Molecular (Marker assisted) breeding and Genetic engineering

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Molecular (Marker assisted) breeding and Genetic
engineering

• 7-27-2008

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Objective

• Industry has experienced significant growth
• Industry production
• Introduction of new products
• Cultivars of existing crops
• New species
• Rapid expansion brings new issues
• Breeders 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
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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

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1. Principles and constraints of marker-assisted
breeding
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Breeding the traditional way

1. Identify source of a trait by phenotypic
   screening of germ plasm collections.
2. Cross source with elite cultivars.
3. Select desirable progeny.
4. Evaluate desirable lines for other agronomic
   traits.
5. Repeat crossing and selection until quality
   reaches variety level.

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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.

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Identification and characterization of QTL
2007
Phenotyping and ...
... Genotyping
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• Two populations with 96 individuals related by
  descent.

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Symptoms of nematode infestation in the field
SSR marker
Nematode cyst on potato root
SNP marker
CAPS marker
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Example potato Integrated map of R loci for
resistance to different pathogens
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Source Gerhard Wenzel, 2006
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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
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Identification and characterization of QTL
2007
FISH
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V
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S

C
Potato pachytene bivalents
Segment Number of observations Length of the segment (SD) µm Molecular size based on FISH (SD) Mb Genetic size cM
Complete chromosome V 4 41.0 4.2 - 70
telomer-GP21 6 4.6 0.6 2.76 0.36 15
GP21-GP179 6 1.6 0.4 0.96 0.24 3
GP179-StPto 6 4.2 0.5 2.52 0.3 5
L
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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)

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Pyramiding resistance
Source Gerhard Wenzel, 2006
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Screening for resistance loci with molecular
markers
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Inter-locus interactions (P lt 0.001)
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Constraints

• Linkage disequilibrium
• Markers specific for an allele in a
  population
• Several markers necessary for QTLs
• DNA sequence is required
• Epistasis

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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
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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
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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
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Principles and constraints of Genetic engineering
(e.g. Poinsettia)
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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
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Example Resistance in Poinsettia
www.gene-quantification.de/siRNA-mechanism.png
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Strategy

1. Agrobacterium strain and RNA constructs
2. Selection and regeneration of transgenic plants
   (resistance gene)

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3. Screening (PCR)
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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!
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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.
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The Rose
Creation of blue rose was achieved introducing
blue color-related enzyme gene from pansy.
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Facts on GMOs
www.ers.usda.gov/Data/BiotechCrops/
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Facts on GMOs
Global area of transgenic crops
Source Gerhard Wenzel, 2006
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Facts on GMOs
Source Gerhard Wenzel, 2006
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Facts on GMOs
Time scale of genetically modified characters in
crops
Source Gerhard Wenzel, 2006
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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)

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