Sequence analysis and ploidy determination of Cryptococcus neoformans var. neoformans

Purpose. The aim of the present study was to clarify the relation between serotype A, D and AD isolates of Cryptococcus neoformans var. neoformans. Materials and methods. Thirty-four isolates of C. neoformans var. neoformans were serotyped by Crypto Check (Iatron Labs., Japan) and genotyped by CR fingerprinting with (GACA)(4) primer, and their DNA content was determined by flow cytometry. In addition, 14 strains were selected to establish the nucleotide sequences of four PCR-fingerprinting major bands (420, 475, 540, and 800 bp). Results. By PCR-fingerprinting analysis the 34 strains were subdivided into three groups: 11 serotype D strains were identified as genotype VN1, seven serotype A strains as genotype VN6, and 16 serotype A, D or AD isolates as an intermediate group, including VN3 and VN4 genotypes, in which different combinations of the major bands characteristic of the VN1 and VN6 genotypes were present. The 540 bp band was observed in all the strains selected for sequencing. Sequence analysis of this band from four strains of the intermediate group demonstrated points of heterozygosity at 27 sites where VN1 and VN6 sequences differed. Flow cytometry revealed that all VN6 and all but one VN1 strains were haploid, like the reference strains NIH B3501 and NIH B3502. On the contrary, VN3 and VN4 isolates had a double DNA content. The results of this study suggest that VN3 and VN4 isolates are diploid.