Plant Physiol, December 2000, Vol. 124, pp. 1477-1480
Seed and Molecular Resources for Arabidopsis
Randy L.
Scholl,*
Sean T.
May, and
Doreen H.
Ware
Department of Plant Biology, Ohio State University, 1735 Neil
Avenue, Columbus, Ohio 43210 (R.L.S., D.H.W.); and School of
Biosciences, Plant Science Division, Life and Environmental Sciences
Building University Park, Nottingham NG7 2RD Nottingham, United Kingdom
(S.T.M.)
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INTRODUCTION |
Arabidopsis, because of its
diminutive size, small genome, and rapid life cycle, has become the
premiere research organism in plant biology. The power of Arabidopsis
as a model system will become even greater now that all five of its
chromosomes have been sequenced.
Before 1990, extensive collections of Arabidopsis mutants were
maintained by individual scientists, most notably Drs. A. R. Kranz, G. P. Rèdei, and Maarten Koornneef. It is fortunate
that it was recognized early on that the establishment of seed stock centers would greatly expedite the dissemination of Arabidopsis research material to the international research community, and thus
three major facilities were established. These include the National
Science Foundation-supported Arabidopsis Biological Resource Center
(ABRC) in the United States, the Biotechnology and Biological Sciences
Research Council-supported Nottingham Arabidopsis Stock Centre (NASC)
in the UK, and Sendai Arabidopsis Seed Stock Center (SASSC) in Japan.
The role of a seed stock center is to collect, propagate,
preserve, and distribute seed lines which are of use to researchers. Mutant lines are the primary core of a seed collection. Arabidopsis researchers are fortunate to have access, through the stock centers, to
approximately 1,000 mutants affecting several hundred separate gene
loci. As we enter the post-genomic era this number will be augmented
greatly. The mutant lines currently available possess a wide range of
interesting characteristics useful to modern biological research. The
genes affected by many of these mutations have been cloned and
characterized, and there is potential benefit to be derived from the
use of appropriate mutants in all areas of plant research.
To date, over 4,000 scientists of diverse backgrounds have utilized the
stock centers. The numbers of stocks distributed from and users
accessing the stock centers have increased steadily since their
inception. ABRC currently sends out 50,000 seed samples and 20,000 DNA
stocks annually, and NASC sends 25,000 stocks per year. Distribution
statistics for the previous five years are shown in Figure
1. Among seed stocks 5,000+ samples of
mutant lines are sent annually and 25,000 samples of T-DNA lines are distributed by ABRC.
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Figure 1.
Number of stocks sent annually, by stock type, for
ABRC seed stocks (A), NASC seed stocks (B), and ABRC DNA stocks
(C).
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SEED STOCKS |
Diverse resources are available to Arabidopsis researchers from
public and private sources. Various molecular resources are also
provided: ABRC maintains and distributes a comprehensive collection of
DNA stocks and NASC distributes plant DNA for reverse genetic applications.
Various single and group stocks, which aid in genetic analyses
associated with basic investigations and gene cloning, have been
developed. Mapping resources include mapped mutants, multiple marker
stocks, four populations of recombinant inbred lines, trisomic lines,
and a population organized by tetrads. Transposon and T-DNA populations
with random insertions throughout the genome have been produced which
enable a line to be identified by phenotype and utilized to identify
and characterize the associated insertion point. Sets of randomly
generated T-DNA lines have been donated so that the current holdings of
T-DNA stocks exceed 100,000. Large numbers of characterized lines,
having transpositions to random locations, are available from the stock
centers. A population with transposed Ds elements is maintained by Dr.
R. Martienssen, Cold Spring Harbor Laboratory (Cold Spring Harbor, NY)
from which lines of interest can be obtained. In addition to the above,
lines transformed with specific transgenes and molecular tags,
transposon parental stocks for generation of new transpositions by
researchers, and several related species are carried by ABRC and NASC.
Populations of seeds mutagenized by chemicals or radiation and bulk
quantities of several ecotypes can be purchased from Lehle Seeds (Round
Rock, TX).
Natural variants ("ecotypes") collected in the wild from around the
world have proven to be useful tools for genetic analyses of
Arabidopsis. The availability of these from ABRC, NASC, and SASSC has
enabled researchers to characterize variation associated with diverse
environmental factors and to clone novel genes.
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MOLECULAR RESOURCES |
DNA Stock Centers
The DNA resources available from ABRC are distributed in different
forms including single characterized plasmids, expressed sequence tags
(ESTs), libraries, and isolated plant DNA. The characterized clones
include known genes, expression marker constructions, and binary
vectors used for plant transformation. A large collection of EST cDNAs
derived from several different EST projects is maintained. Analysis of
the clones suggest that the ESTs currently represent approximately
one-third of the genes of Arabidopsis, and the representation is
expected to increase in the near future as new collections are added. A
minimized EST collection is available as a set on microtiter plates or
as colony blots of clones onto nylon hybridization filters. ESTs from
similar collections are also available from Incyte Corporation (Palo
Alto, CA).
Various cDNA and genomic libraries can be obtained from ABRC. The cDNA
libraries have been isolated from different sources including whole
plants, seedlings, and flowers. Some libraries have been size-selected
to facilitate isolation of larger clones. Depending on the vector,
these libraries may be used for DNA hybridizations, antibody screening
for expressed peptides, and protein-protein interactions in the case of
two hybrid libraries. Genomic libraries available from the ABRC include
phage, cosmid, yeast artificial chromosomes, bacterial artificial
chromosomes (BACs), and plant-transformable BACs (transformable BACs
and binary BACs). Available BACs include those utilized by the
Arabidopsis Genome Initiative public genome sequencing projects.
The BACs are useful for positional cloning. The BAC libraries are
spotted onto nylon so that the filters can be ordered, hybridized to a
probe of interest, and the positive clone(s) obtained from the Center.
The individual transformable BACs can be used for complementation
testing. The available BACs and ESTs, in conjunction with the mapping
resources of the stock centers, provide a powerful set of resources for
positional gene cloning, complementation, expression, and protein characterization.
In conjunction with the seeds available for forward genetics, DNA pools
are offered by the stock centers for reverse genetics and can be
assayed in the user's laboratory for insertion in their specific
gene(s). These DNA samples complement the "knockout" services of
the Arabidopsis Knockout Facility (AKF), which conducts PCR screens
using primers provided by participating laboratories. DNA isolated from
12,000 T-DNA lines is currently available from ABRC and will be
expanded to a total of 40,000+. NASC has DNA for 20,000 transposon
lines, which will be expanded to 40,000+, including some lines
identified by sequences flanking the insertions.
Service Centers
NASC is currently developing a microarray hybridization service
similar to the type offered by the Arabidopsis Functional Genomics Consortium (AFGC). This service will have similar
organizational details and is described in the accompanying article on
microarray analysis. ESTs used in the generation of AFGC microarrays
are available from ABRC.
The AKF, which is part of AFGC, conducts PCR screens using primers
provided by participating laboratories. This, in conjunction with the
DNA samples distributed by the stock centers, provides substantial
populations for reverse genetics. The follow-up seeds for the AKF
service are distributed from ABRC.
An antibody service available from Rose Biotechnology (Winchendon,
MA), a private company, is currently being established. This is
in collaboration with Secant Chemicals (Winchendon, MA) and Rose
Scientific (Winchendon, MA). A few antibodies are currently available, including ones raised against vesicular proteins, essential components of the secretion pathway in plants. An additional 15 antibodies will shortly be made available as markers for a range of
cellular processes and organelle-specific antibodies. An Antibody Distribution Center is planned that if supported by donations from the
community will provide an infrastructure for redistribution of
antibodies developed in academic laboratories.
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ORDERING OF STOCKS AND BIOINFORMATICS |
The bioinformatics components of the stock centers are
focused on providing detailed stock information and ordering
facilities. The ABRC site, AIMS
(http://aims.cse.msu.edu/aims/), is maintained by Dr. Sakti
Pramanik and associates of the Computer Science and Engineering
Department at Michigan State University. AIMS is a relational database
which is utilized for processing stock orders, checking progress of
orders, and examining the order histories of individual stocks. The
NASC web site (http://Arabidopsis.org.uk/) has a
similar ordering facility, stock information, and an insert sequence
blast facility. In addition, a public mapping service derived from the
Lister and Dean RI lines resides at the NASC site as part of a
synteny-driven database of genetic information for Arabidopsis and crop
plants
UK Cropnet (http://ukcrop.net/).
All stocks of ABRC and NASC can be ordered through the web sites
(Table I). The ordering pages can be
found as links to the home pages and used to enter patron information
and request stocks. AIMS requires a login ID and password which can be
obtained by request through the web site. Users may track the progress
of their orders in AIMS. For NASC, an order form linked to the home page can be filled out and is automatically entered in the
database.
In addition to placement of orders, scientists can use the stock center
databases to locate stocks, obtain detailed stock information, and
communicate with center staff. In AIMS there are separate search pages
for seed and DNA stocks. The simplest search mechanism is the
"keyword" search. This window, linked to the main seed and DNA
search pages, queries all stock categories relative to any single-word
term. Thus "apetala" could be used to find all stocks of this gene
class, or "apetala1" could be used to locate all stocks relevant to
the ap1 locus. A phenotypic term such as "glabrous"
could be entered, as could the name of a potential stock donor such as
"Sundaresan." Once a list of stocks is found, any combination of
items can be selected for ordering. The search window for stocks in the
NASC database operates in much the same way. The NASC server
incorporates a "shopping cart" mechanism for collecting found
stocks for ordering purposes. Another search mechanism of the AIMS and
NASC databases is the "text-based" search. This search page allows
more advanced searches to be executed. A simple example would be the
entry of "apetala" in the "gene name" windowthis query
identifies stocks that carry this gene name and excludes cases where
the term may be mentioned in the general description of the stock.
Entry of terms in each of two windows of the page for a search utilizes
these jointly, connected by a logical "and" in AIMS and a logical
"or" in NASC.
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FEES ASSOCIATED WITH STOCKS |
The ABRC and NASC provide stocks to Arabidopsis users
for fees that are less than the production/distribution costs. Prior to
1997 the stock centers' only outside source of support were the
granting agencies, but in recent years partial recovery of operating
costs has been achieved through a user fee system. Two types of fees
are levied: annual laboratory user fees, and individual fees for
aggregate stocks. The annual academic usage fee is $250 (ABRC) or £168
(NASC). Bulk seed and DNA stocks such as sets of T-DNA lines,
recombinant inbred populations, and libraries have associated
individual fees, which are levied in addition to the annual laboratory
user fees. Details of other usage levels and individual stocks are
described on the web sites (Table I).
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FUTURE PLANS |
As plant research moves to a "post-genomic" era, the stock
centers face the challenge of adapting to the accelerating pace of
science, including the incorporation of increasing numbers of novel
resources. In some cases choices may have to be made and resources
organized to maximize their usefulness and allow the greatest number of
items to be distributed. However, over time, an organized set of
loss-of-function mutants corresponding to each Arabidopsis gene will
effectively replace the random insertion populations. Novel mutational
approaches are also being applied and the resulting strains and/or
populations will be sought. New collections of natural variants,
related species, and mapping populations will also be added. For the
DNA collection it is expected that a complete set of ESTs will be
received, with most if not all of these being in the form of
full-length clones. The collection of BACs will be expanded to
encompass greater numbers of transformable BAC clones. The stock
centers will continue to improve the information resources associated
with the stocks, specifically by achieving higher degrees of
integration with The Arabidopsis Information Resource and other public
databases. Information regarding donation and ordering of stocks can be
obtained from the web sites listed in Table I. Donations of seed,
clone, and antibody stocks are welcomed from all researchers.
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FOOTNOTES |
Received September 12, 2000; accepted September
24, 2000.
*
Corresponding author; e-mail scholl.1{at}osu.edu; fax
614- 292-0603.
© 2000 American Society of Plant Physiologists
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INTRODUCTION
SEED STOCKS
