CLEMSON, SOUTH CAROLINA

Clemson University Genomics Institute

 Construction and characterization of a maize bacterial artificial chromosome (BAC) library for the inbred line LH132 --Tomkins, JP, Frisch, DA, Byrum, JR, Jenkins, MR, Barnett, LJ, Wicker, T, 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 and BAC end sequencing technologies, 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 maize LH132 Dekalb inbred line and is suitable for constructing a comprehensive physical framework of the maize genome. HindIII was used as the cloning enzyme because complete digests with maize DNA produced fragments £ 30 kb. The library consists of 427,392 clones stored in 1,113 384-well microtiter plates. A negligible amount of clones (0% based on 347 samples) do not contain inserts as judged by random analysis of BACs sampled from the library. A random sampling of 347 BACs taken from the library indicated an average insert size of 118 kb with a range of 50 to 390 kb. Insert size estimates were based on a very conservative molecular weight marker (Midrange I, NEB). Because the corn genome, like most monocots, has a high percentage of Not1 sites, many insert fragments 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 20.2 maize genome equivalents.

To determine the size distribution of BAC clones in the library, the 347 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 this analysis, 76% of the clones in the library have an average insert size equal to or greater than 100 kb.

The maize LH132 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 LH132 will be constructed by HindIII fingerprinting and BAC end sequencing all clones in the BAC library. Reagent costs for these efforts will be maximized due to a negligible amount of BACs not containing inserts.

Construction of the LH132 maize BAC library was performed at the Clemson University Genomics Institute through funds received from the Monsanto Corp. The library is the property of Monsanto and requests concerning its availability and use should be directed to Joe Byrum (email: [email protected]).
 
 

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