Polymorphism of rDNA fragments identified by PCR-RFLP method in yeast isolates from cheeses.
Malgorzata Robak, Xymena Polomska, Michal Piegza, Wojciech Barszczewski, Maria Wojtatowicz
Biotechnol.&Food Microbiol., Agricultural University, Norwida 25, Wroclaw, 50-375, Poland
The rRNA genes represent the most widely used sequences to study phylogenetic relationships of fungal taxa as well as for yeast identification at the species level. Gene sequencing is an inappropriate tool for the analysis of the yeast biota, because it is an expensive, as well as labour and time-consuming procedure. PCR-RFLP analysis of internal transcribed spacer sequences (ITS1 and ITS2) appears to be a useful and appropriate method for the correct characterization of yeast strains used in food processing. The aim of this work was to study the polimorphism of rRNA genes by the application of PCR-RFLP of rDNA with NS3 and ITS4 primers and MspI, HaeIII and ScrFI restriction enzymes. Tested yeast strains belonged to three species: Yarrowia lipolytica, Debaryomyces hansenii and Geotrichum candidum. They were isolated from different soft and semi-soft cheeses. Y. lipolytica and D. hansenii strains demonstrated high level of polymorphism in the length of amplified regions. After restriction with HaeIII all tested Y. lipolytica isolates showed similar pattern while strains of D. hansenii gave three types of patterns. All G. candidum strains gave similar size of PCR products and profiles with enzyme Msp I. However, their patterns with HaeIII and ScrFI enzymes were much more diverse. Two G. candidum strains revealed similar patterns after restriction with HaeIII enzyme but completely different after using ScrFI.