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Toward a Pinus pinaster bacterial artificial chromosome library

Construction d’une banque BAC pour le pin maritime (Pinus pinaster)

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Annals of Forest Science Aims and scope

Abstract

Conifers are of great economic and ecological importance, but little is known concerning their genomic organization. This study is an attempt to obtain high-quality high-molecular-weight DNA from Pinus pinaster cotyledons and the construction of a pine BAC library. The preparation incorporates modifications like low centrifugation speeds, increase of EDTA concentration for plug maintenance, use of DNase inhibitors to reduce DNA degradation, use of polyvinylpyrrolidone and ascorbate to avoid secondary metabolites, and a brief electrophoresis of the plugs prior to their use. A total of 72 192 clones with an average insert size of 107 kb, which represents an equivalent of 11X pine haploid genomes, were obtained. The proportions of clones lacking inserts or containing chloroplast DNA are both approximately 1.6%. The library was screened with cDNA probes for seven genes, and two clones containing Fd-GOGAT sequences were found, one of them seemingly functional. Ongoing projects aimed at constructing a pine bacterial artificial chromosome library may benefit from the methods described here.

Résumé

Les conifères présentent un intérêt économique et écologique de premier plan mais restent très mal connus du point de vue de l’organisation de leur génome. Cette étude présente une tentative réussie de construction d’une banque BAC de séquences d’ADN de haute qualité et de poids moléculaire élevé à partir de cotylédons de Pinus pinaster. Le protocole de préparation se base sur des ajustements comme une baisse de la vitesse de centrifugation, une augmentation des concentrations d’EDTA dans les culots, l’utilisation d’inhibiteurs des ADNases pour limiter la dégradation de l’ADN, l’utilisation de polyvinylpyrrolidone et d’ascorbate pour éliminer les métabolites secondaires, et de brèves électrophorèses des culots. Un total de 72 192 clones a été obtenu, d’une dimension moyenne d’inserts de 107 kb et représentant l’équivalent de 11X du génome haploïde de pin. La proportion de clones dépourvus d’inserts ou contenant de l’ADN chloroplastique était de 1.6%. La banque a été testée avec des ADN complémentaires de 7 gènes, et deux clones contenant la séquence de la Fd-GOGAT ont été détectés. Des projets visant à construire une banque bactérienne artificielle (BAC) de chromosome de pin tireront bénéfice de l’utilisation de cette méthode.

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Authors and Affiliations

  1. Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias & Institute Andaluz de Biotecnología, Universidad de Málaga, 29071, Málaga, Spain

    Rocío Bautista, David P. Villalobos, Sara Díaz-Moreno, Francisco R. Cantón, Francisco M. Cánovas & M. Gonzalo Claros

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  1. Rocío Bautista
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  2. David P. Villalobos
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  3. Sara Díaz-Moreno
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  4. Francisco R. Cantón
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  6. M. Gonzalo Claros
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Correspondence to M. Gonzalo Claros.

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Bautista, R., Villalobos, D.P., Díaz-Moreno, S. et al. Toward a Pinus pinaster bacterial artificial chromosome library. Ann. For. Sci. 64, 855–864 (2007). https://doi.org/10.1051/forest:2007060

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