Biotechnologies for crop improvement
Introduction on the role of biotechnologies for crop improvement, genetic engineering, in vitro cultures of plant cells and tissues, technologies for diagnostic and traceability, study of genetic variability to study phylogeny, variety characterization, germplasm management, technics to manipulate protoplast and obtaining interspecific hybrids.
Molecular markers and genetic analysis
Concept of genetic marker and introduction to the analysis of molecular polymorphism.
Biochemical markers. Molecular markers, DNA hybridization and PCR, RFLP and VNTR markers, RAPD, SCAR, CAPS, AFLP, SSR, ISSR and SNP markers.
Use of molecular markers
Formal genetics and development of genetic maps;
search for markers linked to loci controlling qualitative and quantitative characters;
positional cloning of Mendelian genes and QTLs;
marker assisted selection in plant breeding;
linkage disequilibrium and association mapping;
search for mutants in experimental populations (“gene machines” and TILLING);
analysis of genetic variability, genetic distances and clustering methods;
variety characterization and analysis of genic flux.
Topics of practical lectures
In silico search for microsatellite markers and primer design.
Search for single nucleotide polymorphisms from EST libraries.
Growing of an F2 population segregating morphological and molecular markers, phenotyping, genotyping (DNA extraction, PCR, electrophoresis), analysis of segregation data.

Testi di riferimento
Barcaccia & Falcinelli - Genetica e genomica Vol. III - Liguori editore.
Material provided during the course.

The course starts with the basic principles of recombinant DNA illustrating the mechanism of action of of restriction and modification enzymes, and gene cloning through host-vector systems.
The mechanism of action of standard and quantitative PCR is presented in detail, but particular importance is given to applications of such methodologies, through specific examples.
Among the different methods of DNA sequencing only the Sanger method is reported, both in its manual and automated versions.
The various techniques of analysis of nucleic acids and proteins, including electrophoresis on agarose and polyacrylamide and through blotting (Southern, Northern and Western blotting), and the different fields of application of these techniques are presented.
In regard to libreries, both the construction process, and the analysis of the genomic, cDNA and expression libraries are shown, with an emphasis on information obtained from their use.
The Arabidopsis thaliana and Homo sapiens genome projects are presented, explaining the criteria of choice of model organisms, and information obtained by the complete knowledge of these genomes.
Structural, functional and comparative genomics, as well as the technologies related to each of these branches, are presented. In particular, technologies related to structural genomics, through analysis of the genomic libraries for the sequencing of extended portions of DNA, and to functional genomics, through the main techniques of analysis of transcriptome and proteome, are explained.
Finally, genetically modified plants are presented: how they are realized through the two main systems (biolistic and Agrobacterium), by illustrating advantages and disadvantages of both these methods; how a genetic construct for expression in plants is made, which genetically modified plants are present on the market, with hints related to the issue of their acceptability.

Practical classes:
• Bacterial transformation using a recombinant plasmid vector, which is extracted in a subsequent practical class and characterized by agarose gel electrophoresis
• Standard PCR to determine the presence of a specific transgene in plants of GM wheats
• SDS-PAGE to analyze the protein composition of wheat kernels
• The use of the spectrophotometer to determine the amount of DNA and proteins

If the students are more than 25, practical classes are performed in turns.

Brown T. Molecular Biotechnology
Slides and other materials will be made available to students on Moodle platform