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Polymerase chain reaction – restriction fragment length polymorphisms for assessing and increasing biodiversity of Frankia culture collections

Canadian Journal of Botany (Impact Factor: 1.4). 12/1999; 77:1261-1269. DOI: 10.1139/b99-083

ABSTRACT During the last few years, some Frankia culture collections that maintained a large number of unidentified and uncharacterized Frankia strains were closed because of funding shortages. To reduce the costs of maintenance, we evaluated the biodiversity of half of the Frankia strains from our collection, by polymerase chain reaction – restriction fragment length polymorphisms (PCR-RFLPs) of nifD–nifK intergenic spacer and 16S–23S rDNA intergenic spacer regions. In this way we were able to reduce the number of strains without reducing the biodiversity of the whole collection. In general the nifD–nifK target proved to be more polymorphic than the rrn target. From 51 isolates of Elaeagnus frankiae, PCR-RFLP results allowed us to detect 13 identical strains, and to predict that the genomic species P8 of Akimov and Dobritsa (1992) very likely agrees with genomic species 5 of Fernandez et al. (1989). Moreover, we revealed genomic groups not yet described, as well as intraspecific variability. For Alnus frankiae, the polymorphisms shown by both the nif and the rrn PCR-RFLPs revealed three host plant species-specific subgroups inside Frankia alni. An expandable data base was created to serve as reference for future biodiversity evaluations on both culture collections and unisolated Frankia populations. It will be accessible by Internet at the International Frankia Website (http://www.unifi.it/unifi/distam/frankia/international.html). Résumé : Ces dernières années, plusieurs collection de cultures de Frankia contenant un grand nombre de souches non identifiées et non caracterisées ont été abandonnées par manque de fonds. Dans le but d'obtenir une réduction des coûts de maintien de notre collection, nous avons évalué la biodiversité existante dans la moitié des nos souches de Frankia. En analysant par la méthode réaction en chaîne de la polymérase – polymorphismes de la longeur des fragments de restriction (PCR-RFPLs) les régions intergéniques nifD–nifK et rrn 16S–23S, nous avons pu reduire le nombre de souches en culture, sans réduire la biodiversité de la collection. En général, l'étude de la région nifD–nifK a donné plus de polymorphisme que celle de la région ribosomale. Entre les 51 Frankia isolés d'Elaeagnus, on a pu détecter 13 souches identiques et montrer que les espèces génomiques P8 de Akimov et Dobritsa (1992) et 5 de Fernandez et al. (1989) pourraient être une même espèce. En plus, on a détecté des groupes génomiques pas encore décrits et une certaine diversité intra-spécifique. Pour les souches isolées d'Alnus, les polymorphismes montrés par les deux regions génomiques ont permis de mettre en evidence, chez espèce Frankia alni, trois sous-groupes spécifiques pour chaque espèce hôte. Pour faciliter les travaux futurs sur la biodiversité des Frankia cultivés ou des populations non isolées, on a construit une banque de profils PCR-RFLP qui sera accessible via l'Internet dans le site Web International sur Frankia (http:

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