Polymerase chain reaction - Restriction fragment length polymorphisms for assessing and increasing biodiversity of Frankia culture collections

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).