Development of an affordable typing method for Meyerozyma guilliermondii using microsatellite markers

• Microsatellite markers have been analyzed for the first time in Meyerozyma guilliermondii.
• A single typing method has been developed for intra-specific discrimination in Meyerozyma guilliermondii species.
• High reproducibility and specificity of the microsatellite tool for M. guilliermondii.
• Three microsatellite markers were sufficient to produce an intra-specific differentiation.
• One microsatellite-marker amplified M. caribbica.

Despite previously published methods, there is still a lack of rapid and affordable methods for genotyping the Meyerozyma guilliermondii yeast species. The development of microsatellite markers is a useful genotyping method in several yeast species. Using the Tandem Repeat Finder Software, a total of 19 microsatellite motifs (di-, tri-, and tetra- repetition) were found in silico in seven of the nine scaffolds published so far. Primer pairs were designed for all of them, although only four were used in this work. All microsatellite amplifications showed size polymorphism, and the results were identical when repeated. The combination of three microsatellite markers (sc15F/R, sc32 F/R and sc72 F/R) produced a different pattern for each of the Type Culture Collection strains of M. guilliermondii used to optimize the method. The three primer pairs can be used in the same PCR reaction, which reduces costs, in tandem with the fluorescent labeling of only the forward primer in each primer pair. Microsatellite typing was applied on 40 more M. guilliermondii strains. The results showed that no pattern is repeated between the different environmental niches. Four M. guilliermondii strains were only amplified with primer pair sc32 F/R, and subsequently identified as Meyerozyma caribbica by Taq I-RFLP of the 5.8S ITS rDNA. Most out-group species gave negative results even for physiologically similarly species such as Debaryomyces hansenii. The microsatellite markers used in this work were stable over time, which enables their use as a traceability tool.