title: Intraspecific variations of Dekkera/Brettanomyces bruxellensis genome studied by capillary electrophoresis separation of the intron splice site profiles.
abstract: In enology, "Brett" character refers to the wine spoilage caused by the yeast Dekkera/Brettanomyces bruxellensis and its production of volatile phenolic off-flavours. However, the spoilage potential of this yeast is strain-dependent. Therefore, a rapid and reliable recognition at the strain level is a key point to avoid serious economic losses. The present work provides an operative tool to assess the genetic intraspecific variation in this species through the use of introns as molecular targets. Firstly, the available partial D./B. bruxellensis genome sequence was investigated in order to build primers annealing to introns 5' splice site sequence (ISS). This analysis allowed the detection of a non-random vocabulary flanking the site and, exploiting this feature, the creation of specific probes for strain discrimination. Secondly, the separation of the intron splice site PCR fragments was obtained throughout the set up of a capillary electrophoresis protocol, giving a 94% repeatability threshold in our experimental conditions. The comparison of results obtained with ISS-PCR/CE versus the ones performed by mtDNA RFLP revealed that the former protocol is more discriminating and allowed a reliable identification at strain level. Actually sixty D./B. bruxellensis isolates were recognised as unique strains, showing a level of similarity below 79% and confirming the high genetic polymorphism existing within the species. Two main clusters were grouped at similarity levels of about 46% and 47%, respectively, showing a poor correlation with the geographic area of isolation. Moreover, from the evolutionary point of view, the proposed technique could determine the frequency of the genome rearrangements that can occur in D./B. bruxellesis populations.
authors.affiliation: Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi di Milano, via Celoria 2, 20133, Milan, Italy.
authors.forename: Ileana
authors.initials: I
authors.lastname: Vigentini
authors.forename: Gabriella
authors.initials: G
authors.lastname: De Lorenzis
authors.forename: Claudia
authors.initials: C
authors.lastname: Picozzi
authors.forename: Serena
authors.initials: S
authors.lastname: Imazio
authors.forename: Annamaria
authors.initials: A
authors.lastname: Merico
authors.forename: Silvia
authors.initials: S
authors.lastname: Galafassi
authors.forename: Jure
authors.initials: J
authors.lastname: Piškur
authors.forename: Roberto
authors.initials: R
authors.lastname: Foschino
mesh_terms: D055168:Brettanomyces; D017931:DNA Primers / Q000235:genetics; D055167:Dekkera / Q000235:genetics* / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods*; D005506:Food Contamination; D005516:Food Microbiology; D014644:Genetic Variation; D007438:Introns / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D022821:RNA Splice Sites; D012326:RNA Splicing; D015203:Reproducibility of Results; D014920:Wine / Q000032:analysis / Q000382:microbiology*; D015003:Yeasts / Q000235:genetics
chemical_list: D017931:DNA Primers; D022821:RNA Splice Sites