Computational protocol: Scleroderma areolatum ectomycorrhiza on Fagus sylvatica L.

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Protocol publication

[…] Characterization of ectomycorrhizae using a molecular approach was based on PCR amplification and sequencing of the complete internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (Gardes and Bruns ). DNA extraction was performed with a DNeasy Plant Mini Kit (Qiagen, Germany), and the ITS region was amplified with the ITS 1f and ITS 4 primer pair (Gardes and Bruns ). After separation and excision of the amplified DNA from the agarose gel and purification of the amplified fragments with Wizard SV Gel and PCR CleanUp System (Promega), sequencing was performed at a commercial sequencing laboratory (Macrogen Inc., Seoul, South Korea). Species, genus or family of ectomycorrhizal fungi were determined by comparing the sequence with the GenBank (http://www.ncbi.nlm.nih.gov) and UNITE (Abarenkov et al. ) databases. Phylogenetic position of the Scleroderma ectomycorrhizae was assessed by comparison to selected Scleroderma sequences from the International Nucleotide Sequence Databases using the stand-alone freeware version of the MAFFT programme (http://align.bmr.kyushu-u.ac.jp/mafft/software/) with the E-INS-i aligning strategy (Katoh et al. ). The MEGA 6.06 package was used for a maximum parsimony phylogenetic reconstruction, with a close-neighbour-interchange on random trees maximum parsimony search method using all sites and 2000 bootstrap replications. The maximum likelihood analysis is based on 1000 bootstrap replicates using a Tamura 3-parameter model with a gamma distributed rates among sites with invariant sites with all sites used. Phylogenetic trees were drawn in MEGA 6.06 and edited in InkScape 0.91. […]

Pipeline specifications

Software tools MAFFT, Inkscape
Applications Miscellaneous, Phylogenetics
Diseases Scleroderma, Localized
Chemicals Lactic Acid