The maize genome is comparable in size to that of humans. Its complexity is even higher because of the many families of retroelements distributed throughout the genome, estimated to be over 80% of the genome content. Fragmenting such a genome, fingerprinting the BAC fragments, and assembling the fragments requires deep coverage accompanied by pegging with markers to confirm and to intercalate the assembled contigs. The markers should be anchored to the genetic map insofar as feasible. This is our central strategy.
The Genetic Map. The goal is a densely marked, high-resolution genetic map, constructed for the intermated B73/Mo17 (IBM) population. We have developed a map containing >1850 markers. The parents of the population, B73 and Mo17, represent the two major heterotic groups of U.S. maize germplasm, and B73 is the inbred line used for the BAC libraries. The IBM population consists of 302 recombinant inbred lines that underwent four rounds of random mating at the F2 stage. The additional meioses result in a three-fold expansion of the genetic map (Liu et al., Genetics 142:247-258, 1996). The combination of a large number of lines and the map expansion generate a map resource with ~17 times the order-resolving power of our previous maize maps (UMC 98 map, Davis et al., Genetics 152:1137-1172, 1999; BNL 96 map, Burr et al., Genetics 118:519-526, 1988). The IBM map now contains >1000 RFLP loci, of which 185 are with new PstI probes developed in the MMP, 46 with new Limagrain (lim) probes, and 129 with nonmaize probes. It contains >800 SSR loci, of which 583 were developed in the MMP.
To contribute to map development, we have produced a number of new single copy RFLP probes, SSR primer sets, and markers for Mutator insertion sites. Screening images for RFLP probes, primer and screening information for 1800 maize SSR loci, and mapscore data for the IBM population are being made available in the Maize Genome Database (MaizeDB, http://www.agron. missouri.edu/). Twelve hundred new SSR loci have been defined in this project (Sharopova et al., Plant Mol Biol 48, 2002) on IBM or either of two other mapping populations. Comparison maps with SSR loci for IBM and the two other populations have been constructed and are displayed at http://mnl.maizegdb.org/ body/ssr.html#mapfiles. SSRs in process are expected to reach the goal of 2000 public SSR loci, which are compiled and tabulated at http://mnl.maizegdb.org/ssr.html.
Seed of the IBM lines, or of a subset of 94 of them, can be obtained from the Maize Genetics Cooperation Stock Center, Urbana, IL. In addition to making seed available, we have elected to aid and promote the use of the IBM map by distributing DNA of the 94 lines in microtitre plate format for individual investigators to use to map target genes of their choice (see http://www.cafnr. missouri.edu/mmp/dna_kits.htm). Web entry for submission of map score data, from which the resulting locus positions are returned to the investigator, is available at http://www.cafnr. missouri.edu/mmp/ibm_resources.htm. Our intention is to provide the community of maize investigators with resources by which all can contribute to comprehensive information on maize gene map position.
To map unigenes that are pegged to BAC contigs but are not on IBM, SNP and INDEL markers are being developed from unigene sequences (see below). Genomic sequencing for regions corresponding to 480 unigenes across 12 inbred lines has been completed. SNP assay is by primer extension and detection on the ABI 3700, and the data are automatically genotyped and processed for mapping by the Laboratory Information Management System (LIMS). The target is to add 2000 genetically mapped SNP pegs.
Mapping of MITEs and AFLPs, which will add to pegs on BAC pools (see below), is also in progress. IBM DNA has been prepared and validated, multiplexed genotyping has been optimized, selective amplifications are completed, and data are ready for evaluation.
The LIMS for the project includes automated genotyping and validation of RFLP, SSR, and INDEL scoring, and preparation of mapping files.
A viewer interface for genetic maps, which provides for selection of various maps and the ability to compare and align any two maps between mapping populations or between chromosomes, or with the 3,267-marker RGP maps for rice, has been deployed and is viewable at http://mnl.maizegdb.org/cMapDB/ cMap.html. This utility has been derived from INE of the Japanese Rice Genome Program (http://rgp.dna.affrc.go.jp/giot/ INE.html), and was made available to modify for maize applications through the cooperation of Drs. Takuji Sasaki and Bal Antonio. Aligning comparisons with sorghum are to be added as available.
A BLAST server is being made available to return a map location if known.
Mutant mapping has placed 412 mutants to bins with high-throughput SSR protocols.
The Physical Map. Our proposed goal was to derive a 10X public BAC library with one enzyme, and to fingerprint and assemble the BACs into contigs to the extent possible. We have succeeded in expanding beyond this goal to genomic DNA libraries derived with HindIII (12.6X) and EcoRI (7X), plus a third library with MboI (7X), produced in collaboration with Jo Messing. Six-filter sets (6X for HindIII, 7X for EcoRI, 7X for MboI) are made available by CUGI at http://www.genome.clemson.edu/orders/. BACs of the HindIII library may be obtained from CUGI, and of the EcoRI and MboI libraries from CHORI at http://www.chori.org/ bacpac/home.htm).
Filter hybridizations of complex markers (telomere, centromere, knob, ribosomal probes) have been scored and are being summarized. Data have been incorporated in the FPC presentation at CUGI.
Filter hybridizations of selected single-copy RFLP markers, including the core markers, have been carried out to peg and anchor BAC contigs to the genetic map. The data have been incorporated in the FPC presentation at CUGI.
Filter hybridizations of RFLP markers that have been mapped in sorghum, maize, and rice are being carried out at the University of Georgia with overgo probes to peg and anchor BAC contigs to cross-species maps. The data will be incorporated in the FPC presentation at CUGI and will be incorporated into MaizeDB.
Following the innovations of Klein et al. (Genome Res 10:789-807, 2000) for pegging with mapped AFLP markers in pools of sorghum BACs, we have prepared 288 pools from six dimensions in a 48 x 48 x 48 array (110,592 BACs, 6X) for analysis with AFLPs, MITES, and selected SSRs. Quality checks on the pools with RFLPs and SSRs have been completed and are positive. So far 100 genetically mapped SSRs have been applied to the pools, and successful tests of deconvolutions of the pools have been done.
On a larger scale, pegging of ESTs has been done in a partnership of the Maize Mapping project with DuPont and Incyte, initiated in April 2001 and completed in November 2001. Over 10,000 EST unigene consensus assemblies have been filter-hybridized to 165,000 BACs (~10X) with overgos. The Public Cornsensus and Unigene set developed by Dupont has been submitted to GenBank. The data have been incorporated in the FPC presentation at CUGI. An ongoing summary of EST documentation is presented at http://mnl.maizegdb.org/ests.html.
Because each library is from the inbred line B73, this enables the project to proceed apace with fingerprinting and assembly of the entire robust resource using the functions of FPC. Fingerprinting progress, including pegging with markers, is viewable at http://www.genome.clemson.edu/projects/maize/fpc/. As of December 23, 2001, 164,701 fingerprints (8X) and 7,760 markers form 9,462 contigs and 7,437 singletons at cutoff 10-12.
A viewer interface is prepared to display the IBM map and BAC contigs with popups for dynamic marker links. This utility has been derived from INE of the Japanese Rice Genome Program (http://rgp.dna.affrc.go.jp/giot/INE.html), and was made available to modify for maize applications through the cooperation of Drs. Takuji Sasaki and Bal Antonio. The viewer, termed iMAP, is being populated with data preparatory to deployment.
Educational Links
Interactive Maize Plant is presented at http://www.maizegdb.org/IMP/.
Methods for Controlled Pollinations of Maize/Corn are presented at http://www.maizegdb.org/IMP/WEB/pollen.htm.
Information about the Life Science Undergraduate Research Opportunities
Program at Missouri is provided at http://www.
lsurop.missouri.edu/PGI.
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