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

• Published in: Functional & integrative genomics Vol. 5; no. 2; pp. 97 - 103
• Main Authors: Isidore, E, Scherrer, B, Bellec, A, Budin, K, Faivre-Rampant, P, Waugh, R, Keller, B, Caboche, M, Feuillet, C, Chalhoub, B
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer 01.04.2005; Springer Nature B.V; Springer Verlag
• Subjects: bacterial artificial chromosomes; Bacteriology; barley; Biological and medical sciences; Botany; chromosomes; Chromosomes, Artificial, Bacterial; clones; Cloning; Cloning, Molecular - methods; Comparative analysis; Contig Mapping; disease resistance; DNA libraries; Fundamental and applied biological sciences. Psychology; genes; Genes, Plant; genomic libraries; Genomic Library; Hordeum - genetics; Hordeum vulgare; Life Sciences; loci; Methods; Microbiology; Miscellaneous; molecular cloning; Nucleic Acid Hybridization; polymerase chain reaction; pooled bacterial artificial chromosome libraries; Quantitative genetics; Rph7 gene; Rph7 locus; rust diseases; Barley; Monocotyledones; Construction; Targeting; Hordeum vulgare; Targeting genes of interest; Genotype; Gene library; Artificial chromosome; Cereal crop; Polymerase chain reaction; Screening; Gene; Gramineae; Angiospermae; Bacteria; Isolate; Isolation; Spermatophyta; Library; Genome; Pooled BAC library; Cultivar
• ISSN: 1438-793X; 1438-7948
• DOI: 10.1007/s10142-004-0127-9

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.