Theoretical and Applied Genetics

, Volume 101, Issue 7, pp 1093–1099

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

Authors

  • Y. Yu
    • Clemson University Genomics Institute, BAC/EST Resource Center, Room 100 Jordan Hall, Clemson, SC 29634, USA e-mail: jtmkns@clemson.edu e-mail: rwing@clemson.edu
  • J. P. Tomkins
    • Clemson University Genomics Institute, BAC/EST Resource Center, Room 100 Jordan Hall, Clemson, SC 29634, USA e-mail: jtmkns@clemson.edu e-mail: rwing@clemson.edu
  • R. Waugh
    • Scottish Crop Research Institute, Invergowrie, Dundee, DD2 2DA, UK
  • D. A. Frisch
    • Clemson University Genomics Institute, BAC/EST Resource Center, Room 100 Jordan Hall, Clemson, SC 29634, USA e-mail: jtmkns@clemson.edu e-mail: rwing@clemson.edu
  • D. Kudrna
    • Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA
  • A. Kleinhofs
    • Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA
  • R. S. Brueggeman
    • Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA
  • G. J. Muehlbauer
    • Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA
  • R. P. Wise
    • USDA-ARS, Corn Insects and Crop Genetics Research Unit, Department of Plant Pathology, Iowa Stae University, Ames, IA 50011-1020, USA
  • R. A. Wing
    • Clemson University Genomics Institute, BAC/EST Resource Center, Room 100 Jordan Hall, Clemson, SC 29634, USA e-mail: jtmkns@clemson.edu e-mail: rwing@clemson.edu
Original Article

DOI: 10.1007/s001220051584

Cite this article as:
Yu, Y., Tomkins, J., Waugh, R. et al. Theor Appl Genet (2000) 101: 1093. doi:10.1007/s001220051584

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 4×4 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.

Key words BarleyBAC libraryP-loop genesResistance-gene analog (RGA)

Copyright information

© Springer-Verlag Berlin Heidelberg 2000