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The Multinational Science Steering Committee:
Committee Chair: Gerd Jürgens, University of Tübingen,
Germany
Michael Bevan, John Innes Centre, Norwich, United Kingdom
Michel Caboche, Lab. Biol. Cellulaire, INRA, Versailles, France
Daphne Preuss, University of Chicago, Chicago, IL, USA
Joseph Ecker, University of Pennsylvania, Philadelphia, PA USA
Fernando Migliaccio, CNR, Monterotondo, Italy
Kiyotaka Okada, Kyoto University, Kyoto, Japan
David Smyth, Monash University, Clayton, Australia
Marc Van Montagu, University of Ghent, Belgium
Preface
Overview of Genome Analysis
Stock Center Resources and Data Bases
National and Transnational Projects
Appendix 1: NSF Arabidopsis Genome Meeting Report
Appendix 2: Summary of December 1998 AGI Meeting at CSHL
Appendix 3: Database Workshop (Madison, WI 1998) Report
Appendix 4: "Arabidopsis thaliana Information Resource Project"
Announcment
The "Multinational Coordinated Arabidopsis thaliana Genome
Research Project" was established in 1990 to promote international
cooperation in basic and applied research with Arabidopsis, a
model plant species amenable to experimental manipulation in the
laboratory. The primary objective of this project has been to
understand the molecular basis of plant growth and development
and to address fundamental questions in plant genetics, physiology,
biochemistry, cell biology, and pathology. Initial plans were
outlined in a publication (NSF #90-80) drafted nine years ago
by an ad hoc committee of nine scientists from the United States,
Europe, Japan, and Australia. In recent years, this project has
become a model for widespread participation and effective coordination
of multinational research efforts in modern biology.
Arabidopsis thaliana, a small plant in the mustard family, was
chosen for this large-scale research effort because it offers
many advantages for detailed genetic and molecular studies. Among
these features are its small size, short life cycle, small genome,
ability to be transformed, availability of numerous mutations,
and prolific seed production. By concentrating research efforts
on a single model organism, detailed information on specific genes
and cellular processes can be readily obtained and rapidly applied
to a wide range of plants relevant to agriculture, health, energy,
manufacturing, and the environment.
Each year since 1990, the scientific steering committee for the
Arabidopsis Genome Project has prepared a progress report summarizing
recent advances in Arabidopsis research. This is the seventh annual
progress report published by the steering committee in conjunction
with the U.S. National Science Foundation. Three years ago the
report was a color brochure designed to explain the value and
significance of Arabidopsis research to a wide audience. Two years
ago the report presented a detailed overview of recent advances
in research with Arabidopsis, along with technical information
for use by members of the Arabidopsis community. The sixth report
presented an updated vision statement for the future to stimulate
further advances in the use of Arabidopsis as a model system for
the analysis of complex organisms.
This report covers progress for the seventh and eighth years of
the project. It is focused on the large-scale analysis of the
Arabidopsis genome. Specifically, this report is designed to make
the available information accessible to the scientific community
in a hands-on format. At the current rate of progress, the genome
sequencing project can be expected to be completed within two
years. The 1998 genome issue of Science (Meinke et al. 1998) featured
Arabidopsis prominently.
Multinational cooperation and communication continue to be an important feature of the Arabidopsis genome project. A brief overview of Arabidopsis research efforts in a number of participating countries is therefore included in this report. Additional information can be obtained through recent publications, electronic news groups and databases, and biological resource centers devoted to Arabidopsis research. As with any document that attempts to summarize the contributions of many individuals, this report may fail to include or misrepresent some significant achievements. The steering committee hopes that members of the Arabidopsis community will overlook such shortcomings and will communicate any concerns to committee members so that future reports will be as accurate as possible. We thank all members of the Arabidopsis community for their many contributions to the success of the initial phase of the Multinational Coordinated Arabidopsis thaliana Genome Research Project.
| 1983 | Publication of first genetic map |
| 1988-89 | Publication of RFLP maps |
| 1990 | Multinational Coordinated Arabidopsis thaliana Genome Research Project initiated |
| 1991 | Arabidopsis Stock Centers at Ohio State (USA) and Nottingham (UK), as well as the Arabidopsis Data Base (AtDB), were established |
| 1991 | First YAC libraries and anchoring of YAC clones to RFLP map |
| 1992 | Publication of first chromosome walk (local contig) |
| 1993 | Recombinant inbred (RI) map |
| 1994-8 | Collections of cDNA (EST) clones sequenced linking up genetic and cytogenetic with physical maps |
| 1995-6 | CIC-YACs, TAMU-BACs, IGF-BACs, Mitsui-P1, Kazusa-P1 libraries |
| 1995-8 | Physical map of all 5 chromosomes delineated |
| Jan 98 | Publication of 1.9 Mb of contiguous DNA sequence from chromosome 4 |
| June 98 | 29 Mb of genomic DNA sequenced |
| Oct. 98 | Arabidopsis featured in genome issue of "Science" |
| Dec 98 | >46 Mb of genomic DNA sequenced and annotated 90 Mb of genomic DNA in edited BAC contigs >41,000 (of 44,000) BAC ends sequenced >11,000 non-redundant (of >37,000) EST clones |
| 2000 | Completion of genome sequencing (expected date) |
Two genetic maps were independently developed: a classic map of
mutations (Koornneef et al., 1983) and a recombinant inbred (RI)
map of molecular markers (Lister and Dean, 1993). As an increasing
number of genes originally identified by mutation has been cloned
and converted to molecular markers mapped onto the RI map, the
two maps are beginning to merge into a unified genetic map. Map
distances differ between the two maps, presumably because of the
different genetic backgrounds. In addition, map distances are
calculated with the Mapmaker program, resulting in local inaccuracies,
such as relative order of closely linked markers. These problems
will eventually be resolved by physical mapping.
The RI map is now commonly used as the standard reference, enabling
new genes identified by mutation to be easily mapped by PCR markers
(SSLP, CAPS). The current RI map (November 1998) contains ca.
800 markers which fall into 3 different categories: "framework"
(fixed reference location), "unique" (defined location
on the map) and "multiple" (several possible locations).
RI markers were also used to map a collection of YAC, BAC and
P1 clones from which physical maps of the 5 chromosomes were initiated,
thus linking genetic and physical maps from the very beginning.
Several physical maps have been established for all 5 chromosomes.
Initially, contigs of large YAC clones were assembled and anchored
to RI markers (e.g. Schmidt et al., 1997; Bouchez et al., 1998).
Corresponding BAC and P1 clones were identified by hybridisation
with YAC clones. For chromosome 5, a nearly complete physical
map was established by P1 and TAC clone contigs (Kazusa homepage;
Kotani et al., 1997). BAC contigs have also been established at
the global scale by fingerprinting and by hybridisation
with BAC endprobes. For example, 9 Mb constituting the bottom
arm of chromosome 3 have been covered by a single BAC contig (see
http://www.genoscope.cns.fr/externe/English/Projets/projetsindex.html).
In addition to whole-chromosome physical mapping with YAC, BAC
and P1 clones, chromosome walks in several chromosome regions
have yielded local contigs up to 2 Mb long (e.g. Hardtke &
Berleth, 1996; Wang et al., 1997; Thorlby et al., 1997), and several
hundred EST clones have been PCR-mapped onto YAC clones (Agyare
et al., 1997).
Fingerprinting data of BAC clones were used to assemble contigs
with FPC software, followed by manual editing to join the initial
contigs. At present, ca. 70 BAC contigs encompass ca. 90 Mb of
estimated 121 Mb total sequence (M. Marra & M. Sekhon, Washington
University, St. Louis; M.A. Marra et al.,1997). High throughput
BAC-endprobe hybridization was used as a complementary approach
to assemble contigs (Mozo et al., 1998). Information gathered
from 2995 hybridization data (including 272 mapped markers) was
manually edited after application of the probeorder computer
program and integrated with the fingerprint data to generate a
complete BAC-based physical map consisting of 27 contigs distributed
over the 10 chromosome arms that covers approximately 124 Mb (see:
http://www.mpimp-golm.mpg.de/101/bac.html). As the genome sequencing
project is progressing, many RI markers are mapped physically,
resulting in an excellent alignment of genetic and physical maps
(see AtDB; see also integrated contig tables by Daphne Preuss
and colleagues at the CSHL website). This integration will undoubtedly
facilitate gene isolation by map-based cloning.
In addition to the unique-sequence regions of the chromosome arms,
both rDNA repeats (NORs on chromosomes 2 and 4) and centromeric
regions have been mapped genetically and physically. The centromeric
regions were mapped by tetrad analysis (Copenhaver et al., 1998)
and localized by in situ hybridization (Brandes et al., 1997).
Thus, an outline of the physical organisation of the nuclear genome
has emerged.
Agyare FD, Lashkari DA, Lagos A, Namath AF, Lagos
G, Davis RW, Lemieux B (1997) Mapping expressed sequence tag sites
on yeast artificial chromosome clones of Arabidopsis thaliana
DNA. Genome Res. 7: 1-9.
Brandes A, Thompson H, Dean C, Heslop-Harrison JS
(1997) Multiple repetitive DNA sequences in the paracentromeric
regions of Arabidopsis thaliana L. Chromosome Res. 5: 238-246.
Camilleri C, Lafleuriel J, Macadre C, Varoquaux F,
Parmentier Y, Picard G, Caboche M, Bouchez D (1998) A YAC contig
map of Arabidopsis thaliana chromosome 3. Plant J. 14:633-642.
Copenhaver GP, Browne WE, Preuss D (1998) Assaying
genome-wide recombination and centromere functions with Arabidopsis
tetrads. Proc. Natl. Acad. Sci. USA 95: 247-252.
Hardtke CS, Berleth T (1996) Genetic and contig map
of a 2200-kb region encompassing 5.5 cM on chromosome 1 of Arabidopsis
thaliana. Genome 39: 1086-1092.
Kotani H, Sato S, Fukami M, Hosouchi T, Nakazaki
N, Okumura S, Wada T, Liu YG, Shibata D, Tabata S (1997) A fine
physical map of Arabidopsis thaliana chromosome 5: construction
of a sequence-ready contig map. DNA Res. 4:371-378.
Marra MA, Kucaba TA, Dietrich NL, Green ED, Brownstein B, Wilson RK, McDonald KM, Hillier LW,
McPherson JD, Waterston RH (1997) High throughput
fingerprint analysis of large-insert clones. Genome Res. 7:
1072-1084.
Meinke, DW, Cherry JC, Dean C, Rounsley SD, Koornneef
M (1998) Arabidopsis thaliana: A model plant for genome analysis.
Science 282: 662-682.
McPherson JD, Waterston RH (1997) High throughput
fingerprint analysis of large-insert clones. Genome Res. 7:1072-1084.
Mozo T, Fischer S, Maier-Ewert S, Lehrach H, Altmann
T (1998) Use of the IGF BAC library for physical mapping of the
Arabidopsis thaliana genome. Plant J. 16, 377-384.
Round EK, Flowers SK, Richards EJ (1997) Arabidopsis
thaliana centromere regions: genetic map positions and repetitive
DNA structure. Genome Res 1997 Nov;7(11):1045-53
Sato S, Kotani H, Hayashi R, Liu YG, Shibata D, Tabata
S (1998) A physical map of Arabidopsis thaliana chromosome 3 represented
by two contigs of CIC YAC, P1, TAC and BAC clones. DNA Res.5:163-168.
Schmidt R, Love K, West J, Lenehan Z, Dean C (1997)
Description of 31 YAC contigs spanning the majority of Arabidopsis
thaliana chromosome 5. Plant J. 11: 563-572.
Thorlby GJ, Shlumukov L, Vizir IY, Yang CY, Mulligan
BJ, Wilson ZA (1997) Fine-scale molecular genetic (RFLP) and physical
mapping of a 8.9 cM region on the top arm of Arabidopsis chromosome
5 encompassing the male sterility gene, ms1. Plant J. 12: 471-479.
Wang ML, Huang L, Bongard-Pierce DK, Belmonte S,
Zachgo EA, Morris JW, Dolan M, Goodman HM (1997) Construction
of an approximately 2 Mb contig in the region around 80 cM of
Arabidopsis thaliana chromosome 2. Plant J. 12: 711-730.
More than 37,000 partial cDNA (EST) sequences have been deposited
in the public databases while the total number of genes is most
likely about 20,000. Building EST "contigs", i.e. larger
cDNA sequences from overlapping ESTs, reduces the number of ESTs
to those representing different genomic sequences (Rounsley et
al., 1996; Cooke et al., 1997). The current estimate of non-redundant
ESTs is about 11,000 or approximately half the total number of
genes.
Large-scale high-throughput genomic sequencing makes use of the
physical maps and the available BAC (TAMU, IGF), P1 and TAC (Mitsui,
Kazusa) libraries (see AGI). BAC, TAC and P1 clones are mapped
onto YAC, and their ends are sequenced to determine minimum tiling
paths for sequencing large regions. More than 41,000 BAC ends
(of a total of 22,000 BAC clones) have been sequenced, yielding
stretches of ca. 400 bp every 4 kb on average (total sequence
ca. 14 Mb). The largest contiguous region sequenced to date is
nearly 1.9 Mb long (Bevan et al., 1998). This region around FCA
on chromosome 4 contains 389 genes of which 46% could not be assigned
a putative function by sequence comparisons with the databases.
On average, one gene (ORF) was found every 4.8 kb, and similar
values were observed for other genomic regions (Quigley et al.,
1996; Sato et al., 1997; Kotani et al., 1997). For many ORFs no
corresponding EST was found in the databases. To identify expressed
genes within contig regions, a novel cDNA selection method has
been proposed (Seki et al., 1997).
Bevan M et al. (1998) Analysis of 1.9 Mb of contiguous
sequence from chromosome 4 of Arabidopsis thaliana. Nature 391:
485-488.
Cooke R, Raynal M, Laudie M, Delseny M (1997) Identification
of members of gene families in Arabidopsis thaliana by contig
construction from partial cDNA sequences: 106 genes encoding 50
cytoplasmic ribosomal proteins. Plant J. 11: 1127-1140.
Kotani H, Nakamura Y, Sato S, Kaneko T, Asamizu E,
Miyajima N, Tabata S (1997) Structural analysis of Arabidopsis
thaliana chromosome 5. II. Sequence features of the regions of
1,044,062 bp covered by thirteen physically assigned P1 clones.
DNA Res. 4: 291-300.
Quigley F, Dao P, Cottet A, Mache R (1996) Sequence
analysis of an 81 kb contig from Arabidopsis thaliana chromosome
III. Nucl. Acids Res. 24: 4313-4318.
Rounsley SD, Glodek A, Sutton G, Adams MD, Somerville
CR, Venter JC (1996) The construction of Arabidopsis expressed
sequence tag assemblies. Plant Phys. 112: 1177-1183.
Sato S, Kotani H, Nakamura Y, Kaneko T, Asamizu E,
Fukami M, Miyajima N, Tabata S (1997) Structural analysis of Arabidopsis
thaliana chromosome 5. I. Sequence features of the 1.6 Mb regions
covered by twenty physically assigned P1 clones. DNA Res. 4:215-230
Seki M, Hayashida N, Kato N, Yohda M, Shinozaki K
(1997) Rapid construction of a transcription map for a cosmid
contig of Arabidopsis thaliana genome using a novel cDNA selection
method. Plant J. 12: 481-487.
The AGI was established on August 20-21, 1996 when representatives
of six research groups (3 from USA and one each from EU, Japan
and France) committed to sequencing the Arabidopsis genome met
in Arlington, VA to discuss strategies for facilitating international
cooperation in completing the genome project. In order to avoid
duplication of efforts, the six groups of the Arabidopsis Genome
Initiative (AGI) agreed to focus on different regions of the genome
(Bevan et al., 1997, Plant Cell 9:476-487). In July 1998, the
members of the AGI met again in Arlington, VA to discuss progress
to date, to anticipate barriers to timely completion, and to establish
an oversight committee for the U.S.-based labs (see Appendix).
At present, the major sequencing domains of the AGI groups have
been assigned as follows:
| Chromosome 1 (30 Mb) | SPP group (Stanford, PennU, PGEC) | Chromosome 2 (14 Mb) | TIGR group | Chromosome 3 (top - 13.5 Mb) | Kazusa group | Chromosome 3 (top - 5 Mb) | TIGR group* | Chromosome 3 (bottom - 9 Mb) | EU project chrom3 (coordinated by Genoscope) | Chromosome 4 (top - 4 Mb) | CSHSC (CSH-WU-ABI group) | Chromosome 4 (bottom - 13 Mb) | EU group (ESSA I, II, III) | Chromosome 5 (top - 9 Mb) | EU group (ESSA III) | Chromosome 5 (top + middle - 4 Mb) | CSHSC (CSH-WU-ABI group) | Chromosome 5 (top + bottom - 17 Mb) | Kazusa group |
|---|
Sequencing is being done on BAC and P1 clones. Two different strategies
are pursued. Both the SPP group and the TIGR group have selected
nucleating sites ("seed BACs") around which BAC contigs
have been established by using BAC end sequences to select adjacent
clones with minimum overlap. This sequential sequencing procedures
involves 32 and 16 starting points on chromosomes 1 and 2, respectively.
The other sequencing strategy adopted by CSHSC, ESSA and Kazusa
involves building of BAC or P1/TAC tiling paths with minimum overlap
of adjacent clones ("sequence ready maps"). This procedure
requires more preparative work but once established, large regions
can be sequenced in parallel, e.g. by the several sequencing groups
within the ESSA group.
Lists of clones selected for sequencing can be found on the web
sites of the sequencing groups. Start dates for sequencing are
indicated and it is agreed that the finished sequences will be
released within 4-6 month after the start of sequencing (for details,
see Appendix). The current state of genome sequencing is as follows
(for overview by chromosome region, see AtDB / Arabidopsis Sequencing
View and the homepages of the AGI groups):
| Chr. | Est. Size | Completed | ||||||||
| (Target) | Clones | Mb | Clones | Mb | Clones | Mb | Clones | Mb | ||
| 1 | 30 Mb | 52 | 5.52 | 16 | 2.02 | 16 | 1.7 | 84 | 9.25 | |
| 2 | 17 Mb | 107 | 10.29 | 45 | 4.32 | 27 | 2.64 | 179 | 17.25 | |
| 3 | 22.2 Mb | 13 | 1.09 | 29 | 2.5 | 27 | 1.2 | 69 | 5.8 | |
| 4 | 18.5 Mb | 153 | 11.71 | 51 | 5.2 | 28 | 2.6 | 231 | 19.4 | |
| 5 | 29.2 Mb | 208 | 14.62 | 15 | 1.2 | 38 | 2.7 | 261 | 18.54 | |
| Total | 120 Mb | 534 | 43.32 | 156 | 15.2 | 136 | 11.8 | 826 | 70.3 | |
Note that the total sequence entered into AtDB and summarised above includes overlaps between adjacent clones (except for those submitted by ESSA and WashU, which have overlaps almost all removed). For this reason the total number of clones sequenced is a better estimate of progress. With 10% overlap, 120 Mb will require 1,390 BAC clones. On 31 December 1998 the following finished clones had been deposited in Genbank:
SPP 50 BAC clones
TIGR 105 BAC clones
CSHSC 60 BAC clones
ESSA 117 BAC and cosmid clones
Kazusa 202 P1, TAC and BAC clones
Total 534 clones (approx. 39% of the total genome)
As of 31 December 1998, the AtDB Sequencing View displays 46 Mb
(39% of estimated 120 Mb genome size) of complete sequence. This
figure is 17 Mb higher than that given at the end of June 1998,
indicating that the current rate of sequencing is close to 3 Mb
per month for the entire AGI project. Taking into account the
sequences that have not been released, the actual amount of sequence
information is close to 55 Mb (almost 50% of the unique sequences).
It is thus a realistic goal to finish the sequence of the Arabidopsis
genome (excluding telomeric and centromeric regions as well as
NORs) by the end of the year 2000.
Completion of the sequence is defined as each chromosome arm between
subtelomeric repeats and centromeric repeats consisting of a single
fully sequenced contig. This excludes the rDNA repeats (NORs on
chromosomes 2 and 4 each of which accounts for ca. 3.5 Mb) and
other internal tandem repeat regions. For these regions, it will
be sufficient to sequence one repeat unit and to estimate the
repeat number at each site. By these criteria, sequencing of chromosomes
2 (14 Mb) and 4 (17 Mb) can be expected to be complete before
the end of 1999.
As sequencing is reaching the closing phase, boundaries between
sequencing domains have to be defined precisely to avoid duplication
of efforts by different sequencing groups. This difficulty has
already been encountered by all the sequencing groups, resulting
in duplication of sequences and mismapped clones (see table).
For example, on chromosome 4 both CSHSC and ESSA sequenced two
different but overlapping clones and had to reassign remaining
projects in a common region of ca. 900 kb. TIGR and SPP have abandoned
or mismapped at least 4 BACs and a chimaeric YAC, while Kazusa
has sequenced several duplicate clones on chromosome 5. Depending
on different rates of progress, it may seem advisable, in the
interest of the Arabidopsis community, to reallocate genomic regions
between the sequencing groups (see Appendix 1 and 2). The fingerprint
map constructed at Washington University and the hybridisation-based
map constructed by T. Altmann have the potential for delineating
these regions before they are sequenced, and will probably be
used for this purpose.
The seed stocks currently available from the two centers include
mutant lines (600), T-DNA lines and pools (30,000+), mapping strains,
the G. P. Rédei collection of mutants and research lines
(300+), the A. R. Kranz collection of mutants and ecotypes (700+),
transposon/transposase lines (100+), RI lines (3 populations),
ecotypes (400+), transgene lines and related species. The genetic
mapping resources of the centers and the T-DNA and transposon
resources complement the AGI sequencing efforts and the current
research focus on functional genomics.
DNA stocks of ABRC include cloned genes (200), RFLP mapping clones
(300+), expressed sequence tagged (EST) clones (30,000+), cDNA
libraries (7), a phage genomic library, YAC libraries (6), BAC
and P1 libraries used in genome sequencing (3) and two-hybrid
libraries (2). In addition, filters of BACs, P1s and YACs for
hybridization and isolated DNA from T-DNA populations (12,000
lines) are available.
The EST collection has been organized so that a set of 11,000,
non-redundant based on the sequences available to TIGR, is being
used by AGI. The 3' sequences of these clones are being analyzed
by the MSU EST project to further eliminate redundancy. Copies
of BAC and P1 clones, for which sequences have been published,
are being sent to many research laboratories. In this connection,
ABRC requests that all sequencing projects adhere, if at all possible,
to the agreed clone-naming conventions when publishing sequences
so that researchers can identify, without confusion, the proper
clones to obtain.
NASC and ABRC are working to enlarge the collections of characterized
mutants and clones. In addition, it is expected that large numbers
of T-DNA lines will be received so that, within the next year,
the available T-DNA lines will represent essential saturation
of the genome. In connection with the accumulating genomic and
cDNA sequence information, these resources will prove invaluable
to the research community. In addition, new transposon-tagged
populations, recombinant inbred mapping populations, a tetrad
mapping populations and GFP lines are being incorporated into
the collections.
The Nottingham Arabidopsis Stock Centre (NASC) curates the Lister
and Dean RI maps that were originally developed and maintained
by Clare Lister and Caroline Dean (JIC, Norwich). NASC also offers
a weekly community mapping service. Anyone can submit data to
NASC for mapping using the specially designed data submission
form. The positions of all markers mapped at NASC are made publicly
available through the NASC WWW server, the Arabidopsis Genome
Resource and AtDB. For private mapping, all the marker scores
are available from NASC. However, the aim for the community is
to have as many markers as possible placed on the canonical map
and so the submission of mapping data for inclusion on the RI
map is appreciated.
The Arabidopsis node of the BBSRC funded UK-Crop Plant Bioinformatics Network (UK-CropNet) based at NASC has established the Arabidopsis Genome Resource (AGR). AGR is being developed as a repository of Arabidopis data of value in the comparative analysis of plant genomes and as an essential tool to aid in the cloning of homeologous genes of agronomic importance.
Comparative analysis in plants relies upon genetic and physical mapping of common probes between species. To this end AGR has made available the YAC physical maps of chromosomes IV and V (from C.Dean, R.Schmidt, M. Stammers). AGR also includes the Recombinant Inbred Maps from NASC integrated with the AGI sequence template clones (locations provided through AtDB). Arabidopsis nucleotide sequences are also included within AGR.
Integrating these data sets is the next key step in the development
of AGR. Sequence overlap between completed AGI clones define contigs
of BACs and P1s. These contigs will be fixed to the YAC physical
maps using the results of BAC-YAC hybridisations. Contigs may
be anchored on the RI maps through the nearest marker information
from individual clones. RI maps and YAC physical maps are to some
extent integrated through the use of some RI markers as probes
in YAC physical mapping.
In collaboration with Martin Trick (John Innes Center), these
data will be used to generate comparative map displays between
Arabidopsis and the Brassicas.
Contact Persons
Randy Scholl, ABRC email: scholl.1@osu.edu
Mary Anderson, NASC email: arabidopsis@nottingham.ac.uk
Web sites
| ABRC: | http://aims.cps.msu.edu/aims/ | NASC: | http://nasc.nott.ac.uk/ | AtDB: | http://genome-www.stanford.edu/Arabidopsis/ |
|---|
The Arabidopsis Data Base (AtDB) is, at this time, located at Stanford University, Mike Cherry, P.I. The explosion of data, both genomic and biological, makes it clear that the data base as it now exists is operating at a minimal, not an optimal, level. The recognition that the community had to express its needs in a more concrete way resulted in two workshops addressing the issues of database composition and management. One was held in 1993 in Dallas, TX and that report can be accessed at http://genome-www.stanford.edu/Arabidopsis/db/dallas.report.html.
However, a more recent workshop on the same topic was held at
the international meeting at Madison, WI in 1998 and that report
is attached as an appendix. The needs are for a central database
with links to other useful databases and information which is
organized in a user-friendly fashion. Recognition of the needs
of the Arabiopsis community as well as other interested communities
has resulted in a call for proposals to the NSF titled "Arabidopsis
thaliana Information Resource Project (AtIR)" The deadline
date is March 22, 1999 and a copy of that announcement is attached
to this report as an appendix.
Recommendation on information management
Large-scale genomic sequencing has reached a critical stage, with
about half the genome in hand. Although the AGI sequencing groups
provide information for specific regions of chromosomes, it is
difficult and time-consuming for the Arabidopsis community to
retrieve the relevant information. To take full advantage of all
the progress that has been made in the analysis of the Arabidopsis
genome, it will be necessary to establish a well-funded unified
genome database that displays sequence and related features together
with biological information in a user-friendly way.
Australia
Arabidopsis research in Australia is focused on building an understanding
of fundamental aspects of plant biology. There is no direct commitment
to large scale genome sequencing at this stage.
Among recent highlights, Liz Dennis, Jim Peacock and colleagues
from CSIRO Division of Plant Industry in Canberra have discovered
a second nonsymbiotic leghemoglobin gene from Arabidopsis (Proc.
Nat. Acad. Sci. US 94, 12230-12234, 1997). They propose that
all plants have two classes of leghemoglobins, as exemplified
by the two genes in Arabidopsis. In the evolution of symbiosis,
the product of one or other of the genes has been recruited on
different occasions to play a new role in association with the
symbiont. In most cases class 1 gene products have been involved,
but the newly discovered class 2 proteins are also potentially
symbiotic.
Another highlight has been the discovery of a gene encoding the
catalytic subunit of cellulose synthase (Science 279, 717-720,
1998). Tony Arioli and colleagues in Richard Williamson's research
group in the Research School of Biological Sciences at ANU in
Canberra have walked to the locus of a temperature sensitive mutant
that leads to root swelling (RSW1). The gene that complements
the mutant phenotype is related to a cellulose synthase subunit
gene from cotton. In the mutant there is widespread accumulation
of beta-1,4-glucan but it is not crystallised into microfibrils,
suggesting such assembly is a role of the RSW1 gene product.
Other active programs include studies of various aspects of flowering,
from induction through floral organ morphogenesis to fertilisation
and seed development. Also topics as diverse as aspects of photosynthesis,
analysis of effects of abiotic stresses including heavy metals
and UV, epigenetic effects of cytosine methylation, and the roles
of the MYB gene family are being actively investigated.
A major commitment is being made to host the 10th International
Conference on Arabidopsis Research in Melbourne from 4-8 July
1999. A Regional Advisory Committee, with colleagues from Japan,
South Korea, Singapore and New Zealand, has been set up to give
the meeeting a Western Pacific focus. This will be the first
time the Arabidopsis community has met outside Europe and North
America, and we look forward to welcoming scientists and students
to Australia where plant science continues to thrive.
Contact Person: David Smyth, Monash University, Melbourne
E-mail Address: David.Smyth@sci.monash.edu.au
Belgium
As Belgium is a federal country we have both federal and Flemish initiatives to support research using Arabidopsis thaliana as the experimental organism.
A Flemisch project is running on the isolation and characterization of new ethylene mutants in Arabidopsis thaliana. This project aims at the isolation of a new series of mutants in the ethylene signal transduction pathway. A combined morphological, physiological and molecular-genetical approach will elucidate a number of previously unknown elements and will provide a better insight in the control of plant development by this hormone.
Belgian governement also stimulates interactions between the different
universities. In this frame a project is running between the universities
of Gent, Antwerp, Brussels and Liège on the growth and
development of higher plants. Many external factors such as light
intensity, light quality, temperature, the availability of nutrients
and the interaction with pathogenic organisms influence to a great
extent, growth and development of higher plants. The current knowledge
on the molecular processes that control growth and development
is still very limited. The national network aims at making a contribution
to developmental biology by studying a limited number of aspects
of plant development. Wherever possible, Arabidopsis thaliana
will be used as a model plant. Keyprojects include the identification
and cloning of key regulatory genes involved in leaf morphogenesis,
the molecular analysis of the formation of syncytia (=large feeding
cells) in nematode infected Arabidopsis roots. The Flemish community
also supports these projects.
Contact Person: Nancy Terryn /Marc Van Montagu, University of Ghent
E-mail Address: nater@gengenp.rug.ac.be
China
Research using Arabidopsis as a model system was further
established in China at national research institutes and universities
in the past year. The research areas mainly include biosynthesis
of amino acids, signal transduction and metabolism of plant hormones,
cell wall formation, seed storage proteins, response to environmental
stresses, isolation of various mutants affecting growth and development,
and characterization of transposable elements. Interests in reverse
genetics and functional genomics are also greatly increased with
the focuses on gene-targeting, constructing a large transgenic
population with mapped Ds randomly distributed at a high density,
developing an expression library to transform in planta and establishing
cDNA array to monitor gene expression and identify functional
genes. Grants to support the research projects mentioned above
are mainly from National Natural Science Foundation of China,
Chinese Academy of Sciences and Hong Kong Research Grant Council/UPGC
Grant HKU.
Contact Person: Jiayang Li, Institute of Genetics, Chinese Academy of Sciences
E-mail Address: jyli@ss10.igtp.ac.cn
Genome sequencing
During the last year, three French laboratories (M. Delseny/Perpignan,
M. Kreis/Orsay and R. Mache/Grenoble) have systematically sequenced
three BACS (300 kb) as part of the EU-ESSA II Program. Delseny's
group has also continued to sequence cDNA clones corresponding
to the 60kbp locus, Em1, on chromosome 3. A French sequencing
center, Genoscope CNS has been created and part of its activity
is devoted to sequencing the Arabidopsis genome. In collaboration
with TIGR and Upenn, Genoscope is generating end sequences from
all 23,000 BAC clones from the TAMU and IGF libraries to expedite
the selection of clones with minimal overlap with those already
sequenced. They are also coordinating a new EU project aimed at
sequencing the lower arm of chromosome 3 (9Mb). This project involves
16 sequencing groups. The goal for Genoscope and three academic
French laboratories is about 2 Mb.
Synteny with other genomes
A program was developed between INRA Rennes and Versailles groups
to identify consensus markers between rapeseed and Arabidopsis
for a number of agronomically relevant genes. A collaboration
between laboratories in Perpignan, Davis and Poznan has found
synteny between five adjacent genes in the chromosome 3 Em locus
of Arabidopsis and genes in B. oleracea, B. nigra and B. rapa.
The EU program EuDicotMap has started to select highly conserved
ESTs of rice and Arabidopsis and to map them in Arabidopsis as
well as important European crops in order to identify synteny
blocks between different families.
Generation of insertion lines and reverse genetics screenings
INRA-Versailles has now generated more than 38,000 T-DNA mutant
lines. Screening of the collection is being done via a coordinated
effort between INRA, CNRS and various European laboratories. Out
of approximately a hundred target genes selected for the screen
insertions were identified in 50% of them. The systematic characterization
of flanking sequences tags of insertions in over a thousand mutants
has now begun. About 11,000 lines will be donated to NASC by the
beginning of next year.
A summary of Arabidopsis genes under study
Research in many areas of plant genetics and biology is being
actively pursued in French laboratories. Plant hormone and signal
transduction, cell wall, secreted and membrane proteins, metabolism,
development, and plant pathogen interactions are being investigated
in laboratories throughout France.
Contact Person: Michel Caboche, INRA Versailles
E-mail Address: caboche@tournesol.versailles.inra.fr
Germany
Arabidopsis research is still increasing in scope at universities
and research institutions. The national research program on "Arabidopsis
as a model for analysing plant development" is in its
final two-year funding period. Because its tremendous success,
an initiative has been made by Arabidopsis researchers to establish
a new program focusing on plant cell biology. Another six-year
national research program on plant hormones to start in 1999 includes
several groups working on Arabidopsis. Beside these programs,
Arabidopsis research is funded within European projects
and by DFG grants on an individual basis or as part of local research
programs.
Several Arabidopsis projects are related to genome research.
ZIGIA, a program operated at the Max-Planck-Institut in Cologne,
aims at the functional analysis through gene inactivation by transposon
insertion. High throughput endprobe hybridization of BAC clones
from the IGF library was done at the Max-Planck-Institut in Golm.
These data were integrated with information made available by
other groups to assemble a complete BAC-based physical map of
the Arabidopsis genome. Projects on transcript profiling
have been initiated at the DKFZ in Heidelberg, the MPI in Golm
and the IPK in Gatersleben. The Federal Ministery of Education
and Science (BMBF) has made a call for proposals within a newly-established
Plant Genome Analysis program (GABI). A joint Arabidopsis proposal
involving 32 projects from 27 different institutions has been
submitted, aiming at a functional analysis of the genome.
An EMBO (European Molecular Biology Organisation) Course held
at the Max-Planck-Institut in Cologne in May 1998 entitled "Molecular
and Biochemical Analysis of Arabidopsis" was attended by
16 participants representing 13 European countries. The course
covered the theoretical and practical aspects of forward and reverse
genetics, genetic and physical mapping, transformation, transient
gene expression, in situ hybridisation, cell biology, physiology,
the yeast two-hybrid system, complementation of yeast mutants
and bioinformatics over an eleven-day period. EMBO Course seminars
from ten invited speakers were integrated with a two-day meeting
of the national Arabidopsis research program.
Contact Person: Gerd Jürgens, Universität Tübingen
E-mail Address: gerd.juergens@uni-tuebingen.de
Italy
Research in Italy with Arabidopsis is growing. About twenty
laboratories are presently attending to researches regarding this
model system. Investigations cover: plant pathogen relationships,
expression of PG and PGIF genes, role of rolB and rolD in plant
differentiation, HD-ZIP transcription factors in plant morphogenesis,
complementation of yeast by Arabidopsis genes, selection of Ca2+
and K+ transport mutants, genes involved in heat and cold resistance,
myb transcription factors, genes of the polyamine pathway, induction
of noduline genes in plants by Rhizobium, use of antisense RNA
to inhibit nitrogen transport, study of agravitropic mutants in
earth and micro g conditions (ESA-ASI projects). Financial support
for the researches is coming from different sources, e.g. the
National Research Council, the Ministry of Agriculture, the European
IV Frame Programs, the ESA-ASI Space Programs, and a few other
National Agencies. Research groups are located both in universities
and in National Institutes (National Research Council, ENEA, National
Institute of Nutrition). The Italian association of researchers
interested in Arabidopsis (ARABITALIA) met for the first time
in September 1997 in Abbadia di Fiastra (Macerata, central Italy).
In this occasion the scientists present to the meeting furnished
a report of their Arabidopsis investigations and projects, and
a booklet carrying the information about research on Arabidopsis
in Italy was also distributed. In this occasion some young Italian
researchers, who are working in foreign countries (USA, and UK)
also reported about their recent investigations. The 1998 annual
Meeting was held at the end of September in Viterbo (central Italy)
in the occasion of the EUCARPIA Symposium on plant breeding.
A document is in preparation about the state of Arabidopsis research
in Italy, and about the actions that can be started to obtain
the financial support that is needed to foster it.
Contact Person: Fernando Migliaccio, CNR (Monterotondo)
E-mail Address: miglia@nserv.icmat.mlib.cnr.it
Japan
Arabidopsis research is well-established in Japan. The
number of laboratories using the model plant for research and
education is still increasing gradually in universities, national
institutes, and private companies. Areas of research are widely
spread from developmental biology, metabolic regulation, gene
expression, environmental stress signaling, and DNA methylation,
to large scale DNA sequencing. The results of the researches
were reported in international meetings such as the " International
Congress of Arabidopsis Research" in Madison, WI, the "Joint
Meeting of Japanese and American Societies of Plant Physiologists"
in Vancouver, BC and in national meetings, especially in the "Workshop
on Arabidopsis Studies", an annual meeting. The 8th workshop
was organized by Kazuo Shinozaki, Minami Matsui, Yuji Kamiya,
and Richard E. Kendrick from October 11 to 13, 1997, at Riken
Institute at Wako city, Saitama. The workshop was joined with
Frontier Research Forum, "Recent Progress of Plant Hormone
Research in Arabidopsis". We had nearly 250 participants,
20 poster presentations, and 37 speakers including 7 guest speakers
from abroad. The 9th workshop was held in Kazusa Academia Center
from Nov. 19 to 20, 1998. The workshop organized by Satoshi Tabata
had nearly 300 participants, 40 poster presentations and 11 presentations.
Topics of the presentations included systemic genome analyses,
patent, and postgenome tactics, as well as mutant analyses, gene
cloning, and newly-developed techniques.
The Japanese Arabidopsis communication network, nazuna-net,
started in January 1995, now includes 442 members (Sept. 1998)
from 99 organizations including 17 private companies (contact:
Dr. Takayuki Kohchi: kouchi@bs.aist-nara.ac.jp). A large-scale
genome sequencing project showed extensive progress at Kazusa
DNA Research Institute in coordination with the Multinational
Arabidopsis Genome Initiative (contact: Dr. Satoshi Tabata: tabata@kazusa.or.jp).
Nearly 12.5 Mb covering 174 P1 clones have been sequenced and
reported in the journal "DNA Research" (contact:
http://www.uap.co.jp),
on a homepage (
http://www.kazusa.or.jp/arabi/). The Sendai Seed
Stock Center (SASSC) is operated by Dr. Nobuharu Goto (n-goto@ipc.miyakyo-u.ac.jp)
since 1993.
Contact Person: Kiyotaka Okada, Kyoto University
E-mail Address: kiyo@ok-lab.bot.kyoto-u.ac.jp
The Netherlands
The Dutch Arabidopsis groups organized their annual meeting in
Utrecht on February 19, which was attended by approximately 80
participants. Arabidopsis groups are located at the Universities
of Leiden, Utrecht and Wageningen and at CPRO-DLO in Wageningen.
Important research topics are in Leiden (Hooykaas) recombination,
auxin action and apoptosis, in Utrecht sugar sensing (Smeekens),
root development (Scheres) and acquired resistance (van Loon),
in Wageningen embryogenesis (de Vries, van Lammeren) and flowering
and seed- development (Koornneef), transposons, genome sequencing,
plant disease resistance genes and developmental biology (Stiekema,
Pereira, Angenent, Groot all CPRO-DLO). The groups collaborate
through their involvement in graduate schools and EU programs.
Contact Person: Maarten Koornneef, Agricultural University Wageningen
E-mail Address: Maarten.Koornneef@BOTGEN.EL.WAU.NL
Spain
No special funding programme supports Arabidopsis research
in Spain. However, more than 20 research groups are currently
active in research with this organism, mainly funded by the National
Biotechnology Programme, Basic Research Programmes, and the European
Union BIOTECH Programme. Some of these groups are involved in
large-scale genome sequencing and function search, specially in
the case of the Myb family of transcriptional factors. Spanish
groups interested in Arabidopsis development are mainly
focused on seed, leaf and flower development, and flowering induction.
This area is seing the incorporation of new groups of Arabidopsis
users, some of them also interested in cell differentiation. In
the area of plant physiology and metabolism some topics that have
seen significant contributions during the year are the study of
secondary metabolism, the identification of new elements in the
signal transduction pathways involved in different environmental
stress responses, and the analysis of sulfur and phosphate assimilation.
Arabidopsis has also being increasingly used for studies
in plant pathogen interactions to identify new elements in the
response signal transduction pathways.
The Spanish Arabidopsis network, funded by the National Biotechnology
Programme, generated a collection of 10000 T-DNA lines that is
being actively used in mutant screenings at both the phenotypic
and DNA levels, in many laboratories. This network that includes
all the Spanish laboratories working with Arabidopsis is
now discussing future join activities. Many more Spanish scientists
are currently involved in Arabidopsis research in other
laboratories around the world. Their succesful integration in
the Spanish R&D system would strongly contribute to steer
the field and increase the contribution of our country.
Contact Person: José Martinez Zapater, Centro Nacional de Biotecnología (Madrid)
E-mail Address: zapater@cnb.uam.es
United Kingdom
There are over 190 projects at present in the UK involving Arabidopsis.
The European Commission continues to be a major source of funding
and the newly announced Framework V programme is due to begin
calls for proposals. Although there are no longer any special
initiatives aimed specifically at Arabidopsis research, The Biotechnology
and Biological Sciences Research Council (BBSRC) funds projects
through competitive grants and special initiatives, contributing
approximately £ 6.8m to Arabidopsis research in the
UK.
An Arabidopsis Gene Function Search Network is currently under
development by Mike Bevan at the John Innes Centre. This is a
network of consortia, groups of labs with a common goal, being
brought together with the aim of doing large scale screening programmes
to reveal the functions of very large numbers of genes being revealed
by the genome project.
The Genetical Society of Great Britain chose Arabidopsis as the subject area for their annual autumn meeting in 1997. The Mendel Lecture was given by Elliott Meyerowitz who was preceded during the day by Mike Bevan, Rob Martienssen, Joe Ecker, Ben Scheres, Caroline Dean, Gerd Jurgens and Brain Staskawicz."Arabidopsis thaliana: Big Ideas from a Small Plant" was such a success that the Society has decided to host a biennial conference on Arabidopsis.
An EMBO (European Molecular Biology Organisation) Course held
at the John Innes Centre in May 1997 entitled "Arabidopsis
as an Experimental Organism" was attended by 12 participants
representing seven European countries. The course covered the
theoretical and practical aspects of mutant screening, genetic
and physical mapping, plant pathology, microscopy, biolistics,
the yeast two-hybrid system, and sequence fragment and data analysis
over a ten day period which also included seminars from ten invited
speakers.
The Chelsea Flower Show judges awarded a prestigious Silver Medal
to the John Innes Centre Science Communication and Education Department
exhibit, entitled "Arabidopsis - a Wonderful Weed".
The exhibit demonstrated how Arabidopsis is used to recognise
genes of agronomic importance in agricultural crops. The public
exposure and media coverage the display attracted in the UK and
abroad has helped to increase awareness of the importance of plant
molecular biology.
In the last year the Nottingham Arabidopsis Stock Centre (NASC)
in collaboration with the Arabidopsis Biological Resource Center
(ABRC) has continued to accumulate the broadest possible range
of stocks to provide the best platform of genetic diversity and
genetic tools for the investigation of this model system. Currently
NASC maintains and distributes over 20,000 accessions of Arabidopsis
to the research community. New stocks generated within the UK
and shortly to be made available include the first 10,000 of the
Sainsbury Laboratory Arabidopsis transposants (SLAT) lines
(Jonathan Jones, Sainsbury Lab, UK), 100 GFP lines (Jim Haseloff,
Cambridge, UK) and a Recombinant Inbred population of Nd (Niederzenz)
x Columbia generated by Eric Holub, Jim Beynon and Ian Crute (HRI
Wellsbourne, UK).
Contact Person: Caroline Dean, John Innes Centre, Norwich
E-mail Address: caroline.dean@bbsrc.ac.uk
United States
Arabidopsis research continues to flourish in both academic and
corporate laboratories in the United States. One of the most obvious
indicators of the value of information that can be gleaned from
Arabidopsis research has been the establishment of several
genomics companies that are exploiting Arabidopsis genetics.
Thanks to continued support from the National Science Foundation (NSF), the
Department of Energy (DOE) and the U.S. Department of
Agriculture (USDA), the Arabidopsis genome
is on track for being completely sequenced by the end of 2000.
A total of 46 Mb of finished sequence had been deposited in public
databases as of January 1999, of which the US sequencing groups
contributed more than 24 Mb. Importantly, the groups in the
US Arabidopsis Genome Initiative (AGI) finished the first phase
of their sequencing effort in less than the original 3 year time
allowed, and could thus begin during 1998 with the second phase
of sequencing ahead of time. In addition to its value for database
mining and other more traditional genomic approaches, the availability
of large amounts of genome sequence together with physical maps
that cover almost the entire genome have begun to eliminate positional
cloning as a bottleneck in Arabidopsis genetics. Much of
this information is conveniently accessed through the
Arabidopsis thaliana database (AtDB) at Stanford University.
The growing importance of Arabidopsis research has also
been evident in the increasing number of participants at the Eight
and Ninth International Conferences on Arabidopsis Research,
which were held in Madison, WI, and drew 817 and 998 participants,
respectively.
Apart from the genome sequencing efforts, important tools are
being developed for reverse genetics and functional genomics.
A significant advance in this area has been an $8.7M award from
the NSF Plant Genome Research Program for a cooperative effort
to provide high-throughput gene expression profiling as well as
gene knock out services to the Arabidopsis community. The identification
of gene knock outs has been made possible through the availability
of large numbers of T-DNA insertion lines, of which 48,500 have
already been deposited with the Arabidopsis Biological
Resource Center (ABRC) at Ohio State University. This number can
be expected to at least double in 1999. The ABRC continues to
be an important resource for the Arabidopsis community. It shipped
29,500 seed and 13,000 DNA stocks in 1997; and 46,500 seed and
16,000 DNA stocks in 1998.
As a direct consequence of the improvements in scientific infrastructure,
significant scientific advances have been made in every area of
Arabidopsis research, including hormone and light signaling,
circadian clock, responses to biotic and abiotic stress and developmental
biology. Some of the most noteworthy discoveries in 1998 included
the discovery of master regulatory genes that protect Arabidopsis
from cold damage and the identification of proteins that transport
auxins.
Contact Person: Detlef Weigel
E-mail Address: detlef_weigel@gm.salk.edu
Contact Person: Jeff Dangle
E-mail Address:dangle@email.unc.edu
NSF ARABIDOPSIS GENOME MEETING REPORT
INTRODUCTION
In 1990, a report entitled "A Long-range Plan for the Multinational
Coordinated Arabidopsis thaliana Genome Research Project"
was published by the National Science Foundation (NSF 90-80).
The report detailed plans made by members of the Arabidopsis
research community in the U.S. and abroad, to collaborate in the
sequencing of the genome of this model plant, and to characterize
the structure, function and regulation of all Arabidopsis
genes. In 1998 it became possible to set a realistic goal of
finishing the sequence by the end of the year 2000.
Since then, a multinational genome sequencing project involving
laboratories in the United States, in Europe, and in Japan, has
been engaged in achieving this goal. This report is the proceedings
of a meeting held to discuss progress to date, to anticipate barriers
to timely completion, and to establish an oversight committee
for the U.S. -based labs. The meeting was held at the National
Science Foundation in Arlington, Virginia on July 9 and 10, 1998.
Participants Representing
Elliot Meyerowitz, California Institute of Technology Chair
Ian Bancroft, John Innes Centre ESSA
Michael Bevan, John Innes Centre ESSA
Ellson Chen, Perkin-Elmer Applied Biosystems CSHSC
Ronald Davis, Stanford University SPP
Nancy Federspiel, Stanford University SPP
Gerd Jürgens, University of Tübingen MSC
Richard McCombie, Cold Spring Harbor Laboratory CSHSC
Rob Martienssen, Cold Spring Harbor Laboratory CSHSC
David Meinke Arabidopsis community
Xiaoying Lin, TIGR TIGR
Curtis Palm, Stanford University SPP
Daphne Preuss, University of Chicago Arabidopsis community
Francis Quetier, Genoscope Genoscope
Steven Rounsley, TIGR TIGR
Marcel Salanoubat, Genoscope Genoscope
Satoshi Tabata, Kazusa Kazusa
Athanasios Theologis, USDA Plant Gene Expression Ctr. SPP
Richard Wilson, Washington University CSHSC
Mary Clutter NSF
Machi Dilworth NSF
DeLill Nasser NSF
James Tavares DOE
Jane Peterson NIH
Adam Felsenfeld NIH
Peter Bretting USDA
Liang-Shiou Lin USDA
STRUCTURE AND PROGRESS
There are six different sequencing consortia participating in
the sequencing phase of the Arabidopsis genome project,
three from the United States, two from the European Community,
and one from Japan. Each is sequencing a different region of
the genome, and each has its own model for distribution of the
necessary work among consortium members. The progress of each
follows, taking them in turn.
TIGR (The Institute for Genome Research,
http://www.tigr.org/tdb/at/at.html)
TIGR has taken on two aspects of the sequencing project. The
first is BAC end sequencing (along with SPP and Genoscope), to
provide one-pass sequences of both ends of the 22,000 BAC clones
that are one type of clone being used for sequencing in the genome
project. The purpose of this is to allow sequential progression
from a single sequenced BAC to the two adjacent genomic regions
with minimal overlap. TIGR has sequenced 16,392 BAC ends from
a total of 9,572 BAC clones, providing a total of 7.34 Mb of BAC
end sequence. The total BAC end sequence from all three groups
is 36,574 BAC ends from 18,746 clones, representing 13.64 Mb.
The second TIGR project is the sequencing of chromosome 2. They
have chosen 16 well-spaced starting points (by use of the Goodman
lab chromosome 2 contig map), and are sequencing BAC clones in
parallel, starting with the original clone in each location, and
proceeding by use of BAC end sequences to adjacent clones with
minimal overlap. The average overlap between adjacent BAC clones
has been 8.2 kb, with a range from 150 bp to 30 kb. At present
4.83 Mb is complete and annotated, 3.25 Mb has shotgun sequencing
or annotation in progress, and 1.38 Mb of BAC clones are in preparation
for sequencing, for a total of 9.46 Mb.
The only problem encountered so far is a gap with no clones to
cross it in present BAC collections, in the m336 large contig.
Fiber FISH done at the University of Wisconsin indicates a gap
size of 500 kb, and the sequence at either side of the gap shows
no special features. There has also been a BAC difficult to close
due to long tandem dinucleotide repeats, but there is no theoretical
barrier to completion of such clones.
The total estimated length of chromosome 2 is less than 14 Mb,
not including an estimated 3.5 Mb of ribosomal DNA tandem repeats
at one end of the chromosome. The current rate of sequencing
in this phase of the project at TIGR is presently 8 Mb per year,
and there is an existing proposal to increase that to 12 Mb per
year. It is estimated that, barring unforeseen problems, chromosome
two, excluding highly repetitive centromeric regions and the rDNA
repeats, will be completed by the end of 1999; if the full capacity
is to be used, clones on other chromosomes will have to be started
by the end of 1998.
SPP (Stanford University, Plant Gene Expression Center, University
of Pennsylvania;
http://pgec-genome.pw.usda.gov;
http://cbil.humgen.upenn.edu/~atgc/ATGCUP.html;
http://sequence-www.stanford.edu/ara/ArabidopsisSeqStanford.html)
These three groups have as a goal completing the sequence of chromosome
1. They have divided some of the preparative tasks, with Stanford
providing automated template preparation, Penn mapping chromosome
1 BACs and providing BAC end sequences to the project, and PGEC
making the sequencing libraries. All groups are involved in sequencing.
The strategy is similar to that of TIGR, whereby seed BAC clones
chosen by the Penn laboratory are used a sequencing origins, and
progress made by use both of BAC end sequences and BAC fingerprints,
to provide minimal overlap. Initially 20 starting points were
used, there are plans to add an additional 20 soon.
SPP has provided 8,936 BAC end sequences to the 36,574 BAC end
total.
The chromosome 1 sequencing done or in progress has so far totaled
5.64 Mb, which is the sequence of 55 BACs and 1 YAC clone. Excluding
overlap between adjacent clones leaves a total unique sequence
in progress or finished of 5.36 Mb. Of this 4.02 Mb are complete,
0.65 Mb in finishing and 0.97 Mb in shotgun phase. Overlap between
adjacent clones has been 2 to 38 kb, with an average less than
7 kb; there has as yet been no failure to find the adjacent clone
from any sequenced BAC.
The total estimated length of chromosome 1 is 30 Mb. Capacity
exists to finish it by the end of 2000, given sufficient funding
- completion will require sequencing approximately 300 BAC clones
in the next 3 years, or 33 BACs per year per participating site.
CSHSC (Cold Spring Harbor Sequencing Consortium;
http://www.cshl.org/arabweb/;
http://genome.wustl.edu/gsc/)
This consortium includes Cold Spring Harbor Laboratories, Washington
University and Perkin-Elmer Applied Biosystems. They are taking
a different approach to choosing the BAC clones to sequence, which
involves HindIII and EcoRI fingerprinting of BAC clones, and from
the clone overlaps inferred from fingerprint identity, producing
deep contigs of overlapping clones. Each contig is then to be
anchored to known chromosomal positions by use of the abundant
public information on BAC clone map positions, or by cross-hybridization
with the YAC contigs already established for chromosomes 4 and
5 at the John Innes Centre in the U.K. Once a genome-wide set
of BAC contigs is available, a minimal tiling path can be calculated
and many clones can be sequenced in parallel. This approach requires
the same degree of preparative work as BAC end sequencing for
a comparable cost, but has the advantages of providing a physical
map to the Arabidopsis community prior to the completion
of the genomic sequence, and also will allow parallel sequencing
of clones rather than the necessarily sequential sequencing using
BAC end sequences. In addition, this method will allow gaps to
be identified in advance of sequencing in the gapped region, and
thus may allow a longer time to close gaps before they become
a critical problem with sequence completion.
So far an estimated 71 MB of the perhaps 120 Mb nuclear genome
is contained in 66 BAC contigs, which contain 10,840 BAC clones.
The chromosome totals are:
Chromosome Mb Contigs
1 22.5 13
2 >4 7
3 17.0 11
4 15.3 8
5 13.4 8
The current rate of BAC clone fingerprinting and editing is 15
Mb per month. It is expected that all 22,000 available BAC clone
will be added to this map by the end of 1998. Concentration at
present is on chromosome 5, where the CSHSC is sequencing, and
chromosome 3, where Genoscope plans to sequence using the CSHSC
contigs.
The CSHSC is committed to sequencing the top of chromosome 4 and
a region of approximately 4 Mb around the centromere and on the
north arm of chromosome 5. Sequence data has been contributed
by all three collaborating partners. Totals finished so far are
690 kb from ABI, 1.22 Mb from CSH and 1.64 Mb from Washington
University, adding up to 3.54 Mb (with overlap subtracted). In
addition to this, approximately 3 Mb of sequencing is in progress,
making a total of more than 6.0 Mb in 61 BAC clones and 1 YAC.
If this rate were to be continued, the proposed chromosome 4
region could be completed by the end of 1998, with chromosome
5 region completion either 1998 or early 1999.
ESSA (European Scientists Sequencing Arabidopsis;
http://muntjac.mips.biochem.mpg.de/arabi/index.html)
The ESSA project is in three phases. Phase I, which is complete,
was to sequence two contiguous regions on chromosome 4. One,
surrounding the FCA genetic marker, is 1.92 Mb (Bevan et al. 1998,
Nature 391:485), the other, around the genetic marker AP2, is
0.41 Mb, for a total completed ESSA I sequence of 2.33 Mb. ESSA
II, which is to be completed in October 1998, has the goal of
completing a 5 Mb region on the long arm of chromosome 4. So
far 3.16 Mb is completed and annotated, an additional 1.73 Mb
completed and in annotation phase, for a total of 4.89 Mb sequenced.
Another 0.24 Mb is nearly complete, for an overall total of ESSA
II complete and nearly complete contiguous sequence of 5.13 Mb.
The ESSA I and ESSA II total of completed and nearly completed
sequence is thus 7.46 Mb.
The two-year ESSA III project begins in August, 1998. Its goal
is to complete the sequence of the long arm of chromosome 4 (estimated
to total 13 to 13.5 Mb) and to sequence two regions of the north
arm of chromosome 5 (with others to be done by CSHSC and Kazusa),
with a total goal of sequencing 9 Mb.
The ESSA procedure is to use the existing YAC contig maps of chromosomes
4 and 5 to group BAC clones in bins according to their YAC cross-hybridization,
then to use SalI digestions and pulsed-field gel electrophoresis
followed by blotting and iterative hybridization with BAC clones
to establish both BAC contigs and an overall SalI restriction
map of both chromosomes. A minimal BAC tiling path is then defined
and called the "sequence ready map,", the clones from
this map are then sent to one of 9 collaborating sequencing laboratories
for nucleotide sequencing. The data are collected and annotated
at MIPS, the Munich Information Center for Protein Sequences.
The only problems encountered so far have been two difficult clones,
one with a large hairpin and the other with a large region of
tandem repeats. Both have been nearly completed, with the tandem
repeats solved by long PCR as a supplement to the shotgun sequencing.
Kazusa DNA Research Institute (
http://www.kazusa.or.jp/arabi/)
The Kazusa Institute is engaged in sequencing the long arm of
chromosome 5 and along with ESSA and CSHSC, portions of the short
arm of this chromosome (totaling 17.2 Mb when complete), and they
are beginning the sequencing of the long (13.2 Mb) arm of chromosome
3.
The clone libraries used are from the Mitsui Plant Biotechnology
Research Institute, and consist of P1 and TAC clones. Clones
from these libraries are initially selected by cross-hybridization
to mapped clone markers. The clones are then anchored on the
YAC contig (for chromosome 5 clones), and fingerprinted as an
integrity check. They are then shotgun sequenced, assembled,
and annotated. A collection of YAC, TAC and P1 clone end sequences
has been made for tiling the chromosome 5 clones, it includes
1254 sequences from 690 CIC YAC clones and 706 sequences from
389 P1 or TAC clones on chromosome 5. Similar methods for chromosome
3 are starting, using the YAC contig map of that chromosome produced
by D. Bouchez and collaborators at INRA. At present, two large
contigs for chromosome 3 exist, one of 13.6 Mb for the long arm,
and one of 9.2 Mb for the bottom arm.
Progress to date has been the release of 8.89 Mb of completed,
annotated sequence, with release of an additional 1.60 Mb scheduled
by August 1. Thus by August 1, 1998, 10.49 Mb will have been
completed and released. 10.15 Mb of this is on chromosome 5,
0.34 Mb on chromosome 3. An additional 2 Mb of chromosome 5 sequencing
is in progress. At current rates of 700 to 800 kb per month,
it is expected that 27 months will be required for completion
of this part of the project, which is estimated to include (in
addition to the 10.49 Mb to be completed by August 1) 7.05 Mb
of chromosome 5 and 13.3 Mb of chromosome 3. Genoscope has proposed
to do 5 Mb of the long arm of chromosome 3 (see below), if they
are able to take this on (a matter now being considered there,
and dependent upon the demand for their resources by human genome
sequencing) the total sequence proposed by Kazusa will be reduced,
and completion will be expected within 2 years.
Genoscope (Centre Nationale de Sequencage;
http://www.genoscope.cns.fr/externe/arabidopsis/Arabidopsis.html)
Genoscope is involved in the second European project. They have
already provided BAC end sequences totaling approximately 11,500
completed end sequences, with plans to provide 2,000 more. Once
this is complete 91% of the 22,000 BAC clones used in the sequencing
project (from the IGF and the TAMU collections) will have available
end sequences.
Their sequencing plan is to use the Bouchez chromosome 3 YAC contigs
to make a minimal BAC tiling path by use of fingerprints done
at Genoscope and at CSHSC, then to sequence the bottom (9 Mb)
arm of chromosome 3. Complete contigs for this region have been
supplied by CSHSC. 16 different European sequencing groups are
receiving the BAC clones from Genoscope, and the data are returned
to MIPS for annotation and entry into a public database. The
sending out of clones is to begin within weeks, and completion
of the 9 Mb region is expected by the end of 2000.
Genoscope has in addition explored with Kazusa the possibility
of sequencing an additional 5 Mb on the top arm of chromosome
3; their ability to do this will depend upon the amount of their
sequencing capacity that will be required to do their part of
human chromosome 14, and their ability to generate extra sequencing
capacity. A decision on whether Genoscope or Kazusa will sequence
this 5 Mb is planned for September, 1998.
Summary of Progress
Chromosome Est. Size (Mb) Complete (Mb) Group
1 ~30 4.02 SPP
2 14 (+rDNA) 4.83 TIGR
3 23 0.34 Kazusa & Genoscope
4 17 (+rDNA) 9.02 ESSA & CSHSC
5 ~30 10.15 Kazusa, CSHSC, ESSA
TOTAL ~114 Mb +rDNA 28.36
In addition, shotgun sequencing libraries are in preparation for
an additional 2.80 Mb, and sequencing is in progress but not yet
complete for an additional 2.98 Mb. Furthermore, 36,574 BAC ends
from 18,746 clones, representing 13.64 Mb, provided by TIGR, SPP
and Genoscope are completed, as are 1254 end sequences from 690
CIC YAC clones and 706 sequences from 389 P1 or TAC clones, provided
by Kazusa.
COMPLETING THE SEQUENCE
Defining completion
In addition to the gene-rich and highly informative regions of
the genome (with one gene every 4-5 kb), there are regions of
repetitive DNA, and perhaps of lower gene density.
One instance is the ribosomal DNA repeats, which are arranged
in two uninterrupted tandem arrays. Each repeat unit contains
a gene for 18S, 5.8S and 25S structural ribosomal RNAs and is
10-10.5 kb in length. The large tandem arrays of repeat units
are found at the top arms of chromosomes 2 (NOR2) and 4 (NOR4).
Each is on the order of 3-3.5 Mb, or 300-350 repeat units.
Centromeric regions are only beginning to be defined at the molecular
level in Arabidopsis, but cloning and chromosome in
situ hybridization studies have shown that these regions contain
multiple tandem repeats of short sequences, a major element of
which is 180 bp repeats and related repeats. In one case (chromosome
1) an estimate of the repeat length is 950 kb. For chromosome
4 the functional centromere is probably on one side of a 180 bp
repeat region, and so far does not seem to be unclonable. There
is some indication that BAC clones from this region may have a
higher amount of repetitive sequence in tandem arrays than other
BAC clones sequenced to date, and one BAC clone from the chromosome
2 centromere region has only 3 genes, a much lower density than
the typical 1 gene per 4-5 kb found elsewhere. Another BAC from
the centromere region of chromosome 4 has a more typical density.
Telomeres and subtelomeric regions in Arabidopsis have
been characterized and appear to be small (totaling perhaps 100
to 200 kb in the genome) and not difficult to sequence so far.
There are also small regions of simple tandem repeats, as for
example as described above in the ESSA project progress report.
This clone, BAC F9F13, contained 10 tandem copies of a 3.5 kb
repeat, as well as 2 additional copies of the same repeat.
Because the exact sequence and number of tandem repeats is not
thought to be consequential for any functional analysis, and in
fact is quite polymorphic between ecotypes, it was decided that
a sufficient characterization of these repeats would be a sequence
of one subunit, and an estimation from blotting or long-range
PCR of the number of tandem copies at each site.
Given this, the complete sequence of the nuclear genome will be
considered to be in hand when each chromosome arm is fully sequenced
as a single contig from subtelomeric repeat to "centromeric"
tandem repeats, with internal tandem repeat regions (including
rDNA repeats) characterized only as far as demonstrating that
they are pure tandem repeats, with the sequence of one repeat
unit determined, and an estimate of repeat number at each site
provided. This characterization already exists for the rDNA repeats
(Copenhaver et al. (1995) Plant J. 7:273-286). This definition
may have to change if unclonable regions are found, or if non-tandemly
organized but nonetheless impossible to sequence (with available
relevant technology) clones are found. To date there is no indication
of either unclonable regions or of clones impossible to sequence
for reasons other than large numbers of small tandem repeats.
Other sequence parameters
Accuracy
All of the participants have agreed before, and continue to agree,
that the standard for sequence accuracy should be one error in
10,000 nucleotides or better, and the projects so far seem to
be achieving this goal. The U.S. groups agreed to a common pair
of tests to monitor sequence accuracy. The first would be using
base calling programs such as Phred (Ewing et al. (1998) Genome
Res. 8:175-185) or TIGR Assembler to assess sequence accuracy
in each sequencing run. The second is to independently determine
the sequence of all regions of overlap between adjacent clones,
and only after sequence finishing to compare them for mismatches.
This serves as an independent method to determine sequence accuracy,
and since all mismatches are to be resolved by further analysis,
this test will in addition indicate the degree of sequence change
due to mutation in the clones being used for sequencing.
The European and Japanese groups have different methods to measure
sequence accuracy, but have the same goal of less than one error
in 10,000 bases.
Annotation
Proper annotation of sequences to indicate the position, structure
and nature of each of the coded genes is a critical component,
and in fact the primary product, of the genome project. It is
clear, though, that initial annotation of sequences is not fully
(or even very) accurate, as the software and algorithms used for
gene recognition can miss exons and introns, and can also indicate
the presence of exons or introns where there are none. This is
as true in animal genome projects as in plant projects. Thus,
annotation will have to be done in stages, with initial annotations
that can be useful, but that must be acknowledged to be flawed.
Each of the sequence groups performs its own annotation, as this
is not only an interesting part of the work, but also helps with
continued sequencing. It was agreed that, to provide the highest
quality initial annotation, each group would use multiple software
programs for gene recognition, and would indicate in its output
the product of each of the programs (something that GenBank cannot
do; thus this requires output to be in a form other than that
sent to GenBank or equivalent public databases). It should be
emphasized that doing this does not remove the requirement for
inclusion of the output in public databases like GenBank or DDBJ.
In addition, experimental means of annotation are to be used
by each group - that is, sequences must be compared with the EST
sequences that are available and that indicate actual RNA sequences,
and must be compared with the genes of known structure that have
been individually studied. Furthermore, feedback from the community
of Arabidopsis researchers should be invited by each group,
to allow correction or improvement of each group's annotations.
As the genome project proceeds, it is important to consider additional
experimental methods for gene recognition, and the application
of such methods should be considered important goals for the project.
Among the experimental methods to be considered is sequencing
of related genomes (such as those of Arabis lyrata or Cardaminopsis
petraea, see
http://www.arabis.net/wild.htm). Because exonic
sequences change more slowly than intronic or intergenic sequences,
this could serve as a very useful indicator of gene location and
exon boundaries. Additional experimental means for improving
annotations include RNA blots and RT-PCR to find if suggested
genic sequences in fact correspond to RNAs, and full-length sequencing
of large numbers of cDNA clones for comparison to genomic sequences.
Maintenance of summary lists of identified genes according to
the type of protein coded (see Bevan et al. 1998, Nature 391:485)
is also an important aspect of annotation.
Because annotation methods and the experimental information on
which they are based is subject to continual improvement, frequent
reannotation is worthwhile. Both the Kazusa and TIGR groups have
plans for systematic reannotation of sequences from all groups.
To facilitate this and, especially, to facilitate community access
to annotations, it was agreed that all groups would work toward
a standardized format for data presentation, and that groups doing
large-scale reannotation would make their data freely available
for mirroring on the web sites of all groups that wish to display
them.
Data release
Each of the U.S. groups sends sequence out unannotated and in
small fragments as soon as it reaches either approximate 2 kb
contigs or 7x average coverage. The sequences from two of the
three groups are sent at this stage to the high throughput genome
sequence (HTGS) part of GenBank, the third group has agreed to
start doing this as well. The sequences are now sent to each
group's own web page, each of which supports BLAST searches, and
are also sent at short intervals to AtDB, the public Arabidopsis
database, where they are also BLAST searchable (
http://genome-www2.stanford.edu/cgi-bin/AtDB/nph-blast2atdb).
The structure of the European projects, where sequence-ready clones
are allocated to many groups, and each group has some discretion
(and rules from their own national government) in how to sequence
and when to submit completed sequence, does not lend itself to
identical release methods or policies. Nonetheless, the groups
agree to collect and distribute through MIPS and AtDB all sequences
as soon as practicable, at latest after completion and before
annotation.
The Japanese group also has its own policies and level of funding
for informatics, which so far have dictated that sequence be released
only after both completion and annotation, and then posted to
DDBJ (DNA Database of Japan) and GenBank. This entails a delay
in public access relative to other groups, as the time from completion
to annotation is about a month, and the time from acquisition
of the earliest data to completion is also appreciable. The Japanese
group will consider mechanisms for earlier release, within the
constraints of policy and of funding for this aspect of the project.
Clone registration (intention to sequence)
One critical aspect of the project is coordination between groups
on the clones to be sequenced, as without tight coordination,
duplication of effort will occur, especially in the closing phases
of the project. In addition, as different groups complete their
assigned regions, reallocation of regions may become necessary
so that groups ahead of their predicted rate can help by sequencing
clones originally assigned to other groups. At present this coordination
has been supplied by direct communication between the groups,
and by the function of an international coordinating committee
of the Arabidopsis Genome Initiative (AGI: see
http://genome-www3.stanford.edu/cgi-bin/Webdriver?MIval=atdb_registry_info.html).
This committee will remain the arbitrator of international sequencing
efforts, but will be supplemented with a new committee that will
allow for closer coordination of the U.S. groups. This new committee
has been mandated by the U.S. funding agencies, as a replacement
for the three separate advisory groups that now exist, one for
each group.
One of the tasks of the U.S. committee will be clone reallocation,
and in addition frequent communication with the members of the
international AGI committee, as a way of stimulating continued
discussion among all groups. As representatives of all groups
will be invited to the meetings of the U.S. committee, these meetings
may also be able to serve as a forum for discussion and decisions
of the AGI committee. This may help the AGI by increasing the
frequency of its considerations.
NEW U.S. STEERING COMMITTEE
Given the important new role of the mandated U.S. Steering Committee
as arbitrator and communication facilitator between the U.S. groups,
and as aid to the AGI committee on the international front, the
role a responsibilities of the committee were discussed and agreed
upon.
The U.S. Steering Committee will have the following responsibilities:
1) Setting boundaries between the U.S. sequencing groups (ideally,
to be defined by sequenced clones) to avoid duplication of effort
in chromosomes where more than one group is working
2) Reallocation of clones or chromosome regions from one group
to another to fit sequencing capabilities to the remaining work.
3) Monitoring and enforcement of the common agreements described
earlier in this report, namely the agreement to work toward a
common annotation format, to provide quality control information
both from base calling programs and from clone overlap regions,
and to monitor sequence release compliance.
4) Providing annual progress reports to the Arabidopsis
community and to the U.S. funding agencies, separate from the
progress reports of each of the individual sequencing groups.
These reports will include a careful consideration not only of
amount of sequence provided by each group, but of progress in
all respects, balanced so that groups taking on difficult clones
to sequence, or who are in closing phase and thus must devote
time to closing gaps, are given full credit for such efforts.
In addition, these reports are to detail progress in the informatics
aspects of the project, including a summary of the progress and
needs of the Arabidopsis database - as an interface between
the database and its advisory committee, the sequencing groups,
and the Arabidopsis community.
5) Provide an interface between the U.S. groups and the international
AGI committee, and act to facilitate the setting of boundaries
and clone reallocation at an international level.
6) The committee should endeavor to meet in person at least once
a year, and have regularly scheduled meetings by electronic mail
or conference call.
The composition of the committee is as follows:
Members:
Ex officio:
The actual members of the committee who have so far agreed to
serve:
Elliot Meyerowitz, chair (U.S. Arabidopsis community)
Daphne Preuss (U.S. Arabidopsis community)
Gerd Jürgens (international Arabidopsis community)
Ex officio:
Joe Ecker, SPP
Dick McCombie, CSHSC
Steve Rounsley, TIGR
Ian Bancroft, ESSA III
Francis Quetier, Genoscope
Satoshi Tabata, Kazusa
Recommendations for the other members were:
Joanne Chory, Pam Green or Detlef Weigel (U.S. Arabidopsis community)
Mark Johnson, Richard Gibbs, John Sulston, Maynard Olsen (sequencing experts)
Mark Boguski (database expert)
Mike Cherry (AtDB representative)
FINAL PROSPECT
Given sufficient funding, which seems very likely, there is no
technical obstacle to the completion of the Arabidopsis
nuclear genome sequence by December 31, 2000. Although the efforts
of the project members must be focused tightly on finishing the
sequencing, it is not too early to begin considering the next
steps, among them experimental methods for annotation, and functional
analyses of genes and gene families.
submitted by:
Elliot M. Meyerowitz July 15, 1998
Summary of December 1998 AGI Meeting at CSHL
1. Daphne Preuss summarized her work on centromeric regions and
presented detailed information on approximate map locations of
BAC contigs and sequenced BACS based on hybridization (Altmann)
and fingerprint (WashU) data. She agreed to make this information
available to the community. Rob Martienssen stressed that individual
clones would need to be compared closely with fingerprint contigs
constructed at WashU because some hybridization data were unreliable.
2. Each group discussed their estimated sequencing capacity and
assigned chromosomal regions for the coming year. Kazusa expects
to finish their assigned regions on III and V by the end of 1999.
ESSA and CSHL/WashU may also complete their assignments on IV
and V at about the same time. SPP is continuing with chromosome
I and was encouraged to avoid starting many additional nucleation
points in order to focus on the same closure issues being addressed
by the other groups. Genoscope has begun sequencing the bottom
arm of III and will continue with this region through 2000. TIGR
expects to finish chromosome II by summer 1999 and will therefore
be the first funded group to run out of an assigned region to
sequence.
3. AGI members discussed the importance of finishing difficult
areas within assigned regions of the genome while also continuing
to make rapid progress on other regions to maximize release of
information to the community.
4. Both TIGR and Kazusa proposed to begin sequencing the "unassigned"
top 5-6 Mb of chromosome III during 1999. After considerable
discussion, both at the AGI meeting and later in the conference
when Satoshi Tabata arrived, a consensus was reached to have
TIGR begin sequencing this region of chromosome III during the
spring of 1999 with the aim of finishing this region by January
2000.
5. Starting in January 2000, TIGR, Kazusa, CSHL, and ESSA will likely have residual sequencing capacity ready to shift to centromeric regions and portions of chromosome 1 that have not yet been completed. By this time a minimal tiling path based on fingerprint data should be available to facilitate assignment of remaining BACs to AGI members. SPP has funding to complete most or all of chromosome I but recognizes that the entire genome
may be completed more rapidly if other groups contribute in the
year 2000 to sequencing portions of this chromosome (or possibly
part of the bottom of chromosome III depending on progress made
by Genoscope) after their own assigned regions have been essentially
completed.
6. Marcel Salanoubat and Francis Quetier led a discussion of
the Genoscope policy for sequence release. While it was clear
that the informatics capabilities of the individual laboratories
in their program varied significantly, there was a general agreement
that the group should strive for immediate release of sequences
(at least for the bigger laboratories within their program).
7 . Rob Martienssen and David Meinke discussed the status of the
CSHL/WashU consortium plans to continue sequencing and fingerprinting
efforts. NSF has now received all of the necessary paperwork
for continued funding of this consortium and expects to make an
award at a level sufficient to enable sequencing another 2.4 Mb
per year starting early in 1999. In addition, NSF has recommended
funding an informatics person at WashU to finish editing of fingerprinted
contigs and establishment of an interactive version of the BAC
physical map that can be accessed via the Internet. This person
will work closely with AtDB to avoid duplication of effort.
8. The CSHL/WashU group has agreed to release to other sequencing
groups all of their edited contig information and fingerprint
database through their ftp site no later than the end of January,
1999. The SPP and TIGR groups are particularly anxious to make
use of this information in order to avoid repeating the contig-building
steps that have already been completed elsewhere. Rob Martienssen
agreed to provide as soon as possible a minimal BAC tiling path
for regions of the genome that may require coordination during
the final year of the project..
9. Joe Ecker and David Meinke discussed a proposal by Hiroaki
Shizuya at Caltech to fingerprint and end-sequence a new BAC library
with large inserts (180 kb average). The general consensus was
that although this library might be very useful in regions of
the genome with minimal coverage and could reduce the overall
cost of sequencing other regions by reducing overlaps, it was
unlikely that many AGI participants would immediate move away
from using TAMU and IGF clones for the bulk of their sequencing
efforts. NSF is willing to discuss further the potential value
of this library with interested AGI members.
10. Rob Martienssen agreed to serve as the next AGI chairperson.
There was general agreement that AGI members should meet again
in summer 1999, perhaps at the next Arabidopsis meeting in Australia,
to assess progress and make specific plans for the future.
Joe Ecker, AGI chairperson
I. VENUE AND PARTICIPANTS
To assess the current and future database needs of the Arabidopsis
community, an NSF-supported workshop on this topic was convened
in Madison Wisconsin on June 28, 1998. The workshop participants
included the following individuals:
Rick Amasino, University of Wisconsin
Mary Anderson, Nottingham University
Mike Cherry, Stanford University
Joanne Chory, Salk Institute
Maarten Chrispeels, University of California San Diego
Jeff Dangl, University of North Carolina
Keith Davis, Ohio State University
Allan Dickerman, National Center for Genome Research
David Flanders, Stanford University
Pam Green, Michigan State University
Bertrand Lemieux, University of Delaware
David Meinke, Oklahoma State University
Larry Parnell, Cold Spring Harbor Laboratory
Daphne Preuss, University of Chicago
Ralph Quatrano, Washington University
Ernie Retzel, University of Minnesota
Steve Rounsley, The Institute for Genomic Research
Randy Scholl, Ohio State University
Chris Somerville, Carnegie Institution of Washington and Stanford
University (chair)
Desh Pal Verma, Ohio State University
The following individuals provided valuable written comments prior
to the meeting (Appendix I):
Jean Greenberg, University of Chicago
Katie Krolikowski, Harvard University
Russell Malmberg, University of Georgia
Jose Martinez-Zapater, Biology Molecular y Virologia Vegetal,
CIT-INIA
Natasha Raikhel, Michigan State University
Pierre Rouze, Flanders Institute of Biotechnology
Chris Town, Case Western Reserve University
Desh Pal S Verma, The Ohio State University
In addition, the workshop was attended by the following observers:
Peter Bretting, USDA/ARS National Program Staff
Greg Dilworth, Department of Energy
Machi Dilworth, National Science Foundation
Margarita Garcia, Stanford University
Paul Gilna, National Science Foundation
Xiaoying Lin, The Institute for Genomic Research
Bob MacDonald, US Department of Agriculture
DeLill Nasser, National Science Foundation
II. GOALS
The general goals of the workshop were to examine the present
and future database needs of the Arabidopsis community and to
outline in general terms the main issues which should be addressed
in any future proposals concerning the development of new or expanded
Arabidopsis databases. The discussions were intentionally focused
on biological and community issues and there was no attempt to
define or specify issues which are related to specific computer
hardware or specific database programs. In particular, no assumptions
were made concerning continued government funding of any current
Arabidopsis database activities.
A previous workshop with these goals was held on June 5th and
6th, 1993. A copy of the published summary that workshop was provided
to all participants and served as a reference to earlier views
and objectives of the Arabidopsis community. [1993
Dallas Workshop Report] In addition, participants were
provided with a draft summary of a BBSRC-USDA bilateral plant
bioinformatics and coordination meeting held at Llangollen Wales,
March 22-24, 1998. A copy of a memorandum, dated February 26,
1998, from the North American Arabidopsis Steering Committee to
the curators of AtDB, concerning the current Arabidopsis community
database needs was also provided. [NAASC
Memorandum] Finally, in preparation for the meeting,
written comments solicited from the community on the Arabidopsis
electronic newsgroup were provided to the participants before
the meeting. A copy of the solicitation and written comments are
appended as Appendix I.
III. RATIONALE FOR AN ARABIDOPSIS DATABASE
The genomes of higher plants, such as Arabidopsis, contain approximately
25,000 genes. During the next several years, the sequence of the
Arabidopsis genome will be completed and extensive sequence information
will become available for many other species, including many plants.
Most or all of the Arabidopsis genes will be used to develop gene
chips or microarrays that permit simultaneous measurements of
the expression (mRNA levels) of all of the genes. These will be
used to generate information about the expression of all the genes
in the organism in response to a wide variety of treatments and
genetic backgrounds. Each experiment could have as many as 25,000
data points for each time point or treatment of each genotype!
Comprehensive libraries of insertional mutations will permit the
isolation, by reverse genetics, of null mutations in any Arabidopsis
gene. Extensive collections of enhancer-trap or promoter-trap
lines are being developed that permit sensitive analyses of the
spatial patterns of gene expression down to the single-cell level.
Thousands of new classes of mutants will be isolated by selecting
for suppressors or enhancers of existing mutations. The corresponding
genes will be cloned by very high resolution mapping of the mutations
so that a limited number of candidate genes which are evident
in the delimited region of genomic sequence can be directly tested
for complementation. This will depend on the development of very
high resolution maps. It seems likely that high resolution proteomics
methods will become important for identifying the substrates of
the thousands of kinase genes that form many of the regulatory
networks in Arabidopsis and other plants. Additionally, extensive
genomic-based work in other plant species will produce a flood
of sequence information. The value of much of that information
will be greatly enhanced by comparison with the aggregate information
available in Arabidopsis. Thus, we are entering an era of explosive
growth of knowledge about Arabidopsis in particular, and plants
in general. Most of the data generated by the projects described
above will never appear in printed journals and will only be available
to the community through electronic databases.
Because Arabidopsis is one of the most intensively studied organisms, and is a direct model for 250,000 closely related species, we believe that it is appropriate to undertake a major investment in developing new information retrieval tools (IRTs) for Arabidopsis in particular and plants in general. By this we mean that because we will know everything about Arabidopsis, it is a suitable object on which to focus the building of a comprehensive database or set of linked databases. However, because the value of Arabidopsis derives from its utility in understanding other plants, it would be desirable to build a structure that permits facile high resolution linking of specific information about Arabidopsis to all other plants.
Looking into the future more generally, it is apparent that scientific
publishing is undergoing a much needed revolution. All of the
major journals will be electronic within a few years and once
that transition is complete, scientists will develop new tools
for interacting with data. The complexity of biological knowledge
in many fields is such that new mechanisms for integrating data
are required. The development of computer programs that calculate
genetic maps "on the fly" from currently available data
is an early example of what will become a more general mechanism
for integrating data. Integrated graphical representations of
patterns of gene expression in individual cells of three dimensional
models of organisms at various developmental stages is another
example that is under development. With such a model it will be
possible to find relationships between objects (eg., genes) and
processes that would be difficult or impossible with current information
retrieval technologies.
Because of the changes taking place in publishing, there may be an opportunity to develop databases that will eventually be self supporting in the same way that journals are self supporting. As the distinction between the format blurs, the concept of paying for a database subscription will become commonplace. However, there are many complex issues associated with imposing charges for database use and the question is largely academic at present.
There are many challenges in developing a new generation database.
Perhaps the foremost is the difficulty in collecting information
from the thousands of scientists who produce primary information
for conventional publication in journals.
IV. CURRENT PUBLICLY SUPPORTED DATABASE ACTIVITIES
The principal publicly supported Arabidopsis database activities
are the AtDB database at Stanford University and the stock center
databases maintained by the Arabidopsis resource centers at Ohio
State University and the University of Nottingham. In addition,
the University of Minnesota supports an EST database for all plants,
and each of the Arabidopsis genome sequencing groups provides
database access to genomic sequences, including BAC end sequences.
The AtDB goal is to provide the plant-biology research community with convenient and correlated access to the publicly available results of Arabidopsis research. This includes published and otherwise freely available information about the genome, the genes it contains, the gene products, their positions on genetic and physical maps, as well as DNA sequences. The users of the database are very diverse, ranging from Arabidopsis molecular biologists to biologists focusing on any other organism. The members of the AtDB project are currently shared with the Saccharomyces Genome Database, and the database administrator is shared with the Expression Microarray database and Genetic Footprinting database projects, all located at the Department of Genetics at Stanford University. In an effort to minimize wasteful duplication of effort, the AtDB project uses much of the same software and staffing structure as the Saccharomyces Genome Database (SGD). The combined SGD and AtDB groups thus benefit from an economy of scale by sharing computing and human resources.
At a meeting of the Arabidopsis genome community in 1992 at the
Cold Spring Harbor Banbury Center, a consensus was reached that
AtDB should take responsibility for providing centralized access
to Arabidopsis databases, a recommendation that has been repeatedly
endorsed by the North American Arabidopsis Steering Committee.
Since that time AtDB has been supported by a grant from the National
Science Foundation. However, the annual level of support for AtDB
has been only a small fraction of the support provided for database
activities for similarly advanced models such as Drosophila, yeast
and mouse.
V. SUMMARY OF CONCLUSIONS AND RECOMMENDATIONS
The highest priorities for database content are:
VI. WHAT SHOULD BE IN THE DATABASES?
The long-term goal is to provide interconnected access to all
information about Arabidopsis. However, certain classes of information
should have a higher priority for immediate inclusion and also
require a high degree of curation in order to be most useful to
the community.
A. Map-Based Information
At present, many laboratories are engaged in cloning genes by
map-based cloning methods. The use of map-based cloning is expected
to continue indefinitely and to become the most widely used method
of cloning genes in the future. The ease with which this can be
accomplished is directly proportional to the availability of information
about genetic and physical maps, polymorphisms, and large clones.
Thus, the greatest current need is a unified genetic and physical
map that incorporates all available information about polymorphic
markers (eg. CAPS, SSLPs, RFLPs), mutations, BAC and YAC clones,
mapped clones and insertions or other modifications of the genome.
Because of the pending completion of the genomic sequence, the
state of the genetic map is expected to change dramatically during
the next several years as sequence-based markers become anchored
on the genomic sequence. The availability of the sequence information
will enhance the value of the integrated map because it will stimulate
map-based cloning efforts which will remain dependent on a high
density of polymorphic markers. The integration of the genetic
and physical maps should be undertaken by a group with appropriate
expertise in both genetic and physical maps and database management
and curation.
Ready, access to primary mapping data should be given highest
priority in database development. Map information should be collected
and presented in a manner that allows the user to determine what
is known, plus what remains questionable or unresolved with respect
to map locations of genetic and molecular markers in combination
with a complete physical map anchored to the complete nucleotide
sequence. In constructing the database, it should be remembered
that recombination data generally provide only rough estimates
of map location, and that mapping data may differ widely in quality
and reliability. Therefore, some database users may prefer direct
access to primary mapping data in order to compare their results
with those obtained in other laboratories. A database that provides
options for visualizing several different maps constructed with
different mapping functions or subsets of markers and primary
mapping data would be particularly valuable to the Arabidopsis
community.
Any proposal for database development should also discuss in some
detail how the integrity of these maps would be verified and maintained.
Some mutations and cloned genes are likely to be known by several
different names. It will therefore be important to establish a
database that will accommodate multiple changes in nomenclature.
Other plant databases are moving toward the use of standard gene
names as described in the Mendel database. The Arabidopsis databases
should also adopt this policy to ensure compatibility with other
databases.
Provisions should also be made to add new types of information
to genetic and physical maps as they become available (break points
of chromosomal aberrations; regions of extensive heterochromatin;
regions with a high/low degree of sequence homology to related
plants; etc.).
B. Sequence information
The value of the genomic sequence will depend on the quality of
the annotation. The goal for the quality of annotation should
be similar or identical to that of other higher organisms. It
should be possible to arrive at an integrated map of a gene by
various routes. A user should be able to begin a query with a
sequence, a gene name, a keyword or a genetic map location. A
user should be able to highlight a region of the genome on a graphical
display and move to increasingly higher levels of resolution with
the click of a mouse. For example, one might start with a whole
chromosome, then move to a ~10 cM region which shows the contigs
of BACs and YACs, the mapped mutations, the sites of insertional
mutations or launching pads for transposons. Next the user should
be able to visualize a ~1 cm region showing all of the above features
plus the locations of open reading frames (theoretical and verified),
ESTs, polymorphic markers, potentially polymorphic markers (ie,.
SSLPs). Finally, at the next level of resolution the user should
be able to visualize the DNA sequence, the various putative open
reading frames indicated by gene finding programs, experimentally
verified genes, ESTs, BAC and YAC end sequences, polymorphisms,
mutations and other known aberrations. The open reading frames
should be linked to information about gene expression, experimentally
verified information about gene function, mutant phenotypes associated
with classical mutations or over or under expression, theoretical
information about gene function based on inference from other
organisms, subcellular localization of the gene product, known
or predicted modifications of the gene product. If there are other
genes of similar structure in the genome, the presence of these
genes should be indicated. Similarity to genes from other plants
should be indicated with a link to the appropriate databases.
The control regions of the genes should be annotated with known
or predicted motifs and with information about the identity of
other genes with similar motifs.
The sequence information should not simply be a link to raw sequence
in GenBank because the level of annotation and tools to manipulate
that sequence do not directly support the kinds of queries made
by most biologists. Thus, the sequence should be directly available
from a specialized database which provides useful tools for manipulating
the sequence. It should be possible to retrieve from the database
sequence information based on map position, type of sequence,
or other specific requirements. All information should be linked
to publications describing the data when possible.
Because the sequencing groups are not expected to have the resources
to provide continued annotation, there will be a need for a group
to take responsibility for continued upgrading of the annotation
of the genomic sequence as information about the sequence becomes
available from direct experimentation and from computational analyses
based on experimental results obtained with other organisms.
C. Expression information
The use of microarrays and gene chips are expected to provide
a massive amount of new information. Most or all of the Arabidopsis
genes will be used to develop gene chips or microarrays that permit
simultaneous measurements of the expression (mRNA levels) of all
of the genes. These will be used to generate information about
the expression of all the genes in the organism in response to
a wide variety of treatments and genetic backgrounds. Each time
point or treatment could have as many as 25,000 data points. Because
the experiments are technically straightforward, it seems likely
that a common type of experiment will be to prepare mRNA from
a mutant and a wild type and to compare the consequences of the
mutation on the expression of all the genes in the organism. In
addition to simply archiving the raw data it should be possible
to query the data in various ways. For instance, as data from
different treatment accumulates, it will become possible to search
for genes that are coregulated with a gene. This kind of query
may provide insights into the identity of otherwise anonymous
genes or reveal the existence of networks. It should also be possible
to identify all the factors that cause altered expression of a
gene, to identify all genes that specifically respond to certain
treatments, to identify mutations that cause similar effects on
gene expression. For these kinds of queries it will be necessary
to have software that can identify data sets that are most similar
from among hundreds or thousands of different data sets produced
by different treatments.
There is also a large need for a repository for information about
spatial aspects of gene expression. There are now many transgenic
lines which exhibit specific spatial patterns of reporter gene
expression, and cloned genes which confer such patterns. In the
short term a database with a controlled vocabulary for the various
cell and tissue types and linked images of the patterns of gene
expression would meet immediate needs. In the longer term, it
would be useful to have graphical tools that would integrate the
patterns of gene expression into an organismic model.
D. Phenotypic Information
Because of the diversity of processes that are being analyzed
by a mutational approach in Arabidopsis, there is a need for facile
access to information about gene function as it relates to the
organism. One aspect of the problem involves determining the genetic
basis for a phenotype. In this case it should be possible to enter
a description of a phenotype and obtain a ranked list of probable
genetic alteration that could give rise to the phenotype. Conversely,
it would be very helpful to be able to enter a gene name and obtain
a description of the corresponding mutant. This capability will
greatly enhance the efficiency with which new mutations will be
studied as the number of known mutations begins to plateau. It
is expected that we will soon have saturating collections of transposon
mutants, so having ways of describing these phenotypes, and making
them accessible, will be important. No capability of this kind
currently exists.
One strategy may be to use organizational schemes as entry points
(phenotypic indexes, so to speak). One such index is the genetic
map position. Knowledge of this provides an entry point to other
mutants and papers. Another possible organizing scheme could be
based on the EcoCyc database format of metabolic pathways, so
that biochemical phenotypes could be correlated, or the knowledge
of existing pathways could be queried. The user would click on
a pathway and learn what was known about this. Another way of
indexing and accessing the data for development might be to have
a standardized Arabidopsis growth animation - at appropriate times
during the growth animation, a user could click on a graphic representation
of an organ or other feature, and then this would lead to additional
information. Clicking on a rosette leaf might lead to various
types of leaf cells or indexed leaf morphologies.
E. Stock-Based Information
The databases maintained by the two Arabidopsis resource centers
at Ohio State University and the University of Nottingham provide
excellent access to information on the availability of biological
and chemical materials related to Arabidopsis research. These
databases have implemented many of the recommendations of the
1993 workshop report and should continue to assume responsibility
for descriptive information concerning seed stocks, clones, vectors,
libraries, cDNAs, oligonucleotides, and any other materials that
may require distribution to the Arabidopsis community. Emphasis
should be placed on careful documentation of biological materials,
controlled vocabularies, and maximal utilization of sophisticated
graphics to display plant phenotypes, molecular hybridization
patterns, and other data where appropriate.
With respect to seed stocks, it should be possible to search the database by general phenotype, not just by gene symbol, in order to obtain a broad listing of ecotypes and mutant lines with similar features. Information on phenotypes, screening methods, growth conditions, and differences between alleles should be included for all mutants available through the stock centers. It should also be possible to obtain information on additional mutants or alleles that have been isolated in specific laboratories but are not available from the stock centers.
Individuals should be able to search for specialized libraries,
vectors, transgenic lines, and molecular reagents (antibodies,
purified proteins, unusual compounds, and biochemical standards)
required for Arabidopsis research.
The stock center databases should be directly linked to a central
Arabidopsis database so that queries about the properties of a
gene or mutant can lead directly to a query about the availability
of the resources used to study these or related aspects of the
biology.
F. Community-Based Information
During the past several years there has been a proliferation of electronic resources that provide easy access to information on a wide range of community issues. For instance, it is now relatively easy to retrieve contact information for colleagues or previous postings on the Arabidopsis newsgroup, the abstracts for meetings are available on line and there is an electronic Arabidopsis journal, Weeds World, which provides a forum for discussion of methods and problems and publication of short papers. Many laboratories have mounted web pages that provide detailed information about specialized methods, specialized databases or collections of genetic materials. The curators of AtDB have provided convenient access to these diverse resources by providing a web page that facilitates connection to these resources.
While it is desirable to continue having one group take responsibility for maintaining a centralized launcher or "data warehouse" for Arabidopsis-related web sites, this should be a relatively inexpensive activity and should not require significant public financial support. The distinction betwee