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Executive summary

2003 Workshop program

2003 Workshop summary

The aim of the workshop held in Washington DC 10-11 November, 2003, was to integrate the advice from other genome projects and develop a strategy for sequencing the wheat genome. Sixty-three scientists, including 45 from the U.S. and 18 from 12 foreign countries participated in the workshop (the full program can be viewed at http://www.ksu.edu/IGROW). Recent conferences, such as the ITMI (Winnipeg, Canada; June 2002), the USDA-CSREES Stakeholders (Washington, DC; November 2002; http://www.aspb.org/downloads/stakreport.pdf), the Plant and Animal Genome (San Diego, CA; January 2003), and the 10th International Wheat Genetics Symposium (Paestum, Italy; September 2003) that was attended by over 500 scientists from 50 countries, have confirmed a high level of interest worldwide in the analysis of the wheat genome. A summary of resources for wheat genome analysis is provided in Table 1.

Wheat is the most widely grown crop, comprising 17% of all crop acreage, and is a staple of 40% of the world’s population providing 20% of the calories consumed. To meet human demands by 2050, grain production needs to increase at an annual rate of 2% on an area of land that will not increase much beyond the present level. This implies that significant advances in the understanding of the wheat plant and grain biology must occur in order to increase absolute yield as well as protect the crop from 25% loss due to biotic (pests) and abiotic stresses (heat, frost, drought, and salinity). Genome sequencing is a widely accepted mechanism for obtaining the knowledge required to tackle significant challenges facing the growing of a crop such as wheat, because it leverages similar work from other crops and plants. Sequencing of the wheat genome is feasible due to the abundance of cytogenetic, molecular, and human resources, and the successes in sequencing several other plant and animal genomes.

The workshop first reviewed national and international collaborative research in wheat genomics and the lessons learned from other genomics projects and model organisms. The workshop accepted some general concepts, including the principle that the genomes of major crops should be sequenced if we are to interpret the details of how gene networks function; that it is not possible to use one (e.g., rice) as a surrogate for the others due to the common occurrence of deletions and rearrangements when cereal genomes (rice, wheat, barley, etc.) are compared; that major differences in gene structure, expression (tissue specificity and timing), and function exist between cereal species; and that gene annotation of orthologous genes between species remains a major challenge. It also was evident that the wheat genome at 16,000 Mbp is likely to be the largest genome ever to be sequenced and will provide a model for structure/function changes that accompany polyploidy, a phenomenon that is common among plants.

Next debated were questions such as: Do we need to sequence the wheat genome? What are the scientific needs for the sequence that cannot be met with existing cereal sequence resources? Is it time to sequence now? What genome should be sequenced (diploid or hexaploid)? What type of sequence should be generated (whole genome shotgun, selected BACs, etc)? What strategies could be used to yield the type of sequence needed? What timetable should be followed for the next steps? How will the broader community be engaged?

CONCLUSIONS FROM THE WORKSHOP

Do we need to sequence the wheat genome?; What are the scientific needs for the sequence that cannot be met with existing cereal sequence resources?
D. Van Sanford emphasized that a wheat sequence will provide perfect markers for difficult traits, harness genetic diversity, enhance quality, increase yield in drought prone areas, and help design varieties for sustainable food production. C.O. Qualset cited the benefits of a wheat sequence for human health and nutrition, such as celiac disease and biofortification, for enhanced nutrition especially in developing countries, world food security, and crop adaptation to climate change.
B. Keller and J.D. Faris provided detailed examples of their analyses in the Lr10 region on chromosome 1AS and the Q gene and Tsn1 gene regions on the group 5 chromosomes (see Table 1), respectively, to indicate the need for genome-level sequencing in wheat. The data provided striking examples of gene discovery in wheat and confirmed the complexity involved in comparing the rice and wheat genome sequences. Studies described by N.L.V. Lapitan (the Dn7 locus encoding resistance to Russian wheat aphid) provided further information on the challenges of utilizing rice-wheat synteny to define the genes responsible for Russian wheat aphid resistance. Although the rice genome sequence can contribute to the investigation of the wheat genome, the details of genes underpinning key features of wheat cannot be defined without genome sequencing.

K.S. Gill, J.P. Fellers, and J. Dvorak provided information on the possible structure of the wheat genome and elaborated on data that is beginning to appear in the literature indicating the existence of gene islands separated by tracts of retrotransposable element-rich DNA. The genome structure of wheat is different from animal (and other plant) genomes and insufficient information is available to analyze the functional significance of the differences without extensive sequencing of the wheat genome.

Is it time to sequence now?
Current investment in the U.S. and internationally have placed wheat in the top tier of plants in genomic resources such as ESTs (www.ncbi.nlm.nih.gov/), chromosome-bin maps of EST loci (http://wheat.pw.usda.gov/NSF/), wheat–rice comparative maps (http://www.gramene.org/), and a BAC contig map of the D genome of wheat (http://wheat.pw.usda.gov/PhysicalMapping/). Many agronomic and ploidy-related genes have been allocated to the gene-rich regions (Fig. 3) and several have been cloned (see Table 1). More than 6 Mb of the wheat genome has been sequenced as a part of various gene discovery and comparative genome-mapping projects. The wheat community needs genome sequencing to be initiated as quickly as possible as an internationally coordinated activity because it is essential for gene discovery and also it is moving very quickly toward saturating the value of the rice genome sequence as a surrogate source of genomic information.

What genome should be sequenced (diploid or hexaploid)?
Comparative analyses on the structure of plant genomes, particularly the recent results from the maize genome project presented by J. Messing and R. McCombie, indicate they are more dynamic than the genomes of animals (discussed by E. Green). Given the polyploid nature of wheat and its immense economic significance, the available information argues strongly for the hexaploid genome to be the main target for a wheat genome project with supporting analyses coming from related cereals such as rice, Brachypodium, barley, and diploid and tetraploid wheat.

What type of sequence should be generated (whole genome shotgun, selected BACs, etc)? What strategies could be used to yield the type of sequence needed?
J. Bennetzen, C. Whitelaw, and J. Messing provided detailed assessments of the shotgun sequencing of the maize genome. The sequencing of products from methyl filtration (MF) and high Cot (HC) fractionation procedures are providing gene sequences that are being integrated into 2-Mb BAC assemblies compiled for the maize genome. The assembly of the maize genome is utilizing the rice genome as a template for confirming mega contigs, as well as utilizing detailed genetic maps. T. Sasaki, C.R. Buell, and L. Stein emphasised the importance of detailed genetic maps to guard against false assemblies of BAC contigs based only on the fingerprinting procedures (even if these utilize high-resolution analytical procedures). J. Bennetzen and R. McCombie noted that shotgun sequencing could produce a draft sequence but that this draft could only be completed by the further analysis of a BAC contig assembly across the entire genome. C.R. Buell emphasized the importance of full-length cDNA sequences to be determined sooner, rather than later, to help interpret the drafts of the genome sequence. Work on full-length cDNA sequences in wheat in Japan and China was reported by Y. Ogihara and J. Jia, respectively.

What timetable should be followed for the next steps? How will the broader community be engaged?
Given the scale and complexity of the wheat genome-sequencing proposal, a staged approach was considered most appropriate, with several pilot projects leading the way to investigate technical issues and establish a BAC contig assembly across the entire genome.

A pilot project building upon the results of J. Dolezel and B. Chalhoub on the analysis of sorted chromosomes was widely considered to be the most viable approach to defining a unit of the wheat genome and obtaining much needed information about the large-scale genome structure as well as specific gene content. J. Dolezel and B. Chalhoub described the chromosome 3B BAC library (68,000 clones, average insert size approximately 100 kb) and others for chromosomes 1BS, 1D, 4D, and 6D (see Table 1). The 3B chromosome-specific BAC library and those of 3A and 3D under consideration by J. Dolezel and B. Chalhoub could form the basis for producing a sequence-ready BAC contig assembly of homoeologous group 3. The production of a sequence-ready BAC contig assembly would make use of detailed genetic maps and the contig assembly for chromosome 3D (from the diploid D genome of wheat, described by J. Dvorak; http://wheat.pw.usda.gov/PhysicalMapping/). These pilot studies will facilitate the assessment of physical map-building strategies for hexaploid wheat. J. Dvorak emphasized that a wheat physical map would have an enormous strategic value for the project. Chromosome 3B is a particularly attractive target because it contains numerous economically important genes, and, as pointed out by R. Ward, would attract the support and complement the project already funded in the U.S. to identify genes conferring resistance to Fusarium head blight. The sequence of genic regions of a homoeologous group of chromosomes would provide information about the large-scale genome structure as well as specific gene content, to guide the planning for sequencing the entire wheat genome.
Another pilot project that was discussed would carry out the sequencing of products from MF- and HC-fractionation procedures from hexaploid wheat. B.S. Gill and W. Li presented preliminary data on genome-specific tags (GSTs) from hexaploid wheat prepared using MF and HC procedures as a means for analyzing the gene-rich regions of the genome. This technology needs careful assessment and would need to be complemented by large-scale sequencing of the EST libraries from diploid and tetraploid wheats in order to provide a database for distinguishing between homoeologous genes from the A, B, and D genomes. In addition, genome sequence-specific, repetitive DNA could be used to assign contigs from shotgun procedures to the genomes of hexaploid wheat.
The value of preliminary sequencing was discussed. J. Bennetzen emphasized the importance of sequencing orthologous regions in the wheat A, B, and D genomes and their putative diploid sources. B. Chalhoub described a pilot study recently funded in France on comparative sequencing of 15 selected regions (100 BACs) from wheat species of different ploidy levels (2x, 4x, and 6x). C.R. Buell argued that preliminary sequencing of random BACs would greatly enhance the understanding of the structure of wheat genomes and gene distribution.

A number of countries and their scientific representatives (R. Appels, Australia; D. Somers and B. Crosby, Canada; J. Jia, China; J. Dolezel, Czech Republic; B. Chalhoub and F. Quetier, France; N. Stein, Germany; S. Nagarajan, India, personal communication; A. Maggio, Italy; Y. Ogihara, Japan; A-M. Botha-Oberholster, South Africa; B. Keller, Switzerland; and I. Bancroft, UK) represented at the workshop indicated their commitment to focusing on certain regions of the genome, with the aim of joining their sequencing efforts into a larger wheat genome-sequencing project. This staged approach would build on the resources already established by large investments in the U.S., as well as investments in other countries, and contribute to specific new pilot projects established in the U.S. Leadership of the wheat genome project is clearly crucial, because contributions from large projects also have to integrate contributions from smaller projects in order to establish an international effort and ensure that accurate sequencing/interpretation is provided to extend the sequence of a particular region of the genome.

RECOMMENDATIONS FOR A WHEAT GENOME-SEQUENCING PROJECT

There was a strong consensus among the workshop participants for a sequencing project in hexaploid wheat because of its economic importance, its historic role as a polyploid genetic model, the availability of extensive genetic and molecular resources, and a large and vibrant global wheat genetics community. Based on the extensive discussion as summarized in the previous section, an international wheat genome-sequencing project could be established through the following steps:

1. Construct an accurate, sequence-ready, global physical (BAC-contig) map anchored to the high-resolution genetic and deletion maps of the 21 chromosomes (see item 4 below) of hexaploid wheat genotype Chinese Spring.
2. Explore the use of flow-sorted chromosome and arm-specific libraries towards the assembly of the global physical map and in preparation for sequencing the genic regions of homoeologous groups of chromosomes.
3. Identify genomic sequence tags (GSTs) using gene-enrichment procedures such as high Cot or methyl filtration, expressed sequence tags (ESTs), and full-length cDNAs of 2x, 4x, and 6x wheats for an accurate estimation of a wheat unigene set.
4. Leverage rice sequence and wheat–rice gene synteny, comparative genetics, and wheat unigenes towards the development of high-resolution genetic and deletion maps of the 21 chromosomes of Chinese Spring wheat.
5. Use the EST and GST unigene set-based overgo probes on high-density filters of the Chinese Spring wheat BAC library to assist in the assembly of global physical map and development of wheat gene filter for the wheat genetics community.
6. Identify a random set of 100-300 BACs, preferably from a range of genotypes, and genic BACs from the global physical map, for sample sequencing and refining the technology for assembling sequences with a high repetitive sequence content.
7. From the global physical map, identify all genic BACs for a 4x skim or full sequencing for the entire wheat genome.
8. Integrate bioinformatics at every step for project management, data analysis, dissemination of data, and improved methods of sequence annotation.
9. Engage on a global (developed and developing countries) basis wheat stakeholders, educational institutions (K-12) in all aspects of the research, technology transfer, manpower training, and promotion of science.
10. All data, materials, and resources must be in the public domain and free of IPR.

Many issues related to the international collaborations and coordination of research will be discussed in follow-up meetings, and a full plan will be in place by the summer of 2004. It is difficult to arrive at an accurate budget for the project, because clear physical mapping and sequencing strategies will not emerge until after the pilot project. The estimated cost will be shared among the dozen countries participating in the project. The project duration is tentatively estimated at 6 years (2004-2010) to be completed in three phases.

Phase 1 (2004-2006): A pilot project will aim to construct a global physical map of a selected homoeologous group of wheat anchored to a high-resolution genetic and deletion map using chromosome- or arm-specific libraries, gene-enrichment methods such as high Cot and methyl filtration, and other relevant resources such as full-length cDNAs, EST, and GSTs from different ploidy wheats. Sample sequencing of 100-300 random BACs also will begin in 2004. Targeted sequencing of gene-rich BACs from regions of biological and economic importance will begin in 2005.

Phase 2 (2006-2008): An efficient method based on pilot experiments will be used to finish a global physical map anchored to the high-resolution genetic and deletion maps of the 21 chromosomes of wheat by 2008. Sequencing of gene-rich BACs will continue.

Phase 3 (2008-2010): Large-scale sequencing of genic BACs will begin in 2008 and an annotated sequence of the expressed portion will be finished by 2010.

The outcomes from a significant, wheat genome project will reach across the global agricultural, research, and education communities. This project will represent an unprecedented international collaboration of wheat researchers to meet the responsibility of providing food for much of the world's population. Scientific outcomes include tools for marker-assisted selection for wheat improvement, insights into how a polyploid genome is organized and functions, and a stimulus for further national and international collaboration.

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