1. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Chapter 19 Genomics and Agriculture Applications of genomics approaches to agriculture

2. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Contents  Background  Agriculturally related sequencing projects  Crop plant  Farm animal  Pathogen  Genomics applied to trait improvement  Breeding  Transgenics and clones

3. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Background  Agriculture looks to genomics for the next “green revolution”  Reasons:  Pace of traditional breeding  Identify genes for useful traits  Relate genetic and physical maps  Protect food chain

4. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics applied to agriculture  Approaches  Similar for plants and animals  Relating traits to genes  Relating genetic maps to physical maps  QTL analysis  DNA sequence  Problem of genome size  Syntenic relationships  ESTs

5. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Sequencing of plant genomes  Reference plant: Arabidopsis thaliana  No agricultural value  Related to cabbage and mustard  Reference for all plants  First plant genome sequenced (Dec. 2000)  Size: 130 Mbp  Number of genes: 28,000  Segmental duplications  Evidence for past increase in ploidy

6. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Sequencing of crop-plant genomes  Reasons for sequencing rice first  Importance as crop  Largest food source for poor  Feeds half of world’s population  Demand likely to increase dramatically

7. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Rice genome  Smallest grass genome  Few repetitive elements  Synteny with other grasses  Genetic and physical maps  Genomic resources  ESTs  Efficient transformation

8. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Efforts to sequence the rice genome  Different efforts  Public: IRGSP/Beijing Genomics Institute  Private: Monsanto/Syngenta  Public performed 10 x coverage  Two strains: Indica and Japonica  Gold standard for other cereal genomes  Microarray of rice used on maize RNA

9. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Facts about the rice genome  Size: 430 Mbp  Number of genes: approximately 60,000  Repetitive elements: Most in intergenic regions

10. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Rice and Arabidopsis genomes  No large areas of synteny  80% of Arabidopsis genes have homologs in rice  Only 50% of rice genes have homologs in Arabidopsis Rice Arabidopsis

11. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics of other cereals  Maize: 3,000 Mbp  Wheat: 5,000 Mbp  Barley: 16,000 Mbp  Genome organization  Genic islands in sea of retroposons

12. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Sequencing strategies for grass genomes  Alternative approaches to genome sequencing  Methylation based  Hybridization based  EST collections

13. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics of other crop plants  Tomato, potato, soybean  EST collections  Woody species  Poplar and pine  Genome organization  No genic islands  Candidate-gene approach

14. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics of farm animals  Livestock farming = 30–40% of world agriculture  Poultry and livestock sales in United States > $70 billion  Disadvantages of genomics  Large sizes of farm-animal genomes  Long gestation times  Difficulty of doing genetics

15. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics of farm mammals  Pig, cow, sheep  Draft sequencing  Compare to mouse, rat, and human  BAC libraries  Physical maps  EST libraries

16. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics of poultry  No reference genome  BAC libraries  > 300,000 ESTs  Microarrays

17. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Sequencing of agricultural pathogens  Animal pathogens  Brucella suis  Infects animals, but can affect humans  Genome revealed to be similar to plant pathogen  Plant pathogens  Problem: large size of genomes

18. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Bioterrorism issues  Intensive agriculture raises risks of disease spread  Example: outbreak of foot-and-mouth disease  Knowledge of pathogen genomes  Helps identify disease agent  Could be used in rapid- detection technologies

19. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Breeding  Domestication of plants and animals selected for valuable traits  e.g., temperament  Later, other traits selected for  e.g., milk production  Traits controlled by several genes  For each gene, different alleles

20. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Quantitative traits  Major traits in continuous gradient  Controlled by QTLs  Infinitesimal model  Many genes, each with small effect  Major-gene model  A few genes, each with large effect  Genomic nature of QTLs

21. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 QTL analysis  QTL analysis requires genetic and physical maps  Similar to association mapping in humans  Relate traits to markers  Or cross two subspecies with different traits  Both domesticated  Wild plus domesticated

22. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genome scan for QTLs  Genome scan for QTLs in progeny  Relate trait to markers  Identifies interval on chromosome

23. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 QTL for tomato fruit size

24. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Marker-assisted breeding  Once a QTL is found, it can be used to assist breeding  Even if the nature of gene is unknown  Markers on either side of the QTL can be followed during the breeding program  Introgress the QTL from one subspecies into another  Markers have to be very closely linked

25. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 QTL to gene  If genome sequenced:  Candidate genes in interval  If genome not sequenced:  Find syntenic region in sequenced genome  To confirm identity:  Look for mutations  Microarrays and 2-D gels  Transfer gene and determine consequences

26. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics tools for breeding  Polymorphic markers  Microsatellites  SNPs  Expression approaches  Microarrays  2-D gels  Bioinformatics  Databases

27. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Improvement of plant traits  Stress resistance  Abiotic  Salinity  Drought  Biotic  Pathogens  Increased yield  Decreased fertilizer utilization  Improved value-added traits

28. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Examples of plant breeding  Hybrid vigor  Corn with improved yield  Compare inbred parental lines with hybrids  Size of tomato fruit  Comparisons of wild relatives with crop plants

29. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Improvement of animal traits  Growth rate  Meat quality  Disease resistance  Reproductive performance  Behavior

30. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Example of animal breeding  Meat quality in pigs  Meat-to-fat ratio  Cross Chinese Meishan pigs with European Large White pigs  Meishan much fatter than European variety  Identified QTL for lean meat

31. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Transgenic technologies  Goal: rapid modification of genes responsible for traits in plants and animals  Gain of function:  Overexpression  Ectopic expression  Loss of function:  Homologous recombination  Antisense or RNAi

32. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Transgenic plant technology  Agrobacterium- mediated transformation  Gene inserted into Ti plasmid  Agrobacterium cocultivated with plant  Ti plasmid transferred into plant genome  Selection with antibiotics Origin of replication Ti plasmid Nopaline utilization T-DNA Transfer functions T-DNA Tumor production Nopaline synthesis Agrobacterium gall on a cherry tree

33. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Transformation of rice

34. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Transgenic animal technologies  Introduction of genes directly into nucleus  Microinjection  Used to produce transgenic pigs, cattle, and sheep  Problems: inefficiency, chromosomal insertion

35. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Gene knockout techniques  Homologous recombination  RNAi  Can be combined with nuclear transfer AGL5 KO construct AGL5 wt genomic agl5 KO genomic Kan R Deleted in KO

36. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Nuclear transfer  “Nuclear transfer” or “cell nuclear replacement” better than “cloning”  Transfer of nucleus from adult cell to unfertilized egg with nucleus removed  Or fusion of adult cell with enucleated egg  Problem: abnormal development

37. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Animal cloning Scottish Blackface Finn Dorset Udder cellEgg cell Remove nucleus from egg cell Fuse cells with electric shock Fused cell grows into an embryo Cloned lamb is born Dolly Finn Dorset Embryo is placed in foster mother

38. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Pharmaceutical proteins  Alpha-1-antitrypsin  Applications in emphysema and cystic fibrosis  Problems in isolating from humans, yeast, or bacteria  Target in cell and then perform nuclear transfer

39. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Summary I  Need for genomics approaches in agriculture  Genomic sequencing  Crop plant  Rice  Farm animal  Livestock and poultry  Pathogens  Bioterrorism

40. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Summary II  Genomics and breeding  QTLs  Traits  Transgenic technologies  Plant  Animal