• Open Access

Optimizing TILLING populations for reverse genetics in Medicago truncatula

Authors

  • Christine Le Signor,

    1. Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses à Graines (UMR-LEG), Institut National de la Recherche Agronomique (INRA), BP 86510, F-21065 Dijon, France
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  • Vincent Savois,

    1. Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses à Graines (UMR-LEG), Institut National de la Recherche Agronomique (INRA), BP 86510, F-21065 Dijon, France
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  • Grégoire Aubert,

    1. Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses à Graines (UMR-LEG), Institut National de la Recherche Agronomique (INRA), BP 86510, F-21065 Dijon, France
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  • Jérôme Verdier,

    1. Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses à Graines (UMR-LEG), Institut National de la Recherche Agronomique (INRA), BP 86510, F-21065 Dijon, France
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  • Marie Nicolas,

    1. Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses à Graines (UMR-LEG), Institut National de la Recherche Agronomique (INRA), BP 86510, F-21065 Dijon, France
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  • Gaelle Pagny,

    1. Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses à Graines (UMR-LEG), Institut National de la Recherche Agronomique (INRA), BP 86510, F-21065 Dijon, France
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  • Françoise Moussy,

    1. Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses à Graines (UMR-LEG), Institut National de la Recherche Agronomique (INRA), BP 86510, F-21065 Dijon, France
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  • Myriam Sanchez,

    1. Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses à Graines (UMR-LEG), Institut National de la Recherche Agronomique (INRA), BP 86510, F-21065 Dijon, France
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  • Dave Baker,

    1. John Innes Genome Laboratory, Norwich NR4 7UH, UK
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  • Jonathan Clarke,

    1. John Innes Genome Laboratory, Norwich NR4 7UH, UK
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  • Richard Thompson

    Corresponding author
    1. Unité Mixte de Recherche en Génétique et Ecophysiologie des Légumineuses à Graines (UMR-LEG), Institut National de la Recherche Agronomique (INRA), BP 86510, F-21065 Dijon, France
      * Correspondence (fax +33-380-693-263; e-mail thompson@dijon.inra.fr)
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* Correspondence (fax +33-380-693-263; e-mail thompson@dijon.inra.fr)

Summary

Medicago truncatula has been widely adopted as a model plant for crop legume species of the Vicieae. Despite the availability of transformation and regeneration protocols, there are currently limited tools available in this species for the systematic investigation of gene function. Within the framework of the European Grain Legumes Integrated Project (http://www.eugrainlegumes.org), chemical mutagenesis was applied to M. truncatula to create two mutant populations that were used to establish a TILLING (targeting induced local lesions in genomes) platform and a phenotypic database, allowing both reverse and forward genetics screens. Both populations had the same M2 line number, but differed in their M1 population size: population 1 was derived from a small M1 population (one-tenth the size of the M2 generation), whereas population 2 was generated by single seed descent and therefore has M1 and M2 generations of equal size. Fifty-six targets were screened, 10 on both populations, and 546 point mutations were identified. Population 2 had a mutation frequency of 1/485 kb, twice that of population 1. The strategy used to generate population 2 is more efficient than that used to generate population 1, with regard to mutagenesis density and mutation recovery. However, the design of population 1 allowed us to estimate the genetically effective cell number to be three in M. truncatula. Phenotyping data to help forward screenings are publicly available, as well as a web tool for ordering seeds at http://www.inra.fr/legumbase

Ancillary