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Construction of a subgenomic BAC library specific for chromosomes 1D, 4D and 6D of hexaploid wheat

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Abstract

The analysis of the hexaploid wheat genome (Triticum aestivum L., 2n=6x=42) is hampered by its large size (16,974 Mb/1C) and presence of three homoeologous genomes (A, B and D). One of the possible strategies is a targeted approach based on subgenomic libraries of large DNA inserts. In this work, we purified by flow cytometry a total of 107 of three wheat D-genome chromosomes: 1D, 4D and 6D. Chromosomal DNA was partially digested with HindIII and used to prepare a specific bacterial artificial chromosome (BAC) library. The library (designated as TA-subD) consists of 87,168 clones, with an average insert size of 85 kb. Among these clones, 53% had inserts larger than 100 kb, only 29% of inserts being shorter than 75 kb. The coverage was estimated to be 3.4-fold, giving a 96.5% probability of identifying a clone corresponding to any sequence on the three chromosomes. Specificity for chromosomes 1D, 4D and 6D was confirmed after screening the library pools with single-locus microsatellite markers. The screening indicated that the library was not biased and gave an estimated coverage of sixfold. This is the second report on BAC library construction from flow-sorted plant chromosomes, which confirms that dissecting of the complex wheat genome and preparation of subgenomic BAC libraries is possible. Their availability should facilitate the analysis of wheat genome structure and evolution, development of cytogenetic maps, construction of local physical maps and map-based cloning of agronomically important genes.

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Acknowledgements

We thank our colleagues Arnaud Bellec, Michaela Libosvárová, Stephanie Pateyron, Radka Tušková and Jitka Weiserová for excellent technical assistance. This work was supported by research grants from the Grant Agency of the Czech Republic (522/03/0354 and 521/04/0607), Ministry of Agriculture of the Czech Republic (QC 1336), research grants from the French Ministry of Research and Agriculture, and by a collaborative project ‘Barrande’ (reg. no. 2003-035-2) between the Ministry of Education, Youth and Sports of the Czech Republic and the French Ministry of Foreign Affairs and Education. J. Janda acknowledges the receipt of a Marie Curie Ph.D. Student Fellowship at URGV-INRA.

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Authors and Affiliations

  1. Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská 6, 77200, Olomouc, Czech Republic

    Jaroslav Janda, Jan Bartoš, Jan Šafář, Marie Kubaláková, Miroslav Valárik, Jarmila Číhalíková, Hana Šimková & Jaroslav Doležel

  2. Department of Cell Biology and Genetics, Palacký University, Šlechtitelů 11, 78371, Olomouc, Czech Republic

    Marie Kubaláková, Jarmila Číhalíková & Jaroslav Doležel

  3. Laboratory of Genome organization, Unité de Recherches en Génomique Végétale (INRA-URGV), 2 rue Gaston Crémieux CP 5708, 91057, Évry Cedex, France

    Michel Caboche & Boulos Chalhoub

  4. Génétique Moléculaire des Céréales, UMR INRA-UBP, Domaine de Crouelle, 234 Avenue du Brézet, 63039, Clermont-Ferrand Cedex 2, France

    Pierre Sourdille & Michel Bernard

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  1. Jaroslav Janda
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  2. Jan Bartoš
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  3. Jan Šafář
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Correspondence to Jaroslav Doležel.

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Communicated by P. Langridge

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Janda, J., Bartoš, J., Šafář, J. et al. Construction of a subgenomic BAC library specific for chromosomes 1D, 4D and 6D of hexaploid wheat. Theor Appl Genet 109, 1337–1345 (2004). https://doi.org/10.1007/s00122-004-1768-8

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