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Molecular (Marker assisted) breeding and Genetic engineering

Molecular (Marker assisted) breeding and Genetic engineering. 7-27-2008. Objective. Industry has experienced significant growth Industry production Introduction of new products Cultivars of existing crops New species Rapid expansion brings new issues Breeder’s rights

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Molecular (Marker assisted) breeding and Genetic engineering

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  1. Molecular (Marker assisted) breeding and Genetic engineering 7-27-2008

  2. Objective • Industry has experienced significant growth • Industry production • Introduction of new products • Cultivars of existing crops • New species • Rapid expansion brings new issues • Breeder’s rights • Grower confidence in cultivar identity • Improved plant quality • Disease and insect resistance • Heat and drought tolerance • Longer shelf life Source: James W. Moyer Dept. of Plant Pathology North Carolina State University, Raleigh, NC

  3. Outline Part I Principles and constraints of marker-assisted breeding Breeding by design Part II Principles and constraints of Genetic engineering (e.g. Poinsettia) Facts about Genetic engineering

  4. 1. Principles and constraints of marker-assisted breeding

  5. Ch. Gebhardt MPIZ Breeding the traditional way • Identify source of a trait by phenotypic screening of germ plasm collections. • Cross source with elite cultivars. • Select desirable progeny. • Evaluate desirable lines for other agronomic traits. • Repeat crossing and selection until quality reaches variety level.

  6. Ch. Gebhardt MPIZ Marker-assisted pedigree breeding for mono- and polygenic traits: • Identify source of the trait by phenotypic screening of germ plasm collections. • Cross source with elite susceptible cultivar. • Select by phenotypic screening progeny with and without the trait of interest. • Identify marker(s) linked to the gene conferring the trait of interest. • Use the marker(s) for MAS in crosses with the source or its descendants.

  7. Ch. Gebhardt MPIZ

  8. Boxplots illustrating the variation of phenotypic data for late blight resistance: Identification and characterization of QTL 2007 Phenotyping and ... ... Genotyping 1 2 • Two populations with 96 individuals related by descent. 3 4 5 Symptoms of nematode infestation in the field SSR marker Nematode cyst on potato root SNP marker CAPS marker

  9. Ch. Gebhardt MPIZ

  10. VI II III IV V I TG5 GP79 GP180 rbcS-1 GP186 CP46 GP23 GP303 Rx2,Nb CT229 TG31 GP21 TG231, TG97 GP519 Stm3016 TG432 GP1-a rbcS-c MBF CosA R1 GP501 CT242 CP48-a TG123 GP25 GP179 R2 TG24 GP508 TG118 CP100 CP6 GP221 CP108 CP18 GP26 Ny TG74 GP184 CT184 tbr GP17-a TG62 CP11 GP276 GP216 GP226 TG20-b TG134 CP19 CP12 GP88 H1 CP113 CD78 CP62 CP109 GroV1 TG22 TG69 GP76 AGPaseS-a TG53 TG115 SbeI GP295 GP172 GP22 CP132 X XII IX VIII XI VII R3,R6,R7 Pat-a CP44 GP185 CP52 CP47 TG105-a Ry-f GP122 sto Ns TG28 TG43-a TG36 TG61 TG45 PLRV.4 TG254 TG105-b TG16 CP117 RB GP92 GP91-c CP53 R TG35 ber Gro1 TG63 CP56 TG68 Rpi1 GP129 GP40-a TG20-a Nx GP125 Gpa2 CP49 Ry,Na GP101 Rx1 CP58 GP34 Ch. Gebhardt MPIZ CT220 GP219 GbssI CP60 Sen1 R Mc1 Example potato: Integrated map of R loci for resistance to different pathogens

  11. Source: Gerhard Wenzel, 2006

  12. Construction of genetic linkage maps 1. Mapping population - Polymorphisms between parents • F2 or backcross populations 2. Population size (1cM= 1 recomb./100 plants) 3. Relationship between DNA marker and cytogenetic map a) aneuploid (trisomics), translocation b) in-situ hybridization Source: Cregan et al. 2001 SSR marker analysis from a Triplo F1 Hybrid in Soybean

  13. Identification and characterization of QTL 2007 FISH 1 V 2 3 4 5 S C Potato pachytene bivalents L

  14. Has MAS a comparative advantage (time and money) versus phenotypic screens? • Increased reliability (phenotypic assays are affected by Environment, heritability, number of genes...) • Increased efficiency (application at seedling stage, screening of many recombinants) • Reducing cost (in general PCR less expensive than phenotypic assay) • Exceeds the limits of classical breeding (e.g. Removal of linkage drag, pyramiding resistance genes, polygenic traits, exotic germplasm)

  15. Pyramiding resistance Source: Gerhard Wenzel, 2006

  16. Ch. Gebhardt MPIZ Screening for resistance loci with molecular markers

  17. Ch. Gebhardt MPIZ Inter-locus interactions (P < 0.001)

  18. Constraints: Linkage disequilibrium Markers specific for an allele in a population Several markers necessary for QTLs DNA sequence is required Epistasis

  19. DNA Fingerprinting and Molecular Markers • DNA fingerprinting is a useful tool in crop genetics to meet recent challenges: • Cultivar identification • Maintenance of breeding lines • Protecting breeders’ rights • Molecular markers can facilitate the identification and introgression of genes for cultivar improvement • Methods for generating genetic markers include: • AFLP • SSR Source: James W. Moyer Dept. of Plant Pathology North Carolina State University, Raleigh, NC

  20. Fingerprinting in Poinsettia • Poinsettia database: • 117 cultivars • 41 AFLP fragments • Successfully distinguishes most cultivars • Multiple plants from representative cultivars used for validation studies • Plants from the same breeding family cluster together • Color sports cluster together as the same cultivar Source: James W. Moyer Dept. of Plant Pathology North Carolina State University, Raleigh, NC

  21. 2.The breeding by design concept Understanding the genetic basis of all agronomically important characters and the allelic variation of those loci, the breeder would be able to design superior genotypes in silico. Peleman and van der Voort, 2003

  22. Principles and constraints of Genetic engineering (e.g. Poinsettia)

  23. Prerequisite of GE • Availability of the trait to be transferred as cloned DNA • Availability of a powerful transfer system (e.g. A. tumefaciens) • Availability of a reliable regeneration system predominantly from a single transformed cell Source: Wenzel, 2006

  24. Example: Resistance in Poinsettia www.gene-quantification.de/siRNA-mechanism.png

  25. Strategy: Agrobacterium strain and RNA constructs Selection and regeneration of transgenic plants (resistance gene)

  26. 3. Screening (PCR)

  27. 4. Southern Blot analysis (transgene integration and copy number) 5. Northern Blot analysis (detect transgene derived siRNA molecules) 6. Virus inoculation and detection (DAS-ELISA) 7. Evaluation of the transgenic plants Result: Transgenic PnMV resistant plants!

  28. More examples on GMOs • Bt- corn Source: The science creative quarterly Development of insect resistance in crops such as maize by incorporating a gene from Bacillus thuringiensis.

  29. The Rose Creation of blue rose was achieved introducing blue color-related enzyme gene from pansy.

  30. Facts on GMOs www.ers.usda.gov/Data/BiotechCrops/

  31. Facts on GMOs Global area of transgenic crops Source: Gerhard Wenzel, 2006

  32. Facts on GMOs Source: Gerhard Wenzel, 2006

  33. Facts on GMOs Time scale of genetically modified characters in crops Source: Gerhard Wenzel, 2006

  34. Constraints of GE Isolation of the gene of interest and the understanding of the biochemical pathways and knowledge in the field of metabolomics Reluctance towards gene technology The presence of marker genes may complicate future commercialization (antibiotic resistance markers) From model plants (e.g. A. tumefaciens) to crops. Growth in the open environment is legally controlled and substantially restricted (gene flow). Cultural problems (e.g. Golden Rice)

  35. Acknowledgements Sadanand Dhekney Christiane Gebhardt Diane Mealo Todd Perkins Manfred Mehring- Lemper Jose Chaparro Santiago Brown Sven van den Elsen Theresa Mosquera

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