Kelly Ramig

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Development of a Framework Map to Identify
Candidate Genes Involved in Carbon Allocation and
Partitioning in Populus

• Kelly Ramig
• Knox College, Illinois
• Mentor Lee Gunter
• Environmental Sciences Division

2
Long-Term Project Interest Enhancing
Sequestration of Atmospheric Carbon Dioxide

• Fossil fuel use and deforestation account for
  emissions to the atmosphere of between 7.1-7.6
  GtC/yr
• Oceans and terrestrial biomass accumulate only
  about 4 GtC/yr of excess carbon emissions
• Due to imbalance, the atmosphere accumulates
  about 3.0-3.6 GtC/yr

From Equity Watch A Climate Newsletter from the
South
3
Long-Term Project Interest Enhancing
Sequestration of Atmospheric Carbon Dioxide

• Carbon Sequestration The ability of plants to
  take CO2 from the atmosphere and store it in
  sinks such as leaves, stems, and roots
• Carbon Allocation Determination of where the
  carbon is stored
• Genetic improvement of poplar plantations could
  result in storage of up to 1.2 GtC/yr

From Environmental Protection Agency
4
Why Populus?

• Widely accepted as genetic model for woody,
  perennial plants
• Most species hybridize and produce fertile
  progeny
• Closely related to Arabidopsis
• Small genome size roughly 520 Mbp
• Genome sequence, BAC library, and Linkage Maps
  for several species and pedigrees available

Cottonwood Tree Populus trichocarpa
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Goal of Immediate Project

• Analyze segregation patterns of genetic markers
  in a large pedigree to determine marker position
• Consolidate marker position information for a
  framework map allowing for future
  characterization of loci involved in carbon
  sequestration and allocation

6
Project

• Extracted nuclear DNA from frozen leaf samples
• Amplified specific genetic marker loci through
  PCR
• Fluorescently labeled PCR product denatured and
  genotyped
• Data analyzed with genotyping software

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Pedigree

• Mother (52-225) is a hybrid cross between P.
  trichocarpa and P. deltoides
• Father (D124) is a pure individual from the
  species P. deltoides

P. deltoides
P. trichocarpa

• This pedigree contains gt1,000 progeny available
  for genetic studies

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Genetic Markers

• Genetic markers are anonymous DNA segments used
  to determine genetic variability
• Microsatellites A heritable and highly
  polymorphic segment of DNA consisting of a short
  repeated sequence (1 to 10 bp)
• Microsatellite loci chosen based on segregation
  and position on map of related pedigree (Family
  13)

Example of Microsatellite Allele Segregation
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Summary of Results

• Of the 64 primers tested
• 82 amplified
• 9.4 showed no segregation
• 91.6 showed segregating alleles
• Of the 51 segregating loci
• 59.3 were fully informative
• 37.0 maternally informative
• 3.7 paternally informative

Non-segregating
No amplification
Segregating
Amplification
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Summary of Results
Locus P571 Parental Cross CC ( ?) x AB (?)
Locus O-202 Parental Cross AB (?) x AN (?)
Expected Mendelian Segregation of Alleles
A 50 B 50 C 100
A 75 B 25 Null 50
Observed Segregation Results
A 51.6 B 48.4 C 100
A 53.6 B 78.6 Null 46.4

• Of the 51 loci observed 25 alleles in 13 of
  the loci show significant segregation distortion
• (based on the Chi-squared statistical test for
  significant difference)

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Genetic Map Development

• Segregation among genetic loci allows
  determination of chromosomal position
• This linkage map was constructed for Family 13
• Arrows indicate some of the loci mapped with new
  pedigree

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Conclusions

• High percentage of amplification displays marker
  fidelity
• High percentage of segregation shows a high
  degree of consistency across pedigrees
• Segregation distortion most likely due to small
  sample size of progeny tested
• Initial work completed for an eventual dense
  genetic map of a large pedigree
• Information collected from 63 out of the gt200
  markers needed for a dense genetic map
• 44 of the gt1,000 progeny tested

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Future Work

• Continue mapping other microsatellite markers in
  order to make a dense genetic map
• Compare the map from this pedigree to other maps
  in order to assess marker fidelity
• Use markers for future characterization of genes
  involved in carbon sequestration and allocation

14
Acknowledgements

• This research took place over the fall semester
  of 2003 at the Oak Ridge National Laboratory.
  Thank you to the Environmental Sciences Division,
  especially the Plant Genomics Group for creating,
  organizing, and funding this project. Sincere
  thanks especially to Lee Gunter, Gerald Tuskan,
  Steve Difazio, Ton Ming Yin, and Sara Jawdy for
  their technical expertise.