93966 - Genetics and Biotechnology for Resilient Crops

Learning outcomes

At course completion, the student possesses knowledge on: the potential of biotechnology based genetic improvement to develop resilient cultivars suitable for sustainable agricultural systems; the molecular genetic control of the main features of agronomic interest including the response to abiotic and biotic stresses, the efficient use of water and nutrients, and host-pathogen interaction; genetic improvement methods that integrate assisted selection, phenotyping high-throughput, genetic engineering and genomic editing. In particular, the student possesses the skills to: participate in the management of genetic improvement programs aimed at varietal development in seed and nursery companies; evaluate and incorporate the appropriate biotechnological tools into genetic improvement programs; recognize and manage the positive aspects and critical issues of varietal innovation in agricultural systems, considering the entire production chain.

Course contents

Knowledge and skills to be achieved

The student will acquire knowledge on principles of

Agricultural Genetics,

Plant Breeding,

Practical and Theory of Breeding for sustainable agriculture, including the integration of precision agriculture methodologies.

Practical and Theoretical basis of Genomics and Biotechnology applied to plant breeding for a more efficient management and use of genetic resources.

The student will learn about the selection methods for self-pollinating, allogamous and vegetative propagation herbaceous plants, the genetic structure of the cultivated varieties. The students will know how to apply the basis of Mendelian Genetics, Quantitative genetics, population genetics, genomics and biotechnology to practical professional and research applications.

In particular, the students will be able to understand, recognize and choose the most appropriate genetic and biotechnological methodologies for the management and promotion of sustainable agricultural practices and the use of agricultural production inputs including improved seeds, fertilizers and crop protection chemicals to machinery, irrigation and knowledge.

Employment opportunities: management, conservation, characterization and use of Plant Genetic resources, either stored in Gene Banks or in open environment contexts; conduction of plant breeding programs as professional plant breeder or researcher; plant varietal choice decisions in specific agricultural systems and environments, particularly as integrated into high value agro-food value chains that target sustainable, high value products and that make use of precision agriculture and identity-preserved productions.

CONTENTS

1. PREREQUISITES NECESSARY TO ACCESS THE COURSE

The student who accesses this course must have a good knowledge of the fundamentals of mathematics, chemistry, plant biology, agronomy, crop biology and physiology, plant pathology as well as a knowledge of the fundamentals of statistical analysis (sample, mean, variance and standard deviation). These prerequisites are provided either by the basic courses delivered during the bachelor and the during the first year of PASA course.

Most importantly, students must have already a clear and good knowledge of the fundamentals of Agricultural Genetics. These prerequisites are already provided by the courses of the three-year degrees of class L-25 - AGRICULTURAL AND FOREST SCIENCES AND TECHNOLOGIES and by the similar courses of the bachelor in Agricultural Sciences and Technologies.

It is highly recommended / necessary to revise the AGRICULTURAL GENETICS COURSE before / at the beginning of the course to fully understand, acquire and retain the applied lessons that will be provided with this course.

More specifically, the PREREQUISITES of knowledge in GENETICS are:

1. Structure and organization of chromatin, DNA and RNA. RNA types: messenger RNA, transfer RNA, ribosomal RNA. DNA replication. Structure of chromatin and chromosomes. Caryotype. Euchromatin and Heterochromatin blocks and their effect on gene regulation, centromeres and distal arms. Significant sequences /functional elements present in chromosomes: Coding sequences, genes, gene content of a genome, repetitive elements, transposons and retrotransposons. The central dogma of molecular biology (one gene, one transcript and one protein, DNA-RNA-PROTEIN FLOW) and modern addendum about the regulation of the flow.

Gene structure, fundamental components of a gene, promoter and his regulatory elements (TATA box), ATG start codon, coding portion, codons sequence (triplets coding for aminoacids, AA), Degeneration or redundancy of the genetic code, exons and introns, STOP signals, untranslated regulatory regions.

2. Transcription and Translation and their regulation mechanisms. Messenger RNA, primary transcripts and mature transcripts. Alternative splicing mechanism and role. Post-translational modifications of active proteins (phosphorylation, glycosylation, acetylation). Protein degradation system, Ubiquitin-proteasome.

Transposable elements (transposons and retrotransposons) families, Transposition mechanisms. Transposons effect on gene regulation as a consequence of transposition (excision and insertion in gene regions). Regulatory elements in cis and in trans.

3. Meiosis and its relevance in determining novel genetic diversity. Meiosis as compared to mitosis. Generation of novel genetic diversity combinations in gametes by cross-over events (intra-chromosome) and by the random assortment of chromosomes in gametes at the first meiotic division.

4. Classical or Mendelian Genetics. Mendelian concepts in the genetic improvement of plants. Phenotypes/traits of SIMPLE GENETIC CONTROL. Direct relationship between one trait and one factor. Small or no influence of the environment on the expression of the phenotype. Mendel’s laws. Mendelian postulates. Genotype and phenotype. From the phenotype to the genotype. Definitions of genetic factor, locus, gene content of the locus, alleles. Dominance and recessivity.

Epistasis and pleiotropy.

Prediction of genotype from phenotype and vice versa. Proof of progeny (test-cross, progeny test). Haploid and double haploid. Allogamy and Autogamy. Hybrid vigor. Artificial hybridization. Generation of F2, BC Backcross populations and population types for farming. Types of crosses and artificial populations.

5. Chromosomal theory of inheritance, Morgan school and Genetic mapping Morgan school and their most relevant experiments in Drosophila. The first proof that factors are associated to chromosomes (phenotype-to sex association experiment). The Recombination Frequency (RecFreq, r) as a genetic measure of map distance between two concatenated loci in a chromosome. the definition of Morgan and CentiMorgan as genetic map units. The Morgan experiment of ordering five concatenated loci. Genetic maps in crops species.

6. DNA analysis in molecular biology lab. DNA extraction and analysis. Molecular markers. Concept and use of molecular markers to construct detailed genetic maps. Genetic mapping of Mendelian loci by molecular markers (extension of Morgan theory)

7. Quantitative genetics. Transition from a simple trait to traits controlled by two, three or more genes. Basic experiments of Johannsen and Nilsson-Ehle on quantitative traits of beans and wheat. Quantitative traits have poli-genic inheritance and are highly affected by the environments. Most relevant experiments of Johannsen and Nilson-Hele in beans and in wheat. Gaussian or Normal distribution of a quantitative trait. Concept of Quantitative trait locus (QTL). Genetic mapping of QTL by molecular markers.

The Basic Genetic Notions and Lessons are found on:

- SLIDE LESSONS LOADED ON VIRTUALE by the teacher lesson by lesson

- TEXT BOOKS:

1. Slides of the course provided by the teacher.

1. iGenetics. Essential by Peter J. Russell. Benjamin-Cummings Publishing Company
2. Genetica Agraria (Peter J. Russell, Wolfe, Hertz, Starr, McMillan), EdiSES editor, 2016. (In Italian, however wery well done)
3. Principles of Plant Genetics and Breeding, 2nd Edition George Acquaah. ISBN: 978-0-470-66475-9 October 2012 Wiley-Blackwell 760 Pages.
4. Plant Biotechnology and Agriculture. Prospects for the 21st Century (Arie Altman and Paul Michael Hasegawa Academic Press) ISBN: 9780123814661 eBook ISBN: 978012381467

B) TEACHING UNITS (two hours each)

The course is divided into the following units.

1. Introduction to genetic improvement in the agri-food and agro-industrial chain

Introduction to genetic improvement in the agri-food and agro-industrial chains. Objectives of breeding. History of Plant Breeding. Contribution of genetic improvement to the increase and sustainability of primary plant production and to the improvement of the healthiness of plant food products. Main critical factors for planning a genetic improvement project according to the objectives and the biology of the species. Definitions of Genomics and Biotechnology. Impact of novel Genomics and Biotechnology techniques applied to modern plant breeding. Breeding version 4.0.

2. Genetic constitution of materials used in ancient and modern agriculture: wild plants, land-races, ecotypes, modern varieties, F1 Hybrid

Physiological, adaptation, plant architecture and productive potential differences and genetic modifications occurred during the domestication, evolution and genetic improvement differentiating: a) wild plants, b) populations of landraces, c) modern varieties. Definition and features of landraces versus varieties, cultivars, pure lines. Varieties in open pollination and self-pollinating species. F1 Hybrids.

3. Population genetics I.

Concept of population and gene pool. Allele(gene) and genotype frequencies in populations. Hardy-Weinberg equilibrium - Diploid equilibrium - and perturbating factors affecting the allelic frequencies over generations (population size, migration, drift, mutations, selection). FITNESS concept and formula. Types of selection (directional, positive, negative or purifying selection, diversifying selection, stabilizing or balancing selection). Gametic equilibrium or haploid equilibrium. Linkage disequilibrium concept. Random mating and inbreeding and their effects on allogamous (open-pollinating) and authogamous (self-pollinating) crops. Concept of Inbreeding, genetic load, and purifying selection. Heterosis and F1 Hybrids.

4. Reproductive systems, authogamy, plant breeding and natural populations.

Power of artificial crossing in plant breeding to achieve the desired objectives. Population improvement as a combination of artificial crossing followed by and inbreeding to facilitate selection. Artificial populations.

Crossing between adapted parents and generation of segregant F2 populations. Pros and constraints of the F2 populations. Donor non-adapted and recipient adapted parents and backcross technique. Generation of advanced F6 Recombinant Inbred Lines for field evaluation of quantitative traits in inbred conditionand genetic mapping. Haploidy and Double haploids. Introgression breeding from non-adapted or wild donors.

Mendelian traits of interest in agricultural genetics. Nazareno Strampelli and Norman Borlaugh lessons as pioneers of modern breeding. The perfect cross made by Strampelli. The Wheat Battle and the Green Revolution. The worldwide impact of CGIAR centers. The semi-dwarf reduced height genes example. Plant architecture mutants of agricultural relevance. Spike and grain mutants relevant for production potential.

Development of artificial populations for genetics research and for plant breeding. Wide crosses, using wild relatives as donors. Clonal propagation. In vitro culture.

5. Genetic variation relevant for adaptation at the molecular level. The mutation landscape as the driver of genetic diversity

Mutation types at the Genomic (polyploidy), chromosomal and gene level. Genic or point mutations. Genetic diversity generated at meiosis and crossing over. Genetic diversity induced by transposable elements.

Where, how, when and why mutations occur. Their biological impact on transcript and protein functionality. Impact at the level of qualitative traits (strong mutations) and quantitative traits (weak mutations).

Neutral, deleterious and positive mutations. Natural selection. Effects on genetic diversity.

Mutation models. Infinite site and Infinite allele model and effects on genetic diversity. Ancestral variant and derived variant (bi-allelic model). Organization of point mutations at the gene level: genic haplotypes as the main drivers of natural variation and phenotypic diversity.

6. Genetic variation relevant for adaptation at the gene level.

Main categorization of genes into structural, housekeeping genes as enzymes in main cell metabolic chains and pathways (photosynthesis, respiration, nitrogen cycle and organication, nitrogen fixation) and regulatory genes who regulate the expression of other genes, namely Transcription Factors, master switches. How genes work in gene networks. Repressor (brake) and activator (transcription accelerator) regulatory genes. Impact on adaptation to the environment and on the response to changes in environmental factors and to abiotic and biotic stresses.

Examples of agricultural interest. Transcription Factors and gene network governing the vegetative and reproductive growth of the plants. Response to Vernalization, photoperiod, and temperature (earliness per se) and related genes common to many plant species. Vernalization genes as an example of pleiotropy

Signal perception and cascade amplification signal. Response to frost as an example and CBF transcription factors. Senescence master switches (NAC genes). Useful alleles discovered by breeders and selected for various environments. Epistasys associated to phenology regulators. (2 hours)

7. Germplasm for breeding. Plant genetic resources; germplasm collections and their managements. Principles underlying the genetic structure of natural and artificial populations and their methods of analysis. Methods of collection and conservation of plant genetic resources. Role of biodiversity in genetic improvement programs. Molecular methods to estimate population genetic structure. Empirical and molecular methods to estimate genetic relationships among individuals / accessions.

8. Plant domestication. implication for plant breeding and modern molecular concepts. Domestication syndrome. Genetic basis of domestication. The Brittle rachis gene. Diversity erosion and bottleneck effects as a consequence of domestication. The importance of genetic resources from wild species stored in germplasm banks to recover useful genetic diversity. Recent concept of Fast / accelerated domestication.

Polyploidy. Auto- (Potato example) and Allo-polyploidy (Wheat example). Polyploidy advantages over diploids. Importance of Allo-polyploidy in natural and agricultural systems.

Generation of novel synthetic species by artificial induction of polyploidy through colchicine (Triticale and synthetic wheat example).

9. Introduction to population genetics II. Genetic basis of quantitative traits and components of the phenotypic variance: genetic and environmental variance. Estimation of heritability. Use of heritability in predicting the response to selection and its limits. Selection for multiple characters and for adaptation to different environmental conditions. Decision making in breeding. Selection methods. Gene action and plant breeding. Breeding trial experimental design for single and multiple environment assessment. Response to selection. Recurrent selection.

10. Breeding objectives. Ideotype concept. Plant architecture. Deconvolution of complex trait into component of simple genetic basis to increase the heritability and to improve the dissection of the inheritance. Proxy-trait concept. Breeding for yield in relation to the environment. Breeding for sustainability. Target population environment concept.

11. Breeding for self-pollinated species. Genealogical selection method ("pedigree"). Variants and modifications including single seed descent, bulk method, double haploids, speed breeding. Breeding for cross pollinated species. (1 hour). Hybrid cultivars. Heterosis and adaptation to environment. Combinatory objectives of hybrid cultivars.

12. Phenotyping and phenomics aided by precision agriculture techniques in breeding and genetics

Phenotyping F2 individuals or advanced lines in field. Structure of a breeding field. Phenomics and Envirotyping concepts. Detailed characterization of the phenotypes and the environment using modern precision agriculture techniques. Use of technologies (soil and plant sensors, hand-held instruments, UAV and RGB, near infrared and multispectral cameras mounted on drones to increase the phenotyping. Hand-held instruments and remote sensing. Phenomobiles and Drones. RGB, multispectral and hyperspectral imaging. Vegetation indexes. Phenomics concept to increase the heritability of traits and to facilitate the genotype-to-phenotype link.

13. Molecular marker technology I. Objectives. Use of molecular markers for A) genetic diversity estimate in germplasm and for genetic improvement. Critical evaluation of advantages and limitations. B) Identification of QTLs C) Marker-assisted selection (MAS). D) Marker-assisted backcross (MABC).

14. Molecular marker technology II. Methods.

Molecular markers and DNA sequencing, cloning and genomics as fundamental tools for advanced plant breeding and biotechnology. Main technologies for identifying sequence polymorphisms and develop DNA marker assays useful in varietal characterization, germplasm and for genetic improvement. Critical evaluation of advantages and limitations. Sequencing of DNA and RNA techniques. Next generation sequencing. RNAseq. Genomics databases.

15. Mapping of genes. Mapping of genes and mapping of QTL by molecular markers. How to do. Linkage mapping in artificial populations. Genome-Wide Association mapping in cultivated or natural germplasm collections/panels bas. Definition of the locus space, gene content, candidate genes and causal gene, causal mutation and quantitative trait nucleotide. The three steps leading to the causal gene isolation: 1) Coarse mapping, 2) Fine mapping, 3) candidate genes identification and functional genomics for understanding which is the causal gene and its functions.

Candidate gene and causal gene (of the phenotype) concepts, alleles (functional variants with effect on the phenotypic trait) and molecular variants. Ancestral wild-type allele and strong (silenced) or partial effect mutant allele.

16. Molecular Breeding. Marker assisted selection. Concept and practice by simple markers and haplotype markers. Concept of identification and selection for the perfect marker obtained from the causative nucleotide. Breeding-by-design. Backcross and marker assisted backcross. Concept of introgression breeding using molecular marker. Negative Concept of linkage drag and how to control it with molecular markers.

17. Artificial Mutagenesis. Methodologies. Mutagenesis based on forward- and reverse genetics concepts. Direct or forward mutagenesis. Chemical and physical agents. Reverse mutagenesis by TILLING.

18. Transgenesis. Definition. Transgene construct components. Ballistic and Agrobaterium transformation methods. Transgenics for resilience to abiotic stresses and resistance to plant diseases and pests. Transgenic cassettes. Gene editing. Cisgenesis. (1 hour). Introduction to Editing techniques and induced modifications. Implications for the product registration and cultivations. Examples of editing for resistance to diseases and for fast domestication.

19. Basis of resilience to abiotic stresses (drought, heat, salt, nitrogen availability). Mostly taken from Tardieu et al Annual Review of Plant Biology, 2018 and from Garbowski et al Restoration Ecology 2020

The main physiological mechanisms / adaptive features to cope with stress: escape, avoidance, tolerance mechanisms. From resource-use efficiency plants concept to efficient use of resource plant concept. Relevance of Feed-back mechanisms in the short and long term response. Resource-conservative versus resource acquisition (uptaking) approaches. Stress recovery concept (re-vegetation).

The timing of water and heat stress and their effects. Heat stress effects. Salt stress effects. Role of the root and shoot apparatus in drought tolerance. Similarity between water and nitrogen shortage induced stress effects. Use of vegetation indexes and infra-red cameras throughout the plant cycle to monitor settlement, biomass development and senescence.

20. Breeding for resistance to disease and insect pests. Genetic basis and Breeding for resistance to pathogens and viruses: PTI and ETI. R genes evolution, in parallel with the effector gene evolution in pathogens. Nucleotide Binding Sites R genes and their multiple alleles evolution. Difference between biotrophic, emi-biotrophic and necrotrophic pathogens and relative host-response strategies (R genes versus QTL). Seedling versus adult plant resistances. Major loci for vertical resistance versus quantitative / adult plant resistances and effects of resistance durability. Examples of non-durable R genes and of durable resistance loci. Marker assisted selection and cisgenic gene cassette examples.

Exercitation units (three-four hours each)

E1. Plant genetics applications in the field of genetic improvement. Domestication and breeding effects. (4 hours). Genetic and phenotypic variation. (video collections previously prepared)

E2. Molecular Markers Laboratory. Expertise in the identification of DNA molecular markers that can be used for varietal identification and selection. Directly in the lab with teacher and tutors.

E3. Genomic databases utilization. In the Informatic room with teacher and tutors.

E4. Computer Exercises on calculation of genetic similarities and building of a linkage map with molecular markers and target loci for phenotypic traits of interest. using molecular marker-based genotypic data and phenotypes calculation of genetic similarities, dendrogram tree and principal component analysis. Using a data-set of molecular markers, construction of a linkage map performing grouping, ordering and order validation of molecular markers. Informatic room

E5. Computer Exercises on QTL mapping, population structure and Genome Wide Association Analysis (GWAS) Analysis of genetic and phenotypic data for the determination of the structure of the populations and for the mapping of QTL Basic and association mapping by GWAS. Using QTL-tagging markers to anchor the QTL to the genome. Inspection of candidate gene content. Retrieving the candidate gene functions in the expression and genomics databases.

SEMINARS Towards the end of the course the teaching lessons will be complemented by one or two seminars with breeders from cereal and horticulture seed companies. This will help students to be exposed to a direct interaction with breeders and to have a more direct perception of the aspects related to the selection in a seed company.

The teacher and post-doctoral from the Genetics andGenomics area have also prepared video taken directly from the fields (in spring time) regarding the principal topics of the course, using wheat as a reference

Texts / Bibliography

For basic concepts of Agricultural Genetics to be reviewed / refreshed:

1. Essential iGenetics by Peter J. Russell. Benjamin-Cummings Publishing Company
   
   
2. Genetica Agraria (Peter J. Russell, Wolfe, Hertz, Starr, McMillan), EdiSES editor, 2016. (In Italian, however wery well done)

The reference texts of the course:

1. Principles of Plant Genetics and Breeding, 2nd Edition George Acquaah. ISBN: 978-0-470-66475-9 October 2012 Wiley-Blackwell 760 Pages. This is the reference course text book for the first part of the course. The more specific second part of the course will be complemented by specific reviews provided directly through VIRTUALE
   
   
2. Plant Biotechnology and Agriculture Prospects for the 21st Century

   Arie Altman and Paul Michael Hasegawa Academic Press

   Hardcover ISBN: 9780123814661

   eBook ISBN: 978012381467

3. The text book is necessarily Complemented by Didactic material provided by Virtuale:

- Schemes and slides presented in class, to be used as a framework to study

- Review manuscripts on specific topics

Teaching methods

The basis of the course is provided by direct lessons based on power-point presentations.

The course is divided into lessons and practical works. The practical exercises are centered on the analysis of molecular markers and to general and practical aspects of plant breeding in general and breeding for resilience to abiotic stresses / resistance to diseases.

In addition, in the second part of the course, the lessons will be complemented by seminars from plant breeders and researchers in the field

Small Discussion groups (single or two students working together maximum), case-study examinations and preparation of short seminars on the topics will be used to deepen technical topical topics related to the subject of study.

Teacher/student weekly direct meetings for open discussion and lesson revision will be opened in free student hours, preferably the day after lessons.

Methods for verifying and evaluating learning

The assessment test is carried out through a final oral exam. The Student will start the exam by presenting his case-study example / seminar work with a few slides to be commented. The student will then continue the exam with the teacher posing three main questions related to three main and different topics addressed during the course. Additional specific questions will be addressed according to student knowledge and response and capability to connect topics, objectives and methodologies, and disciplines. Questions are aimed at ascertaining the knowledge relating to the basic and application notions developed during the lessons and practice. The duration of the test is about 30 minutes.

Teaching support tools

• power-point slides, discussion of scientific manuscripts, direct observations in the molecular lab, botanic garden, field
• Bibliographic material available at the University Library System and provided on the VIRTUALE platform.
• Blog / forum for teacher-student communications on the IOL platform, accessible only to students of the course.
• Weekly direct meetings with the professor and post-docs of the group.

Readings/Bibliography

Texts / Bibliography

For basic concepts of Agricultural Genetics to be reviewed / refreshed:

1. Essential iGenetics by Peter J. Russell. Benjamin-Cummings Publishing Company
   
   
2. Genetica Agraria (Peter J. Russell, Wolfe, Hertz, Starr, McMillan), EdiSES editor, 2016. (In Italian, however wery well done)

The reference texts of the course:

1. Principles of Plant Genetics and Breeding, 2nd Edition George Acquaah. ISBN: 978-0-470-66475-9 October 2012 Wiley-Blackwell 760 Pages. This is the reference course text book for the first part of the course. The more specific second part of the course will be complemented by specific reviews provided directly through VIRTUALE
   
   
2. The text book is necessarily Complemented by Didactic material provided by Virtuale:

   - Schemes and slides presented in class, as a framework to study

   - Review manuscripts on specific topics

    

3. Plant Biotechnology and Agriculture Prospects for the 21st Century

   Arie Altman and Paul Michael Hasegawa Academic Press

   Hardcover ISBN: 9780123814661

   eBook ISBN: 978012381467

   
   

Teaching methods

The basis of the course is provided by direct lessons based on power-point presentations.

The course is divided into lessons and practical works. The practical exercises are centered on the analysis of molecular markers and to general and practical aspects of plant breeding in general and breeding for resilience to abiotic stresses / resistance to diseases.

In addition, in the second part of the course, the lessons will be complemented by seminars from plant breeders and researchers in the field

Small Discussion groups (single or two students working together maximum), case-study examinations and preparation of short seminars on the topics will be used to deepen technical topical topics related to the subject of study.

Teacher/student weekly direct meetings for open discussion and lesson revision will be opened in free student hours, preferably the day after lessons.

Assessment methods

The assessment test is carried out through a final oral exam. The Student will start the exam by presenting his case-study example / seminar work with a few slides to be commented. The student will then continue the exam with the teacher posing three main questions related to three main and different topics addressed during the course. Additional specific questions will be addressed according to student knowledge and response and capability to connect topics, objectives and methodologies, and disciplines. Questions are aimed at ascertaining the knowledge relating to the basic and application notions developed during the lessons and practice. The duration of the test is about 30 minutes.

Teaching support tools

• power-point slides, discussion of scientific manuscripts, direct observations in the molecular lab, botanic garden, field
• Bibliographic material available at the University Library System and provided on the VIRTUALE platform.
• Blog / forum for teacher-student communications on the IOL platform, accessible only to students of the course.
• Weekly direct meetings with the professor and post-docs of the group.

Teaching tools

Teaching support tools

• power-point slides, discussion of scientific manuscripts, direct observations in the molecular lab, botanic garden, field
• Bibliographic material available at the University Library System and provided on the VIRTUALE platform.
• Blog / forum for teacher-student communications on the IOL platform, accessible only to students of the course.
• Weekly direct meetings with the professor and post-docs of the group (tutors).

Office hours

See the website of Marco Maccaferri