Original Article

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

  • Y. YuAffiliated withClemson 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. TomkinsAffiliated withClemson 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. WaughAffiliated withScottish Crop Research Institute, Invergowrie, Dundee, DD2 2DA, UK
  • , D. A. FrischAffiliated withClemson 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. KudrnaAffiliated withDepartment of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA
  • , A. KleinhofsAffiliated withDepartment of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA
  • , R. S. BrueggemanAffiliated withDepartment of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA
  • , G. J. MuehlbauerAffiliated withDepartment of Agronomy and Plant Genetics, University of Minnesota, St. Paul, MN 55108, USA
  • , R. P. WiseAffiliated withUSDA-ARS, Corn Insects and Crop Genetics Research Unit, Department of Plant Pathology, Iowa Stae University, Ames, IA 50011-1020, USA
    • , R. A. WingAffiliated withClemson 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

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