Genetic Coalescence in North American White Pines

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Genetic Coalescence in North American White Pines

• Kathleen Farrell
• Dr. Aaron Liston, Dr. Richard Cronn,
• John Syring

P. strobus
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Coalescence is the pattern of common ancestry.
Coalescence theory At some point all alleles in
the population will be traceable to a single
common ancestral allele that existed in the
population at time t0.

• Factors
• Gene flow
• Hybridization
• Selection
• Population size

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Objective to find low copy nuclear genes that
accurately demonstrate the relationships
(phylogeny) between closely related species,
North American White Pines.
Why low copy genes? Orthologs vs. Paralogs
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Objective to find low copy nuclear genes that
accurately demonstrate the relationships
(phylogeny) between closely related species,
North American White Pines.
Why low copy genes? Orthologs vs. Paralogs
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Species Sampled North American White Pines P.
albicaulis Whitebark Pine P. ayacahuite Mexican
White Pine P. chiapensis Chiapas White Pine P.
flexilis Limber Pine P. lambertiana Sugar
Pine P. monticola Western White Pine P.
strobiformis Southwestern White Pine P.
strobus Eastern White Pine
P. flexilis
P. lambertiana
P. albicaulis
P. ayacahuite
Outgroups P. monophylla Singleleaf Pinyon P.
longaeva Bristlecone Pine
P. monophylla
P. longaeva
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Methods and Approach
DNA Extraction from the megagametophyte tissue.
megagametophyte (1n)
embryo (2n) shoot end
embryo root tip (radicle)
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Polymerase Chain Reaction (PCR)

• How it works
• DNA Denatures
• Primers Anneal
• Taq Polymerase builds a complementary copy.

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Polymerase Chain Reaction (PCR)

• How it works
• DNA Denatures
• Primers Anneal
• Taq Polymerase builds a complementary copy.

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Polymerase Chain Reaction (PCR)
Results Gel Electrophoresis
IFG8612 Optimization MONTICOLA 02S1
Temperature (C) ? 56 57 58 59 60
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Temperature (C) ? 56 57 58 59 60
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Control
Control
62 63 64 65 66 67 68
62 63 64 65 66 67 68
2.0 mM MgCl
1.5 mM MgCl
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Polymerase Chain Reaction (PCR)
Results Gel Electrophoresis
IFG8612 Optimization MONTICOLA 02S1
Temperature (C) ? 56 57 58 59 60
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Temperature (C) ? 56 57 58 59 60
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Control
Control
62 63 64 65 66 67 68
62 63 64 65 66 67 68
2.0 mM MgCl
1.5 mM MgCl
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Polymerase Chain Reaction (PCR)
Results Gel Electrophoresis
IFG8612 Optimization MONTICOLA 02S1
Temperature (C) ? 56 57 58 59 60
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Temperature (C) ? 56 57 58 59 60
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Control
Control
62 63 64 65 66 67 68
62 63 64 65 66 67 68
2.0 mM MgCl
1.5 mM MgCl
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Cycle Sequencing
Capillary Gel
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Cycle Sequencing
Analyzing Results
Clean Sequence
Sequence Noise
Alignment and Editing
------------------------GTAATT----AGATTTGAATGGCATG
GAATGAATGGAAA -GCaGGAtaTTGTCATTTCTTTCAGTAATT----AG
ATTTGAATGGCATGGAATGAATGGAAA ----GGAtaTTATCATTTTTTT
CaGTAATTAATTAGATTTGAATGGCATGGAATGAATGGAAA -GCaGGAt
ATTGTCATTTCTTTCAGTAATT----AGATTTGAATGGCATGGAATGAAT
GGAAA
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Developing a Phylogenetic Tree

• Export alignments to PAUP
• Search for phylogenetic trees based on maximum
  parsimony using the branch and bound algorithm.
• Calculate bootstrap values (measure of confidence
  for each branch) and branch length (measure of
  relative divergence).

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Results
IFG8898
IFG8612
Cinnamyl Alcohol Dehydrogenase
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Results
IFG8898
IFG8612
Cinnamyl Alcohol Dehydrogenase
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Results
IFG8898
IFG8612
Cinnamyl Alcohol Dehydrogenase
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IFG8612 alignment
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IFG8612 alignment
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IFG8612 alignment
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IFG8612 alignment
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Phylogenetic Tree
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Phylogenetic Tree
Outgroups
Bootstrap Support ()
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Phylogenetic Tree
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Phylogenetic Tree
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Phylogenetic Tree
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Phylogenetic Tree
FLEXILIS JVS
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Phylogenetic Tree
FLEXILIS JVS
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Future Research

• Continue screening low copy, nuclear genes to
  compare phylogenetic patterns across the genome.
• Increase sampling to five individuals from each
  species that represent the geographic
  distribution of the species.
• Extend research to include Asian White Pines.

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Special Thanks to Howard Hughes Medical
Institute Portland Garden Club Undergraduate
Research, Innovation, Scholarship, and Creativity
(URISC) National Science Foundation
P. strobus