Scientific Objectives:
The long term goal of our collaboration is to develop an experimentally tractable and closed model system to globally unravel and understand the evolution, physiology and biochemistry of the genus Oryza. The specific objectives of this proposal are to: 1) Construct DNA fingerprint/BAC-end sequence physical maps from 11 deep coverage BAC libraries that represent the 11 wild genomes of Oryza (830,000 fingerprints; 1,659,000 BAC ends);
2) align the 11 physical maps with the sequenced reference subspecies japonica and indica;
3) construct high-resolution physical maps of rice chromosomes 1, 3 and 10 across the 11 wild genomes using a combination of hybridization and in silico anchoring strategies, and;
4) provide convenient bioinformatics research and educational tools (FPC and web-based) to rapidly access and understand the collective Oryza genome.

Broader Impacts:
The research proposed will provide the first ever closed experimental system to understand the evolution, physiology and biochemistry of a single genus in plants or animals. We will align representatives of eleven wild genomes of rice, including both diploids and tetraploids, to the sequenced and finished O. sativa ssp. japonica AA diploid genome. Such a system will empower the scientific community to address complex scientific questions on a whole genome scale. For example, one would be able to determine the majority of genome rearrangements leading to the present day wild species as compared with the sequenced cultivated rice. Such data can be used to study the dynamics of the evolution of a genus and the impacts of domestication. Another example is that one could move vertically across genomes to explore the diversity and evolution of disease resistance gene clusters and their cis regulatory elements. Such data could be used to rapidly identify new and useful disease resistance genes as well as to define conserved regulatory sequences. This research will not only impact rice genomics but will be useful for understanding monocot biology in general and can serve as a model to establish similar systems in both plants and animals.

The PIs have received continuous NSF support since 1994. Wing is PI/Co-PI on 11 (3 of which will expire in September 2003; 2 of which all experimental objective will have been completed); Jackson on 1 and Stein on 1 active NSF Plant Genome grants. Here we will describe our results from prior support of grants directly related to the proposed work.

C.1.1) Title: “Sequencing Rice Chromosomes 3 and 10”: PI R. Wing, Co-PIs D. McCombie, R. Wilson, C. Soderlund, J. Jiang. NSF Award# DBI-9982594 (09/15/99-09/14/2002); NSF Supplemental # (09/15/02-09/14/03); USDA-CSREES award# 99-3517-8505 (09/15/99-09/14/2002); USDA Supplemental award# (09/15/02-09/14/03); DOE Supplemental award# (09/15/02-09/14/03). Award amount: $5,100,000 ($2,550,000 NSF & $2,550,000 USDA) Supplemental Award amount $1,100,000 ($500k NSF, $500k USDA, $100k DOE).
     The overall objective of this 4 year project was to finish and annotate rice chromosomes 10 and 3. Our consortia ACWW ¹ (Arizona Genomics Institute [Wing, Yu, Soderlund], Cold Spring Harbor Laboratory [McCombie, Stein], Washington University Genome Sequencing Center [Wilson] and the University of Wisconsin [Jiang]) was assigned the top half of chromosome 10 (Mb) and the short arm of chromosome 3 (Mb), while TIGR (Buell) was assigned the lower half of ch10 and the long arm of ch3. Ch10 is finished and published in Science in June 2003 (The Rice Chromosome 10 Sequencing Consortium: Yu et al. 2003). Although 10 is the smallest of the rice chromosomes (22.4 Mb) it is also one of the most heterochromatic, especially the top 12 Mb. Therefore it was a difficult chromosome to finish however, in the end, ACWW finished all but 1 of its BACs without any sequencing gaps to a standard of 1 error in 10,000 bases or more.
     We are now focusing our efforts on finishing our region of chromosome 3. As an interim goal, ACWW and TIGR finished a 10X BAC-by-BAC Phase 2 draft of ch3 in December 2002 (IRGSP 2002). For the ACWW region, all sequence is contiguous, representing 16.7 Mb of the rice genome, except for 3 physical gaps between FPC contigs. To date 112 BACs are finished and are in annotation, 5 BACs are in the Phase 2 stage (3 in 1 contig) and 4 BACs are in phase 1 stage. We therefore do not anticipate any delays in achieving the goal of finishing and annotating rice chromosome 3 by September 15, 2003 if not earlier. This is especially true because the remaining genomic sequence is from the more euchromatic region of the ch3. ACWW sequencing status can be viewed for all chromosomes at www.genome.arizona.edu/shotgun/rice/status, which has link to the sequence location in the webFPC map.
     In addition to the scientific contributions made by this project, it also helped us to develop the talent, infrastructure, methodology and management skills to tackle large scale sequencing and physical mapping projects like the one being proposed here.

Publications resulting from the award (9): 1) Zhao et al. 2002; 2) Chen et al. 2002; 3) Cheng et al. 2001b; 4) Cheng et al. 2001a; 5) Yuan et al. 2000; 6) Wing et al. 2001; 7) Bastide et al. 2001; 8) Soderlund et al. 2002; 9) Yu et al. 2003

C.1.2) Title: “The Oryza BAC Library Project”: PI R. Wing, Co-PI C. Soderlund, Tomkins (Clemson) and S. Jackson (Purdue). NSF Award# DBI-0208329 (09/15/02-09/14/04). Award amount: $600k.
     The long term objective of this project is to construct 11 deep-coverage large-insert BAC libraries from representatives of all the known wild genomes of rice (see Table 1 for species and genomes sizes) and provide affordable access to these resources (clones, filters and libraries) through the Arizona and Clemson BAC/EST Resource Centers. The grant was awarded this October and will provide the raw material for our proposal to align the wild genomes with the cultivated and sequenced A genome diploids.
     Progress: In mid-November 2002, PIs Wing and Jackson traveled to the International Rice Research Institute (IRRI: Philippines) to prepare high molecular weight DNA for BAC library construction. The 4 week trip was very productive and we were able to produce 1-2 DNA preparations from 8 of the 11 species. Unfortunately the DNA samples were not digestible with restriction enzymes most likely due to the poor condition of the plant material. In May 2003 Dr. Meizhong Luo returned to IRRI to prepare megabase-size DNA from much younger tissue and brought these samples back to Arizona. Fortunately, this time the majority of DNA samples were digestible with restriction enzymes and therefore can be used for BAC library construction (see figure 1 below). These samples are now being used to build BAC libraries in Arizona and Clemson. The first library has been cloned, from O. rufipogon, with an average insert size of 134kb (see figure 2 below).We plan to construct the 11 BAC libraries over the next year and 3 months. The Wing laboratory has over 10 years of experience in construction of BAC libraries, including over 10 rice libraries, and we do not anticipate any problems in completing our objectives within the time frame of the project now that we have suitable DNA preparations

FIGURE 1. Enzyme digestions of wild species HMW DNA.

Figure 2. NotI digestion of Oryza rufipogon BAC clones

C.1.3) Title: "Maize Mapping Project": Lead PI: E. Coe (Missouri); Arizona PI: R. Wing, Co-PI C. Soderlund. NSF Award# DBI-9872655 (09/15/98-09/14/03). Total award amount: $1,629,246.
     The primary role of our group, in collaboration with Missouri, was to construct a genetically anchored phase I BAC physical map of the maize genome. Our objectives were to: 1) construct a deep-coverage BAC library of the inbred B73 using the HindIII; 2) fingerprint the HindIII library and an additional EcoR1 BAC library; 3) assemble the fingerprints into contigs using FPC; 4) order and merge as many contigs as possible along the maize genetic map to create a phase I physical map.
     Objectives 1-3 are complete and we are now in the final year of the project. We generated about 300,000 successful fingerprints from the two libraries which assembled into about 4500 contigs using FPC. All fingerprint and anchor data can be downloaded and viewed using WebFPC and WebChrom at the www.genome.arizona.edu/fpc/maize site, where WebFPC displays the FPC contigs, and WebChrom shows the location of genetic markers and the FPC contigs, with links to external web based databases. Integration of the genetic map with the physical map can be viewed using iMAP at Missouri. The contigs cover approximately 2036 Mb of the 2500 MB genome, and the longest contig is approximately 4 Mb. The libraries have been hybridized with about 14,715 probes, 798 of which are genetically mapped.
     Our final year goal is to order and merge as many contigs as possible with a realistic goal of achieving about 3000 contigs in the end, 1000 of which will be genetically anchored. Although not a complete physical map we have been able to develop an important resource for maize genetics that is widely used by the community.

Publications from the award: 1) Tomkins et al. 2002; 2) Coe et al. 2002; 3) Cone et al. 2002

C.1.4) Title: "Comparative genomics of rice: reconstructing rice chromosome 1 in related species.": PI S. Jackson, Co-PI P. SanMiguel (Purdue). NSF# DBI-0227414 (10/01/02-09/31/07). Award amount: $1,630,537.
     The long term objective of this proposal is to use BAC libraries and Overgo technology to reconstruct rice chromosome 1 in 6 related Oryza species to examine chromosome evolution in group of closely related species and develop tools for comparative mapping in plant genomes.
     Progress: We have developed a computational pipeline to sift through genomic sequences to find overgos that will be used for comparative mapping. We have designed and begun testing the first 96 out of several thousand overgos on rice and a wild rice BAC library. A description of these resources and an online database is available at http://rice.genomics.purdue.edu. This proposal has just been funded but we have made tremendous progress and one postdoc, one technician and two graduate students are already on board working on this project.
     We anticipate no problems attaining the goals of this project within the timeline and are collaborating with R. Wing to expand the scope of this project to include the entire rice genome instead of just rice chromosome 1.

D.1.5) Title: "Gramene: A resource for comparative grass genomics" PI: L. Stein; Co-PI: S. McCouch (Cornell). USDA-IFAFS# 2000-04538 (09/01/00-08/31/04). Award amount: $2,098,000.
     Gramene (http://www.gramene.org) is a comparative mapping resource for monocot genomes. It combines the extensive colinearity between the genetic maps of the cereals, with the draft and finished genomes of rice to create an environment in which researchers can move easily from a genetically-defined region in one species to a physical map in another species, and ultimately to an annotated region of the rice genome.
     Gramene then builds on top of this comparative mapping framework by adding a knowledgebase of rice functional genomics. From the Gramene web site, researchers can browse and download an extensive collection of annotated rice mutants and their phenotypes, the gene ontology annotations of rice gene products, protein orthology relationships, and an extensively annotated bibliography of rice biology. Researchers also have access to essential resources for comparative genomics, including an ontology of monocot developmental and phenotypic terms (developed jointly with MaizeDB), assay information for the markers used to develop monocot genetic maps, and outgoing links to stock centers and genomics sites.

Fig. 1: Comparing maize map to genetic and physical maps of rice

overview	Detailed view

Fig. 2: Contig view of selected rice region, showing alignment of expressed sequences from rice, maize, and other monocots

1. Rice genomic sequence data: Draft and finished rice genomic sequence, including both japonica and indica subspecies.
     Fully attributed annotations from the rice sequencing groups. These vary in type and protocol from region to region.
     Uniform annotations that Gramene generates internally. Currently this consists of FGENESH gene predictions (Salamov and Solovyev 2000), aligned ESTs and EST clusters, BAC end sequences from rice and other monocots, and genetic markers from rice and other monocots. In the near future we will be adding BLASTX (Altschul et al. 1990) protein to nucleotide alignments, followed by Genewise (Birney and Durbin 2000) alignments and Ensembl pipeline consensus gene models, as described in the Experimental Plan.

2. Rice protein/gene product data: Gramene provides protein records for all published rice protein sequences and hypothetical gene products that have been submitted by the genomic sequencing groups to GenBank/EMBL.
     To provide researchers with the most up to date information on the putative functions of rice gene products, we cross reference all confirmed and hypothetical products with Interpro (Mulder et al. 2003) to provide electronic Gene Ontology annotations. In addition, we manually inspect all confirmed protein entries in order to produce hand-curated GO annotations.
     Phylogenetic relationship information is provided by a table of precomputed BLAST scores, as well as a link to the BLINK service (Wheeler et al. 2003).

3. Monocot genetic and physical map data: We currently provide access to 22 genetic and physical maps for rice, maize, barley, sorghum, oat and the triticeae. Included in this set is the Oryza sativa japonica BAC physical map developed by R. Wing (Chen et al. 2002), and 11 widely used genetic maps of rice. We select which maps to publish in consultation with our Scientific Advisory Board, collaborators and other representatives of the academic and commercial research communities.
     For any map displayed on Gramene, researchers can find the molecular data required to reproduce the component marker assays. In the case of rice maps, the marker molecular data (e.g. primer pairs) is retrieved directly from the Gramene database. For other species, the molecular data is retrieved indirectly via links to affiliated databases such as MaizeDB (Coe et al. 2002) and GrainGenes [http://wheat.pw.usda.gov/index.shtml].

4. Ontology annotations of rice mutants and strains: Researchers can access a set of ontologies from the Gramene web site. In addition to the standard GO, we have developed several plant-specific ontologies in collaboration with MaizeDB and TAIR. The Plant Ontology (PO) is a collection of concepts to describe plant anatomic locations and developmental stages. The Trait Ontology (TO) is a collection of concepts to describe abnormal traits and phenotypes. All of these ontologies are preliminary, and a major effort for the coming years is to generalize and refine them as we apply them to monocot genome annotation.
     We provide researchers with access to 470 published rice mutants, each of which have been annotated with PO and TO terms. Mutant records are often accompanied by illustrations of the phenotype, and have links to the genome, when known, to the OryzaBase strain database, and to literature references.
     The Gramene architecture is based around the Oracle 9i database, accessed via a large open source middleware layer. We use the Ensembl data model (Clamp et al. 2003) for managing and displaying the rice genome and its annotations, the Gene Ontology Consortium schema for ontologies, and custom software for display of protein annotations, comparative maps, rice mutants, and bibliographic references. With the exception of the Oracle DBMS itself, all software used or produced by Gramene is open source, allowing any group to adapt and use our system.
     The bulk of Gramene's analytic work is producing alignments to the rice genome. We use a version of the Ensembl pipeline called Biopipe, which has recently been released by Elia Stupka's group in the Singapore Fugu Sequencing Project. Biopipe is open source cluster management software that allows us to distribute tasks among multiple machines. The primary workhorse for our alignments is Blat (Clamp et al. 2003), a fast sequence alignment algorithm developed by Jim Kent for use in human genome assembly. The combination of Biopipe and Blat allows us to align 300,000 single reads (ESTs or BAC ends) to the rice genome per hour. We use stringent criteria to ensure that alignments are good ones. These criteria provide an overall alignment success rate of 77% for rice genomic sequence sources, such as BAC ends, and 80% for rice ESTs. ESTs derived from non-rice monocots are aligned unambiguously roughly 70% of the time. These numbers should be viewed in the context of the incomplete state of the rice genome. Extrapolating from the current state of the rice genome to a 99% complete state, we expect to see a success rate of 88% and 91% for rice genomic and EST sequences respectively by the time the IRGSP sequencing project is complete in December 2004.
     Since going on line in January 2002, the community usage of Gramene has increased rapidly and now stands at over 50,000 meaningful hits per month, where a meaningful hit is defined as one that causes a database access rather than the retrieval of a static page or image.

Publications from the award: 1) Ware et al. 2002a; 2) Ware et al. 2002b; 3) McCouch et al. 2002; 4) Jaiswal et al. 2002

     These BAC libraries will be the tools necessary to explore how plants evolve and adapt to variability in genome size and structure, ecological habitats and changes in development and physiology. Within the genus Oryza, genome size varies 5-fold (Table 1), polyploidy exists, there are structural chromosome changes between species (Hass et al. 2003; Shishido et al. 2001) and the habitat adaptation varies from forests to swamps and from Himalayan foothills to Caribbean islands (Vaughan 1994). Developmentally these species show differences in many aspects of plant growth and response to environmental stimuli. Since the central species, O. sativa, is a crop species, the ability to examine crop evolution/domestication at the genic/genome level represents an unprecedented opportunity. Several genes important to rice domestication have been placed on the high-density O. sativa linkage map (Cai and Morishima 2000; Zhou et al. 2001), therefore, the opportunity to study specific genes involved in crop domestication will be available. Because of the depth of genetic information available for O. sativa, this Oryza BAC resource will form a closed system for the study of evolution of specific physiological/developmental genes and large-scale genome events.
     Such work will also lead to rapid isolation of developmentally, physiologically and agriculturally important loci and regulatory regions for comparative functional studies.

Data Transfer between CSHL and AGI/Purdue: We will set up a secure incoming FTP site at CSHL that allows AGI and Purdue to deposit fingerprint maps and BAC end sequences. BAC end sequence data will include the quality scores produced by Phred (Ewing and Green 1998; Ewing et al. 1998) in order to facilitate SNP calling. We will transfer BAC contig data using the .ace format output by the FPC program. Uploads will be checksummed to ensure completeness and acknowledged by the project data manager.
    Analysis results, such as mapping positions and SNPs, will be returned to AGI via an outgoing FTP directory. Both the incoming and outgoing FTP sites will be password protected to avoid casual abuse.

Mapping of BAC End Sequences to the Rice Genome: We will use the Biopipe/Blat mapping pipeline to align BAC end sequences from the 11 wild rice species to the reference japonica and indica genomes. Since it is estimated that these genomes different from each other at one position every 200 bp, the polymorphism rate is well below the single-pass sequencing error rate, and the presence of SNPs will not present a significant obstacle to alignment.
     Under our current mapping protocols, 80% of the BAC end sequences are expected to align uniquely to the genome. This means that essentially all BAC contigs will be anchorable to the reference genome except for those locations where the genome sequence will contain gaps due to centromeric repeats or unclonable regions
     An important internal quality control metric will be contiguity. In the ideal case, we expect all BAC end sequences in a contig to map to a contiguous region. In the real case, we will have to deal with three confounding factors:

1. Incorrect mapping of one or more BAC ends. An error in mapping a BAC end sequence will lead to outliers in the distribution of map positions in the contig. This can be recognized easily because the mismapped ends will be in the minority and uncorrelated by their position within the contig.

2. An FPC contig misassembly. Typically this will appear as a "chimeric" contig in which the BAC ends in two or more segments of the contig map to different areas in the genome. Within a segment, the map positions will be correlated. Such cases will be reported back to AGI for manual inspection.

3. A genomic rearrangement. The contig is correct, but there has been a genomic rearrangement between a wild species and the reference species since divergence from their common ancestor. For large rearrangements that are not spanned by the BAC contig, this case will often be indistinguishable from (2). For small inversions and rearrangements in which both endpoints of the rearrangement are captured by the BAC contig, we will be able to distinguish and flag this event. In any event, these cases will be reported back to AGI for manual inspection. Cases that cannot be confirmed to be the result of a BAC contig misassembly will be returned to CSHL for entry into the database as a putative rearrangement event.

Evolution of repetitive DNA sequences in the wild Oryza genomes (SanMiguel and Jackson): Repetitive DNA sequences constitute a majority of most plant genomes. In rice, repetitive elements comprise nearly 40% of the sequenced genome. Previous studies have shown that there is some variation in copy numbers of certain repeats in the Oryza genomes and that this may have contributed to the variation in genome sizes (Uozu et al. 1997). However, in comparison to other cereals (i.e. wheat or maize) there is little variation in genome size among the Oryzas-except that due to polyploidy. Although there is little variation in genome size, the repeats that constitute a major portion of each Oryza genome do differ from each other (reviewed in, Uozu et al. 1997). Furthermore, we know that transposable elements insert and mutate beyond recognition in less than 10 million years (SanMiguel et al. 2002) and that there is approximately 15 million years of evolution represented in the genus Oryza (E. Kellogg, personal communication). The BAC end sequence data representing nearly 10% of each genome presents an opportunity to sample the repetitive fraction of each genome.
     Studies of the transposable element fraction of orthologous segments of grain genomes (For example, in maize and sorghum (Tikhonov et al. 1999) and in wheat and barley (SanMiguel et al. 2002) found no conserved transposable elements that could convincingly be shown to have inserted before the divergence of their respective species. One class of transposable elements, retrotransposons, can be dated as to when they inserted by determining the sequence divergence of their 5' and 3' long terminal repeats (LTRs) (SanMiguel et al. 1998; SanMiguel et al. 2002) The maize and wheat sequences compared were composed largely of these type of elements. Most of these retrotransposons that could be dated, had inserted in the last 3 million years. None that could be dated had inserted more than 6 million years ago.
     Evidence of insertions that occurred longer than 6 million years ago appears to be lost through deletion via an illegitimate recombination mechanism (Devos et al. 2002) Hence, studying orthologous sequences in species that diverged longer than 10 million years ago does not allow one to study orthologous transposable elements. Given the more limited temporal scope of the species studied in this proposal, orthologous transposable elements should be founC.
     A BAC-end sequence that is composed entirely of a repetitive sequence will confound the anchoring of that BAC-end on the Nipponbare genomic sequence on the basis of sequence similarity. However, it might be anchored as a member of a fingerprinted BAC contig. Further, a sequence that is repeated many times in the entire genome may be present only once in a segment of the Nipponbare genome to which a contig is anchored. In this limited segment of the genome, the sequence read would no longer be repetitive and in many places could be placed unambiguously. The majority of such repeats would likely be transposable elements.
    The insertion times of numerous retrotranspons have been estimated. But these times are based on the speculation that retrotransposon sequence will mutate at a rate approximately equal to other sequences not under selection. That is, synonymous site bases in codons or introns. The mutation rate of retrotransposons (and other transposable elements) may well be much higher. In cases where BAC end sequences from several Oryza species can be shown to both overlap a retrotransposon and are positioned orthologously, mutation rates for this type of DNA can be surmised. As the BACs will be constructed using a few restriction enzymes, the chance of any given read of the 0.1x coverage of each species lining up with other non-Nipponbare BAC-end reads is substantially increased.
     A repeat database will be constructed for each Oryza genome structured to map each repeat class onto a complete transposable element, where possible. Further, it will be noted for each repeat discovered whether it has been reliably mapped to a certain position in the Nipponbare genomic sequence. The repeats present in each genome will be broken down into classifications by presumed transposition mechanism (RNA or DNA) and further sub-classified where appropriate. Each repeat db will be compared against another in pair-wise comparisons focusing especially on sequences present at orthologous locations. In addition, conserved classes of repeats will be used to infer genome phylogeny.
     To further understand how these repetitive elements evolve and mold genomes, select repetitive elements will be mapped to chromosomes and extended DNA fibers to get a picture of their long-range (chromosomal) organization. Together with the sequence data this will present a picture of the copy numbers of repetitive DNA sequences, their sequence arrangement and conservation as well as the chromosomal distribution and evolution of these repeats. This approach will provide a unique, almost complete, snapshot of how repetitive DNA sequences evolve in a family structure and how they mold genomes/chromosomes.

Databasing of Results (Stein): Data from FPC contig mapping and SNP calling activities will be entered into the Gramene project database. Our schema does not currently handle deep sets of variations among a collection of genomes.
     To accommodate this, we will modify our schema so that it follows a data model originally developed by the Neomorphic Corporation for use in the Celera annotation of Drosophila, and since adapted and used for the Chado modular sequence database schema [http://www.gmod.org/chado]. In this data model, a region of the genome is represented as a set of triplets S=[[r₁,s₁,e₁],[r₂,s₂,e₂]...] where r is the ID of the reference coordinate system (e.g. the contig), and s and e are the start and end of the sequence using interbase coordinates. An additional pointer provides access to the variant nucleotide sequence itself. This data model provides sufficient flexibility to represent a deep set of variations including single nucleotide polymorphisms, indels, inversions, and rearrangements.

Analysis of Conserved Regions (Stein): The alignment of multiple wild rice species to the reference genome will present us with an opportunity to proofread and improve the rice genome annotations. For example, an Oryza sativa pseudogene that was misannotated as a protein coding gene in the reference sequence, can be caught if it is missing entirely in the wild rice species or has accumulated one or more frameshift or nonsense mutations. In other cases, an unannotated region of the genome where the polymorphism rate drops off significantly may indicate a missed functionally important region, such as a protein-coding gene or RNA. We will systematically examine the genome for such regions.
     Although the evolutionary closeness of the wild and domestic strains diminishes the information available to such tools, we will experiment with using recently-published software that uses sequence conservation to improve predictions of protein-coding and RNA genes. We will scan for protein-coding genes using the TwinScan software (Korf et al. 2001) package, which was recently used to improve gene predictions in regions of human/mouse orthology (Flicek et al. 2003). This software performs ab initio gene prediction on two genomes simultaneously, taking into account such factors as the increased likelihood of seeing polymorphisms at the wobble base of coding sequence. We will also scan the genome for putative functional RNA genes using QRNA (Rivas and Eddy 2001). This software identifies putative RNA genes by searching for coordinated mutations across the axes of symmetry introduced by RNA stem-loop structures.

Data Presentation (Stein): The results of these analyses will be presented on the Gramene web site. The physical maps of the wild rice strains will be displayed side-by-side with each other and with the reference maps using the comparative map viewer CMap. This will provide an intuitive visualization of rearrangements among the various wild genomes of rice. The contig maps displayed on the Gramene viewer will be linked to the WebFPC viewer at AGI for the convenience of researchers wishing to examine the underlying fingerprint data.
     BAC end alignments that support the alignment of FPC contigs to the reference genomes will be displayed using the Gramene sequence viewer. Researchers will be able to "drill down" into the data to the individual nucleotide level, where they will be shown a multiple alignment of the reference genome and one or more aligning wild strains.
     We will provide specialized query tools in addition to the standard search tools already on the Gramene web site. One query tool will allow researchers to search for and download sets of SNPs selected by map position and type, such as coding region SNPs. Another tool will allow researchers to retrieve data on all wild strain contigs that overlap a particular region of the reference genome.
     For the convenience of researchers wishing to design assays for selected SNPs, we will provide a primer-picking interface to the PRIMER3 (Rozen and Skaletsky 2000) program.
     All data analysis results will be downloadable from the Gramene web site as flat files and in XML format. The data will also be available for remote query and browsing using the Distributed Annotation System (Dowell et al. 2001), an XML-based protocol for sharing genomic annotations. Further, all fingerprint data will be downloadable from the AGI web sites to construct and manipulate FPC maps locally

Rod Wing: Wing will serve as overall project director for OMAP. Members of his laboratory will serve as Project Manager (Dave Kudrna), direct the fingerprinting and assembly (Ed Butler), direct BAC-end sequencing (Yeisoo Yu). In years 3-4, the Wing lab will hire a Postdoc and Graduate Student to work on the global alignment and chromosome reconstruction experiments in collaboration with Lincoln Stein and Scott Jackson. The AGI will also serve as a mentor for 2 high school teachers during each summer and several UBRP students throughout the year.

Lincoln Stein: Stein will oversee all aspects of integrating OMAP data into Gramene and coordinating with the other project PIs and Senior Personnel to update the global alignment and chromosome reconstruction experiments during the course of the project. The Stein lab will also conduct the SNP data mining analysis and present the results though Gramene and in publications. The Stein lab will also serve as a mentor for 2 high school teachers and 1 UBRP intern during each summer.

Scott Jackson: Jackson will oversee all aspects, bioinformatics and experimental, of the chromosome reconstruction experiments in collaboration with the Wing and Stein labs. The Jackson and SanMiguel labs will also serve as a mentor for 2 high school teachers, several MARC/AIM students and 1 UBRP intern during each summer.

Phillip SanMiguel: SanMiguel will oversee all aspects of BAC-end sequencing at the Purdue sequencing center. SanMiguel will also lead the repeat analysis efforts in collaboration with the Jackson lab.

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