Summary of sixth lesson

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Summary of sixth lesson

• Janzen-Connol hypothesis explanation of why
  diseases lead to spatial heterogeneity
• Diseases also lead to heterogeneity or changes
  through time
• Driving succession
• The Red Queen Hypothesis selection pressure will
  increase number of resistant plant genotypes
• Co-evolution pathogen increase virulence in
  short term, but in long term balance between host
  and pathogen
• Complexity of forest diseases primary vs.
  secondaruy, modes of dispersal etc

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Summary of seventh lesson

• SEX the great homogenizing force, and also
  ability to create new alleles
• INTERSTERILITY/ MATINGgt SOMATIC COMPATIBILITY
• NEED TO USE MULTIPLE MARKERS SC does that,
  otherwise go to molecular markers
• PCR/ RAPDS

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How to get multiple loci?

• Random genomic markers
• RAPDS
• Total genome RFLPS (mostly dominant)
• AFLPS
• Microsatellites
• SNPs
• Multiple specific loci
• SSCP
• RFLP
• Sequence information
• Watch out for linked alleles (basically you are
  looking at the same thing!)

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Sequence information

• Codominant
• Molecules have different rates of mutation,
  different molecules may be more appropriate for
  different questions
• 3rd base mutation
• Intron vs. exon
• Secondary tertiary structure limits
• Homoplasy

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Sequence information

• Multiple gene genealogiesdefinitive phylogeny
• Need to ensure gene histories are comparable
  partition of homogeneity test
• Need to use unlinked loci

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DNA template
Reverse primer
Forward primer
Thermalcycler
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Gel electrophoresis to visualize PCR product
Ladder (to size DNA product)
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From DNA to genetic information (alleles are
distinct DNA sequences)

• Presence or absence of a specific PCR amplicon
  (size based/ specificity of primers)
• Differerentiate through
• Sequencing
• Restriction endonuclease
• Single strand conformation polymorphism

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Presence absence of amplicon

• AAAGGGTTTCCCNNNNNNNNN
• CCCGGGTTTAAANNNNNNNNN

AAAGGGTTTCCC (primer)
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Presence absence of amplicon

• AAAGGGTTTCCCNNNNNNNNN
• CCCGGGTTTAAANNNNNNNNN

AAAGGGTTTCCC (primer)
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Result series of bands that are present or
absent (1/0)
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Root disease center in true fir caused by H.
annosum
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Ponderosa pine
Incense cedar
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Yosemite Lodge 1975 Root disease centers
outlined
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Yosemite Lodge 1997 Root disease centers
outlined
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Are my haplotypes sensitive enough?

• To validate power of tool used, one needs to be
  able to differentiate among closely related
  individual
• Generate progeny
• Make sure each meiospore has different haplotype
• Calculate P

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RAPD combination1 2

• 1010101010
• 1010101010
• 1010101010
• 1010101010
• 1010000000

• 1011101010
• 1010111010
• 1010001010
• 1011001010
• 1011110101

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Conclusions

• Only one RAPD combo is sensitive enough to
  differentiate 4 half-sibs (in white)
• Mendelian inheritance?
• By analysis of all haplotypes it is apparent that
  two markers are always cosegregating, one of the
  two should be removed

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AFLP

• Amplified Fragment Length Polymorphisms
• Dominant marker
• Scans the entire genome like RAPDs
• More reliable because it uses longer PCR primers
  less likely to mismatch
• Priming sites are a construct of the sequence in
  the organism and a piece of synthesized DNA

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How are AFLPs generated?

• AGGTCGCTAAAATTTT (restriction site in red)
• AGGTCG CTAAATTT
• Synthetic DNA piece ligated
• NNNNNNNNNNNNNNCTAAATTTTT
• Created a new PCR priming site
• NNNNNNNNNNNNNNCTAAATTTTT
• Every time two PCR priming sitea are within
  400-1600 bp you obtain amplification

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AFLPs are read like RAPDs (0/1)
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Dealing with dominant anonymous multilocus markers

• Need to use large numbers (linkage)
• Repeatability
• Graph distribution of distances
• Calculate distance using Jaccards similarity
  index

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Jaccards

• Only 1-1 and 1-0 count, 0-0 do not count
• 1010011
• 1001011
• 1001000

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Jaccards

• Only 1-1 and 1-0 count, 0-0 do not count
• A 1010011 AB 0.6 0.4 (1-AB)
• B 1001011 BC0.5 0.5
• C 1001000 AC0.2 0.8

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Now that we have distances.

• Plot their distribution (clonal vs. sexual)

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Now that we have distances.

• Plot their distribution (clonal vs. sexual)
• Analysis
• Similarity (cluster analysis) a variety of
  algorithms. Most common are NJ and UPGMA

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Now that we have distances.

• Plot their distribution (clonal vs. sexual)
• Analysis
• Similarity (cluster analysis) a variety of
  algorithms. Most common are NJ and UPGMA
• AMOVA requires a priori grouping

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AMOVA groupings

• Individual
• Population
• Region
• AMOVA partitions molecular variance amongst a
  priori defined groupings

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Now that we have distances.

• Plot their distribution (clonal vs. sexual)
• Analysis
• Similarity (cluster analysis) a variety of
  algorithms. Most common are NJ and UPGMA
• AMOVA requires a priori grouping
• Discriminant, canonical analysis

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Results Jaccard similarity coefficients
P. nemorosa
P. pseudosyringae U.S. and E.U.
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P. pseudosyringae genetic similarity patterns are
different in U.S. and E.U.
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Results P. nemorosa
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Now that we have distances.

• Plot their distribution (clonal vs. sexual)
• Analysis
• Similarity (cluster analysis) a variety of
  algorithms. Most common are NJ and UPGMA
• AMOVA requires a priori grouping
• Discriminant, canonical analysis
• Frequency does allele frequency match expected
  (hardy weinberg), F or Wrights statistsis

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The scale of disease

• Dispersal gradients dependent on propagule size,
  resilience, ability to dessicate, NOTE not
  linear
• Important interaction with environment, habitat,
  and niche availability. Examples Heterobasidion
  in Western Alps, Matsutake mushrooms that offer
  example of habitat tracking
• Scale of dispersal (implicitely correlated to
  metapopulation structure)---

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Have we sampled enough?

• Resampling approaches
• Saturation curves
• A total of 30 polymorphic alleles
• Our sample is either 10 or 20
• Calculate whether each new sample is
  characterized by new alleles

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Saturation curves
No Of New alleles
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
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If we have codominant markers how many do I need

• IDENTITY tests probability calculation based
  on allele frequency Multiplication of
  frequencies of alleles
• 10 alleles at locus 1 P10.1
• 5 alleles at locus 2 P20,2
• Total P P1P20.02

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White mangroves Corioloposis caperata
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Distances between study sites
White mangroves Corioloposis caperata
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Forest fragmentation can lead to loss of gene
flow among previously contiguous populations.
The negative repercussions of such genetic
isolation should most severely affect highly
specialized organisms such as some
plant-parasitic fungi.
AFLP study on single spores
Coriolopsis caperata on Laguncularia racemosa
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Using DNA sequences

• Obtain sequence
• Align sequences, number of parsimony informative
  sites
• Gap handling
• Picking sequences (order)
• Analyze sequences (similarity/parsimony/exhaustive
  /bayesian
• Analyze output CI, HI Bootstrap/decay indices

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Using DNA sequences

• Testing alternative trees kashino hasegawa
• Molecular clock
• Outgroup
• Spatial correlation (Mantel)
• Networks and coalescence approaches

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Pacifico
Caribe
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From Garbelotto and Chapela, Evolution and
biogeography of matsutakes
Biodiversity within species as significant as
between species