Plant & Animal Genome IX Conference

Poster: Large Insert Libraries, Gene Isolation, Etc.
P02_34.html

A HALF MILLION CLONES BACTERIAL ARTIFICIAL CHROMOSOME (BAC) LIBRARY OF DURUM WHEAT

¹ Dip. Biologia e Chimica Agroforestale e Ambientale, Universita' degli Studi di Bari, Italia.

² Institut National de la Recherche Agronomique, Station d'Amelioration des Plantes, Domaine de la Motte, BP 29, 35653 Le Rheu Cedex, France.

³ USDA Western Reg. Research Center, 800 Buchanan St., Albany, CA 94710, USA.

⁴ Dept. Agronomy and Range Science, University of California, One Shields Avenue, Davis CA 95616-8515, USA.

We report here the construction of bacterial artificial chromosome (BAC) library of tetraploid wheat Triticum turgidum var. durum and the progress of its characterization. The genotype used for this library is a recombinant substitution line of Langdon carrying a 30 cM segment of chromosome 6BS from T. t. var. dicoccoides. This segment includes a gene for high-grain protein content identified by L. Joppa and G. Hart. DNA from this line was partially digested with restriction enzyme HindIII, subjected to double size selection, electroeluted and cloned into the pINDIGO536 BAC vector. The library consists of 516,096 clones, picked and sorted in 1344 384-well plates, with an average insert size of 120-kb and less than 2% empty clones, based on the evaluation of each ligation. These characteristics are being evaluated with 500 clones (approximately 0.1% of the library) randomly chosen in the library. Excluding the empty clones, the coverage of this library will be between four and five haploid genome equivalents. The clones are being organized in 28 high-density filters with 18,432 double spotted clones using a Q-bot. Plasmid DNA has been extracted from bulk cultures of each 384-well plate for PCR screening (e.g. microsatellites). The screening of the library by hybridization and PCR will be started in the beginning of year 2001. For current status of the library see: http://agronomy.ucdavis.edu/Dubcovsky/BAC-library/BAC_Langdon.htm.

The authors AC and NC contributed equally to this research