A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the identification of clones containing putative resistance genes

Modern cultivated barley is an important cereal crop with an estimated geno me size of 5000 Mb. To develop the resources for positional cloning and str uctural genomic analyses in barley, we constructed a bacterial artificial c hromosome (BAC) library for the cultivar Morex using the cloning enzyme Hin dIII. The library contains 313334 clones (816 384-well plates). A random sa mpling of 504 clones indicated an average insert size of 106 kbp (range=30- 195 kbp) and 3.4% empty vectors. Screening the colony filters for chloropla st DNA content indicated an exceptionally low 1.5% contamination with chlor oplast DNA. Thus, the library provides 6.3 haploid genome equivalents allow ing a >99% probability of recovering any specific sequence of interest. Hig h-density filters were gridded robotically using a Genetix Q-BOT in a 4x4 d ouble-spotted array on 22.5-cm(2) filters. Each set of 17 filters allows th e entire library to be screened with 18432 clones represented pel filter. S creening the library with 40 single copy probes identified an average 6.4 c lones per probe, with a range of 1-13 clones per probe. A set of resistance -gene analog (RGA) sequences identified 121 RGA-containing BAC clones repre senting 20 different regions of the genome with an average of 6.1 clones pe r locus. Additional screening of the library with a P-loop disease resistan ce primer probe identified 459 positive BAC clones. These data indicate tha t this library is a valuable resource for structural genomic applications i n barley.