TITLE: Probe Newsletter, complete issue,
July 1993-July 1994, Vol. 4, No. 1/2.
PUBLICATION DATE: July 1994
ENTRY DATE: January 1995
EXPIRATION DATE: None
UPDATE FREQUENCY: As needed
CONTACT: Plant Genome Data and Information Center
pgenome@nalusda.gov
DOCUMENT TYPE: Text
DOCUMENT SIZE: K
Table of Contents
USDA's Office of Agricultural Biotechnology
Summary of the 1993 Plant Genome Awards
RBNET (Electronic Mail Network for Rice Biotechnologists)
New Capabilities and Connections for Plant Genome Database
Plant Genome II Conference Report
Soybase News
The Class of 1993 Plant Genome Grant Recipients
A Primer on Images and the Internet
Maize Genome Databse, a USDA-ARS Plant Genome Database
Introducing Dr. Edward Kaleikau
Announcing Plant Genome III Meeting
Calendar of Upcoming Genome Events
Survey of Upcoming Genome Events
Plant Genome Analysis by Single Arbitrary Primer Amplification
Distinctive Biology of Forest Trees Highlighted at Sixth Internation. Meeting
USDA'S OFFICE OF AGRICULTURAL BIOTECHNOLOGY
Jean A. Larson, M.A.
Office of Agricultural Biotechnology
U.S. Department of Agriculture
Washington, D.C.
Can bioengineered organisms of agricultural importance be released safely into the environment and the marketplace? Will consumers' desires for labeling of biotech products impact on the proposed Food and Drug Administration's policies? What will be the impact of BST on the dairy industry? Will biotechnology technologies help revitalize rural America? Can this new technology be useful in preventing and detecting food safety problems? These and other questions are routinely being addressed by the USDA Office of Agricultural Biotechnology.
The Office of Agricultural Biotechnology (OAB) was established in 1986 by a Secretary's Memorandum 1020-27. Its role is to coordinate the development of consistent biotechnology policies and procedures within USDA. Current OAB functions include:
Under a Presidential Initiative, OAB is the USDA action
office for a multi-year Federal initiative on
biotechnology research.
OAB staffs a Federal advisory
committee, the Agricultural
Biotechnology Research Advisory
Committee (ABRAC). This Committee
provides a public forum for issues
in agricultural biotechnology.
OAB staffs the Committee on Biotechnology in Agriculture
(CBA), composed of six USDA Agency Administrators and two
Assistant Secretaries, and the Biotechnology Council,
composed of senior agency staff.
OAB has provided leadership for the development of
-biotechnology guidelines for agricultural research;
-scientific exchanges involving biotechnology;
-environmental assessments for transgenic fish;
-performance standards for research with transgenic
fish and shellfish;
-advice when requested by regulatory agencies;
-a biotechnology consumer information plan for USDA;
-coordinated responses on regulatory and research
issues to other Departments;
-international conferences/workshops on animal and
plant biotechnology, including three
international conferences on "The Biosafety Results of
Field Tests of Genetically Modified Plants and Microorganisms";
-staff papers and speeches for the Office of the Secretary.
Since the implementation of the January 31, 1992 Presidential Initiative on Biotechnology Research, OAB has been the action office for USDA. Twelve Federal agencies participate in the activity. Their efforts on the biotechnology crosscut are coordinated by the Biotechnology Research Subcommittee (BRS) of the Committee on Fundamental Science and Engineering Research and Development of the National Science and Technology Council. As a participant, OAB has collected research program and budget data from the USDA's Agricultural Research Service, Cooperative State Research Service, Economic Research Service and the Forest Service and eight non-USDA agencies which are involved in agriculturally related programs and assembled data into a consistent format for reporting.
The ABRAC consists of 15 experts from academia, industry, government, and public interest groups with knowledge and experience in one or more of the following areas: recombinant DNA research in plants, animals, and microbes; ecology and environmental science; agricultural production practices; biological containment and field release; applicable laws and regulations; standards of professional conduct and practice; public attitudes; public health/epidemiology; and occupational health and ethics. Fifteen ABRAC members have been recently appointed by the Secretary of Agriculture.
The purpose of ABRAC is to advise the Department, through the Assistant Secretary for Science and Education, with respect to policies, programs, operations, and activities associated with questions of biosafety, the development of guidelines and performance standards for research with genetically modified organisms, and , in response to a specific request, the development of recommendation for the food safety evaluation of transgenic livestock.
The most important issue that will be addressed by the new Committee in the coming months will be to complete the development of performance standards for outdoor research with genetically modified fish and shellfish. Other important issues may include: management of resistance to biopesticides in crop plants; production of pharmaceuticals in plants and animals; use and effects of synthetic sequences in organisms of agricultural importance; risk management and risk communications; and public attitudes, perceptions, and acceptance of genetically engineered products
The members of the Committee as of August 12, 1994 are:
Dr. Walter A. Hill Dr. Ronald R. Sederoff School of Agri. & Home Economics Dept. of Forestry Tuskegee University North Carolina State Univ. Dr. Anne R. Kapuscinski Dr. James Lauderdale Dept. of Fisheries and Wildlife The Upjohn Company
University of Minnesota
Dr. Pamela G. Marrone Dr. Susan Harlander
Entotech, Inc. Director, Research & Development
Land O'Lakes
Dr. Deborah K. Letourneau Dr. Stanley Pierce
Board of Environmental Studies Rivkin, Radler, Bayh, Hart, &
Univ. of California Kremer
Dr. Rudy Wodzinski Dr. Fernando Osorio
Dept. of Molecular Biology and Department of Veterinary
Microbiology Biomedical Sciences
Univ. of Central Florida Univ. of Nebraska
Dr. Roy Fuchs Dr. H. Alan Wood
Monsanto Agricultural Company Boyce Thompson Inst. for Plant
Research
Dr. James Tiedje Dr. Walter Reid NSF Center for Microbial Ecology World Resources Institute
Michigan State Univ.
Dr. Paul Thompson
Center for Biotechnology Policy and Ethics
Texas A&M Univ.
Minutes from the ABRAC meeting are published and available from the OAB on request.
In addition to the scientific aspects of modern biotechnology, OAB clearly recognizes the public relations dimension of biotechnology. A well-informed public is better able to participate in the decision-making process about biotechnology. Toward this end, OAB shares information with the media, participates in public affairs activities around the country, and contributes articles on biotechnology to the general as well as the scientific press. The Office publishes a monthly newsletter, Biotechnology Notes. The newsletter highlights activities on biotechnology issues at USDA and in the private sector. The dissemination of the newsletter is via mail, through USDA's Computerized Information Delivery System, and on Internet through the National Agricultural Library's Biotechnology Information Center. From time to time the Office has sponsored national and international conferences and workshops on biotechnology topics.
The international and trade implications of agricultural biotechnology are of growing concern to many U.S. economic planners and policymakers. On the international front, OAB is involved in studying research and technology transfer programs in competing nations; promoting international consensus on the scientific principles that underlie the environmental and human safety of agricultural biotechnology; and working with the U.S. Trade Representative, the Food and Drug Administration, and USDA's Foreign Agricultural Service to provide information to trading partners regarding U.S. food safety procedures.
Needless to say, the OAB program is a very dynamic Office that maintains timely responsiveness to the everchanging biotechnology scene.
All the above activities are done with a small core of permanent staff, but OAB Director Dr. Alvin Young says that "much of my staff work is done by individuals on temporary assignment to OAB from other USDA agencies." Specialists in agricultural research, extension/technology transfer, regulations, environmental impact, economics, public relations, and international affairs have been supplied to OAB by cooperating agencies. At the completion of their assignments, says Young, "these individuals take their new biotechnology knowledge and experience with them back to their agencies and everyone benefits."
For additional information or for requesting OAB published materials, contact the OAB at (703) 235-4419.
Dr. Ed Kaleikau, Program Director
National Research Initiative Competitive Grants
Cooperative State Research Services, USDA
Washington, D.C. 20250
In 1993, Congress appropriated $97.5 million for the National Research Initiative Competitive Grants Program (NRICGP), of which $12.1 million was made available for Plant Genome Research. The Plants Division of the NRICGP in USDA's Cooperative State Research Service administers the plant genome grants. In addition to the NRICGP allocation, $3.67 million was appropriated to the USDA Agricultural Research Service (ARS) for program management, setting targets for genome mapping research, and database development for agriculturally important plants. After program administration costs, the net amount available for all plant genome research totalled $15.1 million: $3.0 million for the ARS and $12.1 million for the NRICGP.
Mission-oriented research proposals that address the goal of improving agronomic qualities through genomic research were submitted to the NRICGP by scientists from the research community. Each proposal was peer-reviewed by experts in the area of genomic research and was judged on its scientific and technical merit, qualifications of proposed personnel, and relevance to sustainability and stated research objectives in the solicitation for proposals.
In 1993, the majority of plant genome funds supported awards made in two programs in the NRICGP Plant Division: the Plant Genome Program and the Plant Genetic Mechanisms Program. A small portion of funds also supported genome-related research in other NRICGP programs. Research projects being supported in FY 1993 are summarized in the accompanying tables. A total of 214 proposals were submitted to the program, and 91 awards were made to scientists from 34 states (See article "The Class of 1993 Plant Genome Grant Recipients" in this issue).
Nineteen agronomic, horticultural, and forest tree species are undergoing genetic and physical mapping procedures (table 1). Fifty-six genes and gene systems are being studied (table 2), as well as genetic phenomena in various plant species (table 3). Many new molecular techniques are being pursued (table 4). The data were supplied by the NRICGP staff, and compiled into tables by Dr. G.S. Smith and Dr. J.P. Miksche from ARS. RBNET (Electronic Mail Network for Rice Biotechnologists)
With support from the Rockefeller Fooundation, an electronic mail network has been established to increase communication among the international rice biotechnology community. This network (RBNET) is being managed by Professor Verma at the Ohio State Biotechnology Center, 1060 Carmack Road, Columbus, Ohio 43210 USA. To address all users of the network you may send a message to: rbnet@magnus.acs.ohio-state.edu. To add your name to the RBNET mailing list, please send your E-Mail address to: dverma@magnus.acs.ohio-state.edu.
For those colleagues in countries where electronic mail service is not currently available, attempts are being made to deliver a message by fax. If any one of you are in this situation and wish to communicate to the RBNET users, you may fax your message to the attention of Professor Verma at: (614) 292-5379 and your message will be posted on the RBNET for distribution.
Stephen M. Beckstrom-Sternberg
Plant Genome Data and Information Center
National Agricultural Library, USDA
Beltsville, MD 20705-2351
The Plant Genome Database has been considerably augmented in the past few months. A number of new connections and capabilities have been added to the World Wide Web (WWW) and gopher interfaces, and a text-based Lynx client has been added to provide access to those with slower Internet connections and/or low resolution graphics. The following is a summary list of recent changes to the Plant Genome Database:
1). WWW Interface to ACEDB Databases; 2). New Query Capabilities for Plant Genome Data; 3). Links to external data (Agricola, GenBank, Swiss-Prot, Images); 4). New Gopher Services; 5). Telnet access to WWW Lynx Client; 6). Collaborator's Page summarizes Collaborator's Services; 7). New Draft for Plant Genome III Meeting; 8). New Solanaceae Database Shows Syntenic Relationships
WWW Interface to ACEDB Databases
The Plant Genome Database Project is now offering a collection of ACEDB-based databases for both plant and non-plant genomes. The databases have been installed behind the "Moulon" ACEDB-WWW server and can be browsed and queried interactively. The various databases are listed below:
Plants
AAtDB--Arabidopsis
SoyBase--soybeans
RiceGenes--rice
MaizeDB--maize
GrainGenes--wheat, barley, rye, relatives
TreeGenes--forest trees
SolGenes--Solanaceae
Other organisms
ACeDB--C. elegans
AceMap--Human Chromosome X
FlyDB--Drosophila melanogaster
MycDB--mycobacteria
AGsDB--A Genus species DataBase
New Query Capabilities for Plant Genome Data
The ACEDB-based databases can be queried using the ACEDB query language, query-by-example (QBE) and a query-building (QB) tool. The ACEDB query language requires an understanding of syntax, but QBE and QB do not. In addition, all the data from the ACEDB databases has been indexed for searches using WAIS. Finally, data can be searched using agrep--a tool which allows you to make approximate matches (aka, fuzzy searching).
Links to external data: Agricola, GenBank, SwissProt, Images
HTML links are increasingly being used to connect plant genome data with data from external sources, for example AGRICOLA, GenBank, and SwissProt. This allows us to take advantage of the division of labor between databases specializing in different tasks. For example, the receptor kinase sequence object from the Arabidopsis database AAtDB contains a link to GenBank labeled "M80238". Clicking on it will retrieve a GenBank record from a computer at NIH.
New Gopher Services
The Plant Genome Information on the Agricultural Genome Gopher has been reorganized. It can be used to access the same information that is presented by the WWW interface to the genome databases, both plant and non-plant.
Telnet access to WWW Lynx Client
Users who are restricted to VT100 or VT200 terminals or emulators can access the WWW services using Lynx, a text-only client (see below for access details). The client is set up so that only NAL data (and data from a few other sources) is available--it cannot be used to surf the Internet (sorry).
Collaborator's Page summarizes Collaborator's Services
A page has been set up with links to resources maintained independently by the plant genome collaborators. The services include ftp archives, gophers and access to databases.
New Draft for Plant Genome III Meeting
There is a new version of a draft describing the next Plant Genome Meeting in San Diego in mid-January 1995 (gopher and WWW).
New Solanaceae Database Shows Syntenic Relationships
SolGenes, a database for pepper, tomato and potato, now shows the syntenic relationships between chromosomes from these members of the Solanaceae. The information is presented as an on-line image of a genetic map. The image responds to mouse clicks enabling one to find out more about a particular locus by clicking on it, then selecting "Show as text". Figure 1 is an example comparing chromosome 9 of tomato and potato. Access to these graphically depicted relationships from the Agricultural Genome home page is via the following sequential menu selections (WWW Interface to ACEDB Data, Browse Solgenes, and Multimap).
Ways to Access the Agricultural Genome Databases
WWW: http://probe.nalusda.gov:8000/
Gopher: gopher probe.nalusda.gov
Lynx: telnet probe.nalusda.gov
login: lynx
password: (none - hit return)
Anonymous FTP: probe.nalusda.gov
Plant Genome II Conference Report
Susan McCarthy, Coordinator
Plant Genome Data & Information Center
National Agricultural Library, USDA
Beltsville, MD 20705
Plant Genome II featured applications of genome mapping and analysis to solve existing problems and uncover answers to fundamental questions relating to the plant genome and its evolution. The meeting attracted 553 participants from 22 countries.
According to Steven Oliver, University of Manchester, Manchester, United Kingdom, we are entering a stage where the taxonomy of gene function will be essential in efficiently identifying new genes. This new stage will require a multidisciplinary approach encouraging the collaboration of physiologists, geneticists, biochemists, and plant breeders. He defined this era as a new voyage of the Beagle.
The message was reinforced by Dr. James Cook, USDA, ARS and also Senior Scientist at CSRS, who pointed out that now is the time to bring plant breeders together with molecular biologists to conduct a gene hunt for agronomically important genes.
New Insights
Understanding plant genome structure and organization can lead to interesting and relevant discoveries, as highlighted by Dr. Richard Flavell, Director, John Innes Institute, Norwich, United Kingdom. According to Flavell, understanding the role of epigenetic regulation, gene order, and in situ homology sequence searching will ultimately help in the practical application of biotechnology. Plants have had to defend themselves from foreign DNA over the millennia and as a result have developed strategies -- including gene silencing -- to cope with transposon selection pressures. The plant's ancient art of anti-sense technology may take advantage of gene location. Gene location would determine epigenetic DNA methylation events, which in turn would regulate gene expression.
In all, Flavell points out, concerted evolution in the long term helps to maintain high levels of conservation across the chromosome both in terms of sequence and gene order or synteny.
Thomas Bureau, University of Georgia, detected evidence of ancient transposon and retrotransposon events. Extensive sequence similarity searches were performed on the GenBank and EMBL nucleotide sequence databases. These "database mining" experiments have identified over 100 normal plant gene sequences showing evidence for a member of either the "Tourist" or "Stowaway" family of transposons. The location of several elements corresponds to previously reported cis-acting regulatory elements. Significantly, a "Tourist" element was found to serve as the promoter for the maize auxin-binding protein (abp1). The first plant retrotransposon, Bs1, was found to contain a cellular gene fragment; this provides the first evidence for transduction by a retrotransposon in plants.
Progress in Rice
The Japanese Rice Genome Program reported significant advances. Dr. Nori Kurata, NIAR/STAFF, Japan, described a genetic map with 1,400 RFLP and RAPD markers. Over 7,500 clones from callus tissue at different developmental stages have been sequenced. Of those sequenced 1,800 are clones of known function. Dr. Kurata reports that an expression map has been constructed using cDNA mapping as a base. This map includes information on tissue-specificity, distribution of isozyme genes, gene families, and functionally related genes in the genome, such as ribosomal protein genes and the histone gene family.
Physical mapping in the Japanese Rice Genome Program will be used to identify economically important genes. Two YAC and three cosmid clone libraries have been developed, representing about 20% of the rice genome. Ordered libraries will be prepared from these clone libraries. To date, 120 YAC end-clones have been isolated and end-clone mapping is underway. Typical YACs have 400 kb inserts, which, when finished, the Japanese expect will cover the rice genome six times over.
Chromosomes 1, 4, 6, and 11 are being given high priority. It is known that a number of important resistance genes reside on Chromosome 6. Mapping data from the Japanese program have been entered onto two versions of an internal database called RiceBase. One version contains mostly cDNA information, while the other version has the physical map data.
International collaboration of rice mapping efforts was encouraged by an informal workshop held in conjunction with the conference. Dr. Susan McCouch, Cornell University, and Dr. Gou-fan Hong, Director, Chinese Rice Genome Program, co-chaired the session. Highlights included Dr. Kurata's announcement that the Japanese mapping data should be made public later this year. Five prime sequence data for several hundred markers are currently available. Pamela Ronald, University of California, Davis, CA, announced the public availability of a variety of libraries, including those on Bacterial Artificial Chromosomes and cosmids.
Physical Mapping
Physical mapping was again highlighted in the Arabidopsis workshop. Caroline Dean (John Innes Institute, Norwich, UK) and Howard Goodman (Massachusetts General Hospital) reported that chromosomes 4 and 5 are nearing completion in their joint effort to integrate the two YAC and cosmid maps. A new YAC library developed by David Bouchez should help in developing the integrated physical map. Several thousand Arabidopsis cDNAs have been sequenced by the French EST project. Michel Delseny, (CNRS, Perpignan, France) who reported on the project, indicated that the cDNA sequences have been deposited in the public database EMBL.
Resources
Plant Genome II provided participants with information on useful technologies and resources. The latest developments in the plant genome databases were outlined, as well as computational tools for mapping and sequence analysis. Database demonstrations with a live Internet link were available throughout the meeting, allowing handson experience for interested researchers. Electronic BIOSCI newsgroups were the focus of several workshops organized by Dave Kristofferson (Intelligenetics, Mountain View, CA).
QTL Experimental Design
Quantitative trait loci (QTL) analysis was examined with attention to experimental design and analysis. Dave Webb, Pioneer Hi-Bred, Johnston, IA, looked at soybean cyst-nematode resistance; one soybean introduction was found to have more resistance than any other soybean tested to date. Three resistance loci were identified; with this information, the effect of population size in detecting the traits was tested. Large sample populations were found to be essential in finding and mapping these traits. The minimum sample population size is 200.
The need for large sample populations was again emphasized by Karl Lark, University of Utah, Salt Lake City. Lark found that specialized statistical methods and graphing were needed to identify many important loci. Specifically, Lark identified in interacting traits a condition called epistasis. One trait measured on its own had no effect on plant height. This same trait was found to interact with another plant height QTL and could explain 25% of the plant height variation. The basis of Lark's technique is to use large population sizes and to conduct pairwise comparisons of loci in plants with extreme phenotypes. The results are graphed and epistatic interactions are then identified.
According to Thomas Cheesbrough, South Dakota State University, Brookings, this type of analysis will be essential to studying the genes of such metabolic pathways as oil production, because each enzyme is highly interdependent on the gene products of the entire metabolic chain.
Mapping Technologies
Mapping technologies were featured in several talks and posters throughout the conference. Perry Cregan, USDA, ARS, Beltsville, MD, and others reported on the continued success with simple sequence repeats (SSR). The SSRs are small sequence patterns which are repeated at variable lengths. The variable length of the repeats provides a means to identify varieties and individuals; tools needed by crop breeders and geneticists. In addition to SSR technology, amplified fragment length polymorphism (AFLP), a related new technology, was reported by Drs. Pieter Vos and Marc Zabeau, KeyGene, Wageningen, The Netherlands.
AFLP will provide markers for those map regions which other markers have not successfully bridged. The AFLP technique has the capacity to exploit multiple forms of variation within the genome. The new technology described by Vos is still a long way from direct application by plant breeders, as discussed at the International Triticale Mapping Initiative meeting held in San Diego in conjunction with the Plant Genome II conference.
Plant Genome III will be held January 15-19, 1995, in San Diego, CA. Sessions will address all aspects of mapping, from QTLs to the latest molecular marker technologies, instrumentation, and gene isolation. For more information or program suggestions, contact Jerome Miksche or Stephen Heller, USDA/ARS, BARC-W, Bldg. 005, Room 331-C, Beltsville, MD 20705 USA. (See article, "Announcing Plant Genome III" this issue.) Soybase News
Dr. Lisa Lorenzen
Department of Zoology and Genetics
Iowa State University
Ames, IA
The Soybase staff has been concentrating on the metabolic portion of the database. Information (current through Summer, 1994) on the enzymes and reactions/pathways involved in Nitrogen Metabolism will be entered and available August 15, 1994. The complete set also will include references for all cited articles/books.
The information on Fatty Acids is 50 percent collected and entered, and is slated for completion early in 1995. In addition, a section on Nodulins has been added.
The Pathology section continues to grow, with downy mildew, powdery mildew, frogeye leaf spot, potato mosaic virus, cowpea mosaic virus, and stem canker being the latest additions. Soybase staff members have collected and entered information from the literature on both quantitative and qualitative trait linkages to molecular markers. We wish to encourage anyone who has data that they want included in Soybase to contact us at the e-mail address listed below. We would be happy to include your data.
Thinking toward an integrated legume database, the Soybase staff has assisted in the initiation of four new ACE-type databases: alfalfa, peanuts, common bean, and cool season food legumes such as lentil and chickpea. These databases are being administered under the supervision of Dr. Daniel Skinner, USDA-ARS, Kansas State University; Dr. Gary Kochert, University of Georgia; Dr. Phil McClean, North Dakota State University; and Dr. Fred Muehlbauer, USDA-ARS, Washington State University; respectively.
For additional information on Soybase, or for instructions on how to contact one of the new legume databases, please send inquiries to curator@mendel.agron.iastate.edu.
G. Agrios, P. Chourey
University of Florida
Molecular and Physiological Genetics of Sucrose Metabolizing
Enzymes in Maize Endosperm
M. Alleman
Duquesne University
Regulatory Mutants of the Maize R Locus
J. Anderson, F. Francl
North Dakota State University of Agriculture & Applied Science
Molecular Mapping of Tan Spot Resistance Genes in Wheat
Z. Avramova
Purdue Research Foundation
Nuclear Matrix and Matrix-Attachment Regions (MARs) in Higher
Plants
P. Baenziger, Y. Yen
Nebraska Agricultural Experiment Station, University of Nebraska
Exploring the Interface of Qualitative and Quantitative Genetics
W. Baird, A. Abbott, R. Ballard, W. Bridges
Clemson University
Chromosome Mapping in Peach and its Application to Fruit Quality
Maintenance
G. Bates
Florida State University
Targeting of DNA Integration to Specific Sites in Plant
Chromosomes
J. Birchler
The Curators of the University of Missouri
Molecular Analysis of Maize Centromeres
T. Blake, L. Talbert
Montana State University
Molecular Markers to Correct Germplasm Deficiencies in Wheat and
Barley
W. Briggs
Gordon Research Center
1993 Gordon Research Conference on Plant Molecular Biology Signal
Transduction and Membrane Proteins
S. Brown, H. Aldwinckle, N. Weeden
Cornell University
Genome Mapping & Gene Tagging in Apple
M. Bustos
University of Maryland, Baltimore County
Hormonal Control of Gene Expression During Seed Maturation
R. Cantrell
Agricultural Experiment Station
Board of Regents of New Mexico State University
Use of RAPD Markers to Determine the Genetic Diversity of
Gossypium Germplasm Derived from Interspecific Hybridization
`What is the use of a book', thought Alice, `without pictures or conversations?' --Lewis Carroll
Introduction
High speed digital networks and computers with graphical capabilities have made it possible to retrieve and view images almost as easily as one views purely textual material. This article is intended to be a quick introduction to this subject with some pointers to other resources.
What is a Digital Image?
Digitally stored images are analogous to a paint-by-number kit. The image is composed of rectangular regions, called pixels, and each pixel is assigned a number. Each number corresponds to a color or shade of grey. In order to reconstruct an image for viewing, one needs both the values for each pixel and a legend which maps the numerical values to a color. Image files on computers typically contain both the array of pixel values and the color map.
Digital images usually contain less information than a corresponding image captured with conventional photographic methods. Photographic film is an excellent repository for information. One 35mm slide can easily hold one hundred million bytes of data. This makes the digital storage of images somewhat problematic. Even if the cost of storage were not a factor, the time involved in retrieving and displaying very large stored images would be prohibitive. All common conversion of photographic data to digital data involves the loss of some information. Once the images are digitized, they are often compressed. Some compression methods result in further information loss. There is always a tradeoff between faithfulness of reproduction and amount of storage space required. Even if storage were free, it would be advantageous to keep the image size small for rapid transferal and viewing. This dynamic has led to many different formats for computer images, each with advantages and disadvantages. The decision about which format to use is highly dependent on the application, and on the hardware that will be used. On the Internet, exchanged images tend to follow two main formats: GIF and TIFF. These formats, particularly the latter, have many variations. JPEG images are a ubiquitous TIFF variant (JFIF).
In addition to these, different computer types can have their own internal formats, customized for their particular hardware. The PICT format on Macintosh is an example of this.
Software
There is software available for Unix, Macintosh and Intel-based platforms that will allow you to view downloaded images. You may retrieve them using anonymous ftp and experiment with them for free. Some of the software is shareware, and the author expects some small compensation if you like the software and continue to use it. (One author, for example, would like you to send a case of beer; another a postcard of your town.) Sources for image viewing software for major platforms will be found later in the article.
Hardware
Workstations such as those from Sun Microsystems or Silicon Graphics were developed with advanced graphical uses in mind. High-end Macintoshes and PC-clones are adequate, however. Monitor screens should be at least 15 inches, and graphics hardware may be helpful or necessary. Image analysis can be slow even on very fast computers.
Typical Image Sizes and Network Bandwidth
Images seen on the Internet can range in size from hundreds to millions of bytes. A four-megabyte file is not uncommon. Internet connections can be characterized by the bandwidth of the connection. Typical bandwidths are 9.6, 14.4, and 56 kilobaud. Major nodes may have one megabaud connections or better. Bytes per second can be approximated by dividing the baud rate by 10. A 56 kilobaud channel will transfer about 5600 bytes per second.
A typical 100 kilobyte image will take at least 1 second to move across a one megabaud channel and more than 17 seconds across one of 56 kilobaud. In addition, the presentation of the image on the computer screen may take seconds once the image has been transferred.
To the user, large images or slow network connections will be seen as delays. An occasional 10-second delay after pressing a key or mouse can be tedious in an interactive environment, arguing for very high speed network connections or using smaller image files. Compression techniques can be very useful, particularly when animated images are transferred.
Image Capture
There are at least two sorts of devices which one can use to digitize an image. Both make use of charge-coupled devices (CCD) which use quantum effects to transduce light to a pattern of electrical signals. Scanners have a linear array of CCDs which are mechanically moved across a flat image (in the manner of a xerographic machine). CCD cameras have a two-dimensional array of devices so that the entire image is captured at once. CCD cameras use conventional optics and hence can be used to take pictures of 3-D objects.
CCD devices are susceptible to thermal noise; they will produce small random signals even in absolute darkness. To increase signal-to-noise ratios, the devices can be cooled. Some CCD cameras come with refrigeration units for this purpose. CCD cameras are more expensive, but allow for a higher throughput in a production setting.
Commercial photographic processing labs commonly have equipment to directly digitize color slides. Copy stores will often have scanners available for rental. These work quite well with color or black-andwhite prints.
Where to get Image Viewing Software
All of this software may be retrieved using anonymous ftp.
Macintosh
JPEGView ftp to sumex.aim.stanford.edu
directory: /info-mac/app/
NIH Image ftp to zippy.nimh.nih.gov
directory: /pub/nih-image/
Unix/X11
xv ftp to bongo.cc.utexas.edu
directory: /gifstuff/xwindows/
xloadimage ftp to bongo.cc.utexas.edu
directory: /gifstuff/xwindows/
Intel/Windows3
Lview ftp to oak.oakland.edu
directory: /pub/msdow/windows3/
Intel/MSDOS
cshow ftp to bongo.cc.utexas.edu
directory: /gifstuff/ibmpc/
For More Information
These Usenet electronic conferences are sources of useful discussion:
alt.binaries.pictures.misc
comp.graphics
alt.graphics.pixutils
List of Frequently Asked Questions (FAQ), with answers, for these conferences are available by anonymous ftp from rtfm.mit.edu
Mary Polacco, Database Developer
Curtis Hall, University of Missouri
Columbia, MO 65211
The Maize Genome Database, or MaizeDB, is curated as a Sybase database at the University of Missouri-Columbia and provides user-friendly, Internet access to the maize genome and the biology of maize.
Information includes 142 genetic maps, with 4,864 mapped loci, recombination and map score data (2,164 entries), 986 probes, 1,701 genetic/cytogenetic stocks, 7994 locus variations, 4,662 stock pedigrees, 5,600 bibliographic references indexed to genetic objects, and addresses of maize researchers.
Gene functionality may be queried by mutant phenotype, trait, confirmed
or putative gene products, metabolic pathways, induction conditions and
text descriptions of genes. Work is in progress to document
quantitative trait loci. Users
with Internet connections may access the data by several procedures
that are described more fully below: guest login, gopher, World Wide
Web, or file transfer of a special graphics, ACEDB format.
Data is input from various sources, including specially formatted, electronic lab notebooks of researchers who focus on mapping or mutant characterization. The contributors are international and from academic, government, and industrial research groups. Information, especially regarding gene function and expression, is also taken from the scientific literature, both electronic and printed.
Connecting To Other Databases
Using Mosaic, a type of free software that connects users to information on the World Wide Web (WWW), users connected to MaizeDB may retrieve information, including images and graphics, from other databases around the world, as easily as if the data existed in the MaizeDB records. If desired, they may store the information on their machines.
For example, while browsing the MaizeDB, you may read that the function of maize gene, dps1, was confirmed by transgenic complementation of E. coli mutations in dap1. By clicking on dap1, you would retrieve the record from the E. coli Stock Center at Yale University. Clicking on the EC number for gene products that are enzymes connects you to the ENZYME database. ENZYME describes the reaction, and in turn, connects to all Swiss-Prot entries corresponding to that EC number, as well as to OMIM (On-line Mendelian Inheritance in Man).
WWW connectivity requires precise matching of records in MaizeDB to records in other databases around the world. It permits curators of distinct datasets to combine data in a seamless fashion without actually importing the data. The ability to extract distinct formats of data from MaizeDB makes preparing files of matching identifiers relatively easy, so that external databases may use the WWW to connect to us, as occurred in June 1994 with SwissProt.
WWW/MaizeDB currently accesses external data in the following databases:
GenBank ....... nucleotide sequences
SwissProt ..... protein sequences; connects to Prosite (motifs, signature sequences), MedLine, EMBL
dbEST.........random cDNA's partial sequences, with periodically updated homology searches
E. coli Stock
Center ....... E. coli genetic stocks, map
ENZYME .......reactions, comments; connects to SwissProt, OMIM
AAtDB ........ Arabidopsis Genome Database
Accessing MaizeDB - Requirements
Guest login only requires that your machine have Internet connectivity, direct or indirect. Modem connections are supported, as are connections using any computer, including PC, Macintosh, and Unix. guest login protocol:
telnet teosinte.agron.missouri.edu
login: guest
password: corncob
Guest login provides
access to:
it does not support mouse-capability
Guests are encouraged to leave comments on the Note form of the database. While not required, leaving your e-mail address will permit us to contact you directly for further clarification.
NOTE: Users with X-Windows (this is not the same as Microsoft Windows)
software will enjoy the most user-friendly access to the database. If
connecting by modem, the X-Window will not function, and users should
select the vt100 emulation.
NOTE: If using the vt100 emulation of MaizeDB/Sybase, type "r" while
holding down the "control" (aka "CTRL") key to access the commands
required to query or browse the database. The command utilities are
described in more detail in the "help" option that appears or after
successful login as a guest.
Gopher
Gopher makes available hierarchical collections of information across the Internet. Gopher client (user) software provides easy access to all gopher data servers. All words in a record, except commonly used words, are indexed and thus may be used to query records. Free gopher client software for Unix, PC, or Macintosh machines is available by anonymous ftp (file transfer protocol)* from boombox.umn.edu. Once installed, open server teosinte.agron.missouri.edu, port 70 or use gopher to find us by location in Columbia, Missouri. On-line help is provided by the gopher software and is in a file on the MaizeDB gopher server.
World Wide Web (WWW)
WWW is a hypermedia retrieval system which allows users to traverse on-line documents by clicking on hyperlinks-terms, icons, or images that point to other related documents. Hyperlinks permit retrieval of any document anywhere on the Internet. Retrieved "documents" may include text files, graphics, and videos. Connecting to the WWW currently works best if users have access to Mosaic software installed on a Unix machine. Macintosh and PC Mosaic software are rapidly approaching the capability of the Unix version.
Users without WWW software may access the WWW-linked format of MaizeDB by selecting the Lynx option after "guest login."
Mosaic software supports mouse capability and is available without charge by anonymous ftp from ftp.ncsa.uiuc.edu. The Unix version, but not the Macintosh or PC version, requires an X-Window on the user's machine; it will require a systems administrator to install. To access MaizeDB from Mosaic software, use our WWW address, otherwise known as URL or uniform resource locator:
http://teosinte.agron.missouri.edu/top.html
The WWW formatted data is dynamically extracted from the most current version of the database, which is continuously updated.
ACEDB Format
This is a special graphical format and requires a UNIX machine. The database may be retrieved by anonymous ftp from the National Agricultural Library, probe.nalusda.gov in directory pub/maize. This format is static, and periodically extracted from MaizeDB. It does not support the robust queries of the Sybase database, accessible by the guest login service.
ANONYMOUS FTP requires that the user have ftp or file transfer software to connect to another machine. Once connected, login as "anonymous" and use your e-mail address as the password. If using a Unix machine, type: cd pub/maize, and to transfer the database, type: get mace.tar.Z
History Of MaizeDB Design: Some Landmarks
Fall 1991
First prototype MaizeDB operational. Some 24,000 records created the first 6 months, largely from data summaries in the Maize Genetics Cooperation Newsletter (MNL), volume 65. The database currently contains over 78,000 records.
December 1992
First public access to the data, a gopher server established. First access was 100-200 connections/month, and has grown to over 1,000 connections/month.
March 1993
Maize Gene List, MNL, vol 67, pp. 134-15, extracted from MaizeDB Version 2 of MaizeDB implemented.
June 1993
ACEDB formatted data extracted from MaizeDB.
August 1993
Tool developed for loading references from PC and Macintosh reference manager formats.
December 31, 1993
Guest login to MaizeDB established.
Winter 1994
MaizeDB placed on the World Wide Web; currently there are 600-1,100
connections/week.
WWW connections made to external databases, listed above.
March 1994
Genetic indexing of 1993 references extracted from the MaizeDB, published as hardcopy in MNL, vol 69. pp 148-153. Information was indexed to chromosome, gene or allele and trait.
June 1994
SwissProt connects to MaizeDB using a file extracted from MaizeDB per specifications of SwissProt curators.
Developers and Curators of the Database Include:
E.H. Coe (PI), P. Byrne, G. Davis, D. Hancock, M. Polacco (Columbia,
MO)
M. Berlyn, S. Letovsky (New Haven, CT)
C. Fauron (Salt Lake City, UT)
S. Rodermel, C. Wetzel (Ames, IA)
M. Sachs (Urbana, IL)
For further help in accessing the database, please e-mail db_request@teosinte.agron.missouri.edu or contact Denis Hancock, (314) 882-1722 (phone), (314) 874-4063 (FAX).
Dr. Edward K. Kaleikau has assumed responsibility as program director for the Plant Genome Program of the USDA National Research Initiative Competitive Grants Program (NRICGP). In this position, Dr. Kaleikau coordinates the competitive grant review process for the Plant Genome Program. His responsibilities include selecting and working with members of the review panel in conjunction with the panel manager, as well as handling other review assignments as needed.
In addition, Dr. Kaleikau is co-chairman of the Plant Genome Steering Committee along with Dr. Jerome Miksche, and serves on the USDA Biotechnology Research Subcommittee.
Dr. Kaleikau, a native of Hawaii, received his B.S. degree in biology/chemistry (1981) from Graceland College in Lamoni, IA and Ph.D. degree in plant genetics (1988) from Kansas State University. His Ph.D. dissertation investigated the inheritance and chromosomal mapping of genes controlling in vitro tissue culture response in wheat.
Dr. Kaleikau developed his interest in plant genetics during an internship at the Arco Plant Research Institute in Dublin, CA, after graduation from college. He gathered further experience as a technician for Advanced Genetic Sciences in Manhattan, KS.
Prior to joining the NRICGP, Dr. Kaleikau received postdoctoral training at Stanford University, where he was awarded fellowships from both the National Institute of Health and National Science Foundation to study the regulation of transcription initiation and termination of rice mitochondrial genes.
Dr. Kaleikau can be contacted as follows:
Internet: EKLEIKAU@DARTH.ESUSDA.GOV
Phone: (202) 401-5114
Address: USDA/CSRS/NRICGP
901 D Street, SW
Aerospace Bldg, Rm 323
Washington, DC 20250-2241
Building on the successes of Plant Genome I and II, we are pleased to announce that the Plant Genome III meeting will be held on January 15-19, 1995, in San Diego, CA.
Session Topics:
In addition to the formal sessions and posters during the week, Sunday afternoon will feature a computer workshop on Genome Information Tools and Resources. The workshop will provide a view of some of the existing software used to maintain and analyze genomic information. The talks at this workshop are designed to give the average plant molecular biologist an idea of available resources and their capabilities. "Hands on" computer sessions will also be available throughout the week. Don't miss this opportunity for individualized training.
Additional Workshops:
Sunday, January 15: 9:00 am - 6:00 pm
International Consortium for Sugar Cane Biotechnology
Organized by James Irvine (JIRVINE@TAMU.EDU) Tuesday, January 17: 3:30 pm - 6:00 pm
Organized by Dave Neale (DBN@S27W007.PSWFS.GOV)
Organized by Susan McCouch
(SUSAN_MCCOUCH@QMRELAY.MAIL.CORNELL.EDU)
Organized by Dave Kristofferson (KRISTOFF@NET.BIO.NET)
Organized by Caroline Dean (ARABIDOPSIS@BBSRC.AC.UK
Howard Goodman (GOODMAN@FRODO.MGH.HARVARD.EDU)
Organized by Patrick Hayes (HAYESP@CSS.ORST.EDU) Tuesday, January 17: 7:30 pm - 10:00 pm
Organized by Dave Neale (DBN@S27W007.PSWFS.GOV)
Organized by Jeff Bennetzen (MAIZE@BILBO.BIO.PURDUE.EDU)
Michael Gale (JEFFERY@BBSRC.AC.UK)
Organized by Carl Price (PRICE@MBCL.RUTGERS.EDU)
Ellen Reardon (REARDON@CCIT.ARIZONA.EDU)
Organized by Sriyani Rajapakse
(SRIYANI_RAJAPAKSE@QUICKMAIL.CLEMSON.EDU)
Organized by Dave Kristofferson (KRISTOFF@NET.BIO.NET Wednesday, January 18: 3:30 pm - 6:00 pm
1. Maize
Organized by Ed Coe (ED@TEOSINTE.AGRON.MISSOURI.EDU)
2. Legumes
Organized by Randy Shoemaker (RCSSHOE@IASTATE.EDU)
3. ITMI
Organized by Calvin Qualset (ITMI@UCDAVIS.EDU)
Olin Anderson (OANDERSON@PW.USDA.GOV)
Pat McGuire (ITMI@UCDAVIS.EDU)
Michael Gale (JEFFERY@BBSRC.AC.UK)
4. Tagging Genes for Abiotic Stress
Organized by Henry Nguyen (806-742-1622)
5. Cotton
Organized by Andrew Paterson (AHP2343@BIOCH.TAMU.EDU)
Abstract Deadline:
Abstracts submissions are due by November 1, 1994. All submitted
or invited poster talks will be one-page long, using forms
provided by the PG-III conference organizer, Scherago
International. The PG-III abstracts will be available online
prior (and after) to the meeting at probe.nalusda.gov via gopher
and the WWW.
Student Travel Grants:
The International Society for Plant Molecular Biology is again
sponsoring four student travel grant awards. For details please
contact Dr. Stephen Heller by E-mail.
Location: Town & Country Hotel
500 Hotel Circle North
San Diego, CA 92108
Phone: (619) 291-7131
FAX: (619) 291-3584
Cost: $300 advance registration up to December 1, 1994
$350 after December 1, 1994 and on-site
$100 Student (Pre-Ph.D) registration
(Requires a letter of certification from
department chairperson)
All registrations include one copy of the printed conference abstracts, Monday-Thursday continental breakfasts, Sunday evening opening reception, Monday evening wine & cheese reception, and Wednesday evening dinner.
PG-III Co-Chairpersons:
Stephen Heller, USDA/ARS, Beltsville, MD, USA
(SRHELLER@ASRR.ARS.USDA.GOV)
Jerome Miksche, USDA/ARS, Beltsville, MD, USA
(JMIKSCHE@ASRR.ARSUSDA.GOV)
Michael Gale, John Innes Centre, Norwich, UK
(JEFFERY@BBSRC.AC.UK)
Susan McCouch, IRRI, Philippines
(SUSAN_MCCOUCH@QMRELAY.MAIL.CORNELL.EDU)
Conference Co-sponsors:
USDA, Agricultural Research Service
USDA, National Agricultural Library
Rockefeller Foundation
International Society for Plant Molecular Biology
John Innes Centre
To Register Contact:
Darrin Scherago
Scherago International, Inc.
11 Penn Plaza, Suite 1003
New York, NY 10001
Phone: (212) 643-1750
FAX: (212) 643-1758
E-mail: SCHERAGO@BIOTECHNET.COM
September 18-22, 1994: 16th International Congress on Biochemistry and Molecular Biology, New Delhi, India. Contact: K. Kooraram, Magnet World Travel, 18-30 Clerkenwell Rd., London, EC1M 5NN, UK.
September 19-20, 1994: Drug Discovery & Commercial Opportunities in Medicinal Plants, Arlington, VA. Contact: IBC USA Conferences Inc., 225 Turnpike Rd., Southborough, MA 01772-1749. PHN: (508) 481-6400, FAX: (508) 481-7911.
September 19-22, 1994: John Innes Symposium: Biochemistry of Development, Norwich, England. Contact: John Innes Centre, Norwich Research Park, Colney, Norwich, Norfolk, England NR4 7UH. PHN: 44 603 52571, FAX: 44 603 56844.
September 25-27, 1994: Harnessing Apomixis: A New Frontier in Plant Science, College Station, TX. Contact: Dr. David M. Stelly, Dept. of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843-2474. PHN: (409) 845-2745, FAX: (409) 862-4733, EMAIL: monosom@rigel.tamu.edu.
September 30-October 5, 1994: Structural Molecular Biology Conference, Mont St. Odile, France. Contact: Dr. Josip Hendekovic, European Science Foundation, 1 Quai Lezay-Marnesia, F-67080 Strasbourge, Cedex, France. PHN: (88) 76 71 35, FAX: (88) 36 69 87, TELEX: 890440.
October 2-6, 1994: 22nd Aharon Katzir-Katchalsky Conference: Plant Molecular Biology--Potential Impact on Agriculture and the Environment, Koln, Germany. Contact: Secretariat 22nd AKK Conference, Aharon KatzirKatchalsky Center, Weizmann Institute of Science, Rehovot 76100, Israel. PHN: 972-8-342148, FAX: 972-8-474425.
October 2-6, 1994: 1994 Second International Symposium on the Applications of Biotechnology to Tree Culture, Protection, and Utilization, Minneapolis, MN. Contact: Edith Franson, Executive Secretary, Tree Biotechnology Symposium, Forestry Sciences Laboratory, P.O. Box 898, Rhinelander, WI 54501. PHN: (715) 362-7474, FAX: (715) 362-7816.
October 7-10, 1994: Genetic & Biochemical Approaches for Studying Cell Death, American Society for Biochemistry and Molecular Biology Fall Symposia 1, Granlibakken, Lake Tahoe, CA. Contact: ASBMB Fall Symposia Office, Room 3206, 9650 Rockville Pike, Bethesda, MD 20814-3998. PHN: (301) 530-7010, FAX: (301) 530-7014.
October 14-17, 1994: Mechanisms of Regulated Intracellular Protein Degradation: American Society for Biochemistry and Molecular Biology Fall Symposia 2, Whistler, British Columbia, Canada. Contact: ASBMB Fall Symposia Office, Room 3206, 9650 Rockville Pike, Bethesda, MD 20814-3998. PHN: (301) 530-7010, FAX: (301) 530-7014.
October 16-21, 1994: Recombinant DNA Biotechnology III Conference, Deauville, France. Contact: Engineering Foundation, Room 303, 245 East 47th St., New York, NY 10017. PHN: (212) 705-7837, FAX: (212) 705-7441.
October 28-31, 1994: Oligonucleotide Selection and Molecular Diversity: American Society for Biochemistry and Molecular Biology Fall Symposia 3, Granlibakken, Lake Tahoe, CA. Contact: ASBMB Fall Symposia Office, Room 3206, 9650 Rockville Pike, Bethesda, MD 20814-3998. PHN: (301) 530-7010, FAX: (301) 530-7014.
November 1-4, 1994: Cucurbitaceae 94: Evaluation and Enhancement of Cucurbit Germplasm, South Padre Island, TX. Contact: Dr. James R. Dunlap, Texas Agricultural Experiment Station, 2415 East Highway 83, Weslaco TX 78596. PHN: (210) 968-5585, FAX: (210) 968-0641, EMAIL: jdunlap @tamu.edu
November 13-16, 1994: Third International Symposium on the Biosafety Results of Field Tests of Genetically Modified Plants and Organisms, Monterey, CA. Contact: Ms. Pat Day, University of California, DANR, 300 Lakeside Dr., 6th Flr., Oakland, CA 94612-3560 OR USDA, Office of Agricultural Biotechnology. PHN: (703) 235-4419, FAX: (703) 235-4429.
November 17-19, 1994: 1994 San Diego Conference: The Genetic Revolution, San Diego, CA. Contact: Scherago International, 11 Penn Plaza, Suite 1003, New York, NY 10001. PHN: (212) 643-1750, FAX: (212) 643-1758, EMAIL: Scherago@Biotech.Net.Com.
November 21-24, 1994: Brighton Crop Protection Conference: Pest and Diseases, Brighton, UK. Contact: Conference Associates and Services Ltd., 55 New Cavendish St., London W1M 7RE, UK.
WORKSHOPS AND COURSES
September 26-30 or October 31-November 4, 1994: Polymerase Chain Reaction Methodology Workshop, Columbia, MD. Contact: Exon-Intron, Suite 130, 9151 Rumsey Rd., Columbia, MD 21045-1929. PHN: (301) 730- 3984, FAX: (301) 730-3983.
October 3-7, 1994: RNA Isolation & Characterization Workshop, Columbia, MD. Contact: Exon-Intron, Suite 130, 9151 Rumsey Rd., Columbia, MD 21045-1929. PHN: (301) 730-3984, FAX: (301) 730-3983.
October 10-14, 1994: Advanced Course on Molecular Biology Workshop, Leiden, Netherlands. Contact: Dr. L.A. van der Meer-Lerk, Institute of Biotechnology Studies Delft, Kluyver Laboratory, Julianalaan 67, 2628 BC, Delft, Netherlands. PHN: 015-78 51 40, FAX: 015-78 23 55.
October 17-20, 1994: Polymerase Chain Reaction Techniques and DNA Sequencing Lecture Course, Lake Tahoe, NV. Contact: Director, Center for Advanced Training in Cell and Molecular Biology, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064. PHN: (202) 319-6161, FAX: (202) 319-4467, EMAIL: millerm@cua.edu.
October 17-20, 1994: Recombinant DNA Methodology and DNA Sequencing Lecture Course, Lake Tahoe, NV. Contact: Director, Center for Advanced Training in Cell and Molecular Biology, Catholic University of America, 620 Michigan Ave., NE, Washington, DC 20064. PHN: (202) 319-6161, FAX: (202) 319-4467, EMAIL: millerm@cua.edu.
December 14-17, 1994: International Symposium on Plant Molecular
Biology and Biotechnology Workshop, New Delhi, India. Contact: G.
Chatterjee, International Centre for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. PHN: (011)
6867356, FAX: (011) 6862316.
January 7-13, 1995: Plant Cell Biology: Mechanisms, Molecular
Machinery, Signals, and Pathways: Keystone Symposium, Taos, NM.
Contact: Keystone Symposia, Drawer 1630, Silverthorne, CO 80498. PHN:
(303) 262-1230, FAX: (303) 262-1525.
Future Events
January 15-19, 1995: Plant Genome III, San Diego, CA. Contact: Plant Genome III, c/o Scherago International Inc., 11 Penn Plaza, New York, NY 10001. PHN: (212) 643-1750, FAX: (212) 643-1758, EMAIL: scherago@biotechnet.com
February 4-9, 1995: Advances in Gene Technology: Protein Engineering and Structural Biology: Miami Bio/Technology Winter Symposium, Ft. Lauderdale, FL. Contact: Miami Bio/Technology Winter Symposia, P.O. Box 016129 (M823), Miami, FL 33101. PHN: (800) 642-4363, FAX: (305) 324- 5665, EMAIL: mbws@mednet.med.miami.edu
March 5-9, 1995: XVIII Eucarpia Symposium: Ornamental Plant Improvement, Classical and Molecular Approaches, Tel Aviv, Israel. Contact: Dan Knassim Ltd., P.O. Box 57005, Tel Aviv, 61570 Israel. PHN: (972) 3-5626470, FAX: (972) 3-5612303.
April 23-27, 1995: 3rd International Union of Biochemistry and Molecular Biology Conference: Molecular Recognition, Singapore. Contact: 3rd IUBMB Conference Coordinator, Ken-Air Destination Management Company, 35 Selegie Rd., 09-19 Parklane Shopping Mall, Singapore 0718. PHN: (65) 336-8857/8, FAX: (65) 336-3613.
May 13-17, 1995: Ninth International Biotechnology Meeting & Exhibition, San Francisco, CA. Contact: Biotechnology Industry Organization, 1625 K St., NW, Suite 1100, Washington, DC 20006-1604. PHN: (202) 857-0244, FAX: (202) 331-8132 or (202) 857-0237.
July 4-7, 1995: 9th International Rapeseed Congress, Cambridge, England. Contact: Denis Kimber, 44 Church St., Haslingfield, Cambridge, CB3 7JE, England.
July 14-19, 1995: 15th International Conference on Plant Growth Substances, Minneapolis, MN. Contact: Gary Gardner, Dept. of Horticultural Science, University of Minnesota, 305 Alderman Hall, St. Paul, MN 55108. FAX: (612) 624-3606, EMAIL: ggardner@maroon.tc.umn.edu
August 6-11, 1995: 10th International Workshop on Plant Membrane Biology, Regensburg, Germany. Contact: Widmar Tanner, Lehrstuhl fr Zellbiologie und Pflanzenphysiologie, Universitt Regensburg, Universittsstrasse 31, 93053 Regensburg, Germany. FAX: 49-943-3352.
August 6-12, 1995: 20th World Congress of the International Union of Forestry Research Organisations, Tampere, Finland. Contact: Professor Risto Seppala, Finnish Forest Research Institute, IUFRO-95, Secretariat Unioninkatu 40A 00170, Helsinki, Finland.
Julia Bailey-Serres and Sheila L. Fennoy Department of Botany and Plant Sciences, University of California Riverside, CA 92521-0124
The overall bias in synonymous codon usage of a genome is speciesspecific. Analysis of protein coding regions of small samples of plant genes for a number of species revealed codon usage biases.1 The synonymous codon usage of nuclear genes of plants va ries mainly in the bias toward C or G versus A or U in the silent third nucleotide position. Nuclear gene coding regions of monocots are enriched in codons ending in C and G, whereas dicots have a higher frequency of codons ending in A and U.
We used a multivariate statistical analysis to examine codon usage in maize. More biased codon usage was recognized among more highly expressed genes, whereas more random codon usage was observed among more lowly expressed genes. Our work indicates that t he overall codon usage patterns in maize reflect the G+C content of the genome. Codon usage bias of individual genes may not solely reflect the nucleotide compositional bias of a chromosomal region, but may be affected by selection on the silent third nucleotide.2
The accumulation of DNA sequence data for a large number of nuclear genes of plants provided an opportunity to further examine synonymous codon usage. Table 1 shows a summary of codon usage for three plant species, maize (Zea mays L.), soybean (Glycine ma x L.), and Arabidopsis (Arabidopsis thaliana). Non-duplicate protein coding sequences were obtained from the September 1992 releases of GenBank and EMBL databases and the literature, and the relative synonymous codon usage was determined. The synonymous c odons used at a higher frequency in these data sets are indicated with an asterisk.
Information on codon usage is useful for the design of degenerate oligonucleotide primers for PCR amplification of regions encoding conserved proteins. In addition, consideration of G+C content or codon usage appears to be important for high levels of exp ression of bacterial genes in plants.3,4 Further systematic analyses are needed to determine the role of the G+C content and codon usage in regulating gene expression.
REFERENCES
and Fischhoff, D.A. (1991)
Proc. Natl. Acad. Sci. USA, 88, 3324-3328.
Table 1. Summary of relative synonymous codon usage in three plant species. Codon usage was tabulated (N) for maize (100 genes), soybean (71 genes), and Arabidopsis (112 genes). The relative synonymous codon usage (RSCU) is the observed frequency divided by the expected frequency assuming random codon usage. The most frequently used synonym for each amino acid of each plant is marked by an asterisk.
Peter M. Gresshoff
Plant Molecular Genetics
Center for Legume Research and Institute of Agriculture
The University of Tennessee
Knoxville, TN 37901-1071
Molecular genetics approaches have enriched the resolution of plant genome analysis. The ability to clone and sequence specific genome regions has added sequence-based information to our understanding of plant genomes derived from cytogenetics and large-scale DNA analyses (such as reassociation analysis).
While the database of DNA sequences is exponentially growing, methods are needed to investigate plant genomes at a level of complexity above the primary sequence, but below the cytogenetic, karyotypic arrangement.
Single, arbitrary primer-based DNA amplification techniques (DAF, RAPD and AP-PCR) were developed (Caetano-Anolls et al., 1991a; Williams et al., 1990; Welsh and McClelland, 1990), extending the utility of PCR to general genome analysis (fig. 1). Because of a plethora of terms, we proposed the general acronym MAAP (Multiple Arbitrary Amplicon Profiling; Caetano-Anolls et al., 1992b, 1993, 1994).
In essence, MAAP involves the use of a short, arbitrarily chosen oligonucleotide primer which, annealed to DNA, will direct DNA amplification of multiple genome regions (amplicons; Mullis, 1991). Temperature cycling and the use of a thermostable DNA polymerase are common components with the more specific and targeted PCR. In contrast to PCR, MAAP procedures use a single primer which is of arbitrary sequence. MAAP intentionally generates multiple products, which itself would be a rather undesirable result in a PCR reaction. MAAP is general, so that a primer used for one species can be used repeatedly for others, even if evolutionary distances between the template DNAs are large.
Amplification products are separated and recorded by a variety of detection methods; in all cases, a linear array of signals generates a profile, which is representative for the target DNA and specified by the DNA sequence of the primer. Variations in primer sites on the target DNA, length variations between primer sites, and possibly changes in the secondary structure of target DNA between or flanking the primer recognition sites, generate molecular polymorphisms. These amplification polymorphisms define molecular regions of the plant genome and thus can be used as (1) potential sequence tagged sites for positional cloning approaches, or (2) components of profile used in DNA profiling and diagnostics.
Three Techniques
MAAP procedures were developed independently, and apparently concurrently, in three laboratories. Welsh and McClelland (1990) developed AP-PCR, which uses PCR-length primers [18 to 32 nt] of arbitrary sequence to amplify target DNA under low stringency annealing conditions for two amplification cycles. This allows abundant mismatching and the generation of multiple amplification products (equivalent to a PCR reaction having gone wrong). Increased stringency of annealing at later amplification cycles generated reproducible products which were resolved on polyacrylamide gels and detected by autoradiography.
Williams et al (1990) invented the RAPD procedure, in which an arbitrary primer of either 9 or 10 nt produced amplification products after temperature cycling. RAPD products are routinely resolved on agarose gels and visualized by ethidium bromide. This provides a rapid method of scanning a genome. Alternative methods of detection, such as PAGE and silver-staining, coupled with careful optimization of amplification parameters (Collins and Symons, 1993) improved the utility of the approach. RAPD is widely used because of its simplicity and low-cost instrumentation.
Caetano-Anolls et al. (1991a,b) developed DNA Amplification Fingerprinting (DAF). Of all MAAP procedures, DAF utilizes the shortest primers, down to 5 nt in length. The optimal length was found to be 8 nt, a length which does not produce efficient amplification with RAPD. Informative amplification profiles were generated with 5 nt primers (5-mers), using soybean DNA as a template (Caetano-Anolls et al., 1993).
DAF products are routinely separated by thin polyacrylamide gels, backed onto plastic Gel-Bond film. This gel-plastic support, which provides support during the washing steps and helps preserve the original gel, is stained by an improved silverstaining method (Bassam et al., 1991; Caetano-Anolls and Gresshoff, 1994a), which detects DNA at about 1 pg mm-2. Resultant gels are air-dried and kept for permanent record and evaluation.
Pattern Detection
The PAGE/silver-staining technique provides a low-cost, high-throughput analytical method of DAF products. DAF products were also resolved by alternative methods. Agarose gels give clear resolution, but fewer products (Prabhu and Gresshoff, 1994). Fluorochrome labeled octamer primers were generated which then directed amplification of plant DNA (Caetano- Anolls et al., 1992a). The resultant amplification products were separated on an ABI Sequencer using Gene Scanner software. Single nucleotide resolution was obtained for lower sized amplification products. Tests using capillary electrophoresis have been promising (Dr. Patrick Williams, DNA Testing Laboratory, AFIP, Gaithersburg, MD; personal communication), providing separation of single samples in 30 minutes. In general, DAF generates scoreable polymorphisms in the molecular size range from 100 to 800 bp. Recently, we have used the pre-cast and automated PhastGel system (Pharmacia Inc.) to obtain profiles for pathogenic nematodes on soybean (Baum et al., 1994). Bands at higher molecular weight (up to 1500 bp) were scoreable; species and race-specific polymorphisms were detected. Denaturing gradient gel electrophoresis (DGGE) is another method which would help to distinguish polymorphic products of wheat (He et al., 1992).
Genetic Uses of DAF
The ability to detect molecular markers closely associated with genes of agricultural importance makes marker-based breeding an attractive proposition. The need for maintaining large plant populations through advanced breeding cycles can be reduced by detecting heterozygotes. MAAP markers converted through cloning, partial sequence analysis and specific PCR primer synthesis may provide SCARs (sequence characterized amplified regions), which are diagnostic for either a gene region in a plant or a pathogen. Figure 2 cartoons the utility of RFLPs and MAAP markers in generating diagnostic tools. For example, it may be possible to find markers specific for a soybean nematode race (see Baum et al., 1994), to convert it to a SCAR, then use a diagnostic, proactive test on agricultural soil to predict which nematode race is predominant in the field prior to planting.
The ability to generate many amplification products means that DAF is very efficient in scanning the genome of an organism for variable sites. In a survey of 25 primers (all octamers), Prabhu and Gresshoff (1994), working with G. max and G. soja, detected an average of 1.5 AFLPs per primer. Interestingly, RAPD gels of soybean produce an average of 5 to 7 scoreable bands, while DAF in soybean produced an average of 20 to 25 bands. Accordingly, the ratio of scored polymorphism to scoreable band is nearly the same, that DAF is not picking up more AFLPs because of the shorter primer length, but because of the detection method.
DAF markers were shown to be repeatable polymorphisms in different DNA isolations, operators, time periods, and amplifications. They are heritable, as are about 75% of AFLPs between G. max and G. soja segregated as dominant Mendelian markers in F2 populations (Prabhu and Gresshoff, 1994; Caetano-Anolls et al., 1993). Interestingly, the other 25% segregated in a uniparental way, being either maternal or paternal. Maternal inheritance presumably stems from amplification of cytoplasmic replicons. As yet, paternal replication is unexplained, and may represent either highly repeated chromosomal replicons or possibly alterations from normal cytoplasmic inheritance patterns in soybean.
Recombinant Inbred Lines
Several DAF polymorphisms were mapped in recombinant inbred lines of soybean (Prabhu and Gresshoff, 1994). The use of inbred lines is very convenient for DAF, as the lines are predominantly homozygous. Since DAF markers are dominant, it is impossible to distinguish the dominant homozygote from the heterozygote. Accordingly, in normal F2 populations, larger sample numbers are required to obtain data equivalent to data obtained from the analysis of a codominant (e.g., RFLP) marker. In recombinant inbreds, however, DAF and RFLP markers share the same statistical advantages. Figure 3 provides a summary of some RIL mapping data (conducted in collaboration with Dr. Gordon Lark, Utah).
The large number of products allows a high-density genotyping and genotype differentiation (Gresshoff, 1992). This form of fingerprinting is similar to the Universal Product Code, in which bars and spaces define a product. Reliable exclusion is obtained when one or more bands differ between samples. Inclusion is more difficult, as many primers need to be tested, frequency of variation within the sampled species needs to be known, and careful statistical statements need to be generated. One cannot declare with 100% certainty that two things are the same; the statement must always be probabilistic. It is up to the user (society, courts, scientists) to concur on acceptable levels of confidence for such probabilities.
Fingerprint Applications
DAF allowed the easy distinction of variant turfgrass material in commercial plots (Callahan et al., 1993). For example, foundation stock from several geographic locations gave identical profiles for Bermudagrass Tifway 419, while samples analyzed from golf course owners repeatedly showed major variation. The application of DNA tests to the turfgrass industry is a major challenge in an area of repeated vegetative propagation, triploidy and genetic instability. Using DAF markers, Weaver (1994) developed a phylogenetic tree of centipedegrasses.
Sunflower material provided by a seed company was categorized into several groups. Some common bands permitted the suggestion of a possible pedigree. This type of analysis has utility for product verification and plant variety rights.
The determination of genetic identity is also essential for the determination of plant product quality, as many food manufacturers use processes directly optimized for a specific biological feedstock. This industry relies on biological material; it is essential that quality biological feedstock enters the manufacturing process. Often it is impossible to inspect the source plant as one looks at a harvested product. It is for these industrial and related horticultural applications that a new technology was needed. DNA analysis has provided an additional way by which closely related organisms are distinguished for industrial., manufacturing, and retailing purposes.
DAF markers are useful in defining closely linked regions in bulked segregant analysis (Michelmore et al., 1991). The availability of large primer sets and the generation of multiple amplification products result in the efficient screening of the genome.
Induced plant mutations have the advantage of being in near-isogenic background as the genetic difference between parent and mutant is minimal. Using 25 DAF primers, Caetano- Anolls et al. (1993) showed that the induced supernodulation mutant nts382 and its wild- type parent cv. "Bragg" did not show polymorphisms despite the pairwise comparison of nearly 500 amplification products. Only in the use of MAAP, in which the target DNA was predigested with two restriction nucleases (four base cutters) and then amplified with a single octamer, could polymorphisms be detected between mutant and wild-type parent. Only 19 primers were needed to reveal 42 AFLPs. Fourteen of these segregated at 100% with the supernodulation phenotype in G. soja (wild-type) and G. max (mutant ) derived F2 populations. Some AFLPs distinguished between the nts382 and nts1007 alleles. It is likely that these are valuable markers close to the nts locus and their cloning and further characterization will facilitate the isolation and ordering of yeast artificial chromosomes (YACs) in that region.
Mini-hairpin Primers
Funke and Kolchinsky (1994) demonstrated that stable YACs carrying soybean genomic DNA can be constructed, with an average size of about 200 kb (maximum 900 kb). About 7% represented chloroplastic DNA. The combination of clustered molecular markers, the ability to generate medium-sized YAC clones, end-clones and possible contigs, increase the chances of isolating soybean regions carrying developmentally significant genes. Caetano-Anolls and Gresshoff (1994b) used mini-hairpin primers in a DAF reaction to profile such soybean YACs. The mini-hairpin primers are interesting, because they contain on their 5 end a 7 nucleotide fold-back loop (4 nt in stem, 3 nt in the loop). The 3 end can be as short as 3 nt, allowing the generation of a small set of 64 primers, which are useful for the characterization especially of small genomes or genome components such as plasmids or YACs. <bodytext>These findings show that single primer DNA amplification analysis of plant genomes adds a further genetic tool to construct high-density maps needed for positional cloning and marker-based breeding approaches.
DAF Parameters
Primer length: 5 nt minimum; 8 nt optimum Primer 3 end most important for specificity of reaction Primer concentration: 3M for 8-mer primer and up to 30M for 5-mer primer 2mM MgCl2 optimum for soybean genome and 6mM optimum for bacterial genome Taq polymerase produces good amplification results for large fragments Truncated Stoffel fragment should be used to amplify fragments in the 50-200 bp range
Excess template DNA (>25 ng/25 l reaction) reduces intrinsic amplification products
For a more complete discussion of these parameters please gopher to: gopher.nalusda.gov. Select Information Centers from the menu. Next select Plant Genome Data and Information Center. If you would like a hard copy of the paper please contact the Plant Genome Data and Information Center at the address on page.
Abbreviations: PCR=polymerase chain reaction; DAF=DNA amplification fingerprinting; AP- PCR=arbitrary primer-PCR; MAAP=multiple arbitrary amplicon profiling; nt=nucleotide; bp=base pair; PAGE=polyacrylamide gel electrophoresis; RAPD=random amplified polymorphic DNA; RFLP=restriction fragment length polymorphism
References:
Baum, T. J., Gresshoff, P.M., Lewis, S.A. and Dean, R.A. (1994) Characterization and phylogenetic analysis of four root-knot nematode species using DNA amplification fingerprinting and automated polyacrylamide gel electrophoresis. MPMI 7: 39-47.
Bassam, B.J., Caetano-Anolls, G. and Gresshoff, P.M. (1991) A fast and sensitive silver- staining for DNA in polyacrylamide gels. Analytical Biochemistry 196: 80-83.
Caetano-Anolls, G. and Gresshoff, P.M. (1994a). Staining nucleic acids with silver: an alternative to radioisotopic and fluorescent labeling. Promega Notes 45: 13-18.
Caetano-Anolls, G. and Gresshoff, P.M. (1994b). DNA amplificatio fingerprinting using arbitrary mini-hairpin oligonucleotide primers. Bio/Technology 12: 619-623.
Caetano-Anolls, G., Bassam, B.J. and Gresshoff, P.M. (1991a) DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. Bio/Technology 9: 553-557.
Caetano-Anolls, G., Bassam, B.J. and Gresshoff, P.M. (1991b) DNA amplificationfingerprinting: a strategy for genome analysis. Plant Molecular Biology Reporter 9: 292-305.
Caetano-Anolls, G., Bassam, B.J. and Gresshoff, P.M. (1992a) DNA amplification fingerprinting with very short primers. In: Application of RAPD Technology to Plant Breeding. ed. M. Neff. ASHS, publ. (St. Paul, MN). pp 18-25.
Caetano-Anolls, G., Bassam, B.J. and Gresshoff, P.M. (1992b) DNA fingerprinting: MAAPing out a RAPD redefinition. Bio/Technology 10: 937.
Caetano-Anolls, G., Bassam, B.J. and Gresshoff, P.M. (1993) Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease digested DNA: identification of markers linked to the supernodulation locus of soybean. Mol. Gen. Genetics 241: 57-64.
Caetano-Anolls, G., Bassam, B.J. and Gresshoff, P.M. (1994) Multiple arbitrary amplicon profiling using short oligonucleotide primers. Plant Genome Analysis. ed.
P.M. Gresshoff, CRC Press, Boca Raton, FL, pp 29-46.
Callahan, L. M., Caetano-Anolls, G., Bassam, B.J., Weaver, K. MacKenzie,
Collins, G.G. and Symons, R.H. (1993) Polymorphisms in grapevine DNA detected by the RAPD PCR technique. Plant Molecular Biology Reporter 11: 105-112.
Funke, R. P. and Kolchinsky, A.M. (1994) Plant yeast artificial chromosome libraries and their use: status and some strategic considerations. Plant Genome Analysis. ed. P.M. Gresshoff, CRC Press, Boca Raton, FL pp. 125-134.
Gresshoff, P.M. (1992) DNA fingerprinting brings high-tech genetics into commercial greenhouses. Grower Talks (July 92), pp 119-127.
He, S., Ohm, H., and McKenzie, S. (1992) Detection of DNA sequence polymorphisms among wheat varieties. Theor. Appl. Genet. 84: 573-578.
Kolchinsky, A., Landau-Ellis, D., Angerm uller, S., Deckert, J., and Gresshoff, P.M. (1994) Molecular analysis of a polymorphic DNA region tightly linked to the supernodulation (nts) locus of soybean (in review).
Michelmore, R.W., Paran, I. and Kesseli, R.V. (1991) Identification of markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions using segregating populations. Proc. Natl. Acad. Sci. (USA) 88: 9828-9832.
Mullis, K.B. (1991) The polymerase chain reaction in an anemic mode: how to avoid cold oligodioxyribonuclear fusions. PCR Meth. Applic. 1: 1-4.
Prabhu, R.R. and Gresshoff, P.M. (1994) Inheritance of polymorphic markers generated by DNA amplification fingerprinting and their use as genetic markers in soybean. Plant Molecular Biology (in press).
Weaver, K. (1994) MS dissertation. The University of Tennessee, Knoxville.
Welsh, J. and McClelland, M. (1991) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18: 7213-7218.
Williams, J.G.K, Kubelik, A.R, Livak, K.J, Rafalski, J.A. and Tingey, S.V. (1990) DNA polymorphisms amplified by arbitrary primers are useful genetic markers. Nucleic Acids Res. 18 6531-6535.
Claire Kinlaw and David Harry
Institute of Forest Genetics, USDA, Forest Service
Berkeley, CA 94701
Forest trees and their molecular genetics were the focus of a recent conference (Prouts Neck, Maine, USA, May 20-23, 1994) organized by Michael Greenwood and Keith Hutchison (University of Maine, USA). A group of 70 researchers gathered to discuss progress toward understanding and manipulating molecular processes within this diverse group of economically and ecologically important species.
The biology of forest trees provides both distinct challenges and unique opportunities. Compared to previous meetings of this research community, significant progress has been made in several areas. Research teams continue to isolate and characterize new genes, while transgenic plants, especially in Populus, are increasingly being used to study gene function in vivo. Genome mapping has also matured. In earlier meetings, presentations described the construction of genetic linkage maps, while at this meeting maps were presented as tools to identify and dissect quantitative trait loci (QTL).
Advantages of the haploid genetics offered by conifer gametophytes continue to be exploited for mapping work and for population surveys. Recent advances in model organisms continue to influence studies in forest trees. Homeotic genes, for example, are being sought for flower and cone development. Other research focuses on processes that either are unique to trees or are simply more important in trees than in other organisms. For example, lignin and its related biosynthetic pathways are affected by wounding and stress in trees as in other plants, while in trees alone, lignin also is a major component of wood.
Gene Expression Patterns
As highlighted by Olof Olsson (Swedish University of Agricultural Sciences, Sweden), woody angiosperms show fluctuations in gene expression during annual cycles of quiescence and "reesence" in addition to changes observed during development and in response to environmental stresses. With the goal of increasing pulp production efficiency, Wout Boerjan's laboratory (Universiteit Gent, Belgium) has produced transgenic Populus plants containing antisense constructs for the lignin biosynthetic enzymes O-methyltransferase (OMT) and cinnamyl alcohol dehydrogenase (CAD). Work on conifer lignin and its role in development continues despite being hindered by the lack of reliable transformation and regeneration methods. Working on Pinus taeda, John Mackay (North Carolina State Biotechnology Group, USA, directed by Ron Sederoff) described the isolation and characterization of several genes encoding lignin biosynthetic enzymes including phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and 4-coumarate:CoA ligase (4-CL).
A slightly different tack toward understanding xylogenesis is being taken by Mackay's colleague, Malcolm Campbell. Because myb-like genes play an important role in signal transduction pathways in other organisms, such genes may also control conifer xylem development. Campbell has initiated cloning of Pinus taeda homologues. A similar rationale underlies the strategy of Sharon Regan and Bob Rutledge (Petawawa National Forestry Institute, Canada) in their efforts to characterize MADS box homeotic genes controlling cone development in Picea mariana.
With the goal of understanding the role of flavonoids in root formation, Lise Jouanin's laboratory (INRA, Versailles Cedex, France) has produced transgenic Populus and Juglans containing altered levels of chalcone synthase (CHS). Carmen Diaz-Sala, Keith Hutchison, and Mike Greenwood (University of Maine, USA) are investigating the cellular and molecular changes associated with the organization of root primordia in Pinus taeda. In particular, they are addressing how the cytoskeleton orients the plane of cellular divisions and nuclear reorganization.
Engineering for pest resistance using proteinase inhibitors is being done by several groups including those of Ned Klopfenstein (USDA-FS Center for Semiarid Agroforestry, USA), Lise Jouanin, and Seguin Armand (Universite Laval, Canada). Jouanin has found a cysteine protease inhibitor to be particularly effective against pests which contain high levels of cysteine proteases.
Genes responding to major environmental stresses have been identified in stress-specific cDNA libraries. Genes induced by drought stress in Pinus taeda (Shujun Chang et al., Texas A&M University, USA) include caffeoyl CoA, SAM synthetase, chitinase, and a protein similar to an animal skin matrix component. Genes induced by ozone (Dieter Ernst, Institut fur Biochemische Pflanzenpathologie, Germany) in Pinus sylvestris include CAD, stilbene synthase, hydroxymethylglutaryl-CoA-synthase, and polyubiquitin. As found in angiosperms, certain conifer genes appear to be induced by a number of environmental stresses. For example chitinase is induced by wounding, fungal infection, and drought (Haiguo Wu, Craig Echt, and John Davis, University of Florida, USA).
To expand upon the identification of new conifer genes, Claire Kinlaw (Institute of Forest Genetics, USA) has initiated "single-pass" sequencing efforts of Pinus taeda seedling cDNAs used as markers by David Neale and co-workers (Institute of Forest Genetics, USA) for genetic maps. These identified sequences will provide molecular tools for studying conifer genome organization and evolution. Early results have been encouraging in that a variety of genes have been identified including those encoding photosynthetic proteins, translation factors, glycolytic enzymes, and stress-response proteins.
With a systemic point of view, Gary Coleman (Oregon State University, USA) proposed a model to explain how trees regulate autumn nitrogen storage and spring remobilization in response to nitrogen availability and photoperiod. In this model, bark and leaves communicate with each other using a bark storage protein (BSP) and a leaf protein encoded by Win4. During short days or high levels of nitrogen, BSP accumulates in bark parenchyma while the Win4-encoded protein is repressed. During long day shoot growth, BSP is degraded while the Win4-encoded protein accumulates.
Genetic Maps and Quantitative Trait Loci
Genetic maps using two alternative approaches are being used to identify QTLs. Lively discussions of the merits and disadvantages of these two alternate approaches accompanied formal presentations. Mitch Sewell (Institute of Forest Genetics, USA) described the integration of restriction fragment length polymorphism (RFLP)-based linkage maps from two Pinus taeda pedigrees. This work will further efforts by Neale and his co-workers to dissect wood quality traits and to understand conifer genome organization and evolution. Several members of the Forest Biotechnology Group at North Carolina State University (USA) presented RAPD-based maps including those from Eucalyptus (Dario Grattapaglia) for the identification of QTL controlling sprouting and rooting.
New Markers
Several laboratories are exploring the use of length polymorphism among simple sequence repeats (SSRs). Craig Echt, (USDA Forest Service, Rhinelander, USA) has observed that approximately 0.7% of the Pinus strobus genome is comprised of SSRs. Of the primer pairs tested from the flanking sequences of Pinus strobus SSR loci, approximately 65% reliably amplify DNA. A high proportion show size polymorphisms, and a significant number amplify DNA from other conifer taxa. In apparent contrast to these observations, Keith Hutchison (University of Maine, USA) has observed a low level of size polymorphisms among SSR alleles in Larix laricina.
With a similar goal of developing co-dominant PCR-based markers, but using a different approach, David Harry (Institute of Forest Genetics, USA) is designing and testing primers based upon sequences of specific Pinus taeda cDNAs. Approximately 75% of the primer pairs reliably amplify genomic DNA, with a high proportion revealing Mendelian polymorphisms following restriction enzyme digestion. Some primers amplify only hard pines, others amplify all pines, and still others amplify DNA from other conifer taxa. Hisato Okuizumi presented an application of restriction landmark genomic scanning (RLGS) to large genomes by including a restriction trapper. High-speed scanning of entire genomes and the construction of genetic maps of individual trees from a single run with several hundred loci are made possible. As an example, a profile of Pinus koraiensis was shown.
Describing Genome Flux and Evolution
Because seed plants represent an ancient lineage, and because woody
plants have long generation times, mechanisms of genetic mutation and
genome evolution, as well as rates of species evolution, continue to be
important areas of study. In seeming contrast to low levels of observed
SSR polymorphism in Larix laricina, Hutchison and coworkers have found
relatively high levels of sequence variation within genomic regions
encoding proteins.
In addition, they have observed segregation distortion of alleles under
different environments. The apparent contrast between the high levels
of polymorphism among coding regions and low levels of polymorphism
among SSR regions may indicate that conifers have a relatively
efficient mismatch repair mechanism and may thus partially account for
the stability of conifer karyotypes.
Jean Bousquet (Universite Laval, Canada) and his co-workers are investigating ancient events during the evolution of seed plants. After carefully calibrating a molecular clock, they established that modern gymnosperms derived from a single lineage, and they estimated divergence times to have occurred as follows:
liverworts from vascular plants 440 mya ferns from seed bearing plants 400 mya flowering from cone bearing plants 290 mya monocots from dicots 200 mya Pinus from Pseudotsuga 140 mya
Ross Whetten (North Carolina State University, USA) is exploiting the idea that a tree's crown represents a common lineage of shoots with known separation times. Using visible phenotypes in peach, Whetten estimated the somatic excision rate of a transposable element. The rate is relatively higher than rates reported for annual species and varies among meristematic layers. This notion that different shoots within the crown of a tree can be genetically distinct might help explain how long-lived trees endure pathogens and insect pests with shorter generation times.
Genetic Diversity and Population Structure
In addition to their use in mapping, RAPDs have been used by a number of laboratories for estimating genetic diversity and describing population structure. Natalie Isabel (Universite Laval, Canada) compared estimates of genetic variation within and among populations of Picea mariana using RAPDs and allozymes. Results from these two types of markers were similar. Linda DeVerno (Petawawa National Forestry Institute, Canada) surveyed Pinus resinosa using 400 RAPD primers and found no polymorphism. Again this data supports earlier conclusions based upon allozymes.
1995 Meeting
The next tree molecular geneticists meeting will take place at
Universiteit Gent, Belgium, in combination with the IUFRO Somatic Cell
Genetics Working Party. Those wishing more information are encouraged
to contact Wout Boerjan
(Boerjan%research%RUG.genetica@genwet1.rug.ac.be).