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Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) had been used for molecular identification of  Sporothrix  spp., which is the causative fungi of sporotrichosis and the most prevalent deep‐seated dermatomycosis. Also, mtDNA‐RFLP had been used to investigate the molecular epidemiology of sporotrichosis. While the current standard for molecular diagnosis is performed by sequence analysis of the calmodulin gene ( CAL ), correspondence between the results from  CAL  and mtDNA is of diagnostic and epidemiological interest. Here, we investigated the correspondence between  CAL  and mtDNA used for molecular identification of  Sporothrix globosa  and  S .  schenckii , which are two major species. We also investigated and propose molecular markers suitable to describe the epidemiology of  S .  globosa , which is considered as a species with few intraspecific polymorphisms. Eighty‐seven strains morphologically identified as  S .  schenckii sensu lato  were investigated. They were identified as group A (17 types, 17 strains) or B (14 types, 70 strains) by mtDNA‐RFLP. Partial sequences of  CAL , internal transcribed spacer, and spacer between atp9 and cox2 genes of mtDNA of these strains were determined. All group A strains corresponded to  S .  schenckii , and group B to  S .  globosa . The sequences of the amplicons targeted on the spacer region in mtDNA of  S .  globosa  ranged 510–515 bp in length and exhibited 10 molecular variations, whereas  CAL  indicated seven molecular variations. In conclusion, most of the  S .  schenckii sensu lato  strains isolated from Japanese sporotrichosis patients were confirmed as  S. globosa , because group B, which comprised the majority of strains, matched perfectly with  S .  globosa  by the  CAL  sequencing study. We proposed sequence variations in the spacer between atp9 and cox2 genes of mtDNA as a suitable molecular epidemiological marker for  S .  globosa .

Genotyping of intraspecies polymorphisms of Sporothrix globosa using partial sequence of mitochondrial DNA