Coordinated Agricultural Project conference planning workshop, May 22, 2004, Minneapolis, MN
A Coordinated Agricultural Project (CAP) conference proposal was submitted to the USDA-NRI program in March 2004. To conduct preplanning for the CAP conference we held a meeting on May 22, 2004 with a group of barley geneticists and breeders. The goal of this planning meeting was to discuss the strengths and weaknesses of currently available barley genomics tools and to establish a framework to further consolidate these into a toolkit for barley translational genomics. Barley geneticists and breeders attended from the U.S., Canada, and Scotland.
Participants:
Timothy Close, University of California, Riverside
Julie Dickerson, Iowa State University
Ed Kaleikau, USDA
Andris Kleinhofs, Washington State University
Peggy Lemaux, University of California, Berkeley
David Marshall, Scottish Crop Research Institute
Diane Mather, McGill University
Gary Muehlbauer, University of Minnesota
Kevin Smith, University of Minnesota
Brian Steffenson, University of Minnesota
Robbie Waugh, Scottish Crop Research Institute
Roger Wise, USDA-ARS, Iowa State University
Agenda:
(1) CAP conference proposal (Gary Muehlbauer)
(2) NABGP structure and function (Brian Steffenson)
(3) Barley2 GeneChip as a launching point for whole genome analysis (Roger Wise)
(4) Physical mapping (Tim Close)
(5) Bioinformatics; BarleyBase and HarvEST (Julie Dickerson and Tim Close)
(6) Reverse genetics (Peggy Lemaux and Robbie Waugh)
(7) Genomics and barley breeding (Kevin Smith)
(8) Wrap up
Report:
CAP conference proposal. Gary Muehlbauer from the University of Minnesota presented an overview of the Coordinated Agricultural Program conference proposal submitted to the USDA-NRI in March 2004.
· The major objective of the conference proposal is to develop an approach to integrate genetic, physical, trait and expression maps and other key genomics resources into a toolkit to enhance the delivery of measurable outcomes from barley breeding and genetical research. The long-range goal is to improve the efficiency of breeding for biotic and abiotic stress resistance, and agronomic and quality traits.
Muehlbauer described how a major strength of the barley genetics community was the international cooperation among barley geneticists that has led to the development of the currently available genomics resources. Cooperation with our international collaborators will likely continue. Several of these international cooperators attended the meeting.
The overall approach of the conference is to discuss approaches to utilize the Barley1 Affymetrix GeneChip for high throughput mapping. The Barley1 GeneChip was designed and produced with funding from an USDA-IFAFS grant (PIs: Kleinhofs, Wise, Close, Wing and Muehlbauer) and is the first publicly available GeneChip for crop plants. The Barley1 GeneChip represents approximately 22,000 genes. Muehlbauer described the three approaches to gene mapping using the GeneChip that were described in the conference proposal including: using wheat-barley addition lines for physically mapping genes to chromosomes; meiotic mapping using differences in gene expression; and DNA-based single feature polymorphisms (SFPs). Muehlbauer is pursuing the wheat-barley addition line approach and the results to date are exciting. For example, approximately 600 genes have been physically mapped to chromosome 6(6H); 350 on 6HL and 250 on 6HS. Just for comparison, the approximate number of cDNA clones mapped on the entire barley genome is 600. Therefore, the wheat-barley addition line approach will greatly enhance the number of mapped genes in barley. Several groups in the barley genetics community are pursuing gene expression-based meiotic mapping and DNA-based SFP mapping. The preliminary results from these approaches are also promising. High-throughput GeneChip-based mapping is a powerful tool and should greatly increase the number of genes mapped to the barley genome. The Barley 1 GeneChip can also be used to identify genes in deletion mutants as demonstrated by the work of Kleinhofs with the fast neutron induced Rpg1 suppressor mutant.
NABGP structure and function. Brian Steffenson from the University of Minnesota provided an overview of the structure and function of the North American Barley Genome Project (NABGP) grant program. The NABGP grant program has approximately $650,000 on a yearly basis. The NABGP largely funds grants to individual investigators. These grants are focused on mapping, marker assisted selection, genomics tool development, association genetics, microarray studies, and genetic stock and clone maintenance. Since its inception, NABGP has funded 40 PIs from more than 15 different institutions. These grants have provided seed money for larger funded grant proposals to USDA-NRI and NSF. Recent examples include NSF funding of barley physical mapping (PI: T. Close), an USDA-NRI grant to develop BarleyBase (PIs: J. Dickerson and R. Wise), and a USAID Cereal Comparative Genomics Initiative grant to exploit genetic diversity in wild barley (PIs: B. Steffenson, G. Muehlbauer, and K. Smith).
The call for proposals goes out in the fall with a mid December deadline. The committee consisting of Darrell Wesenberg, Brian Steffenson, Mike Davis, Gary Hanning, Cynthia Henson, Andy Kleinhofs, Diane Mather, Richard Horsley and Patrick Hayes meets in early January to evaluate the proposals. If there is insufficient expertise on the committee, a proposal may be sent to an outside reviewer for additional input. The proposals are evaluated for scientific merit and potential for the improvement of barley quality and productivity.
Historically, the structure of NABGP has provided the barley genetics and breeding community with the ability to maintain projects and initiate new ones. Some of the discussion focused on the use of future NABGP funding. One proposal was whether some of the NABGP funding could be used to support a larger genomics grant to several investigators. This would provide a foundation and launching point for large genomics efforts. However, a possible consequence of this type of action could be less funding for more applied projects that directly benefit breeding programs and the development of new varieties. No consensus was reached on this issue.
Barley2 GeneChip as a launching point for whole genome analysis. Roger Wise from USDA-ARS (Ames, IA; Iowa State University) presented an overview of the Barley1 Affymetrix GeneChip development and the extent of the initial experiments conducted using the GeneChip. The Barley1 GeneChip has been a highly commercially successful product for Affymetrix and based on its success, they have agreed to underwrite the design and production of a GeneChip for wheat, soybean, maize, and a variety of other organisms. Affymetrix has also agreed to produce the Barley2 GeneChip free of charge.
Wise presented an approach to capturing 90% of the genes in barley. This approach relies on developing high cot and methylation filtration libraries and sequencing 500,000 reads from each library. Wise estimated that the cost of this activity would be $2 million. The resulting sequence data would likely provide 90% of all the genes in the barley genome. These gene sequences would be used to develop the Barley2 Affymetrix GeneChip. The Barley2 GeneChip would be used for high-throughput mapping of genes on the barley genome and gene expression analyses. This approach would provide the genetic tools to identify and map all or most genes in the genome. The mapped genes could be used for marker-assisted selection in breeding programs, genetic studies and other biotechnology applications.
Academically, all of the participants agreed that for barley research in general this was a commendable goal and one which would have universal support were resources not limited. However clearly this is not the case. Discussion of this idea therefore focused on the cost/benefit of this proposal for applied breeding. We discussed the following questions: Would this be a good use of the limited resources for barley genomics? Do breeders really require 1,000s of markers per chromosome? Well-saturated maps are needed for breeding but what constitutes saturated? In contrast, for basic barley genetics, gene cloning and biology knowledge of every gene in the genome could greatly enhance the efficiency of these activities.
Physical mapping. Tim Close from the University of California at Riverside described the progress on his NSF-funded barley physical mapping project. Close has funding to fingerprint approximately 60,000 bacterial artificial chromosome (BAC) clones and develop contigs. Fingerprinting of an additional 6,000 BAC clones was funded by NABGP prior to the NSF funding. Close is focused on the 6.3X Morex BAC library that is represented by 313,344 clones. Completion of these BAC clones is intended to cover most of the barley “gene space”, estimated to be only 12-20% of the barley genome. Close reported on progress from 21,616 BAC addresses compiled during Fall 2003 from seven sources: Close (US), Kleinhofs (US), Wise (US), Muehlbauer (US), K. Gill (US), Lemaux et al. (US), and Altschmeid & Stein (Germany). Nearly half of these BAC clones were identified using mapped cDNA probes, while most of the others were identified using EST-derived “overgo” probes. Close’s project has fingerprinted this initial set of BAC clones, developed contigs and displayed the current progress on http://wheatdb.ucdavis.edu:8080/wheatdb/index.jsp. The main person responsible for the fingerprinting was Mingcheng Luo at UC Davis. The results of this recently completed first phase of the project were a quite acceptable 73.3% success rate, with 15,513 clones used for FPC assembly, comprising 2,262 contigs and 2,446 singletons. These contigs account for 470 Mb of the barley genome, which is slightly more than the complete rice genome, but only 9.4% of barley. With the observed average contig length of 208 kb and an expected density of 4 genes per 100 kb in gene-dense regions, this would be nearly 19,000 barley genes, or about 1/3 of all barley genes if barley and rice each have about 57,000 genes. Close stated that the remainder of the physical mapping part of his project will occur using 2-3 more batches of BAC clones. Close also stated that during the remaining rounds of probing and fingerprinting, he is willing to design overgo probes for barley genes, send overgos to individual investigators, and fingerprint the BAC clones they identify. These activities are expected to provide a physical map of at least 80% of the Morex barley gene space by the end of 2005.
A point was made that the available BAC libraries in barley are not sufficient to develop a complete physical map, due to gaps that must exist in the existing Morex library, which was produced with only one restriction enzyme.
Peggy Lemaux from the University of California at Berkeley described the current progress toward developing transgenic barley populations containing maize Ds elements that have transposed to new genome locations. Lemaux has developed lines containing the Ac transposase gene and separate lines that contain the nonautonomous Ds element linked to the selectable marker gene, bar. Initial work from the Lemaux laboratory showed that it is possible to tag a gene with a transposon that controls a morphological trait and gives a phenotype. Lemaux went on to describe that by crossing the Ac transposase-expressing lines with Ds-bar lines, activation and movement of the Ds element will occur and following segregation the transposed Ds element will be stably integrated and can be mapped using a PCR-based mapping strategy. This system will provide the opportunity to isolate genes of interest through transposon tagging and to generate new alleles in genes of interest. It also can be used to identify gene-rich regions due to the high likelihood (86%) of the Ds to transpose into genes or putative genes. Under a 5-year NSF Plant Genome grant, Lemaux with P. Hayes and P. Bregitzer are making an effort to develop lines that contain Ds elements throughout the barley genome and mapped to each bin. These lines will be made available along with Ac transposase lines to researchers, who have genes of interest that map close by the transposed Ds element in order to do saturation mutagenesis of the region in close proximity (10 cM) to the Ds element. Approximately 20 Ds insertions have been mapped. A publication describing these lines has been submitted to Theoretical and Applied Genetics and, following publication, these lines will be made publicly available through GrainGenes.
Genomics and barley breeding. Kevin Smith from the University of Minnesota presented a description of the current status of markers in barley breeding and the needs of breeders. Smith described the enormous datasets breeders generate through the course of breeding activities. In general, these data are under utilized, as breeders are mostly concerned with identifying the few lines they will advance. Therefore, there are large datasets that could be utilized for a variety of activities. He also presented a pedigree of the Minnesota barley breeding program showing that there is little variation in the program although genetic gains are still being made. However, he also presented a genome-wide haplotype analysis of the Minnesota breeding germplasm that showed several regions of the genome that are fixed and several that are still segregating, indicating that there are still a few regions of the genome available for selection. Smith also described a general approach that breeders use during the course of developing new cultivars. Most rely on good x good crosses with some new germplasm integrated into the program and subsequent selection, resulting in the development of parents for further crossing. He stated that the primary genomics needs of the barley breeding community include multiple and easy (e.g., SSRs and SNPs) to use markers for all regions of the genome. Multiple markers are necessary to ensure that for every region of the genome there are useful markers.
The description of barley breeding and the discussion that followed was probably the most informative to the group. The challenge for barley genomics is to develop the genetic resources such that the barley breeders can utilize the data in a seamless fashion.
Informal discussions during the ITMI workshop. Informal discussions were held during the ITMI workshop that focused on the objectives and overall outcome of the CAP conference. These discussions focused on identifying the key strategic issues and the appropriate person to develop concepts (e.g., breeding approaches and objectives) that will be highlighted at the CAP conference.
Outcomes:
1. An outline structure for the CAP conference was developed during the proposal preparation. This meeting helped refocus the structure of the CAP conference to the role of genomics in breeding and applied outcomes.
2. Key strategic issues that were identified and need to be highlighted at the CAP conference include: development of breeder friendly genetic maps and markers; database(s) that provide easy access to the genetic maps; database(s) of barley germplasm haplotypes; incorporating trait data into the genetic maps; and dissemination of information to barley breeders through workshops.
3. Additional strategic issues need to be defined/developed in more detail prior to the CAP conference. Smith and Muehlbauer volunteered to develop an overall plan for barley breeding that fully incorporates new genomics technologies and bioinformatics.
4. Continued support and collaboration was 'pledged' by the international contributors n attendance.
5. To maintain the discussion on these topics, Smith and Steffenson will organize a follow-up meeting at the International Barley Genetics Symposium in Brno, Czechoslovakia.