Degree Course: Biotechnologies for agro-food safety and quality Syllabus for A.Y. 2020/2021  

1. Genome size and organization:
• Comparing prokaryotic and eukaryotic genomes: the C-value paradox described through examples of various Angiosperm plants;
• Forces that influence genome size and expansion-contraction mechanisms (unequal crossing-over; illegitimate recombination).

2. Types, abundance and organization of genic and non-genic sequences in the eukaryotic chromosome:
a) Non-gene sequences
• Telomeric, centromeric and intercalary highly repeated sequences; micro- and mini-satellites;
• Transposable elements (TE) in Eukaryotes: comparison with prokaryotic IS elements; classes of TE and relative mode of transposition di ET and abundance in various Eukaryotic genomes: DNA TE (e.g. Ac-Ds) and RNA TE or retroelements, with (retrotransposons) or without LTR motifs (e.g. Gypsy, Copia, Alu, SINEs, LINEs); effect of TE on size and structure of Eukaryotic genomes; amplification of TE through intra- and inter-element unequal crossing-over;
b) Eukaryotic genes:
• Fine structure and structural variability (e.g. intron abundance and size) among taxa; exon re-shuffling and development of novel genes;
• Gene families and super-families: gene duplication and divergence (neo- and sub-functionalization): examples of the histone gene family, of resistance gene families in plants and of the human globin super-family.

3. Genome compartmentalization: mosaic organization (isochores) of the human genome and of plant genomes; “gene-rich” and “gene-poor” regions; TE as main components of inter-genic spaces; comparison between genetic and physical maps: uneven distribution of recombination along the Eukaryotic chromosome (“hot” and “cold” spots of recombination and their correlation with gene density and transcription).

4. Comparative genomics:
• Intergenomic and interspecific micro- and macro-synteny and colinearity; the “circle diagram” of grass genomes; levels of conservation: homoeologous chromosomes and orthologous genes;
• Chromosome rearrangements and disruption of synteny and colinearity: evidence from comparative mapping and comparative sequencing.

5. Evolution and adaptive mechanisms of plant genomes, with particular focus on polyploidy; rapid genome changes following the polyploidization event: maintenance and elimination of duplicated genes/sequences: examples from natural and neo-synthesized Triticum polyploids; gene silencing.

6. Epigenetics and epigenomics: modifications of chromatin structure (chromatin remodelling) mediated by DNA methylation, histone methylation/acetylation, RNA molecules; epigenetic changes associated with polyploidization.

7. Plant genome manipulations aimed at practical applications
• Targets of manipulations for breeding purposes: utilization of genetic variability outside the “target” species
• From creation of amphidiploids to single chromosome addition and substitution lines to targeted interspecific transfer of chromosome segments (chromosome engineering) in agriculturally relevant plant species: strategies and case-studies, with particular focus on Triticeae species (wheat and related species), Solanum spp., Medicago spp., Lolium-Festuca complex.

- Teaching material (e.g. lesson slides) and scientific articles provided by the teacher and available on the institutional website;
- Additional scientific articles on students' request, both for the preparation of the exam test and for the deepening of aspects of specific, individual interest;
- Chapters selected from:
- Barcaccia & Falcinelli – Genetica e genomica (Liguori ed.), vol. I, II e III;
- Hartwell et al. - Genetica - dall'analisi formale alla genomica (McGraw-Hill);
- Russell PJ - Genetica: un approccio molecolare (Pearson)

PROGRAM The program is divided in three modules: 1) Structural Genomics, 2) Bioinformatics and 3) Functional Genomics. 1) STRUCTURAL GENOMICS (10 hours of lesson) - Sequencing methods: 1) Second generation sequencing: ILLUMINA, Pyrosyquencing (ROCHE 454), SOLiD; 2) Third Generation Sequencing: HELICOS (Helicos Biosciences); PacBio (Pacific Biosciences); Nanopore (Oxoford Nanopore); 2) Comparison of next generation and next-netx generation sequencing methods. - Full genome sequencing strategies: hierarchical method and WHOLE GENOME SHOTGUN; - Gene Annotation; - Functional annotation; - Projects for the sequencing of whole genomes in plant species of agricultural interest. - Illustration of major databases (NCBI, EMBL, DDBJ), searches in biological databases (BLAST), sequencing software and for the design of oligonucleotides; 2) BIOINFORMATICS (8 hours of lesson) - Analysis of genomics, transcriptomics, proteomics and metagenomics data using R & RMarkdown. 3) FUNCTIONAL GENOMICS (12 hours of lesson) - Gene expression analysis of single genes (TaqMan, LUX, Molecular Beacons and Scorpions probes) and large scale gene expression analysis (microarray and RNAseq). - The genetic transformation of plant species. Transformation mediated by Agrobacterium or by biolistic method. Preparation of plasmid vectors. - Study of gene function: gene overexpression and knock-out (RNA antisense, RNA interference) in transgenic plants; - Preparation of constructs for the realization of cis-genic plants. - Chemical mutagenesis and TILLING; physical mutagenesis with fast ions and neutrons; insertional mutagenesis: T-DNA and transposons; - Application of mutagenesis for functional studies and breeding programs. - Site-specific modifications. Methods of "genome editing": 1) zinc-finger nucleases (ZFN), 2) transcription activator-like effector nucleases (TALENs), and 3) Clustered Regularly Interspaced Short Palindromic Repeats-associated (Cas) proteins (CRISPR / Cas) - Application of "genome editing" methods for the genetic improvement of species of agrarian interest. Laboratory exercises will focus on the following topics: 1) Search in databases of nucleotide and protein sequences. Use of bioinformatics tools to open sequence files (DNAMAN, FINCH TV, GENEIOUS). Use of the BLAST algorithm to search nucleotide or protein sequences in databases. Alignment of nucleotide and acidic amino acids through the CLUSTAL OMEGA and GENEIOUS programs. Construction of phylogenetic trees (2 hours) 2) Identification of SNPs on genes of interest by TILLING in durum wheat (4 hours) 3) Preparation of constructs for genetic transformation by cis-genic approach: Insertion of the recombinant cassette into a bacterial vector; Transformation of bacterial cells; Extraction and digestion of the recombinant plasmids with restriction enzymes (8 hours) 4) Use of molecular marker to select transgenic plants (4 hours)

GENETICA un approccio molecolare. Quarta edizione Peter J. Russell Edizione italiana a cura di Carla Cicchini e Alessandra Marchetti ISBN:9788865186176 Biotecnologie e Genomica delle Piante. Rosa Rao e Antonietta Leone. Editor IDELSON-GNOCCHI. Power Point presentations and papers provided by the professor.

Introduction – market of food microorganisms and enzymes; EU regulations
Biotechnological applications to the agri-food chains of:
- Bread and bakery products;
- Malt and beer;
- Still and sparkling wines;
- milk, cereals, legumes and dried fruit based fermented foods;
- Coffee;
- Cocoa.

- Slides and course material in pdf electronic format
- Advances in Food Biotechnology, Wiley
- Fundamentals of Food Biotechnology, 2nd Edition, Wiley
- Food Biotechnology, Springer
- Biotecnologie alimentari, Piccin

Program Lectures: Fundamentals of genetic improvement and reproduction biotechnology in farm animals. Artificial insemination, embryo transfer. Practical applications of reproductive biotechnology. The production of transgenic animals. The cloning of pets. Biotechnological methods applied to food safety and quality: biotechnologies applied to supply chain control and genetic traceability of livestock products; analysis of GMO residues; use of molecular markers for the traceability of species, for the traceability of race, and for individual traceability. Use of cell cultures for studies of biology and physiology of farmed animals.

G. Poli. Biotechnology. Know to choose. Ed. Utet, Turin, 2001.
Teaching material provided by the teacher.
Every year the material is made available to attending students and not using as a 'DropBox' archive.
At the beginning of the lessons and also during the course of the year, access is made to a folder in which the material presented in class is made available the day before the lectures are held.

- Introduction, history and aim of plant breeding, concept of ideotype.
- Biology of plant reproduction: floral morphology, macro and microsporogenesis, macro and microgametogenesis, pollination, progamic phase, fertilization, embryogenesis, seed and fruit development. Molecular biology of flowering induction and flower development, genes controlling inflorescence, flower and floral organs identity, ABC(DE) model. Mode of reproduction (sexual reproduction, vegetative propagation, apomixis), sex determination (hermaphroditism, monoecism, dioecism), mating system (autogamy and allogamy), experimental characterization of plant reproductive system and of outcrossing rate. Male sterility (genetic, cytoplasmic and genetic-cytoplasmic, functional and conditional). Self-incompatibility (sporophytic and gametophytic). Apomixis, cytoembryological bases, genetic control, breeding of obligate and facultative apomictic species, perspectives of transferring apomixis to sexual species. Fruit set and development, parthenocarpy, biotechnological approaches to control fruit set. Fruit ripening, mutants affected in ripening and in pigment accumulation.
- Sources of genetic variability: gene pool concept, germplasm collection and storage, seed banks, germplasm evaluation, variability induced by mutagenesis, somaclonal variation.
- Intra and interspecific cross, sexual interspecific barriers, biotechnologies to facilitate interspecific crossing, in vitro fertilization and embryo rescue.
- Theory of selection: principles of theory of selection for monogenic and polygenic characters, penetrance and expressivity.
- Genetic population structure in autogamous, allogamous, vegetatively propagated and apomictic species.
- Breeding schemes for autogamous species: selection in existing populations: mass selection and pure line selection. Selection in segregating populations: pedigree, bulk and single seed descent methods, double haploids. Backcross method to transfer a dominant and a recessive allele, linkage drag, principles of marker assisted selection, multiline varieties, F1 hybrids in autogamous species.
- Breeding schemes for allogamous species: mass selection, recurrent selection, synthetic varieties, F1 hybrids in allogamous species, use of male sterility in hybrid production schemes.
- Principles of genetics of seed production: evaluation of new varieties, subscription to the Register of Varieties, maintenance selection and seed production, isolation, generations of seed multiplication. Legislation related to seed production and marketing. Biotechnologies for the protection of plant varieties (molecular markers for distinctiveness), estimation of genic flux and gene flux containment in conventional and genetically modified varieties.

Seminars: students will be invited to attend one or two seminars focussing on technical and/or scientific aspects related to topics of the course.
Practical activities: practical activity will be dedicated to the knowledge of genetic variability in a crop species, to the examination of mutants involved in reproductive biology, to technicalities to carry out controlled crosses and to a visit to a company or experimental station involved in breeding and/or seed production of crop species.

Barcaccia G. e Falcinelli M., Genetica e genomica, vol. II, 2005, Miglioramento genetico, Liguori.
Lorenzetti F. et al., Miglioramento genetico delle piante agrarie, 2018, Edagricole.
Ciriciofolo E. e Benincasa, Sementi: Biologia, produzione e tecnologia, 2018 Edagricole.
Materiale fornito dal docente tramite il Portale Docente.

The course focuses on the study of different families of natural organic substances derived from the acetate, shikimate and mevalonate pathway. Of each of these, structural aspects, properties, biological activities and applications in agronomy, food, cosmetics, pharmaceuticals and nutraceuticals are explored. Among the classes of secondary metabolites, the phenolic substances and, among the various biological activities, the antioxidant activity are particularly explored. The techniques of extraction, purification and structural characterization of natural substances are also studied. Seminars given by colleagues from outside the University of Tuscia on topics relevant to the program are scheduled during the course. Exercises and/or teaching visits are, in addition, planned.

Medicinal Natural Products. Paul M Dewick, John Wiley & Sons, Ltd

The course is mainly divided into three sections: an initial more general section, a specific section focused on the use of plants for the production of recombinant proteins and a third section where students present in the classroom, in Italian, a scientific paper written in English. Moreover during the course three seminars, taught by guest experts, are planned on specific topics.
In the initial section general aims of pharmaceutical biotechnology and pharmaceuticals obtained through biotechnological production are introduced, in this regard some definitions of Biopharmaceutical are provided together with specific examples and the general picture of their worldwide market. In addition, biotech drugs are compared to traditional drugs with particular reference to new approaches for their realization. In this context also recapitulative concepts on recombinant DNA technology strictly applied to pharmaceuticals are provided. All main biological systems for production are analysed: bacteria especially E. coli, yeasts, insect cells, different types of mammalian cells and chicken eggs. Production issues for large scale issues at both upstream and downstream processing level are also addressed in the light of international quality standards and of "Good Manufacturing Practices" (GMP) procedures. In particular specific lessons are dedicated to biofermentations, product processing, techniques of analysis and purification of biological macromolecules and their formulation for their release on the market. After an introduction to immunity, immunogenicity and the immune system of humans, all the main types of biopharmaceuticals: vaccines, polyclonal antibodies, monoclonal antibodies, immunomodulatory proteins and enzymes, in the context of the pathologies to which these drugs are directed to, are defined and described also using specific case studies. At the conclusion of the general section recent developments of nanotechnology in biomedicine and diagnostics and possible future applications in the biopharmaceutical industry are discussed.
The second section, all centered on the use of plants for the expression of recombinant polypeptides with pharmaceutical activity, starts with the in depth discussion of plant viruses from a molecular point of view for the importance they have had and continue to have in the development of expression systems. In particular the various strategies of expression and replication of the best known plant viruses are addressed and the systems that make use of these viruses for the heterologous transient expression in plants analyzed in detail. Subsequently the most used plants in biopharmaceutical development (tobacco, benthamiana, rice, maize, potato and tomato) and the different types of genetic modification applicable to these species are addressed (stable nuclear transformation, plastid transformation and transient transformation). Numerous case studies concerning plants developed biopharmaceuticals will be presented and analyzed in detail to illustrate the different approaches in relation to the type of biopharmaceutical, the pathology and the receiving species (for human or veterinary use).
The third section focuses on the acquisition of skills in the analysis of scientific international manuscripts and on the acquisition of skills in the presentation and exposure of technical content. To this end, students choose an article from a list of publications selected by the teacher and prepare a power point presentation of about 20 minutes to explain to their class mates, the general theme, the experimental approach, the results and prospects of the work. All selected publications concern the design, implementation and development of biopharmaceuticals in plants.
The three seminars that take place during the course are: i) elements of immunology held by Dr. Selene Baschieri from the ENEA Casaccia research center; ii) principles of vaccinology held by Dr. Chiara Lico from ENEA Casaccia research center.

Biotecnologie farmaceutiche; Crommelin D. J., Sindelar R. D.; Zanichelli; 2000; ISBN: 8808094197.
Elementi di Virologia Vegetale; Giunchedi, Conti, Gallitelli, Martelli; 2007; ISBN: 978-88-299-1838-6.

The course concentrates on consolidating and acquiring familiarity with verb structures and increasing a wide range of academic and subject-specific vocabulary.

National Geographic Life, Level B2
Resources on the Moodle platform (Distulab page)

European policies on food safety; elements and evolution of the concept of food products quality and traceability; the role of the animal feeding and nutrition on the quality and safety of the derived food product. Methods and analytical procedures for the evaluation of the quality and traceability (origin and/or process) of the animal food products. Description of the main animal food production chains (dairy, meat, eggs, beekeeping products) end the related aspects of food quality and safety, traceability and labelling. Climate changes and other environmental factors affecting the quality and safety of animal food products. Regulative frames for the quality, safety, and traceability of animal food products. Process traceability, Control and self-control (via HACCP) in the animal food production chains.

The study materials (slides, normative acts, literature) will be provided by the Professor

European policies on food safety; elements and evolution of the concept of food products quality and traceability; the role of the animal feeding and nutrition on the quality and safety of the derived food product. Methods and analytical procedures for the evaluation of the quality and traceability (origin and/or process) of the animal food products. Description of the main animal food production chains (dairy, meat, eggs, beekeeping products) end the related aspects of food quality and safety, traceability and labelling. Climate changes and other environmental factors affecting the quality and safety of animal food products. Regulative frames for the quality, safety, and traceability of animal food products. Process traceability, Control and self-control (via HACCP) in the animal food production chains.

The study materials (slides, normative acts, literature) will be provided by the Professor

Lectures will be supported by powerpoint presentations of the issues and will be available to students. In addition, novelty articles, which are published during the teaching period, will be distributed and discussed with the aims to advance the knowledge on the topics of the teaching subjects.
Gene network regulation of development and production: branching and tree vigour, changing phase, alternate fruit bearing, flower induction, fruit development and maturation, postharvest fruit regulation.
The genetic network that controls plant interaction with biotic and environmental factors: a) photoreceptors and regulation of shadow avoidance response, phase changing, photoperiodic rhythmic responses; b) temperature receptors system and adaptive responses to high and low temperatures, vernalization and flower development and fruiting; c) flooding and adaptive response; d) water and minerals sensors and regulating plant nutrition and metabolite compounds; e) communication between plants (and scion-rootstock) and plant with other organisms; e) plant gene regulation host/parasite-predators: the case of Erwinia amylovora (Fire blight, Pseudomonas spp; Venturia, etc.).
Plant organs: fruit and leaf as bio-factory of structural and secondary metabolites. Genetic and epigenetics regulation of secondary metabolites synthesis and catabolism: apple and grape mutants cases. Use of bioinformatics for the detection and isolation of functional allelic genes (and orthologs), small RNAs and methylation status of regulatory sequences playing an important role into relevant biological processes.
Overview of isolation, cloning, sequencing of functional genes and/or functional sequences; generation of constructs to apply in improvement biotechnology programmes. Gene transfer in woody plants. Innovative techniques for studying gene expression and epigenetics.
Molecular tools as markers for assisted selection and genetic improvement. Main biotechnological improvement fruit crop plants, examples. The experiences of transgenes in fruit tree species and new system to reduce juvenility and applying of Cisgenome and Editing genome.
The relevant plant and productive traits that require biotechnology and genetic improvement among the main fruit tree species.

Laboratory exercises will take place in the Laboratory of Molecular eco-physiology of woody plants and tissue culture ( rooms 232-234) and 2CFU (16 hrs) will be dedicated.
Practice exercises will be by conducted in the laboratory to complete the acquisition of the technologies: On fruit crops plants, it will run on: nucleic acid extraction, purification and quantitative and qualitative assessment; phylogenetic analysis of the extra- and intra-species diversity applying molecular markers (AFLP, SSR, EST, etc.). Particular attention will be paid to the HRM and ARMS techniques for the analysis of SNPs markers and methylation status (developed in the laboratory), semi-quantitative and quantitative allelic expression analysis; epigenetic analysis of cloned plants for the verification of the genetic identity with random and dedicate procedures (M-SAP, EpiHRMAssay, etc). Finally, bioinformatic tools will be also used to tanscriptome and genome analyses, and biological function analysis.

Biodiversity, Chapman & Hall, London, 1988
G. Valle, M. Helmer Citterich, M. Attimonelli, G. Pesole., Introduzione alla Bioinformatica, Zanichelli, 2003
LITZ R.E., BIOTECHNOLOGY OF FRUIT AND NUT CROPS., CABI PUBLISHING, 2004
Articoli e materiale didattico forniti direttamente dal docente

Definition, general framework and challenges of the bioeconomy. Importance of the bioeconomy for the national economy, with particular reference to the agri-food sector. Use of chemical inputs in the agri-food sector.
Fundamentals of (bio)economics: market supply and demand, choices of consumers and producers, market in perfect competition, market power, technological innovation, value and protection of intellectual property.
Economics of agrifood farms: sustainable production and management choices.
Community strategies and policies: bioeconomy strategies, Green Deal, Farm to Fork, Common Agricultural Policy.
Quantitative methods for analysis in bioeconomy: economic models of producers and consumers choices.
Case studies: illustration and discussion of various case studies with the active participation of students.

1. A sustainable bioeconomy for Europe: strengthening the connection between economy, society and the environment. European Commission, Brussels, 2018.
2. A new bioeconomy strategy for a sustainable Italy. Italian bioeconomy Strategy BIT, 2019.
3. Teaching material made available by the teacher.

The course is organized with a preliminary part during which various crops are illustrated in terms of the relative qualitative aspects, with greater in-depth analysis of wheat, the problems relating to traceability , along with the biochemical and molecular techniques whose knowledge is necessary for the understanding of the specific topics that will be addressed in the second part.
Specifically, this preliminary part of the program includes:
• Definition of crop quality and description of the main qualitative characteristics
• Possibility of intervention to improve quality characteristics
• Legislation in regard to traceability and tracking in supply chains, certification and labeling, individual and collective marks, certification of quality
• Definition of food fraud
• Analytical methods: proteomics, standards and quantitative PCR (RT-PCR), chromatography, ELISA test, molecular markers, including DNA barcoding

In the second part specific problems related to plant-based products are presented. Stable subjects are wheat-based products and genetically modified plants. According to the time necessary to carry out exhaustively these two major issues, other issues are treated, on the basis of the preferences expressed by the students, such as, for example, vegetable oils and wine traceability.
Concerning wheat-based products, bread and pasta, with the relevant legal definitions and the processes that lead to their realization are illustrated. A few typical breads are also described and case studies in which biochemical and molecular techniques are used for their traceability are presented.
Part of the course is devoted to adverse reactions to wheat (in particular allergies, celiac disease and non-celiac wheat sensitivity).
With regard to genetically modified plants, the substantial equivalence issue is developed, along with legislation issues. The main methods of traceability and risk assessment, are discussed by presenting specific case studies.

All case studies are presented in order to stimulate the students critical skills. Every case study is presented by giving particular importance to the aim of the work, the procedure to be followed to achieve the proposed objective, the critical discussion of the results.

The practical classes provided are:
• SDS-PAGE of pasta products to control the varietal composition declared on the label, through the comparison of the electrophoretic profiles obtained by extracting proteins from pasta products, with that from the declared wheat varieties
• SDS sedimentation test for the prediction of dough technological properties. Two durum and two bread wheats with opposing technological properties are usually compared in order to highlight differences
• SE-HPLC for the prediction of dough technological properties. Two durum and two bread wheats with opposing technological properties are usually compared in order to highlight differences
• ELISA test on pasta for celiac people to control the effective absence of gluten using polyclonal antibodies against gluten proteins

The teacher makes the slides and the bibliographic materials available