Objective 5. Bioinformatics: Public web-based databases of libraries and maps.

1. Objectives and coordination structure 2. Integrate contigs withmapped DNA markers, thusplacing contigs onto wheat linkageand deletion maps.Coordinator:Bikram Gill 1. Construct BAC &BIBAC libraries, fingerprintclones, and assemble contigs.Coordinator:Jan Dvořák Assessment ofthe insular organization of the wheat D genome by physical mapping DBI-0077766 5. Bioinformatics: public web-based databases of libraries and maps.Coordinator:Olin Anderson 3. Construct physical mapof the 7 D-genomechromosomes.Coordinator: Mingcheng Luo PI & Project Coordinator:Jan DvořákProject Manager:Patrick McGuire Triticum aestivum2n=42, ABD genomes 4. Assess gene distribution across chromosomesCoordinator:Jan Dvořák Compared to the genomes of model plants Arabi-dopsisthaliana and rice, each wheat genome is an order of magnitude larger. Indirect evidence suggests that in the wheat genomes there may be gene-rich islands (gene insulae) separated by gene-poor or gene-empty regions. The goal of this Project is to obtain a detailed picture of gene distribution in the wheat D genome and arrive at an understanding of the evolution of the global organization of large genomes. To assess gene distribution across the D-genome chromosomes, we exploit the fact that the Aegilops tauschii genome is completely homologous with the D genome of wheat. Physical maps of each of the seven Ae. tauschii chromosomes will be constructed and a large number of gene loci will be placed on them. The physical maps will be integrated with existing wheat linkage and deletion maps. The distribution of loci integrated into the physical maps will be used to assess the distribution of genes in the D-genome and the distribution and characteristics of gene insulae (see Figs. 1 & 2 for work flow). The physical maps of the D-genome chromosomes will be compared with the maize physical map and the rice genomic sequence. These comparisons may shed light on the evolution of the global structure of large cereal genomes. The identification and mapping of gene insulae in the wheat D genome will facilitate the discovery and isolation of economically important genes, ultimately allowing the sequencing of the most relevant regions of wheat genomes. The project is a collaborative effort involving the following: Wheat Genomics Center (University of California, Davis–USDA-ARS-WRRC) Jan Dvorak (PI) Olin Anderson (Co-PI) Mingcheng Luo (Co-PI) Yong Qiang Gu (Collaborator) Karin Deal (Postdoc) Frank You (Bioinformatician) Kansas State University Bikram Gill (Co-PI) Wanlong Li (Postdoc) Texas A&M University Hongbin Zhang (Collaborator) UC Genetic Resources Conservation Program Patrick McGuire (Project manager) Introduction Triticum monococcum, AmAm Triticum urartu, AA Aegilops speltoides, SS Aegilops tauschii, DD Aegilops tauschii2n=14, D genome Triticum turgidum, AABB Triticum aestivum, AABBDD Approaches and status after 24 months (9/1/00–8/31/02) Objective 1. To construct several Ae. tauschii large-insert libraries in BAC and BIBAC vectors, automate the fingerprinting procedure, finger-print the clones, and construct contigs. Objective 2. To integrate the contigs with linkage- and deletion-mapped wheat cDNA clones (ESTs) and other DNA markers, thereby placing the contigs into bins delineated by deletion break-points on the wheat D-genome deletion map. Objective 5. Bioinformatics: Public web-based databases of libraries and maps. (1) A workshop on BAC library construction involving personnel from two participating labs was held at the BAC Center of TAMU in the fall of 2000. (2) A workshop on high-throughput BAC DNA extraction was held at the BAC Center of TAMU in winter of 2001. (3) Two high school students from the UC Davis Junior Academic Science Research Achievement Program for social groups underrepresented in science participated in J. Dvorak’s and M.C. Luo’s laboratories in the summer of 2001. Approach: The Project website will display the following: (1) data on BAC and BIBAC libraries, (2) BAC and BIBAC fingerprints, (3) correspondence between BACs and cDNA (EST) markers, (4) BAC and BIBAC contigs and physical maps with inte-grated markers, ESTs, and traits, and (5) positions of gene islands on the D-genome chromosomes. Status: The Project website has been developed and is publicly accessible at http://wheat.pw.usda. gov/PhysicalMapping/. Its purpose is to disseminate to the science community such information as a detailed project description, lab protocols, data-bases (the BAC library database, the BAC finger-printing database, the FPC database, and the marker-BAC clone integration database), progress, and a roster of personnel involved in the work. A password-protected section has been created to provide Project members with a forum for discus-sion of strategies, techniques, and data as well as detailed information about material sharing and transfer. Algorithms for the editing of BAC finger-print profiles have been developed and are being optimized. This program eliminates substandard profiles, profiles of short-insert or empty clones, as well as any cross-contamination, and control BACs which are inserted into each plate for quality con-trol. In the accepted profiles, it eliminates back-ground peaks caused by partial digests. Project databases have been developed and integrated with the GrainGenes and NCBI databases. All databases are publicly accessible. 1. Five Ae. tauschii BAC and BIBAC libraries. 2. Ae. tauschii BAC and BIBAC fingerprints and contigs of 200,000 clones. 3. Correspondence between Ae. tauschii BACclones and cDNA (EST) and other markers. 4. D-genome physical maps. Approach: Five new genomic DNA libraries of Ae. tauschii ssp. strangulata from Armenia (line AL8/78-2-2) were constructed in a BAC vector and a plant-transformation-competent BIBAC vector utilizing three different restriction sites (EcoRI, BamHI, and HindIII). A total of 200,000 clones (~9-fold genome coverage) will be fingerprinted and DNA fragments will be sized by ABI 3100 capillary electrophoresis DNA sequencers (Fig. 2). Status:The sizes of the libraries range from 54,224 to 76,800 clones of an average insert sizes from 149 to 190 kb (Table 1). Two fingerprinting tech-niques, the TAMU-fingerprinting kit and an ABI SNaPshotTM-based technique, were developed and extensively tested for high-throughput fingerprinting of BACs. In addition, DNA isolation procedures were evaluated for each technique. The SNaPshot-based technique produced fewer artifacts, was more flexible, and, thus, was ultimately chosen for routine fingerprinting of Ae. tauschii BACs (Fig. 3). In collaboration with TIGR, the technique was eval-uated by successful reconstruction of sequenced rice contigs. The Qiagen Large-DNA ConstructTM BAC DNA isolation kit produced consistently high-quality BAC DNAs. As of September 22, 2002, a total of 65,157 BAC clones have been fingerprinted and the fingerprints were deposited on the Project’s publicly available website (URL: http://wheat.pw usda.gov/ PhysicalMapping/). Approach: Because of difficulty with pool hybridiz-ing of the BIBAC clones, we have changed our strategy from using 40,000 each of BAC clones and BIBAC clones to using 73,728 BAC clones. These clones were arrayed onto four high-density mem-branes and are being probed with pools of EST and RFLP clones. A two-dimensional 7 x 7 insert-pooling technique is used to maximize the efficiency of hy-bridization. More than 80,000 wheat ESTs are avail-able and 3,977 ESTs have been mapped to wheat deletion bins by the effort of the Wheat EST Genom-ics Project (NSF DBI-9975989). Over 2,000 RFLP markers have been mapped genetically and about half of them mapped into deletion lines. Thus, an-choring the EST and RFLP clones to BAC contigs will integrate the contigs into the wheat deletion maps. Status: As of September 20, 2002, a total of 427 clones that had previously been placed on Triticeae RFLP linkage maps have been integrated into the 73,728 BAC clones and the results have been made publicly available on the Project website. (Numbers in parenthesis refer to objectives) Resources externalto Project Triticeaegeneticmaps Wheatdeletionmaps Production of BAC andBIBAC libraries (1) RFLP and othermarkers and mappedwheat ESTs Fingerprinting oflibraries (1) Assembly ofcontigs (1) 73,728 BAC clones arrayedonto membranes (2) Membranes hybri-dized with markers and ESTs (2) Contigs integratedinto genetic andphysical maps (2) Figure 1. Project strategy Construction of physical maps of D-genome chromosomes (3) Assessment of genedensity acrosschromosomes (4) BACs DNA isolation Reactions DNA fragments isolated from Ae. tauschii AL8/78 were cloned in a bacterial artificial chromosome vector (BAC) and a plant-transfor-mation-competent vector (BIBAC). A total of 201,216 clones will be characterized by a global finger-printing method and assembled into contigs on the basis of finger-print-sharing among the clones. Fingerprinting of this large number of clones and their assembly into contigs will be accomplished by automation of the fingerprinting process and by employing ABI 3100 capillary electrophoresis units for automated fragment sizing and computer-based contig assembly. A subset of 73,728 BAC clones was arrayed and are being hybri-dized with cDNA and other mar-kers mapped on Triticeae genetic maps. In addition, two thousand wheat ESTs (generated and map-ped on wheat deletion maps by NSF Plant Genome Research Program project DBI-9975989) will be hybridized with the arrayed subset of clones. The mapped marker integration into the BAC population will assist in identifying the position of each contig on each D-genome chromosome. Neigh-boring contigs will be identified and joined to generate the phy-sical map of each of the seven D-genome chromosomes. BAC-marker integration Fragmentsizes areshownabovepeaks Deliverables Fragment sizing Training Data processing Integrated databases Figure 3. A BAC finger-printing profile generated by SNaPshotTM procedure Contig assembling http://wheat.pw.usda.gov/PhysicalMapping/wheatdb.html Figure 2. Work flow of BAC finger-printing and BAC-marker integration Assessment of the insular organizationof the wheat D genome by physical mapping Common wheat, Triticum aestivum, originated by inter-specific hybridization of three diploid species, Triticum urartu, Aegilops speltoides, and Ae. tauschii. The wheat nucleus therefore contains three pairs of genomes, designated AA, BB, and DD.