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First published online March 12, 2004; 10.1104/pp.103.034538

Plant Physiology 134:1317-1326 (2004)
© 2004 American Society of Plant Biologists

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BREAKTHROUGH TECHNOLOGIES

Anchoring 9,371 Maize Expressed Sequence Tagged Unigenes to the Bacterial Artificial Chromosome Contig Map by Two-Dimensional Overgo Hybridization1

Jack Gardiner*, Steven Schroeder, Mary L. Polacco, Hector Sanchez-Villeda, Zhiwei Fang, Michele Morgante2, Tim Landewe3, Kevin Fengler, Francisco Useche, Michael Hanafey, Scott Tingey, Hugh Chou4, Rod Wing, Carol Soderlund and Edward H. Coe, Jr.

Department of Agronomy, University of Missouri, Columbia, Missouri 65211 (J.G., S.S., H.S.-V., Z.F.); Plant Genetics Research Unit and Department of Agronomy, U.S. Department of Agriculture-Agricultural Research Service, Columbia, Missouri 65211 (M.L.P., E.H.C.); DuPont Agriculture and Nutrition—Molecular Genetics, E.I. du Pont de Nemours and Company, Newark, Delaware 19714 (M.M., K.F., F.U., M.H., S.T.); Incyte Genomics, St. Louis, Missouri 63114 (T.L., H.C.); Arizona Genomics Institute, University of Arizona, Tucson, Arizona 85721 (R.W.); and Arizona Genomics Computational Laboratory, University of Arizona, Tucson, Arizona 85721 (C.S.)

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 x 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.

1 This work was supported by the National Science Foundation (grant no. 9872655).

2 Present address: Dipartimento Produzione Vegetale e Tecnologie Agrarie, Universita' Di Udine, Via delle Scienze 208, 33100 Udine, Italy.

3 Present address: Navigen, 4069 Wedgeway Court, Earth City, MO 63045.

4 Present address: Department of Earth and Planetary Sciences, Washington University, St Louis, MO 63130.

www.plantphysiol.org/cgi/doi/10.1104/pp.103.034538

* Corresponding author; e-mail gardiner{at}ag.arizona.edu; fax 520–621–7186.

Received October 7, 2003; returned for revision November 16, 2003; accepted November 16, 2003.




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