Mark–recapture cloning: a straightforward and cost-effective cloning method for population genetics of single-copy nuclear DNA sequences in diploids

Authors

  • N. BIERNE,

    1. Génome Populations Interactions Adaptation, UMR 5171, Université Montpellier II — IFREMER — CNRS, Station Méditerranéenne de l’Environnement Littoral, 34200 Sète, France,
    Search for more papers by this author
  • A. TANGUY,

    1. Equipe Evolution & Génétique des Populations Marines, UMR 7144, UPMC — CNRS, Station biologique de Roscoff, BP. 74, Place Georges Teissier, 29682 Roscoff, France,
    Search for more papers by this author
  • M. FAURE,

    1. Génome Populations Interactions Adaptation, UMR 5171, Université Montpellier II — IFREMER — CNRS, Station Méditerranéenne de l’Environnement Littoral, 34200 Sète, France,
    Search for more papers by this author
  • B. FAURE,

    1. Equipe Evolution & Génétique des Populations Marines, UMR 7144, UPMC — CNRS, Station biologique de Roscoff, BP. 74, Place Georges Teissier, 29682 Roscoff, France,
    2. Laboratoire de Génétique et Pathologie, IFREMER, 17390 La Tremblade, France,
    Search for more papers by this author
  • E. DAVID,

    1. Laboratoire des sciences de l’environnement marin, UMR CNRS 6539, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale 29280 Plouzané, France,
    Search for more papers by this author
  • I. BOUTET,

    1. Laboratoire des sciences de l’environnement marin, UMR CNRS 6539, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale 29280 Plouzané, France,
    Search for more papers by this author
  • E. BOON,

    1. Génome Populations Interactions Adaptation, UMR 5171, Université Montpellier II — IFREMER — CNRS, Station Méditerranéenne de l’Environnement Littoral, 34200 Sète, France,
    Search for more papers by this author
  • N. QUERE,

    1. Equipe Evolution & Génétique des Populations Marines, UMR 7144, UPMC — CNRS, Station biologique de Roscoff, BP. 74, Place Georges Teissier, 29682 Roscoff, France,
    Search for more papers by this author
  • S. PLOUVIEZ,

    1. Génome Populations Interactions Adaptation, UMR 5171, Université Montpellier II — IFREMER — CNRS, Station Méditerranéenne de l’Environnement Littoral, 34200 Sète, France,
    2. Equipe Evolution & Génétique des Populations Marines, UMR 7144, UPMC — CNRS, Station biologique de Roscoff, BP. 74, Place Georges Teissier, 29682 Roscoff, France,
    Search for more papers by this author
  • P. KEMPPAINEN,

    1. Department of Marine Ecology, Tjärnö Marine Biological Laboratory, 45296 Strömstad, Sweden,
    Search for more papers by this author
  • D. JOLLIVET,

    1. Equipe Evolution & Génétique des Populations Marines, UMR 7144, UPMC — CNRS, Station biologique de Roscoff, BP. 74, Place Georges Teissier, 29682 Roscoff, France,
    Search for more papers by this author
  • D. MORAGA,

    1. Laboratoire des sciences de l’environnement marin, UMR CNRS 6539, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale 29280 Plouzané, France,
    Search for more papers by this author
  • P. BOUDRY,

    1. Laboratoire de Génétique et Pathologie, IFREMER, 17390 La Tremblade, France,
    Search for more papers by this author
  • P. DAVID

    1. Centre d’Ecologie Fonctionnelle et Evolutive — CNRS, 34293 Montpellier cedex 5, France
    Search for more papers by this author

Nicolas Bierne, Fax: +33 (0)467463399; E-mail: n-bierne@university-montp2.fr

Abstract

We describe a simple protocol to reduce the number of cloning reactions of nuclear DNA sequences in population genetic studies of diploid organisms. Cloning is a necessary step to obtain correct haplotypes in such organisms, and, while traditional methods are efficient at cloning together many genes of a single individual, population geneticists rather need to clone the same locus in many individuals. Our method consists of marking individual sequences during the polymerase chain reaction (PCR) using 5′-tailed primers with small polynucleotide tags. PCR products are mixed together before the cloning reaction and clones are sequenced with universal plasmid primers. The individual from which a sequence comes from is identified by the tag sequences upstream of each initial primer. We called our protocol mark–recapture (MR) cloning. We present results from 57 experiments of MR cloning conducted in four distinct laboratories using nuclear loci of various lengths in different invertebrate species. Rate of capture (proportion of individuals for which one or more sequences were retrieved) and multiple capture (proportion of individuals for which two or more sequences were retrieved) empirically obtained are described. We estimated that MR cloning allowed reducing costs by up to 70% when compared to conventional individual-based cloning. However, we recommend to adjust the mark:recapture ratio in order to obtain multiple sequences from the same individual and circumvent inherent technical artefacts of PCR, cloning and sequencing. We argue that MR cloning is a valid and reliable high-throughput method, providing the number of sequences exceeds the number of individuals initially amplified.

Ancillary