phenotypes
for selected genotypes, clonal material and control transformed Populus
trees?

(Mid term) What genes affect wood chemistry phenotypes?

(Long term) What are the molecular mechanisms that underlay the
variation in wood chemistry phenotypes? What controls the developmental
mechanisms that affect wood chemistry phenotypes? How early can an
interesting phenotypic trait be detected in transformed tissue?

Short-term Goals:

-   Determine appropriate age(s) of trees for reliable comparisons of
    wood chemistry phenotypes useful for detection of altered phenotypes

-   Identify and develop spectroscopic methods (e.g., NIR, analytical
    pyrolysis, FTIR, Ramen) to rapidly determine and measure changes in
    chemical composition.

-   Identify and develop spectroscopic methods to rapidly determine and
    assign changes in chemical structure (e.g., changes in lignin
    structure such as syringyl to guaiacyl ratio).

-   Develop rapid methods and calibrations for other tree tissues such
    as bark, leaves and roots.

Mid-term Goals:

-   Develop methods necessary to determine associations between wood
    chemistry phenotypes and changes in gene expression

-   Screen populations of transgenic lines for altered wood phenotypes
    with multiple methods and compare the methods for relative strengths
    and weaknesses.

-   Identify genes that affect chemical composition, lignin structure,
    and carbohydrate structure.

-   Develop rapid phenotyping methods to identify changed in metabolic
    profiles related to cell wall chemistry and chemical structure.

Long-term Goals:

-   Develop associations between genetic manipulations, metabolic
    profiles of young transformed tissue (callous tissue or young
    shoots) and adult chemical composition and structure to allow for
    early detection of interesting phenotypic traits.

-   Isolate and obtain proof of genes that underlay wood chemistry QTLs.

**3) Wood anatomy**

Science Question:

Short-term Goals:

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Mid-term Goals:

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Long-term Goals:

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**4) Crown form and architecture**

Science question:

Short-term Goals:

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Mid-term Goals:

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Long-term Goals:

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**5) Leaf expansion and canopy phenology**

Science question:

Short-term Goals:

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Mid-term Goals:

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Long-term Goals:

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**6) Stress physiology**

Science question:

Integrating multiple scales of information that affect phenotype

Short-term Goals:

-   Nutrition

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Mid-term Goals:

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Long-term Goals:

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**7) Pest and Disease Resistance**

Science Question:

Short-term Goals:

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Mid-term Goals:

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Long-term Goals:

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**Scientific Objective #2**

Associate traits of interest as detected in phenotypic studies to gene
expression, metabolic characterization, and protein profiling.

**1) Molecular Phenotyping**

Need to do microarray analyses of gene expression on well characterized
pedigrees and mutants identified in early- and field-detection screens.

**2) Metabolic Phenotyping**

**3) Proteomic Characterization**

**Scientific Objective #3**

Greenhouse, laboratory, and field-level screening of aberrant phenotypes
will generate vast amounts of data that, in order to a valuable
community resource, must not only be archived, but also must be
accessible.

**1) Database**

Databases of phenotypic information from baseline studies and transgenic
populations need to be built and accessible to the community. Linkages
to molecular phenotyping, metabolic profiling, and protein
characterization also needs to be organized

**Conclusion**

Our need for high-throughput phenotyping is certainly not new. Long
before the genomic era, techniques for assessing plant phenotypes were
needed as new cultivars and/or varieties were being developed through
conventional breeding approaches. Today, however, tools specific to the
high-throughput screening of plant phenotypes offers to promote
understanding of gene function and will soon become a valuable component
of the plant genomic toolbox. Although the scientific community
currently relies heavily on visual evaluation of mutants, imaging
techniques that allow immediate and non-invasive detection of altered
plant characteristics, before visual symptoms appear must be developed
for monitoring changes in phenotypic characteristics of transgenic
plants expressing those genes.