U.S. Poultry Species Coordination Activities
Supported by Allotments of Regional Research Funds, Hatch Act
For the Period 1/1/02-12/31/02
Overview: Coordination of Poultry Genome Mapping under the National Animal Genome Research Program (NAGRP) is a joint effort of Michigan State University (MSU) and the USDA, ARS, Avian Disease and Oncology Laboratory (ADOL). CSREES support is allocated via NRSP-8. The NAGRP is made up of the membership of the Animal Genome Technical Committee, including the Poultry Species Subcommittee.
FACILITIES AND PERSONNEL: Jerry Dodgson, Department of Microbiology & Molecular Genetics, MSU, serves as Coordinator with Hans Cheng of ADOL as Co-Coordinator. Both MSU and ADOL provide facilities and support.
OBJECTIVES: 1. Develop high resolution comparative genome maps aligned across species that link agricultural animal maps to those of the human and mouse genomes, 2. Increase the marker density of existing linkage maps used in QTL mapping and integrate them with physical maps of animal chromosomes, and 3. Expand and enhance internationally shared species genome databases and provide other common resources that facilitate genome mapping.
PROGRESS TOWARD OBJECTIVE 1. High resolution poultry genome maps.
The Reference Linkage Map(s). Numerous labs have cooperated in mapping DNA-based polymorphic markers by genotyping samples from the same two international reference crosses, the Compton population (Bumstead and Palyga, Genomics 13, 690-697, 1992), and the East Lansing population (Crittenden et al., Poultry Science 72, 334-348, 1993). This map has been enhanced by genotyping of a third cross, the Wageningen population, by Martien Groenen and colleagues (Groenen et al., Genomics 49, 265-274, 1998). A consensus map based on all three map populations has been published (Groenen et al., Genome Res. 10:137-147, 2000). Updates (Schmid et al., Cytogenet. & Cell Genet. 90:169-218, 2000) bring the number of markers on the consensus map to 1965, placed into 50 linkage groups, covering around 4000 cM. The East Lansing map has expanded to 1230 markers on 43 linkage groups (other evidence places E46 on GGA2 and E66 on GGA5, but there is not enough statistical support in our map alone to establish these linkages). Of these, 514 are microsatellite markers. Over 310 genes have been mapped in the East Lansing map. These provide evidence that gene order is conserved between the human and chicken genomes to a remarkable extent. Several studies have appeared (Burt et al., Nature 402:411-413, 1999; Groenen et al., op. cit.; Waddington et al., Genetics 154:323–332, 2000; Suchyta et al., Anim. Genet. 32:12-18, 2001; Jennen et al., Anim. Genet. 33:205-210, 2002 Buitenhuis et al., Anim. Genet. 33:249-254, 2002) that estimate between 100 and 200 breakpoints in gene order separating the human and chicken genome, with some blocks of conserved synteny (not necessarily gene order) exceeding 100 cM. Thirty of the 38 autosomes are now marked by hybridization tags, and 24 autosomes have been associated with the appropriate linkage group (Schmid et al., op. cit.). Further effort is needed to complete such assignments. Progress on the linkage map has slowed as work has shifted towards physical maps and preparing for full genome sequencing, as described below.
PROGRESS TOWARD OBJECTIVE 2. Physical maps and map integration.
Physical mapping resources, such as chicken large insert bacterial artificial chromosome (BAC) libraries, have been expanded. Zhang, Dodgson and colleagues have generated a library of over 115,000 BACs (~15X) at Texas A&M and have fingerprinted 65,000 of these (Lee et al., Animal Genetics, in press). Preliminary BAC contig building has been done, and these contigs are now in the process of being editted. On-line, interactive contig building for the fingerprinted Texas A&M BACs is now available at http://hbz.tamu.edu. P. de Jong (Childrens Hospital of Oakland Research Institute, CHORI) has generated a ~10X BAC library (CHORI-261) with extra large inserts using DNA we provided from the same bird used for the Texas A&M BACs. CHORI has also prepared a large insert turkey BAC library (CHORI-260) in collaboration with Kent Reed and Dave Harry. The Washington U. Genome Center has been provided copies of the Texas A&M and CHORI-261 chicken BAC libraries, and they are fingerprinting both libraries completely. They are also in the process of building and editting BAC contigs. Dodgson and Cheng are integrating the BAC contigs with the linkage map by screening the libraries for genetic markers using overgo hybridization and 3-dimensional PCR, respectively. Their efforts, plus those of cooperating scientists who've been provided BAC filters (see below), have led to over 1200 BAC assignments to nearly 400 distinct markers or genes. These linkages will be an essential part of the contig and sequence assembly processes. Crooijmans and Groenen are also fingerprinting their BAC library (Crooijmans et al., Mamm. Genome 11:360-363, 2000) and building contigs.
Burnside, Cogburn, Morgan, Cheng, Reed and Neiman in the U.S. and several European groups are generating EST collections (Tirunagaru et al., Genomics 66:144-151, 2000) and beginning to apply them for microarray analysis. Boardman et al. (Current Biology 12:1965-19-69, 2002) announced the sequencing of over 300,000 chicken ESTs from a wide variety of tissues and developmental stages. Data are posted in Genbank and in a searchable format at www.chick.umist.ac.uk. Paul Neiman, Joan Burnside and Dave Burt are in the process of assembling EST clusters and selecting 14,000 probes to use in building a general chicken microarray. This will then be printed and made available at the Fred Hutchinson Cancer Research Center Genomics Facility for an appropriate fee.
Radiation hybrid (RH) panels have been constructed by Vignal and colleagues at INRA (Morisson et al., Genet. Sel. Evol. 34:521-533, 2002), and framework RH maps are in the process of being constructed. Samples of the RH panel DNAs or collaborative screening of them are available through Alain Vignal.
In response to a request from the National Human Genome Research Institute (at NIH), a white paper proposing full sequence analysis of the chicken genome was submitted (2/10/02) by McPherson, Dodgson, Krumlauf and Pourquie. The NHGRI review panel subsequently placed the chicken genome among the "high priority" sequencing targets (the white paper and the NIH response are posted at http://www.genome.gov/page.cfm?pageID=10002154). The Washington U. Genome Center has been provided DNA from the same bird used to build the BAC libraries described above, and they have constructed small plasmid libraries for bulk whole genome shotgun (WGS) sequencing. The actual sequencing reads should be completed in 2003, followed by assembly and annotation of the full genome sequence of the chicken.
PROGRESS TOWARD OBJECTIVE 3: Database and other map resources.
ChickGBASE: The latest version of ChickGBASE is constructed in the comparative mapping Arkdb format. Arkdb was primarily developed by Andy Law, Dave Burt, Alan Archibald, and others at the Roslin Institute. ChickGBASE is available in the Arkdb format at http://www.ri.bbsrc.ac.uk/chickmap/chickgbase/chickgbase.html. A mirror site for the poultry database is being mounted at the Iowa State database site, http://www.genome.iastate.edu/.
WWW Homepage: We maintain a WWW homepage for the Poultry Genome which links to ChickGBASE, the Roslin Institute homepage, and a variety of other genome mapping resources. The Homepage provides the latest EL maps and mapping data, an updated list of published microsatellites, descriptions of available resources, the latest cytogenetic map, and access to a host of other information. It can be accessed at http://poultry.mph.msu.edu.
Interactive BAC Contig Analysis Web Site: On-line BAC contig analysis is now available at http://hbz.tamu.edu. At the moment, one can search for overlapping BACs with any query BAC among the 65,000 that have been fingerprinted at Texas A&M. To access BAC fingerprints and conduct e-chromosome walking, or build overlapping clone path tiles, go to http://hbz7.tamu.edu/homelinks/phymap/chicken/chick_home.htm Scroll down to Chicken Clone -> FPC Hitting Tool and click. Enter a BAC of interest (if it's among those in the fingerprint database listed), select a criterion for overlap significance and search for overlapping BACs. Other aspects of BAC contig on-line analysis are under construction.
Reference Panel DNA: DNA from the East Lansing international reference population has been sent to many laboratories throughout the world.
Primer Kits: Several kits of microsatellite primer pairs have been made available for free distribution. The first of these is the Population Tester Kit. This contains 9 primer pairs which define microsatellites with high polymorphic information content (numerous alleles widely distributed in several populations). Six large Comprehensive Mapping Microsatellite Kits containing a total of 647 primer pairs for markers covering most of the chicken genome have also been available. For those with access to ABI sequencers, they are also fluorescent and can be multiplexed. A new version of a framework primer kit (with 147 well-spaced microsatellite marker primer pairs) called the "Comprehensive Mapping Kit #7" was made available this year (thanks in part to a generous donation from Hy-Line International). One or more kits have now been provided ~120 different labs, worldwide. Two Chicken Gene Primer Pair kits have been made available containing a total of 300 primer pairs to sequenced chicken genes for use in EST mapping and expression analysis.
Physical Mapping Resources: At least three public BAC libraries for chicken and one for turkey are now available. These are described above under Objective 2 progress. Filter arrays of robot-spotted BAC clones are being distributed for both the Texas A&M and CHORI-261 chicken BAC libraries (so far, 41 requests have been filled). PCR-screenable BAC DNA pools from row, column and superplate pools of the first 30,000 clones of the BamHI library have been made by Research Genetics, supported, in part, by Coordination funding and are now available. Samples of these pools or collaborative screening of them are available from the coordinators. Once a clone(s) of interest has been identified, it (they) can be obtained at nominal cost from the Texas A&M GENEfinder BAC Center or CHORI.
Chicken Microarray: As described in objective 2 progress, Neiman, Burnside and Burt are designing a 14,000 element chicken microarray. Poultry coordination funds, along with additional support from Drs. Burnside and Cheng will pay for obtaining and amplifying the necessary EST clone DNAs and the initial array development. Printing costs will be covered by user fees from those who purchase the arrays.
Newsletter: The Poultry Genome Newsletter is published quarterly and is distributed through our WWW Homepage, electronically on the angenmap email discussion group and via hard copy to scientists worldwide. Meetings: Over 1800 scientists attended the joint Plant and Animal Genome X meeting held last January, held jointly with the annual NAGRP meeting. Coordination funds helped support attendance at PAG-X and will do so again for the upcoming PAG-XI in January, 2003. A small amount of support was also provided for the upcoming Gordon Conference on Quantitative Genetics and Genomics in 2003.
PLANS FOR THE FUTURE.
OBJECTIVE 1. High resolution poultry genome maps.
Efforts will be made to improve overall map marker density as a result of the upcoming WGS sequence generation. Comparative chicken/human map development will also continue and QTL analysis should expand.
OBJECTIVE 2. Physical maps and map integration.
One or more full genome physical maps using BAC contig-building strategies and radiation hybrid panels should be available in FY 2003. Additional efforts are underway to fill gaps between contigs, improve map resolution and integrate these physical maps with existing linkage maps. EST and array approaches will continue to expand for both chicken and turkey in the U.S. and in Europe. General purpose chicken microarrays should be available sometime in 2003. RH panel framework mapping has begun and will continue. Bulk sequence generation for the WGS chicken genome sequence should be completed in 2003, although editting and annotation efforts will continue for some time to come.
OBJECTIVE 3: Database and other map resources.
The chicken ChickGBASE database will continue to be updated and improved at the Roslin Institute and the Iowa State mirror site. Discussions are underway about what types of bioinformatic resources will be required to handle the full genome sequence and related data. At least part of this role will be filled by the National Center for Biotechnology Information at NIH, in the same way they now serve the human and model organism genomes. Poultry Genome Newsletter and homepage information will be continue to be distributed and enhanced. We also will continue to distribute reference panel DNAs, microsatellite primer panels, BAC library resources (library, clones, filter sets, PCR screening pools) and Type I gene-based primers. Chicken microarrays will be available through the Fred Hutchinson Cancer Research Center Genome Facility with initial development support from coordination and other funds.
(Prepared 12/12/02)