Genetic Diversity of Frankia Microsymbionts in Root Nodules from Colletia hystrix (Clos.) Plants by Sampling at a Small-Scale

Abstract

The genetic diversity of microsymbiont Frankia from Colletia hystrix (Clos.) plants growing in a restricted area, were investigated by PCR-restriction fragment length polymorphism (RFLP) technique. DNA from field-collected nodules was amplified by PCR with primers targeting two genomic regions; one included a large portion of the 3′ end of the 16S rRNA gene, the intergenic spacer, and the 5′ end of the 23S rRNA gene and the other in the nifD–nifK intergenic region in the nif operon as a means to estimate molecular diversity. A HaeIII digestion of the PCR product allowed us to identify PCR-RFLP groups or haplotypes among the Colletia-infective Frankia strains tested. An exhaustive small-scale sampling permitted us to detect haplotypes with a low frequency in the microsymbiont population and showed that Frankia microsymbionts have a higher genetic diversity than previously reported. Fifteen haplotypes were recognized on the basis of combining the restriction patterns in each region analyzed. The haplotype designated as A3 was found with a high frequency in the five microsymbiont Frankia groups studied indicating a dominant haplotype. This haplotype was also exhibited by strain ChI4, which was isolated in 1991 in the same locality suggesting that it is the most common haplotype in this area and very stable over time.