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Title: CONSTRUCTION AND CHARACTERIZATION OF A DEEP-COVERAGE BACTERIAL ARTIFICIAL CHROMOSOME LIBRARY FOR MAIZE

Author
item TOMKINS, J - CLEMSON UNIVERSITY
item DAVIS, G - UNIV OF MISSOURI-COLUMBIA
item MAIN, D - CLEMSON UNIVERSITY
item YIM, Y - CLEMSON UNIVERSITY
item DURU, N - UNIV OF MISSOURI-COLUMBIA
item MUSKET, T - UNIV OF MISSOURI-COLUMBIA
item GOICOECHEA, J - CLEMSON UNIVERSITY
item FRISCH, D - CLEMSON UNIVERSITY
item Coe Jr, Edward
item WING, R - CLEMSON UNIVERSITY

Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2001
Publication Date: 5/1/2002
Citation: TOMKINS, J.P., DAVIS, G.Y., MAIN, D., YIM, Y., DURU, N., MUSKET, T., GOICOECHEA, J.L., FRISCH, D.A., COE JR, E.H., WING, R.A. CONSTRUCTION AND CHARACTERIZATION OF A DEEP-COVERAGE BACTERIAL ARTIFICIAL CHROMOSOME LIBRARY FOR MAIZE. CROP SCIENCE. 2002. V. 42(3). P. 928-933.

Interpretive Summary: The genome of maize (corn) is about the same size as that of humans, 2.5 billion DNA bases. For effective access to the genes in this singularly important crop, the proven laboratory strategy is to produce fragment libraries in bacterial artificial chromosomes (BACs). These fragments can then be individually characterized and connected to each other by several cross-informative methods. A library of fragments consisting of 247,680 separate fragments has been produced from one of the most agronomically important inbred lines used to produce corn hybrids. The library represents overlapping fragments that cover the genome 13.5-fold, which insures comprehensive coverage. Characterization of the library shows it to be of high quality, with very few empty or undesired fragments. The library is made available for public use, as copies spotted on filters or as clones in plates. The impact of this research is the first high- quality, publicly available resource of clones of the whole genome for cor genomics. It will be a foundation for sequencing of the genome of corn and for in-depth analysis of genes by researchers in the public and private sectors.

Technical Abstract: Modern cultivated maize has an estimated genome size of 2,500 Mb. To develop the resources for genomics in maize, a bacterial artificial chromosome (BAC) library for the inbred line B73 was constructed using the cloning enzyme HindIII. The library contains 247,680 clones (645 384-well plates). A random sampling of 697 clones indicated an average insert size of 136 kb (range = 42 to 379 kb) and 0.4% empty vectors. Screening for chloroplast DNA content indicated an exceptionally low 0.18% contamination with chloroplast DNA. The library provides 13.5 haploid genome equivalents allowing >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically on 22.5 cm**2 filters. Partial screening (6X) of the library with 20 single-copy probes identified an average 7.1 positive signals per probe, with a range of 3-15 positive signals per probe. To evaluate for sequence-tagged-connector (STC) analysis, 768 BAC clones were end sequenced in both forward and reverse directions giving a total of 1415 successful reads. End sequences were queried against SWISS-PROT, GenBank NR, MIPS Arabidopsis, maize genomic sequence dbGSS, and maize cDNA database dbEST. Results are publicly available at the CUGI website (www.genome.clemson.edu/projects/stc/maize/ZMMBBb/).