RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM GROUPS AND PHYSICAL MAP OF MITOCHONDRIAL-DNA FROM FUSARIUM-OXYSPORUM F SP-NIVEUM

Variation in Fusarium oxysporum f. sp. niveum was examined by identifying restriction fragment length polymorphisms (RFLPs) in mitochondrial DNA (mtDNA). Six RFLP groups (RFLPG) were detected among 50 pathogenic isolates by Southern hybridization with cloned PstI mtDNA fragment probes (pFON2a [9.1 kb]-pFON8b [1.0 kb]). A restriction map of mtDNA from a race 0 isolate (FL-60-3A) was constructed by digestion of the mtDNA with four restriction endonucleases: EcoRI, HindIII, HpaI, and PstI, either singly or in selected pairs, as well as by identifying bands in each digestion pattern with each of the pFON clones. Intra-mtDNA sequence homology was revealed by hybridization between the PstI fragment pFON7 (1.5 kb) and two other areas of the mtDNA: pFON2a (9.1 kb) and pFON6 (2.0 kb). Most of the fragment differences that discriminated RFLP groups were detected by the pFON7 probe. Relatedness between RFLP groups was analyzed by the unweighted paired group method using arithmetic means with Jaccard's similarity matrix based on the presence or absence of hybridization bands. The cophenetic correlation coefficient in the dendrogram generated was 0.997. RFLPG I was the most common group containing 34 isolates and was clustered to RFLPG IV with a similarity of 90%. RFLPG II was the second most common group containing seven of the isolates and was clustered to RFLPG III with a similarity of 90% resulting in two main clusters. Both main clusters were grouped to each other with a similarity of 84%. RFLPG VI was the most distant from the other RFLP groups with a similarity of 65%, whereas RFLPG V was intermediate to the other four RFLPGs (I, II, III, and IV) with a similarity of 75%. Two nonpathogenic isolates (SC-A[87] and OK-256[87]) were outgrouped from all pathogenic isolates with a similarity of only 21 and 8%, respectively. The RFLP groups appeared primarily to be a result of base substitutions and/or length mutations within the mtDNA; however, a heteroplasmy of mtDNA was also detected in RFLPG IV. The relationship between RFLP groups of the pathogenic isolates was analyzed by simple empirical analysis to produce an unrooted most parsimonious gene tree with the fewest possible stepwise changes. In the gene tree produced, RFLPG II was located at the center with the fewest numbers of steps to all other groups. RFLPG V was the most distant group and required the most steps (four) to get to RFLPG II.