Abstract
In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)5 and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)5 for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA gene, considered as the gold standard. An array matrix was constructed for each dataset and used as input for clustering with two methods (hierarchical dendrograms and QAPGrid layout). For both yeast datasets, unrelated species were clustered in the same group. The sensitivity score of (GTG)5 MSP-PCR fingerprinting was high, but specificity was low. As a conclusion, the yeast diversity inferred in several previous studies may have been underestimated and some isolates were probably misidentified due to the compliance to this screening procedure.
Highlights
Yeast identification is currently based on sequencing of domains 1 and 2 (D1/D2) of the LSU rRNA gene and/or the ITS1-5.8SITS2 region [1], proposed as a universal barcode for fungi in 2011 [2]
The Microsatellite/Minisatellite Primed (MSP)-PCR Fingerprinting technique has been widely applied in the literature using primers as (GAC)5, (GACA)4, (GTG)5 and M13
We found high intra and inter-specific variability in the fingerprint profiles, with clusters comprising isolates belonging to different species, suggesting a high probability of misidentification when MSP-PCR fingerprinting followed by sequencing of representatives of each profile is applied in yeast diversity studies
Summary
Yeast identification is currently based on sequencing of domains 1 and 2 (D1/D2) of the LSU rRNA gene and/or the ITS1-5.8SITS2 region [1], proposed as a universal barcode for fungi in 2011 [2]. Monitoring the contribution of each species or population, both in industrial microbiology or yeast diversity studies, involves the isolation and analysis of a large number of isolates, which makes the identification of all the isolates by sequencing unfeasible in small research laboratories. In this regard, many molecular techniques have been developed to discriminate between different yeast species. Each study reports different DNA amplification protocols, jeopardizing the comparison of genetic profiles, and making it impossible to share genotype databases among laboratories
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