CONSTRUCTION AND CHARACTERIZATION OF A BARLEY(Hordeum vulgare) cv. MOREX BAC LIBRARY

¹ Clemson University Genomics Institute, Clemson, SC 29634 USA

² Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA Scotland

³ Department of Crop and Soil Sciences and Genetics and Cell Biology, Washington State University, Pullman, WA 99164-6420 USA

BAC libraries containing large genomic DNA inserts are important research materials for map-based cloning, physical mapping and genome sequencing of agriculturally important genes. We have constructed a BAC library from the barley cultivar Morex. Isolated nuclei were embedded in agarose plugs and partially digested with Hind III. DNA fragments(125-350kb) were size selected, ligated into the pBeloBAC11 BAC vector and transformed into E. coli (DH10B) by electroporation. White colonies were picked into 384- well microtiter plates with the Genetix Q-bot robot. The barley BAC library contains 313,344 clones (816 384-well plates). The library was partially characterized by analyzing 500 random clones and by selecting 459 disease resistance-like clones. The disease resistance-like clones were identified by hybridizing with a P-loop motif primer. The average insert size was 106 kb (range 30-195) in both experiments. Based on the random sample, there are 3.4% empty vectors and 1.5% chloroplast clones. Presence of mitochondrial DNA clones was not determined. Assuming haploid genome size of 5,000Mb, this BAC library represents about 6.3 nuclear genome equivalents and provides >99% probability of isolating a specific genome sequence. High density filters were prepared using the Q-bot robot in 4X4 double spot method and distributed to the barley research community. Amplified P-loop clone fragments are being mapped to determine the genomic distribution of the clones.