Polymerase chain reaction-restriction fragment length polymorphisms for assessing and increasing biodiversity of Frankia culture collections. Can J Botany

Canadian Journal of Botany (Impact Factor: 1.4). 12/1999; 77(9):1261-1269. DOI: 10.1139/b99-083


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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    • "These clustering have been confirmed by other molecular studies such as intertranscribed spacers (ITS) 16S-23S rRNA (Ghodhbane-Gtari et al., 2010), gyrB (Nouioui et al., 2011) and glnII (Gtari et al., 2004; Nouioui et al., 2011) gene sequence analysis. Due to the slow growth of Frankia strains and the limited funding for maintaining several collections especially those containing unidentified and uncharacterized strains, there is need for reducing the costs of shortages without risk of losing biodiversity (Lumini and Bosco, 1999). As a result of this, the present study tests the efficiency of some genetic fingerprinting and low cost based techniques for worldwide and routine characterization of Frankia isolates. "

    African journal of microbiology research 02/2014; 8(20):2006-2010. DOI:10.5897/AJMR2014.6826 · 0.54 Impact Factor
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    • "Total nucleic acids can be extracted from cells or nodule tissue using the method described by Lumini and Bosco (1999). Polymerase chain reaction (PCR) amplification and restriction enzyme analysis can be performed on the intergenic region found between the nifD and nifK, and nifH and nifD genes (Hahn et al. 1999; Lumini and Bosco 1999; Nalin et al. 1999). Nucleic acid extraction, sequencing and repetitive extragenic palindromic PCR (REP-PCR) fingerprinting using BOX primer, PCR detection of uncultured Frankia and Southern hybridization using digoxigenin labelling are described by Maunuksela et al. (1999). "
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    Canadian Journal of Botany 03/2011; 85(3). DOI:10.1139/B07-017 · 1.40 Impact Factor
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    • "The nifD-nifK IGS was amplified using primers FGPD807-85 (5 -CACTGCTACCGGTCGATGAA-3 ) and FGPK333 -355 (5 -CCGGGCGAAGTGGCT-3 ) which were designed from the conserved regions of the nifD 3 portion and the nifK 5 portion, respectively (Lumini and Bosco, 1999). PCR amplifications were performed in 50 µL "
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    ABSTRACT: Nodule samples from 90A. nepalensis individuals were collected at five sites in the Hengduan Mountains. PCR-RFLP analysis of IGS betweennifD andnifK genes was directly applied to unculturedFrankia strains in the nodules. Sizes of thenifD-nifK IGS amplicons and genetic distance between the RFLP patterns from these samples were noticeably different, indicating significant genetic variation in theFrankia population. There were some nodule samples, which produced more than one PCR fragment, and compound RFLP patterns, indicating thatFrankia strains with different PCR-RFLP patterns coexisted in the same host plant under natural conditions. Among the 29 restriction patterns obtained, 5 patterns were found in more than one population and occurred in the majority of samples, while each of the other 24 patterns were represented by only one or two samples and were endemic to a particular population. From the calculatedGst and UPGMA cluster analysis, genetic diversity ofFrankia strains was inferred to be related to climate and glaciation history in the Hengduan Mountains.
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