300319 UE Plant Chromosome Structure and Evolution: theory and practice (2023W)

The purpose of the course is two-fold. First, it offers an overview of plant cytogenetic techniques: from traditional chromosome counting to more recently established techniques of chromosome painting (in situ hybridization) and immunocytochemistry. Second, it presents detailed and up-to-date information about plant chromosome structure (centromere, telomere etc.) and main types of DNA sequences used for cytogenetic analyses. The newest methods developed recently to analyse genome composition and to characterize different types of repetitive DNA is plant genomes (high-throughput sequencing) will also be introduced. The course introduces also types and consequences of chromosome rearrangements including polyploidy, in the context of chromosome evolution. The use of cytogenetic data (obtained from both classical and molecular cytogenetic studies) in inferring plant taxa relationships and in evolutionary studies of different plant groups will be discussed. The course will give the up-to-date overview of the state-of-art in the analyses of plant chromosomes.
The practical part of the course will include chromosome preparation in selected groups of plants, as well as classical and molecular chromosome staining methods. The students learn to perform DNA:DNA in-situ hybridization (FISH), in particular to prepare and label the DNA probes (e.g., species-specific repetitive DNA, rDNAs, telomeric sequences), to detect of the hybridization sites and to analyse the obtained data (fluorescence microscopy). The students will acquire practical skills in chromosome handling and will learn to analyse and interpret chromosomal data in evolutionary context.

Aims:
- To present and discuss in details the structure, types and function of chromosomes, and to emphasize their role in evolutionary processes.
- To present most relevant case studies.
- To introduce most important cytogenetic and genomic techniques, including state of the art next generation sequencing, and to discuss their application for evolutionary cytogenetics of plants.
- To introduce modern techniques used nowadays to study the structure and function of plant genomes and to emphasize the importance of such analyses as complementary approach for phylogenetic, evolutionary, or populational studies.
- To introduce methods for in situ-hybridization probe preparation and labelling, chromosome preparation and quality checks, and finally in situ hybridization (FISH).
Students will apply gathered knowledge to analyze and to interpret obtained results (light and fluorescent microscopy).

Course content:
- Planning of the experiments; material pre-treatment and fixation; preparation of chromosome spreads using classical methods; documentation.
- Enzymatic chromosome preparations; DNA probe preparation and labelling (PCR labelling, nick translation); preparation of buffers and solutions for FISH.
- Checking of the quality of DNA probe labelling; FISH (fluorescent in situ hybridization); pretreatments, and hybridization.
- FISH: washing, detection and amplification.
- Analysis of FISH data (light and fluorescence microscopy)