Descripción de puestos de trabajo: Funciones y responsabilidades

4.2.4 DNA extraction and amplification).

4.2.2

Herbarium collection analysis

All species of the Resupinateae possess many taxonomic characters that are useful in identification. Among the most important characters for use in identification are the colour, size and shape of the fruit body; the colour, shape (including ornamentation) and size of surface hairs; the presence, colour and thickness of a gelatinous layer in a vertical

section of the fruit body tissues; basidiospore shape, size and ornamentation; the size and shape of basidia and the presence, shape and size of any other characteristic cells in the hymenium such as cheilocystidia, pleurocystidia, or metuloids.

Notes and measurements were taken of dried collections for fruit body size (in m), colour and shape; location of attachment to the substrate; presence and distribution of surface hairs; and presence or absence of a subiculum or subiculum-like structure. Small portions of fruit bodies of dried collections were rehydrated, and changes in colour of the fruit body and surface hairs were noted (often, fruit bodies are much darker in colour with surface hairs more visible when dry than when fresh). Photographs of the dried

collections and rehydrated portions were taken, then the rehydrated portions of the fruit body were sectioned by hand using a razor blade and squash-mounted on a microscope slide.

Two mounts were made on each microscope slide, in different mountants to emphasize different microscopic characters (Melzer’s reagent for spore, surface hair, and hyphal characteristics, and KOH for basidia and cystidia characteristics; Largent, 1977). Basidiospore, basidium, cystidia and surface hair measurements are represented as a range, with values in parentheses representing the smallest and largest values, and the size range representing values between the tenth and ninetieth percentiles (as per Thorn and Barron, 1986). Other measurements (for example the diameter of hyphae, or the length and width of sterigmata) are represented as a range of values, the smallest and largest observed. A full description of microscopic methods can be found in Chapter 2, section 2.2.2.

4.2.3

Media and cultures

Cultures were obtained from the ICMP culture collection in New Zealand and were maintained in 60 mm Petri dishes of Malt Extract Agar (MEA, 12.5 g malt extract and 15 g agar per liter distilled water; Nobles 1965) and Potato Dextrose Agar (PDA, EMD Chemicals, Inc.). Liquid cultures for DNA extraction were made in malt extract broth (5 g malt extract per liter distilled water). These cultures were allowed to grow to produce

enough biomass for harvesting for DNA extraction (see section 4.2.4 DNA extraction and amplification).

4.2.4

DNA extraction and amplification

DNA was extracted from dried herbarium collections and fresh collections in the same way. Portions of the fruit body were placed on a microscope slide and rehydrated in sterile distilled water. Once the small portions were rehydrated, they were finely chopped using a sterilized razor blade. Fifty milligrams of tissue was put into a micro-bead tube from the PowerLyzer UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, California), and processed according to the manufacturer’s protocol. DNA was extracted from liquid cultures using the same kit with the following modifications. Mycelium was removed from liquid medium, excess media removed and 50 mg transferred into the micro-bead tube. DNA extracts were quantified using a Nanodrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, Delaware) and kept at 4 °C (overnight) or at -20 °C (for extended periods).

The DNA extract was amplified using a PCR protocol in a Biometra T1 Thermocycler (Montreal Biotech) according to Koziak et al. (2007) and the fungal primers ITS1 (5’— TCCGTAGGTGAACCTGCGG—3’; White et al., 1990) and LR5 (5’—

ATCCTGAGGGAAACTTC—3’; Vilgalys and Hester, 1990). This primer pair amplifies ribosomal DNA from the 3’ end of the small ribosomal subunit (SSU) through the ITS1, 5.8S, ITS2, and the 5’ end of the large ribosomal subunit (LSU; including the D1/D2 variable domains). Presence or absence of a PCR product was determined using gel electrophoresis in a 1.5% Agar gel made with TAE electrophoresis buffer containing 0.5 µg/mL ethidium bromide.

Once the presence of the desired size of PCR product was confirmed, the PCR products were cleaned using the QIAquick PCR purification kit (Qiagen, Mississauga, Ontario) or the BioBasic PCR purification kit (Bio Basic Canada Inc., Markham, Ontario). Each DNA extract was amplified in four PCR reactions of 30 µL to ensure a large amount of PCR product was obtained, and these four PCR tubes were pooled into one cleaned PCR

product. The cleaned PCR product was quantified on the Nanodrop 2000 machine and sent for sequencing.

4.2.5

Sequencing of PCR products

Cleaned PCR products were sent for sequencing at the Robarts Research Insitute at Western University using sequencing primers ITS1, LS1 (5’—

ACTACCCGCTGAACTTAAG—3’; Hausner et al., 1993) and LR3R (5’—

GTCTTGAAACACGGACC—3’; Vilgalys and Hester, 1990) on the coding strand and LR5, LR3 (5’—GGTCCGTGTTTCAAGAC—3’; Vilgalys and Hester, 1990) and LS1R (5’—CTTAAGTTCAGCGGGTA—3’; Hausner et al., 1993) on the complementary strand. Once all six regions were sequenced, the sequences were cleaned and assembled in SeqEd v1.03 (Applied Biosystems Software, Foster City, California) for phylogenetic analysis.

4.2.6

Phylogenetic analysis

The sequences generated in this study were aligned with those already available in GenBank (see Appendix B for a full list of sequences used throughout this thesis) using MEGA5 v5.05 or MEGA6 v6.01 for Mac (Tamura et al., 2011 & 2013). When

applicable, alignments were edited to improve the alignment. Species of Calyptella and Hemimycena were chosen to root the tree, as per Thorn et al. (2005). The ingroup used to construct the tree consisted of 45 sequences of 25 different taxa, resulting in an alignment of 1875 characters. The dataset was trimmed to 1213 characters so all sequences were of the same length in the alignment, and the resulting alignment file was exported for further analysis.

Phylogenetic analyses according to maximum likelihood methods were performed in MEGA5/6. Node support was assessed using bootstrapping, with 1000 replicates. The tree was generated using the combined dataset of ITS and LSU sequences and is presented in Figure 4.3.

4.3

Results