Submitted to: Journal of Theoretical and Applied Genetics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/1/2000
Publication Date: N/A
Citation: Interpretive Summary: Modern cultivated barley has a genome size almost twice that of humans. An extensive history of research has resulted in knowledge about the genes that control barley functions, such as malting quality, yield, stress response and disease resistance. Soon, the complete genomic sequencing of two small-genome model plants, Arabidopsis (120-Mb) and rice (430-Mb) will be a reality. In contrast, complete sequencing of large-genome plants is not yet practical. While many important traits have been mapped in barley, the molecular isolation of the genes that encode them has been difficult. This report describes the preparation of a bacterial artificial chromosome (BAC) library that has been made available to the public barley research community. The results described address the goals of National Program 302, Plant Biological and Molecular Processes, and will greatly facilitate the molecular isolation of genes of agricultural importance.
Technical Abstract: Modern cultivated barley is an important cereal crop with an estimated genome size of 5,000 Mb. To develop the resources for positional cloning and structural genomics analyses in barley, we constructed a bacterial artificial chromosome (BAC) library for the cultivar Morex using the cloning enzyme HindIII. The library contains 313,344 clones (816 384-well plates). A random sampling of 504 clones indicated an average insert size of 106 kbp (range = 30 to 195 kbp) and 3.4% empty vectors. Screening the colony filters for chloroplast DNA content indicated an exceptionally low 1.5% contamination with chloroplast DNA. Thus, the library provides 6.3 haploid genome equivalents allowing >99% probability of recovering any specific sequence of interest. High-density filters were gridded robotically using a Genetix Q-BOT in a 4x4 double-spotted array on 22.5 cm2 filters. Each set of 17 filters allows the entire library to be screened with 18,432 clones represented per filter. Screening the library with 40 single copy probes identified 6.4 clones per probe, on the average, with a range of 1-13 clones per probe. A set of resistance gene analog (RGA) sequences identified 132 unique RGA-containing BAC clones representing 20 different regions of the genome for an average of 6.6 clones per locus. Additional screening of the library with a P-loop disease resistance primer probe identified 459 positive BAC clones. These data indicate that this library is a valuable resource for structural genomics applications in barley.