Intraspecific variations of Dekkera/Brettanomyces bruxellensis genome studied by capillary electrophoresis separation of the intron splice site profiles

In enology, “Brett” character refers to the wine spoilage caused by the yeast Dekkera/Brettanomyces bruxellensisand its production of volatile phenolic off-flavours. However, the spoilage potential of this yeast is straindependent.Therefore, a rapid and reliable recognition at the strain level is a key point to avoid serious economiclosses. The present work provides an operative tool to assess the genetic intraspecific variation in this speciesthrough the use of introns as molecular targets. Firstly, the available partial D./B. bruxellensis genome sequencewas investigated in order to build primers annealing to introns 5′ splice site sequence (ISS). This analysis allowedthe detection of a non-random vocabulary flanking the site and, exploiting this feature, the creation of specificprobes for strain discrimination. Secondly, the separation of the intron splice site PCR fragments was obtainedthroughout the set up of a capillary electrophoresis protocol, giving a 94% repeatability threshold in our experimentalconditions. The comparison of results obtained with ISS-PCR/CE versus the ones performed by mtDNARFLP revealed that the former protocol is more discriminating and allowed a reliable identification at strainlevel. Actually sixty D./B. bruxellensis isolates were recognised as unique strains, showing a level of similaritybelow 79% and confirming the high genetic polymorphism existing within the species. Two main clusterswere grouped at similarity levels of about 46% and 47%, respectively, showing a poor correlation with thegeographic area of isolation. Moreover, from the evolutionary point of view, the proposed technique coulddetermine the frequency of the genome rearrangements that can occur in D./B. bruxellesis populations.