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

被引：29

作者：

Isidore E. ^{[1
]}

Scherrer B. ^{[1
]}

Bellec A. ^{[2
]}

Budin K. ^{[2
]}

Faivre-Rampant P. ^{[2
]}

Waugh R. ^{[3
]}

Keller B. ^{[1
]}

Caboche M. ^{[2
]}

Feuillet C. ^{[1
]}

Chalhoub B. ^{[2
]}

机构：

[1] Institute of Plant Biology, University of Zürich, 8008 Zurich

[2] INRA, U. de Recherche/Genomique Vegetale, 91057 Evry

[3] Genome Dynamics, Scottish Crop Research Institute, Dundee DD2 5DA, Invergowrie

来源：

Functional & Integrative Genomics | 2005年 / 5卷 / 2期

关键词：

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

D O I：

10.1007/s10142-004-0127-9

中图分类号：

学科分类号：

摘要：

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. © Springer-Verlag 2005.

引用

页码：97 / 103

页数：6

共 20 条

[1]

Allouis S., Moore G., Bellec A., Sharp R., Faivre Rampant P., Mortimer K., Pateyron S., Foote T.N., Griffiths S., Caboche M., Chalhoub B., Construction and characterisation of a hexaploid wheat (Triticum aestivum L.) BAC library from the reference germplasm 'Chinese Spring, Cereal Res. Commun., 31, pp. 331-338, (2003)

[2]

Arumuganathan K., Earle E.D., Nuclear DNA content of some important plant species, Plant Mol. Biol. Report, 9, pp. 208-218, (1991)

[3]

Brunner S., Keller B., Feuillet C., A large rearrangement involving genes and low-copy DNA interrupts the microcollinearity between rice and barley at the Rph7 locus, Genetics, 164, pp. 673-683, (2003)

[4]

Burke D.T., Carle G.F., Olson M.V., Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors, Science, 236, pp. 806-812, (1987)

[5]

Chalhoub B., Belcram H., Caboche M., Efficient cloning of plant genomes into bacterial artificial chromosome (BAC) libraries with larger and more uniform insert size, J. Plant. Biotechnol., 2004, pp. 181-188, (2004)

[6]

Clarke L., Carbon J., Colony bank containing synthetic ColE1 hybrid plasmids representative of the entire E. coli genome, Cell, 9, pp. 91-100, (1976)

[7]

Feuillet C., Travella S., Stein N., Albar L., Nublat A., Keller B., Map-based isolation of the leaf rust disease resistance gene Lr10 from the hexaploid wheat (Triticum aestivum L.) genome, Proc. Natl. Acad. Sci. USA, 100, pp. 15253-15258, (2003)

[8]

Fu H.H., Dooner H.K., A gene-enriched BAC library for cloning large allele-specific fragments from maize: Isolation of a 240-kb contig of the bronze region, Genome Res., 10, pp. 866-873, (2000)

[9]

Fu H.H., Dooner H.K., Intraspecific violation of genetic colinearity and its implications in maize, Proc. Natl. Acad. Sci. USA, 99, pp. 9573-9578, (2002)

[10]

Graner A., Siedler H., Jahoor A., Herrmann R.G., Wenzel G., Assessment of the degree and the type of restriction-fragment-length-polymorphism in barley (Hordeum vulgare), Theor. Appl. Genet., 80, pp. 826-832, (1990)

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