SUBJECT

Molecular Taxonomic and Philogenetic Methods in Mycology

practical

master

Faculty of Science

Biology MSc

3

Semesters 1-4

Autumn/Spring semester

1. Introduction Methods in molecular taxonomy and phylogenetics. The ground of the used laboratory methods (DNA extraction, PCR, RFLP, sequencing). Short overview of the analyses of the data. DNA extraction: different extraction techniques, kits. DNA extraction from frutibody samples.

2. PCR The overview of the specific aspects of polimerase chain reaction (specific primers, loci in fungal taxonomy and phylogenetics etc.). Set the PCR reaction with target DNA extracted on the 1st practice.

3. What to do with the amplicons? ground of agarose gel electophoresis, cloning, RFLP and sequencing. Check of the PCR from the 2nd practice, set of digestion with restriction enzymes. Set of the cycle sequencing reaction.

4. Analyses of the results of 3rd practice. Analysis of the RFLP. Checking and correction of chromatograms of the sequencing reactions. Staden program package.

5. The Staden program package Database, handle of a database with Staden program package. Analyses of sequences (restriction sites, finding vectors, etc.), annotation.

6. First midterm exam. The GenBank 1st midterm. Using the GenBank for mycological works. Search in taxonomy database. BLAST. Annotation. Collecting of data for phylogenetic analyses.

7. Alignments ground of the multiple sequence alignment algorithms. Programs for multiple alignment (ClustalX, MultAlin). Align of the sequences collected on the 6th practice. The effect of the different parameters. Checking and editing of the alignments (Proseq program). Different file formats.

8. Distance based methods Distance calculation with amino acid sequences, matrixes (PAM, Blossum etc.). Models of nucleotide substitution. Problems of distance calculations (multiple hits etc). Distance calculations with different programs (MEGA, Phylip) using different parameters. The question of the gaps.

9. Phylogenetic trees inferred from distances Neighbor-joining, minimum evolution. Phylogenetic inferring with different programs (MEGA, Phylip). Editing of the phylogenetic trees.

10. Parsimony The ground and philosophy of parsimony. Powers and pitfalls. The effects of the different parameters. Inferring parsimony based phylogenies (MEGA, Phylip).

11. Phylogenetic analyses with Maximum Likelihood methods I. The ground of the Maximum Likelihood methods in the phylogenetic inferring. Test of parameters (Modeltest program)

12. Phylogenetic analyses with Maximum Likelihood methods II. ML methods for phylogenetic analyses with the program PhyML. Testing and comparing different phylogenetic hypotheses by ML.

13. Bayesian methods The ground of the Bayesian methods. Bayesian methods in phylogenetic analyses. Monte-Carlo Markov chains (MCMC). The posterior probabilities. Analyses with the program MrBayes.

14. Statistical analysis of the branches Bootstrap and jackknife methods. Calculation of bootstrap values to test the branches (programs Mega, Phylip, PhyML)

15. 2nd midterm exam II. midterm exam: Solving different problems and doing calculations using the learnt methods.

• · Page, RDM, Holmes, EC 1998. Molecular Evolution. A Phylogenetic Approach. Blackwell Science

• · Nei, M, Kumar, S 2000. Molecular Evolution and Phylogenetics. Oxford University Press.

• · Felsenstein, J, 2004. Inferring Phylogenies. Sinauer Associates