To make sure that I was going down the right path, and to get some insight into what methods Siebel was using in the genetic identification of yeasts, I sent off Wild Yeast 1 on a little agar plate to their lab in Montreal. Looking at the report, it seems they actually outsource their labwork somewhere else, potentially Lallemand.
The results came in a few days ago, and it confirms what I found earlier, “WY1″ is 100% Saccharomyces cerevisiae.
The method applied was sequencing of the domain D1-D2 of the 26s rDNA, as published by Kurtzman CP and Robnett CJ (see FEMS Yeast Res. 2003 Jun;3(4):417-32.) The sequence was then “blasted” agains the NCBI sequence database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Very similar to what I did before, although my focus was on the ITS. The sequencing and PCR of these domains is a real pain because of the repeats. I have only been partially successful in getting good reads focusing on these sequences. rDNA is notorious as well for this, but I have never tried this specific part of the DNA. Worth a shot.
Below the sequence as received from Siebel:
TTAGTAACGGCGAGTGAAGCGGCAAAAGCTCAAATTTGAAATCTGGTACCTTCGGTGCCCGAGTTGTA ATTTGGAGAGGGCAACTTTGGGGCCGTTCCTTGTCTATGTTCCTTGGAACAGGACGTCATAGAGGGTG AGAATCCCGTGTGGCGAGGAGTGCGGTTCTTTGTAAAGTGCCTTCGAAGAGTCGAGTTGTTTGGGAAT GCAGCTCTAAGTGGGTGGTAAATTCCATCTAAAGCTAAATATTGGCGAGAGACCGATAGCGAACAAGT ACAGTGATGGAAAGATGAAAAGAACTTTGAAAAGAGAGTGAAAAAGTACGTGAAATTGTTGAAAGGG AAGGGCATTTGATCAGACATGGTGTTTTGTGCCCTCTGCTCCTTGTGGGTAGGGGAATCTCGCATTTCA CTGGGCCAGCATCAGTTTTGGTGGCAGGATAAATCCATAGGAATGTAGCTTGCCTCGGTAAGTATTAT AGCCTGTGGGAATACTGCCAGCTGGGACTGAGGACTGCGACGTAAGTCAAGGATGCTGGCATAA
A read of 573 nucleotides. A quick Blast indeed revealed 100% S. cerevisiae. Looking at the Genbank sequence it aligns to (GenBank: JQ968601.1) the two primers used to PCR the 26S rDNA are NL1 and NL4 (See O’Donnell, K. 1993. Fusarium and its near relatives, p. 225-233. in The fungal holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematics). A quick search in pubmed resulted in this paper by Joseph Cano and coworkers: Molecular and Morphological Identification of Colletotrichum Species of Clinical Interest, J. Clin. Microbiol. June 2004 vol. 42 no. 6 2450-2454 ) Where they list the primer sequences, and the complete protocol for sequencing and PCR.
So, with this information in hand it should be easy to do genetic profiling in the future.