Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region

Edwige Isidore, Beatrice Scherrer, Arnaud Bellec, Karine Budin, Patricia Faivre-Rampant, Robbie Waugh, Beat Keller, Michel Caboche, Catherine Feuillet, Boulos Chalhoub (Lead / Corresponding author)

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

To isolate genes of interest in plants, it is essential to construct bacterial artificial chromosome (BAC) libraries from specific genotypes. Construction and organisation of BAC libraries is laborious and costly, especially from organisms with large and complex genomes. In the present study, we developed the pooled BAC library strategy that allows rapid and low cost generation and screening of genomic libraries from any genotype of interest. The BAC library is constructed, directly organised into a few pools and screened for BAC clones of interest using PCR and hybridisation steps, without requiring organization into individual clones. As a proof of concept, a pooled BAC library of approximately 177,000 recombinant clones has been constructed from the barley cultivar Cebada Capa that carries the Rph7 leaf rust resistance gene. The library has an average insert size of 140 kb, a coverage of six barley genome equivalents and is organised in 138 pools of about 1,300 clones each. We rapidly established a single contig of six BAC clones spanning 230 kb at the Rph7 locus on chromosome 3HS. The described low-cost cloning strategy is fast and will greatly facilitate direct targeting of genes and large-scale intra- and inter-species comparative genome analysis.

Original languageEnglish
Pages (from-to)97-103
Number of pages7
JournalFunctional and Integrative Genomics
Volume5
Issue number2
DOIs
Publication statusPublished - 1 Apr 2005

Keywords

  • Barley
  • Pooled BAC library
  • Screening
  • Targeting genes of interest

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    Isidore, E., Scherrer, B., Bellec, A., Budin, K., Faivre-Rampant, P., Waugh, R., Keller, B., Caboche, M., Feuillet, C., & Chalhoub, B. (2005). Direct targeting and rapid isolation of BAC clones spanning a defined chromosome region. Functional and Integrative Genomics, 5(2), 97-103. https://doi.org/10.1007/s10142-004-0127-9