LABOULBENIALES - Agentschap voor Natuur en Bos

1. INTRODUCTION1.1. DIFFICULTIES FOR DNA EXTRACTION OF LABOULBENIALESMolecular studies of Laboulbeniales are challenging for the following reasons(WEIR & BLACKWELL, 2001a):The microscopic size of the thalli (length range [35 μm – 2 mm], on average200-300 μm) (SCHELOSKE, 1969; HULDÉN, 1983; SANTAMARÍA, 1998);Thalli require micromanipulation techniques in order to remove them fromtheir hosts;Thalli are heavily melanized and difficult to break open;The outer envelope comprises an adhesive mucilaginous componentinterfering with centrifugation;Recalcitrance to axenic culture.Many different approaches did not succeed in releasing DNA. Differentapproaches were used, most without any success: prolonged boiling of thalli(HANSON, 1992), microwave treatment (GOODWIN & LEE, 1993) and immersion inliquid nitrogen (HAUGLAND et al., 1999). Also direct addition of intact thalli toPCR mastermix was unsuccessful (e.g. pers. comm. DE KESEL, 2009).Beyond this, extra problems arose when working with hosts that are dried orpreserved in ethanol (95 %) (WEIR & BLACKWELL, 2001a).1.2. GENBANK1.2.1. THE GENBANK SEQUENCE DATABAS EDatabases form the basis for most applications in bioinformatics. The GenBanksequence database is an open access, annotated collection of all publiclyavailable DNA sequences. GenBank continues to grow at an exponential rate– in February 2008, there were approximately 82.853.685 sequences inGenBank; in October 2010 this number was already increased to 125.764.384(GENBANK RELEASE NOTES, 2010). This database is part of the InternationalNucleotide Sequence Database Collaboration, which comprises the DDBJ, theEMBL, and GenBank at the National Center for Biotechnology Information(NCBI). These three institutes exchange data on a daily basis.1.2.2. THE SEARCH FOR SEQUEN CES OF LABOULBENIALES/LABOUL-BENIOMYCETES IN GENBANKGenBank only includes 21 representatives of the order of the Laboulbeniales,shown in Table VII below.Of the 27 sequences in GenBank, categorized within the classLaboulbeniomycetes, 19 are the same as with the „Laboulbeniales‟ search.This could be the result of wrongly classified species. However, thisinconsistency can also be the consequence of the two different ways ofclassifying Laboulbeniales. The remaining 8 sequences are shown in Table VIIbelow.P a g e | 32