PROCEDURES:
Objective 1. Develop high resolution
comparative genome maps aligned across species that link agricultural
animal maps to those of the human and mouse genomes.
The
ultimate goal of the animal genome research is to facilitate the
identification, cloning, and characterization of genes responsible for
economically important traits. Linkage maps of polymorphic markers are
being developed in agriculturally important species and are already being
utilized to map ETL. The next step, identifying and cloning genes
responsible for these traits, armed only with knowledge of their
chromosomal localization, is a formidable one. Even in humans and mice,
for which financial resources are comparatively abundant, map-based
cloning is an arduous task. Positional cloning in humans, however, is
rapidly being replaced by "positional candidate cloning," a strategy in
which candidate genes are generated from high-density transcription maps
across the large intervals (potentially containing hundreds of genes) to
which traits can be genetically mapped. This approach and the detailed
maps of functional human genes that support it, also offer special
opportunities for the animal genome research community. The identification
of regions of chromosomal homology between humans (or mice) and
agriculturally important animals will also provide candidate genes on the
human map for many animal ETL of agricultural interest. It then becomes
much easier to locate the homologous animal candidate gene(s) to be
tested. Thus, the development of high resolution comparative maps will
permit animal geneticists to extrapolate candidate genes directly from
human and mouse gene maps to animal ETL maps.
Although the human
genome is destined to be the prototypic genome for comparative mapping
purposes, the mouse genome map cannot be ignored. The designated mammalian
"model organism" of the human genome initiative is also a valuable
laboratory model for identifying both QTL and single genes related to
growth, reproduction and disease. Many of these genes are likely to be
homologous to ETL in livestock genomes. Furthermore, the HGP has already
led to the development of a high quality mouse-human comparative map that
serves as a model for what we hope to achieve with agricultural animal
species.
Comparative maps of economically important animals
relative to humans and mice will be essential for agriculture to directly
benefit from the human genome initiative. However, comparative maps
between agricultural animal species are also needed to derive maximum
benefit from the animal genome effort. Homologous genes related to muscle
composition, reproductive performance or disease resistance, for example,
will be important to more than one animal industry. Especially valuable
will be comparative maps between genomes that are most closely related
evolutionarily, for example, sheep and cattle or chicken and turkey.
Cattle markers are already routinely tested and used in the sheep genome
mapping effort.
The specific aims to be addressed in objective 1 are as follows.
1. Common marker panels. Facilitate the mapping of a common
set of homologous genes in all relevant species. This will involve
prioritization of a common set of genes to facilitate the most efficient
use of resources and the development of techniques that improve the speed
and cost-efficiency of comparative mapping.
2. Comparative map
resources. Facilitate the development and distribution of resources
for high-resolution comparative mapping, for example, ZOO-FISH painting
reagents, radiation-hybrids, reference mapping family DNA panels, and
primer sets with the potential to amplify homologous genes in more than
one species.
3. Comparative map data sharing. Facilitate
the development of comparative genome databases that include
agriculturally important animals. This will include continued development
of species databases in formats compatible with merger and cross-reference
with each other and with human and mouse genome databases.
Since
relatively few animal geneticists regularly work in more than one or two
species, comparative mapping inherently requires sharing results and DNA
probes between investigators and across species. No RR project is set up
to do this. The mechanisms by which NRSP-8 will facilitate comparative
mapping are common to all three objectives and will be discussed below
(Mechanisms section). As will be described later, aims 2. and 3. naturally
interface with some of the aims of objective 3.
Objective 2. Increase the marker density of existing linkage
maps used in QTL mapping and integrate them with physical maps of animal
chromosomes.
Mapping QTL is inherently imprecise, making it
difficult, if not impossible, to localize them more accurately than to
within 5-10 cM intervals. This implies that maps with high resolution and
excellent alignment with those of humans or mice will be required to
understand and fully take advantage of QTL. An integrated genome map
consists of a genetic linkage map with a high density (ca. 1-2 cM) of
widely polymorphic markers that is aligned with a complete physical map
consisting of ordered, overlapping clones from large insert libraries.
Integrated maps of the type and quality described are presently available
in two vertebrate species, human and mouse. Two basic components were
required to assemble those maps: large arrays of markers, a subset of
which can be ordered by genetic linkage mapping, and complete genomic
libraries in large insert vectors, i.e., yeast artificial chromosomes
(YACs) and bacterial artificial chromosomes (BACs). Radiation hybrid
mapping panels can also be of great value in the development of integrated
physical/genetic maps. Significant progress has been made in the
generation of both large insert libraries and useful markers for cattle,
sheep, swine and chicken. However, the existing marker collections are
insufficient, either for whole genome, high resolution QTL mapping or for
the generation of complete integrated maps. A less serious problem is that
existing large insert libraries are relatively few and of limited
availability in some species.
The genetic linkage maps for
agricultural animal species presently available demonstrate a fairly high
level of coverage (ca. 95% of markers in linkage groups) and many of the
markers have widespread utility for animal breeders. However, the average
marker spacing is approximately 3-10 cM, depending on the species in
question, which corresponds to a distance of roughly 2-10 million base
pairs (Mb) of DNA, and there are many gaps much larger than this. Since
even the best large insert libraries have average inserts of 1 Mb or less,
present marker resolution is insufficient for map integration.
Furthermore, many of the available markers are not sequence-tagged site
(STS) loci suitable for physical map assembly and/or are not highly
polymorphic for wide utility in ETL mapping crosses (resource
populations). This is especially true for commercial line crosses that are
likely to be more uniform genetically than the divergent experimental
crosses that have been heavily used to date. Thus, it is clear that better
marker densities are required for both genetic and physical mapping. To
put this in context, the human genetic map is about the same size (in cM)
as those of the major agricultural animal species. Even for the first
generation integrated human genome map, over 15,000 STS markers were used.
In the most general sense, all genome mapping involves binning (or
pooling) a collection of linked markers into ever-smaller subsets such
that a local order is generated. Thus, the goals of genome coordination
are to assist with the use of common marker sets and common binning pools,
such that data can be collected and assembled into shared framework maps
with high resolution, accuracy, and utility. Markers can be binned by
meiotic breakpoints, as in linkage mapping, by radiation-induced
breakpoints, as in radiation hybrid mapping, or by DNA breaks induced in
recombinant DNA library construction, the basis of high resolution
physical maps.
The specific aims to be addressed in objective 2 are listed below.
The means by which they will be achieved are described later in the
Mechanisms section.
1. More mapped markers. Framework
genetic linkage maps should approach a theoretical resolution of about 1-2
cM. This will require that 3,000 (or more) markers be placed on the
framework map for each species.
2. Maps/markers of higher
utility. As much as possible, framework markers should be STS loci,
preferably genetically variable STS markers such as microsatellites. Such
markers are most useful for correlating physical maps with ETL map data.
In addition, STS markers that are within expressed genes (expressed
sequence tags or ESTs) are optimal for comparative mapping as described in
objective 1.
3. Binning pools for mapping. Improved
framework map resolution also requires that markers be distributed in
smaller bins. This involves common use of reference panels with more
meiotic breakpoints, combining reference panel data into consensus maps,
development and use of radiation hybrid mapping panels, and/or physical
mapping of STS markers using high quality, large insert DNA libraries.
Objective 3: Expand and enhance internationally shared species
genome databases and provide other common resources that facilitate genome
mapping.
Coordination and sharing of mapping resources are
vital to streamlining research and to avoiding duplication. In particular,
shared resources allow wider participation of scientists who have limited
local access to equipment and supplies. Researchers and breeders also need
unimpeded access to databases, together with the necessary tools for
queries and analysis in order that increased understanding can be
developed from a synthesis of many individual experiments. Inherent in the
top-down approach of genomics, along with the impressive data generation
power of modern technology, is the need to deal with large, complex data
sets in a meaningful way. A new discipline has emerged focused on this
challenge: Bioinformatics.
Genome databases that underpin the
needs of the livestock genome mapping programs have been developed through
productive international collaborations. The initial model adopted for
pigs, chickens and sheep was based on the mouse genome database - GBASE.
More recently, the bioinformatics group at the Roslin Institute has
developed a revised model for livestock genome databases - Arkdb.
Editorial and curatorial functions are shared by scientists in the United
States and elsewhere. One of the major accomplishments of the NRSP-8 has
been to support the development of databases for each species. This
support assisted in the creation of these databases and their continuing
evolution, such that they now include linkage mapping, physical mapping
and information on individual genes, markers and mapping materials. These
databases also include information on mapping resources and published
references. The continued development and maintenance of these shared
databases (and their expansion to the horse genome project) is central to
the shared activities of NRSP-8.
The specific aims to be addressed
in objective 3 include:
1. Improved databases and
communication. Existing genome databases will be improved based on
rapid developments in bioinformatics technology. New data will be added,
databases will become more fully networked, and new search engines will be
employed. Other communication mechanisms such as newsletters, email
servers, Internet homepages, and the annual NAGRP meeting will be
enhanced.
2. Shared mapping resources. Shared resources
will be maintained and expanded to enhance the cost-effectiveness of
genome mapping and its applications. These include mapping panel DNAs,
large insert libraries, comparative mapping resources, and a variety of
primers for PCR-based marker mapping.
Mechanisms: The ways in which NRSP-8 will support the
three objectives and their individual aims can be grouped in three
categories: Communication, Sharing Materials and Sharing Data.
These will be outlined below, along with examples of their applications to
the individual objectives.
Communication. First, by sharing
plans for future research and results of past research, efficient,
coordinated mechanisms can be developed for improving existing framework
(consensus) maps. One example of this are the chromosome workshops that
have been and are being arranged for many specific swine and bovine
chromosomes. In addition to the single species chromosome workshops held
to date, interspecies comparative chromosome workshops will occur to
ascertain regions of shared gene order. Furthermore, the NRSP-8 Technical
Committee can prioritize common genes to place on maps that will most
effectively answer the critical questions of genome evolution and
comparative map utilization. Technical Committee meetings will facilitate
interaction between groups working in different species, so that they can
develop the cross-species collaborations that are essential for
comparative map generation. NRSP-8 has taken the lead in coordinating its
annual meetings jointly with related RR committees and, more recently,
arranged to conduct these meetings as part of an expanded Plant and Animal
Genome (PAG) meeting. This provides greatly enhanced opportunities for
information and technology transfer between international scientists in
all aspects of agricultural genetics in both the public and private
sectors (attendance was about 600 at the first joint PAG meeting in
January, 1997, and is expected to be 800-1000 in 1998).
Other
communication mechanisms: The publication of newsletters and other printed
material, along with the development of World-Wide Web (WWW) species
homepages and an on-line-computer discussion group for gene mappers
(ANGENMAP) have proven very valuable and popular as ways to share
information. These provide user-friendly ways for the participating
scientists to keep abreast of each others' activities, available
resources, upcoming meetings, and general advances in technology and
research. They encourage better communication and cooperation among the
participating scientists, worldwide. Perhaps even more important,
newsletters and WWW homepages provide very effective outreach functions in
keeping potential beneficiaries in wider agricultural and consumer group
circles aware of and involved in the advancements in agricultural
genetics. Our newsletters and homepages are seen by thousands of
people, many of whom are not gene mappers but simply interested
agricultural scientists, consumers, students and hobbyists. These
activities will continue and expand. As another example, animal
geneticists have recently joined their plant colleagues in generating the
Probe newsletter for agricultural genomics.
Sharing
materials. As described in the Critical Review, the sharing and
distribution of a variety of panels, primers and libraries is already well
underway and has proven very successful. The goal will be to increase the
utility of these resources, especially as scientific advances make new
types of markers and materials applicable. This will speed applications of
the maps to specific agricultural problems. Shared markers will be vital
as enhanced maps provide more and more opportunities for fine mapping of
ETL and tests of marker-assisted selection strategies.
DNA panels
and libraries: Family materials for gene mapping and ETL research are
difficult and expensive to develop. Shared use of a DNA panel adds value
(map information) to that panel for everyone involved. The coordination
effort will be directed to collecting DNA from international and national
mapping and ETL families and sharing that with members of species
committees. This allows for joint genetic framework maps and for joint ETL
analyses. Similarly, somatic cell hybrid panels (including radiation
hybrids) and large insert libraries function analogously to family panels
as binning resources for physical maps rather than linkage maps. Several
BAC libraries for various species have been made at Texas A&M and
elsewhere (see Critical Review). The maintenance and distribution of
mapping resources like BAC or YAC libraries and somatic cell hybrid panels
requires significant effort and expense, and this process will be
facilitated and supported by species coordinators. This avoids duplication
and increases the power of the research at a fraction of the cost. In
addition, large insert DNA clones from contiguous regions (contigs) will
be shared for fine structure comparative mapping to answer more detailed
questions about genome evolution mechanisms.
Primers and other
marker materials: Panels of microsatellite (and other STS) markers will
continue to be developed and enhanced, and the requisite PCR primers will
be provided to interested investigators to increase the resolution,
accuracy, and utility of framework marker sets and maps. Sharing of
primers for ETL analyses and mapping research have proven very valuable. A
total of approximately 1000 primer pairs have been shared with
participating members (typically 30 or more participants/species) at a
fraction of their cost if each station made them individually. In the
future, a variety of comparative mapping probes will also be developed
and/or distributed using coordination funds. These include comparative
anchor tagged sequence (CATS) primer pairs for PCR-based comparative
mapping, Type I gene clone DNAs and/or primers and ZOO-FISH comparative
cytogenetic probes, used in karyotype-based comparative mapping.
Sharing data. For comparative mapping to be most effective,
the individual species databases must be able to directly exchange
information. To some extent this process has begun, with links of existing
databases to human (or mouse) maps and databases. The development of the
next generation of database, named TCAGdb for The Comparative Animal
Genome database, is already well underway (see Swine section of Critical
Review). This will pull together data from the single species databases
for cross-species comparisons, as will be required for true comparative
mapping. TCAGdb is based on the Arkdb model, a generic model applicable
for all relevant species. (Arkdb versions of some of the species databases
have recently been installed, see Critical Review). This advanced database
has all the previous capabilities of providing information on linkage and
physical mapping data. In addition, information on primers and mapping
tools will be greatly expanded. Data entry tools will be improved such
that entry is faster and more complete. Over the next 5 years, we can
expect rapid advances in the bioinformatics field, as it struggles to keep
up with the massive amounts of human, mouse, and model organism genome
data being generated. NRSP-8 support will allow for the judicious
application of the most effective of these advances to the agricultural
animal genome databases. In the Internet environment, the actual
location(s) of the database is not critical. In fact, at least two nodes
for each existing database are desirable, to provide a back-up for one
another and to insure rapid user access. It is envisioned that the
Coordinators for each species will be co-editors (with appropriate
international collaborators) for each of the respective databases.
Editors, with the assistance of curators at the node sites, will receive
and evaluate data from participating scientists, ask for revisions if
necessary (e.g., for database consistency), and approve data for
inclusion. Coordinators (and journal editors)will be responsible for
requiring that map data that support publications are submitted by the
authors. Curators will also be active in technical improvement of the
databases. For maximal cost effectiveness, it is anticipated that two or
more Coordinators will share one primary US node for their databases,
supported by a single curator (see budget).
EXPECTED OUTCOMES:
1. Framework genome maps for
agricultural species will be improved to a resolution (average marker
spacing) of 1-2 cM from the existing 5-10 cM.
2. Physical maps
based on cloned DNA fragments will be developed that cover large
contiguous chromosomal domains or, in some cases, whole chromosomes.
3. Comparative maps will be available which link the genomes of
agricultural animals to those of human and mouse.
4. Together, the
first three outcomes will facilitate the isolation and characterization of
ETL-encoding genes from several species' genomes, most often via the
positional candidate approach to gene cloning (see objective 1). ETL will
be identified that relate to meat, milk, egg, and wool productivity,
disease resistance, and reproductive efficiency. Based on this
information, breeding schemes will be designed to take maximal advantage
of the existing genetic diversity of agricultural animals.
5. New
genetic tests will be developed for productivity and disease resistance
traits. These tests will be applied by the commercial sector to make food
(and other animal products) safer and more economical and to generally
improve animal health and welfare.
6. Internationally-shared
species genome databases will be enhanced such that genomics information
will be readily available and widely used.
7. Marker primer pairs,
DNA mapping panels, large insert libraries and other relevant mapping
resources will be obtained by participating laboratories at little or no
cost.
All the above outcomes will stimulate interest and enhance
expertise in animal genomics to speed its application to problems of
economic interest.
ORGANIZATION:
NRSP-8 supports the National Animal
Genome Research Program (NAGRP), which is organized to link and facilitate
animal genome research efforts with universities, SAES, CSREES, ARS, and
the animal breeding industry.
National Animal Genome Technical
Committee. The Technical Committee is composed of participating scientists
appointed by their SAES Directors or other administrators of each
cooperating organization. The Technical Committee meets annually and
provides guidance to the NAGRP. The members of the Technical Committee are
subdivided into Species Committees to interact and work with the
designated Species Coordinators. Each year the Technical Committee elects
a chairperson and a secretary; the Species Committees also elect their own
chairpersons and secretaries. The chairperson of the Technical Committee
calls and conducts meetings of the Technical Committee, coordinates the
activities of the Species Committees, keeps the NAGRP Program Leader
informed, and prepares and submits an annual report of Technical Committee
activities and accomplishments to the NAGRP Program Leader and
Administrative Advisors. The secretary prepares and distributes minutes,
maintains a record of participating scientists and organizations, and
establishes a permanent list of publications from the project.
Executive Committee. The NRSP-8 Executive Committee is designated
to deal with activities between scheduled meetings of the full Technical
Committee. The Executive Committee is composed of the Technical Committee
Chairperson and Secretary, Species Genome Committee Chairpersons, and
Species Coordinators. The Administrative Advisors and the NAGRP Program
Leader are ex-officio members.
Species Genome Committees. The
Species Genome Committees are composed of members of the NRSP-8 Technical
Committee with active genomic research programs within that species. The
Species Committees identify and establish criteria for the acceptance,
preservation and use of reference and resource families, suggest priority
areas for mapping and ETL research in their species, identify needs for
genetic markers and other shared materials, and aid in the growth and
development of species databases. These activities are planned in concert
with the Species Coordinators.
Species Genome Coordinators. Five
Species Coordinators (Cattle, Sheep, Swine, Poultry, Horse) will be
selected by a competitive process, open to all Technical Committee members
and administered by the NAGRP Program Leader and Lead Administrative
Advisor. They will seek input and advice in doing so from the Species
Genome Committees. Current Species Coordinators (selected in a previous
competition) will be allowed to reapply along with other interested
scientists. The Species Coordinators are closely linked to the NAGRP
Program Leader and the Species Genome Committees. They work directly with
and for their respective Species Committees to coordinate gene mapping
activities for that species. They participate in the establishment of a
genome mapping database, facilitate the entry of genetic information into
the databases, facilitate the summarization and interpretation of genomic
data, periodically revise and distribute maps, serve as a repository for
genetic materials, and distribute reference DNA panels, genetic probes and
markers, and other materials as determined by the Species Committee.
Administrative Advisors. The Regional Associations of Directors in
the North Central, Northeastern, Southern, and Western Regions will
designate their Regional Representatives to NRSP-8. These Administrative
Advisors will provide policy guidance to the Technical Committee and work
closely with the CSREES Representative (NAGRP Program Leader) on
administrative, programmatic, and budgetary matters. The current
Administrative Advisors are: