Monthly Archives: December 2014

ITS-PCR of WLP644 Brettanomyces bruxellensis trois

A couple of days ago, Omega Yeast Labs posted an update on their facebook page, where they stated that they’ve done 5.5S rDNA sequencing on White Labs’ WLP644 Brettanomyces bruxellensis trois strain, and results showed that it was actually a strain of Saccharomyces cerevisiae. Since I have some pure cultures of the yeast strain myself, and am currently doing lots of ITS-PCR for work-related things, I decided to test WLP644 myself. Note, I haven’t done any sequencing, just amplification of rDNA (ITS1, 5.8S and ITS2) using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), and then digestion of amplicons using the HaeIII restriction enzyme. The amplicon size of Saccharomyces yeasts is 840-880 bp, while the amplicon size of Brettanomyces yeasts is around 500 bp. Digestion of the amplicon with HaeIII results in a four-band pattern for Saccharomyces cerevisiae (320, 225, 180, 140 bp), and a two-band pattern for Brettanomyces bruxellensis (380, 100 bp). Anyways here is my gel:


First lane 100 bp ladder, second lane WLP644 amplicon, third lane HaeIII-digested WLP644 amplicon, fourth lane S. cerevisiae A62 amplicon, fifth lane HaeIII-digested S. cerevisiae A62 amplicon, sixth lane negative control, seventh lane 100 bp ladder. I unfortunately didn’t include any positive control for Brettanomyces (500 bp amplicon; 380 and 100 bp HaeIII-digested fragments), as I didn’t have any easily and rapidly accessible, but the results should still speak for themselves. It seems very likely that WLP644 is in fact a strain of Saccharomyces (cerevisiae) and not a Brettanomyces yeast (or at least contains Saccharomyces yeast in case WLP644 is a blend, and the single colony I’ve purified and extracted DNA from was Saccharomyces). This is basically a confirmation of Omega Yeast Labs’ results.

Edit: For more (great) information, check out these posts on Sui Generis and Embrace the Funk!

Pham T, Wimalasena T, Box W, Koivuranta K, Storgårds E, Smart K & Gibson B (2011) Evaluation of ITS PCR and RFLP for differentiation and identification of brewing yeast and brewery ‘wild’ yeast contaminants. J Inst Brew 117: 556-568. Full-text available here.