Construction and characterization of a maize bacterial artificial chromosome (BAC) library for the inbred line B73 --Tomkins, JP, Frisch, DA, Jenkins, MR, Barnett, LJ, Luo, M, Wing, RA The construction of a physical framework for the maize genome requires the use of a large insert genomic library. In order to develop a comprehensive physical framework for maize using fingerprinting technology, a deep coverage BAC library was developed. Methods used were generally similar to those described previously by Tomkins et al. (Plant Molec. Biol. 41:25-32, 1999).

The library was developed from the inbred maize line B73 which is suitable for constructing a comprehensive physical framework of the maize genome. The library is also well suited for map-based cloning and other genomics applications. HindIII was used as the cloning enzyme because complete digests with maize DNA produced fragments £ 30 kb. The library consists of 247,680 clones stored in 645 384-well microtiter plates. A neglible amount of clones (0.4 % based on 3/697 samples) do not contain inserts as judged by random analysis of BACs sampled from the library. A random sampling of 697 BACs taken from the library indicated an average insert size of 136 kb with a range of 42 to 379 kb. Insert size estimates were based on a very conservative molecular weight marker (Midrange I, NEB). Because the corn genome, like most moncots, has a high percentage of Not1 sites, many insert fragments often migrate below 50 kb. It has been our experience that BAC clones from monocot genomes are likely to have inflated insert size estimates when evaluated with a conventional 50 kb lambda ladder. Therefore, we believe that the insert size estimates in the present study are quite conservative. Based on a haploid genome size of 2,500 Mb (Arumaganthan and Earl, Plant Mol. Biol. Rep. 9:208-218, 1991), the coverage of the library is about 13.5 maize genome equivalents.

To determine the size distribution of BAC clones in the library, the 697 BACs analyzed with NotI digests were grouped by insert size and the frequency of each group of clones represented in the library was determined. Based on these data, 88% of the clones in the library have an average insert size equal to or greater than 100 kb.

The maize B73 BAC library is well suited to construct a comprehensive physical framework of the maize genome due to its high redundancy and large average insert size. The physical framework for B73 will be constructed by HindIII fingerprinting. Reagent costs for these efforts will be maximized due to the very low percentage of BACs not containing inserts.

Construction of the maize B73 BAC library was performed at the Clemson University Genomics Institute for the Missouri Maize Project (Ed Coe et al.) through a subcontract as part of a grant received from the National Science Foundation Plant Genome program. Requests concerning availability and use of the library should be directed to David Frisch (email: [email protected]).
 
 
 
 

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