Intragenomic heterogeneity in the 16 S r RNA genes of F lavobacterium columnare and standard protocol for genomovar assignment

Genetic variability in 16 S r RNA gene sequences has been demonstrated among isolates of F lavobacterium columnare, and a restriction fragment length polymorphism ( RFLP ) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16 S r RNA gene sequences (ca. 1250‐bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16 S r RNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within H ae III restriction sites. The occurrence of H ae III restriction sites within the portion of the 16 S r RNA gene used for typing the F . columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F . columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II‐ B and III. Additionally, we describe a new genomovar, I/II.