Construction and characterization of a bacterial artificial chromosome library of apple (original) (raw)

Abstract

A bacterial artificial chromosome (BAC) library has been constructed from apple (Malus_×_domestica Borkh.) using the variety “Florina”, which is resistant to scab (Venturia inaequalis) by virtue of the Vf gene. Since apple leaves are rich in polyphenols, high-molecular-weight DNA was extracted from leaf nuclei with a protocol adapted to apple. The nuclei were then embedded in agarose microbeads and partially digested by varying ratios of _Eco_RI to _Eco_RI methylase. The resulting DNA fragments were size-selected by pulsed-field gel electrophoresis, ligated to the BAC cloning vector pECBAC1 and transformed into Escherichia coli cells by electroporation. A total of 36 864 recombinant clones (BACs) were obtained. The library has an average insert size of 120 kb and represents approximately 5×apple haploid-genome equivalents. It was screened with six cDNA probes using the chemiluminescent DIG system. An average of 4.4 clones was detected for each locus. The apple BAC library will be used to isolate the Vf scab resistance gene through map-based cloning. In this connection the library was screened with a marker closely linked to the Vf gene and six positive clones have been isolated. This library should thus be well suited for map-based gene cloning, in particular for the isolation of the Vf gene and for the construction of a physical map of the apple genome.

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Authors and Affiliations

  1. Dipartimento di Colture Arboree, University of Bologna, Via Filippo Rè 6, I-40126 Bologna, Italy Tel.: +39 051 766425 Fax: +39 051 351500 E-mail: vinatzer@agrsci.unibo.it, , , , , , IT
    B. A. Vinatzer & S. Sansavini
  2. Department of Soil and Crop Sciences and Crop Biotechnology Center, Texas A&M University, College Station, TX 77843-2123, USA, , , , , , US
    H.-B. Zhang

Authors

  1. B. A. Vinatzer
  2. H.-B. Zhang
  3. S. Sansavini

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Received: 19 February 1998 / Accepted: 30 April 1998

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Vinatzer, B., Zhang, HB. & Sansavini, S. Construction and characterization of a bacterial artificial chromosome library of apple.Theor Appl Genet 97, 1183–1190 (1998). https://doi.org/10.1007/s001220051008

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