Mushrooms can reproduce asexually (vegetative), which is the predominant form, and sexually. The fruit body of many fungal species is made up of a stem, cap and gills of which the cap contains the conidia that play a role in the reproduction. The mushroom itself represents a fruit body of the fungus and the major part of the organism is generally found below the surface (the mycelium). 10, 11 Fungi are categorized in different groups and identification of fungi serves many functions. Mushrooms are found in all possible ecosystems and the phylum contains many complex and highly advanced species that feed on predominantly dead organisms. Based on the observed variation in the LSU region, a phylogenetic tree can be readily established, see for instance Liu et al. 9 The amplified gene region is approximately 850 bp. 8 For this reason, the LSU is now more often used in the DNA barcoding of fungi. 7 However, recent research has shown that sequencing only the nrITS marker is not always sufficient for accurate species identification and therefore a combination of DNA sequencing of different markers is advised. The major advantage of nrITS barcoding is the use of well-validated primer sequences, detectability due to the large number of copies of the rRNA clusters and appropriate sequence variation in the nrITS genes between related organisms. 4- 6 The nrITS region of the rRNA gene cluster is the most commonly used target to identify fungi, which comprises a region of 600 bp. 3 To discriminate species, the nuclear internal transcribed spacer (nrITS) and the 28S nuclear ribosomal large subunit (LSU) rRNA marker sequences are generally used. 1, 2 To identify mushrooms, DNA barcoding is seen as one of the most powerful tools since identification based on morphology is not always sufficient. It was first developed by Paul Hebert in 2003 for butterflies, and in 2008, a consortium of institutions joined forces under the name iBOL to start the ambitious task of building reference libraries for barcodes of all life on earth. In addition, the results can be compared to published fungal phylogenetic trees.ĭNA barcoding is a molecular methodology that identifies species using short genetic markers. The method described is very robust, reagents and equipment are readily available, and costs are relatively low. In case of collecting DNA barcodes of an earlier sequenced species, students could upload the data to a repository established for facilitation of future research projects. Students aligned and analyzed the sequences using BLAST and Geneious and subsequently created a phylogenetic tree. Next, DNA extractions and quantitation were performed, PCR amplification was done, and samples were sent out for Sanger sequencing.
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First, fieldwork in the Netherlands was carried out, during which students collected mushrooms under supervision of a professional mycologist with the goal to (a) verify morphologically based identifications with a molecular method and (b) assess phylogenetic relationships of the different species collected. Students perform this project in 6–8 weeks, which also includes preparing a poster, writing a report and presenting a paper related to the work in a journal club format. Here, we present an example of DNA barcoding by sequencing a segment of the 28S nuclear ribosomal large subunit rRNA gene of wild mushrooms and framing the education in a project form for undergraduate students in biology. DNA barcoding is an important molecular methodology for species identification that was developed over the last two decades and it should be covered in the biology bachelor curriculum.
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