**The** ***Populus*** **Genome Science Plan**

Panel on Gene Expression and Microarrays

Panel Members: Peter Nilsson, Wout Boerjan, Jörg Bohlmann, Amy Brunner,

Steve DiFazio, and John MacKay

"Standardization and production of microarrays and analysis tools for
general use in the community as part of a future functional genomics
effort."

**Background**

DNA microarrays have during the last few years made an impressive impact
in the research area of functional genomics. The technology of spotted
cDNA microarrays was pioneered at Stanford University, in the latter
part of the nineties, with the creation of the first whole genome array
for yeast . This was a major breakthrough in microarray technology for
transcript profiling, when the enormous possibilities were realized with
simultaneous analysis of whole transcriptomes. This led to a dramatic
increase in microarray research.

Another factor that affected the breakthrough of microarrays was the
parallel establishment of photolithographically *in situ* synthesized
oligonucleotides and methods to analyze hybridizational signals from
them, developed by Affymetrix, Inc. As more and more sequences became
available through the Human Genome Project and other large-scale
sequencing efforts, it became possible to design short, gene specific
oligonucleotides to be used as immobilized probes. However, this latter
methodology has not yet reached the same broad distribution in academia,
mainly because the costs were initially very high.

Finally, microarrays for transcript profiling based on spotted long
oligos (50-70-mers) have recently been introduced. The primary advantage
of this approach is the reduced effort needed to prepare the probes for
immobilization and the possibilities to design oligos for certain
purposes such as the analysis of splice variants or coverage of closely
related species.

The majority of microarrays for organisms that are not completely genome
sequenced have been based on amplified cDNA. These are usually produced
in the context of EST-sequencing projects, with the creation of
unigene-sets through sequence assembly, preparation of clones by
cultivation, plasmid preparation, PCR, PCR-purification, and transfer to
a suitable spotting plate. For some of the major model organisms, i.e.
those that have been completely sequenced, it is now also possible to
design oligos through bioinformatic efforts and synthesize or buy them
for spotting.

Another alternative is to design gene-specific PCR primers and
individually amplify fragments of all genes, as has been done in the
CATMA project for Arabidopsis.

**Current state of** ***Populus* microarrays**

UPSC/KTH, Sweden

A large scale Tree Functional Genomics program has been run in Sweden by
the Umeå Plant Science Centre in collaboration with KTH, Stockholm. The
first *Populus* arrays that were produced were tissue-specific arrays
with approximately 3000 wood and leaf clones, respectively. The wood
chip has been utilized for a number of studies such as full-scale
analysis of very thin wood-forming tissue sections (*Hertzberg et al,
2001, PNAS*). The very limited amount of starting material for target
preparation led to the development of a methodology for amplification of
minute samples. It is based on capture of short 3'-tags of fragmented
cDNA, which enables non-biased PCR amplification and labeling
(*Hertzberg et al, 2001, Plant J*). Subsequently a unigene collection
based on 33.000 EST sequences was assembled, rendering 13.500 clones
which constitute the POP1 set. More than 2000 POP1 arrays have been
produced at KTH since Nov. 2001 and they are now the basis for many
different experiments for several groups both in Umeå and in Stockholm,
as well as several external collaborators. The next generation, denoted
POP2, will be based on 100.000 ESTs and a unigene set consisting of
25.000 clones. It is now being prepared and is estimated to be ready to
spot in mid 2003.

A research project led by Chung-Jui Tsai at Michigan Technological
University has produced 11,000 ESTs from various tissues of aspen (shoot
tips, young leaves, young stems, root tips) that have been deposited in
GenBank.  6K microarrays are being produced for gene expression analysis
as part of a Michigan Life Sciences Corridor-funded functional genomics
research project to investigate various aspects of growth and
metabolism.  The 6K arrays consist of \>5,000 unique sequences with some
degree of redundancy due to contig-forming overlapping clones.  

Pilot studies using spotted oligos are also underway. Helge Küster,
University of Bielefeld, Germany: 2400 oligos (70-mers from Operon)
selected for their association with the symbiosis between Poplar and
Mycorrhiza. The Forest Biotechnology Group at Oregon State University is
using a 230 oligo (70-mer) array of selected transcription factors and
related regulatory genes to study changes in gene expression associated
with maturation and flowering. Oak Ridge National Laboratory is
producing design software and an array of 1500 70mer oligos to test the
ability to differentiate closely related members of gene families while
still retaining functionality across the genus.

**Scientific objectives**

A whole-genome microarray would be an invaluable resource for the poplar
community, and the most expeditious way to accomplish this is through
international collaboration. The aim is to establish a global collection
of DNA probes for spotting onto microarrays, covering all genes in
*Populus*. Importantly, it should be possible to utilize them for the
majority of the different *Populus* species and subtypes being used for
research. One of the major objectives is also to establish a strong and
active microarray group within the IPGC, working for standardization and
development of analysis tools through a common database. It is also
important that the availability of spotted microarrays is not restricted
to a limited number of research groups. This could be achieved by
assigning some resource centers responsibility for the production and
distribution of microarrays.

**Short-term goals**

The initial aim will be to establish a consensus strategy toward the
common goal of a complete whole-genome array. This will be done within
all research groups with interests in *Populus* microarrays. The aim is
to enable a coherent effort to combine existing and planned
microarray-related *Populus* projects, both in terms of sharing
knowledge and experiences and not the least, physical resources. Limited
local efforts for different species and genotypes could be combined
through exchange of DNA probes and/or sequences. The most important
strategic issue to decide upon is what kinds of array-immobilized probes
to use. The information that will be achieved from the ongoing genome
sequencing and annotation will definitely be a necessary knowledge base
for any attempts to generate a whole genome array. Even if an existing
large-scale EST-sequencing effort has been accomplished to create a cDNA
microarray covering substantial parts of the genome or rather
transcriptome, it is hard to foresee that cDNA will be the ultimate
choice in the end. Instead it is easy to see the advantages of spotted
long oligos, not least in terms of possibilities for complete coverage
and also for the possibilities to design oligos to conserved regions,
enabling a broad usage for the whole *Populus* genus. Problems will of
course appear for the genomes which are not sequenced, but different EST
sequences could possibly aid in the design process. Furthermore, spotted
longmer oligos are advantageous for the flexibility, ease of preparation
and exchange of information regarding oligo sequences.

**Mid-term goals**

The availability of the complete and annotated genomic sequence will
enable the initialization of the bioinformatics process to design
gene-specific oligos. The aim is to collect all publicly available
*Populus* sequence information and also encourage different research
groups in the consortium to provide sequences which are not yet publicly
available. A coherent bioinformatics effort is needed.

A relative high financial investment is initially needed to purchase
complete oligo sets, even though the actual production costs for the
arrays are drastically reduced. However, it is possible that a deal
could be reached with a company to do a large-scale synthesis and sell
small batches to individual groups at a reasonable price.

**Long-term goals**

The long-term goals are to have a solid global organization for all
*Populus* related microarray activities, centralized around the
production and utilization of a common whole genome array.

A complete awareness of the availability of the commonly achieved whole
genome spotted oligo arrays will provide extremely good opportunities
for the whole *Populus* research community to perform many interesting
experiments. An ultimate aim is also to integrate all results in a
common *Populus* expression database, which will enable important
cross-experiment data mining.

**Future applications**

A whole-genome array will be an invaluable tool in poplar functional
genomics for the foreseeable future. The array will continue to provide
a comprehensive view of transcriptome differences among tissue types and
along developmental gradients, allow in-depth characterization of
responses to environmental perturbations, and facilitate the
deconvolution of complex regulatory pathways through the analysis of
mutant lines. In addition, the array will undoubtedly be used in novel
ways such as to derive traits for Quantitative Trait Locus analyses, or
to generate character traits for phylogenetic analyses. As the costs of
array production, labeling, and hybridization continue to fall, the
range of experiments will grow, providing an increasingly detailed view
of the functional genomics of this important model organism.