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

Y. Yu, J. P. Tomkins (Lead / Corresponding author), R. Waugh, D. A. Frisch, D. Kudrna, A. Kleinhofs, R. S. Brueggeman, G. J. Muehlbauer, R. P. Wise, R. A. Wing (Lead / Corresponding author)

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Abstract

Modern cultivated barley is an important cereal crop with an estimated genome size of 5000 Mb. To develop the resources for positional cloning and structural genomic analyses in barley, we constructed a bacterial artificial chromosome (BAC) library for the cultivar Morex using the cloning enzyme HindIII. The library contains 313344 clones (816 384-well plates). A random sampling 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 chloroplast DNA content indicated an exceptionally low 1.5% contamination with chloroplast DNA. Thus, the library provides 6.3 haploid genome equivalents allowing a >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 18432 clones represented per filter. Screening the library with 40 single copy probes identified an average 6.4 clones 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 representing 20 different regions of the genome with an average of 6.1 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 genomic applications in barley.

Original languageEnglish
Pages (from-to)1093-1099
Number of pages7
JournalTheoretical and Applied Genetics
Volume101
Issue number7
DOIs
Publication statusPublished - 7 Dec 2000

Keywords

  • BAC library
  • Barley
  • P-loop genes
  • Resistance-gene analog (RGA)

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    Yu, Y., Tomkins, J. P., Waugh, R., Frisch, D. A., Kudrna, D., Kleinhofs, A., Brueggeman, R. S., Muehlbauer, G. J., Wise, R. P., & Wing, R. A. (2000). A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and the identification of clones containing putative resistance genes. Theoretical and Applied Genetics, 101(7), 1093-1099. https://doi.org/10.1007/s001220051584