LEARNING OUTCOMES:
1. Discuss why DNA is a good source of phylogenetic information.
2. Explain the differences between the dominant and codominant markers.
3. Compare different phylogenetic approaches (Maximum parsimony, Maximum likelihood and Bayesian approach).
4. Provide an overview of cytogenetic methods in phylogeny and systematics.
5. Apply different field sample collecting techniques for for future DNA analyses.
6. Isolate DNA from fresh and preserved tissue and apply methods of SSRs, AFLP and DNA sequencing.
7. Calculate genetic distances of DNA data, produce phylogenetic tree and carry out an analysis of the genetic structure and maximum parsimony.
8. Autonomously use different phylogenetic and genetic computer programs (Gene Mapper, Power Marker, AFLPsurv, Past, Phylip, SplitsTree, Structure, Geneious, ClustalX, TCS, PAUP)
9. Use different databases and computer programs on the internet (eg., GenBank, Structure harvester)
10. Apply all steps of scientific research in practice
COURSE CONTENT:
Molecular systematics is a fusion of traditional systematics (nomenclature, classification, and taxonomy), molecular biology and bioinformatics. It tries to explain the overall biodiversity on Earth in the light of their evolutionary history (phylogeny) using molecular characteristics of organisms (DNA, RNA and proteins) and different phylogenetic methods. The stages of the molecular systematic research are well defined. The research begins with the selection of the problem and continues with research design, sampling, determination of molecular profiles, phylogenetic analyses, determination of phylogenetic relationships, and ends with a discussion and conclusion of the history of the evolution (phylogeny), history of geographical migration (phylogeography), classification, taxonomy, nomenclature etc. Course lecturer and associate teachers (through 15 lectures and 15 laboratory and computer exercises), will make students more familiar to fundamental principles and methods of this very propulsive scientific discipline. The exercises are designed in such a way that at the end of the course they can be assembled in the final practical report that resembles a scientific paper.
LECTURES AND SEMINARS:
1. Course content. Knowledge assessment and evaluation. Seminar topics. Preconditions of origin and development of molecular phylogeny and systematics.
2. Controversies in molecular phylogeny and systematics. Research design. Collecting and storage of tissue.
3. Codominant DNA markers RFLP, SSRs.
4. Dominant DNA markers RAPD, AFLP.
5. Measures of genetic distance.
6. UPGMA and NJ trees. Bootstrap support.
7. Analysis of genetic structure. Bayesian approaches in analysis of genetic structure.
8. Sequencing and alignment of DNA sequences. Phylogenetic trees and their terminology.
9. Phylogenetic methods based on genetic distances. Maximum parsimony.
10. Maximum likelihood. Bayesian methods.
11. Cytogenetics in phylogeny and systematics.
12. Overview of the scientific research of the lecturers and asistents of this course
13. Seminar reports I
14. Seminar reports II
15. Written Examination
PRACTICAL WORK:
Relationships and genetic diversity of some plant groups currently researched in our lab eg. Circum Adriatic species of the genus Campanula, genus Micromeria or genus Salvia.
1. Total genomic DNA isolation.
2. Preparation of the agarose gel, electrophoresis and measuring the concentration of total genomic DNA
3. AFLP restriction and ligation. PCR amplification of nuclear and chloroplast DNA regions
4. AFLP preselective PCR. Agarose gel elektrophoresis of PCR amplified nuclear and chloroplast DNA regions.
5. AFLP selektive PCR. PCR amplication of microsatellite SSR loci.
6. Analysis of selective AFLP and SSR products by capillary electrophoresis. Purification of PCR products of nuclear and chloroplast DNA regions and DNA sequencing.
7. Processing of AFLP data using a computer program GeneMapper 4.0.. Preparation of data for further statistical processing computer program: scanAFLPv1 2.r. The calculation of genetic distances and phylogenetic tree constructions computer programs: AFLPsurv, Past, TreeCon, Phylip, SplitsTree.
8. Determination of genetic structure of populations using AFLP data and computer programs Arlequin, Structure and BAPS.
9. Processing of SSR results using GeneMapper 4.0. computer program. Preparation of data for statistical analysis computer program Convert. Production of genetic distances matrices and phylogenetic tree building computer programs: microSAT, PowerMarker, Phylip, PowerMarker, SplitsTree.
10. Determination of the genetic structure of populations with the help of SSR data and computer programs Arlequin, Structure and BAPS.
11. Analysis and alignment of nuclear and chloroplast DNA sequences computer programs Genious and ClustalX.
12. Preparation of nuclear and chloroplast DNA sequences for phylogenetic analysis using Maximum parsimony approach computer programs PAUP and TNT.
13. Preparation of nuclear and chloroplast DNA sequences for phylogenetic analysis using Maximum likelihood and Bayesian approach computer programs PAUP, RAxML and MrBayes.
14. Student's own analysis of previously published results from our laboratory using above mentioned computer programs.
15. Discussion of obtained results and practicum report writing.
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- Liber Z. 2014. Filogenija I molekularna sistematika. DVD izdanje
Soltis D. E., Soltis S. S., Doyle J.J. 1998. Molecular Systematics of plants II. DNA sequencing. Kluwer Academic Publishers. Boston. Dodrecht. London.
Salemi M., Vandamme A.M. 2003. The Phylogenetic Handbook. A Practical Approach to DNA and Protein Phylogeny. Cambridge University Press, Cambridge.
- Felsenstein J. 2004. Inferring Phylogenies. Sinauer Associates Inc., Sunderland, Massachusetts, U.S.A.
Hall G.B. 2001. Phylogenetic Trees Made Easy_a how to manual for molecular biologist. Sinauer Associates Inc., Sunderland, Massachusetts, U.S.A.
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