title: (GTG)5 MSP-PCR fingerprinting as a technique for discrimination of wine associated yeasts?
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.
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Campus do Vale, Porto Alegre, Brazil; Departamento de Microbiologia, Imunologia e Parasitologia, ICBS, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, Porto Alegre, Brazil.
authors.forename: Mauricio
authors.initials: M
authors.lastname: Ramírez-Castrillón
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Campus do Vale, Porto Alegre, Brazil; Departamento de Microbiologia, Imunologia e Parasitologia, ICBS, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, Porto Alegre, Brazil; Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina, Laboratório de Análises de Vinhos e Derivados, Estação Experimental de Videira, Campo Experimental, Videira, Brazil.
authors.forename: Sandra Denise Camargo
authors.initials: SD
authors.lastname: Mendes
authors.affiliation: Departamento de Ingeniería Informática, Universidad de Santiago de Chile, Santiago de Chile, Chile.
authors.forename: Mario
authors.initials: M
authors.lastname: Inostroza-Ponta
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, ICBS, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, Porto Alegre, Brazil.
authors.forename: Patricia
authors.initials: P
authors.lastname: Valente
mesh_terms: D016000:Cluster Analysis; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D014644:Genetic Variation; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000235:genetics; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014920:Wine / Q000382:microbiology*; D015003:Yeasts / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D012335:RNA, Ribosomal