Overview

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Overview

• Armillaria bulbosa (gallica)
• Known as the Humungous Fungus, or honey mushroom
• Form rhizomorphs, which make up much of the
  humungous part
• Basidiocarp cap 6 cm in diameter, stem is 5-10
  cm tall
• Facultative tree root pathogen

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Life cycle Reproduction

• Sexual
• Basidiocarps release spores (n) after karyogamy
  and meiosis
• 2 mating-type loci, each with multiple alleles in
  the population
• Isolates (n) must have different alleles at two
  mating type loci to be sexually compatible
• Asexual
• vegetative spreading of rhizomorph
• The large mass of rhizomorph that is genetically
  isolated is called a clone

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Building up the question

• By extending the areas sampled in subsequent
  years, we were finally able to delimit the large
  area occupied by this genotype and then go on to
  show that this genotype likely represents and
  individual
• - Myron Smith

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Researchers Question

• The clonal individual is especially difficult
  to define because the network of hyphae is
  underground
• How do you unambiguously identify an individual
  fungi within a local population?

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Approach

• 1. Collect samples
• 2. Check mating type
• - Somatic compatibility test
• - Distrubution of mating-type alleles

• 3. Molecular testing
• - RFLP
• - RAPD
• 4. Statistics
• 5. More testing

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Methods and Materials 1

• 1. Collecting samples
• Researcher collected samples over a 30 hectare
  area by baiting Armillaria with poplar stakes and
  taking tissues and spores
• They then grew the successfully colonized stakes
  in soil taken from the study site
• Each fungal colony cultured was called an isolate.

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Methods and Materials 2
Example (not Armillaria)

• 2. Checking mating type
• - Somatic incompatibilityFor two fungal
  isolates to fuse, all somatic compatibility loci
  must be the same.
• Fusion means theyre clones ?

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Methods and Materials 2

• 2. Checking mating type
• - Distrubution of mating alleles
• Mating occurs only when coupled isolates have
  different alleles at two unlinked, multiallelic
  loci A and B. (They have an incompatibility
  system)
• If fruit bodies had the same alleles at A and B,
  and were collected from the same area, they were
  assumed to be from the same clone

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Result 1

• Somatic compatilbilty
• isolates from vegetative mycelium from a large
  sampling area fused
• Mating alleles
• They had the same mating type

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Result 1

• Clone 1 was found to exceed 500 m in diameter
• Used previously collected mtDNA restriction
  fragment patterns

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Sensitivity of Approach

• Problem These tests alone are not enough to
  distinguish a clone from closely related
  individuals

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Why?

• Q The first two tests were not sensitive enough
  to tell a clone from a close relativeWhy?
• A Spores from same point source have the same
  mating-type alleles, but the offspring they
  produce after inbreeding are genetically
  distinct.

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Methods and Materials 3

• 3. Molecular Testing
• - RFLP analysis at 5 polymorphic, heterozyg. loci
  of mtDNA from Clone 1
• - RAPD analysis at 11 loci

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RAPDS vs. RFLPs

• Use 1 short PCR primer
• When it finds match on template at a distance
  that can be amplified (primer binds twice within
  50 to 2000 bp) RAPD amplicon
• Dominant, annoymous

• Total genomic, vs single locus
• Use endonuclease to digest DNA at specific
  restriction site
• Run digest and see how amplicon was cut
• Single locus is co-dominant

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Result 2

• RFLP
• All 5 loci from Clone 1 were heterozygous and
  identical (both alleles present at loci
  1,1)
• RAPD
• All 11 RAPD products were present in all
  vegetative isolates

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Statistical Analysis

• The probability of retaining heterozygosity at
  each parental locus in an individual produced by
  mating of sibling monospore isolates
• 0.0013
• So they were pretty confident that cloning was
  responsible for their results, not inbreeding

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More testing, just in case

• To be completely confident, they tested
• 1) that nearby Clone 2 was different and lacked 5
  of the Clone 1 heterozyg. RAPD fragments,
• 2) more loci, totaling
• 20 RAPD fragments
• 27 nuclear DNA RFLP fragments
• all were identical in Clone 1

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Sensitivity of RAPDs

• Tested on subset of spores from same basidiocarp
• RAPDs differentiated among full sibs

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Conclusions

• Somatic compatibility, mating allele loci, mtDNA,
  RFLP, and RAPD tests all indicate that a single
  organism could indeed occupy a 15 hectare area

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Conclusions

• The larger individual, Clone 1 was estimated to
  weigh 9700 kg and be over 1500 years old

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Implications

• ?????
• Fungi are one of the oldest and largest organisms
  on the planet
• Recycle nutrientsvery important!
• Armillaria bulbosa also a pathogen its effects
  on forest above may be huge as well.

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HOST-SPECIFICITY

• Biological species
• Reproductively isolated
• Measurable differential size of structures
• Gene-for-gene defense model
• Sympatric speciation Heterobasidion, Armillaria,
  Sphaeropsis, Phellinus, Fusarium forma speciales

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Phylogenetic relationships within the
Heterobasidion complex
Fir-Spruce
Pine Europe
Pine N.Am.
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The biology of the organism drives an epidemic

• Autoinfection vs. alloinfection
• Primary spreadby spores
• Secondary spreadvegetative, clonal spread, same
  genotype . Completely different scales (from
  small to gigantic)
• Coriolus
• Heterobasidion
• Armillaria
• Phellinus

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OUR ABILITY TO

• Differentiate among different individuals
  (genotypes)
• Determine gene flow among different areas
• Determine allelic distribution in an area

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WILL ALLOW US TO DETERMINE

• How often primary infection occurs or is disease
  mostly chronic
• How far can the pathogen move on its own
• Is the organism reproducing sexually? is the
  source of infection local or does it need input
  from the outside

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IN ORDER TO UNDERSTAND PATTERNS OF INFECTION

• If John gave directly Mary an infection, and Mary
  gave it to Tom, they should all have the same
  strain, or GENOTYPE (comparisonsecondary spread
  among forest trees)
• If the pathogen is airborne and sexually
  reproducing, Mary John and Tom will be infected
  by different genotypes. But if the source is the
  same, the genotypes will be sibs, thus related

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Recognition of self vs. non self

• Intersterility genes maintain species gene pool.
  Homogenic system
• Mating genes recognition of other to allow for
  recombination. Heterogenic system
• Somatic compatibility protection of the
  individual.

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Recognition of self vs. non self

• What are the chances two different individuals
  will have the same set of VC alleles?
• Probability calculation (multiply frequency of
  each allele)
• More powerful the larger the number of loci
• and the larger the number of alleles per locus

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Recognition of self vs. non self

• It is possible to have different genotypes with
  the same vc alleles
• VC grouping and genotyping is not the same
• It allows for genotyping without genetic tests
• Reasons behing VC system protection of
  resources/avoidance of viral contagion

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Somatic incompatibility
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More on somatic compatibility

• Perform calculation on power of approach
• Temporary compatibility allows for cytoplasmic
  contact that then is interrupted this temporary
  contact may be enough for viral contagion

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SOMATIC COMPATIBILITY

• Fungi are territorial for two reasons
• Selfish
• Do not want to become infected
• If haploids it is a benefit to mate with other,
  but then the nn wants to keep all other
  genotypes out
• Only if all alleles are the same there will be
  fusion of hyphae
• If most alleles are the same, but not all, fusion
  only temporary

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SOMATIC COMPATIBILITY

• SC can be used to identify genotypes
• SC is regulated by multiple loci
• Individual that are compatible (recognize one
  another as self, are within the same SC group)
• SC group is used as a proxy for genotype, but in
  reality, you may have some different genotypes
  that by chance fall in the same SC group
• Happens often among sibs, but can happen by
  chance too among unrelated individuals

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Recognition of self vs. non self

• What are the chances two different individuals
  will have the same set of VC alleles?
• Probability calculation (multiply frequency of
  each allele)
• More powerful the larger the number of loci
• and the larger the number of alleles per locus

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Recognition of self vs. non selfprobability of
identity (PID)

• 4 loci
• 3 biallelelic
• 1 penta-allelic
• P 0.5x0.5x0.5x0.20.025
• In humans 99.9, 1000, 1 in one million

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INTERSTERILITY

• If a species has arisen, it must have some
  adaptive advantages that should not be watered
  down by mixing with other species
• Will allow mating to happen only if individuals
  recognized as belonging to the same species
• Plus alleles at one of 5 loci (S P V1 V2 V3)

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INTERSTERILITY

• Basis for speciation
• These alleles are selected for more strongly in
  sympatry
• You can have different species in allopatry that
  have not been selected for different IS alleles

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MATING

• Two haploids need to fuse to form nn
• Sex needs to increase diversity need different
  alleles for mating to occur
• Selection for equal representation of many
  different mating alleles

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MATING

• If one individuals is source of inoculum, then
  the same 2 mating alleles will be found in local
  population
• If inoculum is of broad provenance then multiple
  mating alleles should be found

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MATING

• How do you test for mating?
• Place two homokaryons in same plate and check for
  formation of dikaryon (microscopic clamp
  connections at septa)

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Clamp connections
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MATING ALLELES

• All heterokaryons will have two mating allelels,
  for instance a, b
• There is an advantage in having more mating
  alleles (easier mating, higher chances of finding
  a mate)
• Mating allele that is rare, may be of migrant
  just arrived
• If a parent is important source, genotypes should
  all be of one or two mating types

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Two scenarios

• A, A, B, C, D, D, E, H, I, L

• A, A, A,B, B, A, A

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Two scenarios

• A, A, B, C, D, D, E, H, I, L
• Multiple source of infections (at least 4
  genotypes)

• A, A, A,B, B, A, A
• Siblings as source of infection (1 genotype)

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SEX

• Ability to recombine and adapt
• Definition of population and metapopulation
• Different evolutionary model
• Why sex? Clonal reproductive approach can be very
  effective among pathogens

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Long branches in between groups suggests no sex
is occurring in between groups
Fir-Spruce
Pine Europe
Pine N.Am.
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Small branches within a clade indicate sexual
reproduction is ongoing within that group of
individuals
NA S
NA P
EU S
890 bp CIgt0.9
EU F
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Index of association

• Ia if same alleles are associated too much as
  opposed to random, it means sex is not occurring
• Association among alleles calculated and compared
  to simulated random distribution

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Evolution and Population genetics

• Positively selected genes
• Negatively selected genes
• Neutral genes normally population genetics
  demands loci used are neutral
• Loci under balancing selection..

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Evolution and Population genetics

• Positively selected genes
• Negatively selected genes
• Neutral genes normally population genetics
  demands loci used are neutral
• Loci under balancing selection..

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Evolutionary history

• Darwininan vertical evolutionary models
• Horizontal, reticulated models..

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Phylogenetic relationships within the
Heterobasidion complex
Fir-Spruce
Pine Europe
Pine N.Am.
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Geneaology of S DNA insertion into P ISG
confirms horizontal transfer.Time of
cross-over uncertain
NA S
NA P
EU S
890 bp CIgt0.9
EU F
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Because of complications such as

• Reticulation
• Gene homogeneization(Gene duplication)
• Need to make inferences based on multiple genes
• Multilocus analysis also makes it possible to
  differentiate between sex and lack of sex
  (Iaindex of association), and to identify
  genotypes, and to study gene flow

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Basic definitions again

• Locus
• Allele
• Dominant vs. codominant marker
• RAPDS
• AFLPs

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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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Example 2 Distinguishing taxa in the Pleurotus
eryngii (King Oyster Mushroom) complex using
AFLPs (Urbanelli et al., Appl. Microbiol.
Biotechnol. (2007) 74592-600)
Photo The New York Times
Photo Wikimedia Commons
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Distinguishing taxa in the Pleurotus eryngii
(King Oyster Mushroom) complex using AFLPs

• Goal to determine, using multilocus genotypes,
  whether the distinction between Pleurotus
  eryngii, P. ferulae, and P. eryngii var.
  nebrodensis is supported by genetic data
• 90 populations sampled
• 94 AFLP loci scored

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P. ferulae (http//steinpilz.up.seesaa.net)
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Sample AFLP Gel
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AFLP Data Map from Urbanelli et al. (2007)
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AFLP Data Map with UPGMA dendogram from Urbanelli
et al. (2007)
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RAPDS use short primers but not too short

• Need to scan the genome
• Need to be readable
• 10mers do the job (unfortunately annealing
  temperature is pretty low and a lot of priming
  errors cause variability in data)

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RAPDS use short primers but not too short

• Need to scan the genome
• Need to be readable
• 10mers do the job (unfortunately annealing
  temperature is pretty low and a lot of priming
  errors cause variability in data)

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RAPDS can also be obtained with Arbitrary Primed
PCR

• Use longer primers
• Use less stringent annealing conditions
• Less variability in results

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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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WORK ON PINES HAD DEMONSTRATED INFECTIONS ARE
MOSTLY ON STUMPS

• Use meticulous field work and genetics
  information to reconstruct disease from infection
  to explosion
• On firs/sequoia if the stump theory were also
  correct we would find a stump within the outline
  of each genotype

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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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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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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 (rarefaction) 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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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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Example

• SPECIES X 50blue, 50 yellow

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AMOVA example
Scenario 1
Scenario 2
v
POP 1
POP 2
v
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Expectations for fungi

• Sexually reproducing fungi characterized by high
  percentage of variance explained by individual
  populations
• Amount of variance between populations and
  regions will depend on ability of organism to
  move, availability of host, and
• NOTE if genotypes are not sensitive enough so
  you are calling the same things that are
  different you may get unreliable results like 100
  variance within pops, none among pops

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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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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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RAPDSgt not used often now
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RAPD DATA W/O COSEGREGATING MARKERS
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PCA
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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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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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From Garbelotto and Chapela, Evolution and
biogeography of matsutakes
Biodiversity within species as significant as
between species
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Microsatellites or SSRs

• AGTTTCATGCGTAGGT CG CG CG CG CG
  AAAATTTTAGGTAAATTT
• Number of CG is variable
• Design primers on FLANKING region, amplify DNA
• Electrophoresis on gel, or capillary
• Size the allele (different by one or more
  repeats if number does not match there may be
  polimorphisms in flanking region)
• Stepwise mutational process (2 to 3 to 4 to 3 to2
  repeats)

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ACACACACACACACACAC

• MS18 (AC)38 218 bp
• (AC)39 220 bp
• (AC)40 222 bp
• MS43a (CAGA)70 373 bp
• MS43a (CAGA)71 377 bp
• MS43a (CAGA)72 381 bp
• (220-218)2 22
• (222-218)2 42
• (377-373)2 42
• (381-373)2 82
• (39-38)2 12
• (40-38)2 22
• (71-70)2 12
• (72-70)2 22

AMOVA Analysis of Molecular Variance
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Example 1 Origins of the Sudden Oak Death
Epidemic in California(Mascheretti et al.,
Molecular Ecology (2008) 17 2755-2768)
Photo UC Davis
Photo www.membranetransport.org
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Photo Northeast Plant Diagnostic Network
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Spatial autocorrelation
Within approx. 100 meters the genetic structure
correlates with the geographical distance
10 100 1000
Geographical distance (m)
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Spatial autocorrelation
Morans I (coefficient of departure from spatial
randomness) correlates with distance up to
Distribution of genotypes (6 microsatellite
markers) in different populations of P.ramorum
in California
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NJ tree of P. ramorum populations in California
HU-1
MA-1
HU-2
MA-2
SC-2 MO-1 MO-2
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Example microsatellites genotyping of P.
ramorum isolates

• Phytophthora ramorum (Oomycete)
• causal agent of Sudden Oak Death (SOD) first
  reported in California in 1994
• SOD affects tanoak (Lithocarpus densiflora),
  coast live oak (Quercus agrifolia), Californian
  black oak (Quercus kelloggii), and Canyon live
  oak (Quercus chrysolepis)
• P.ramorum also cause a disease characterized
  mostly by leaf blight and/or branch dieback in
  over 100 species of both wild and ornamental
  plants, including California bay laurel
  (Umbellularia cailfornica), California redwood
  (Sequoia sempervirens), Camellia and
  Rhododrendron species

Collection of infected bay leaves from several
forests in Sonoma, Monterey, Marin, Napa,
Alameda, San Mateo
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Microsatellites (I)mating type A1 (EU) and
mating type A2 (US)

• A2 (US) A1 (EU)
• Locus 29 325/ - 325/337
• -/337
• Locus 33 315/337 325/337
• Locus 65 234/252 236/244
• 220/222

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Microsatellites (II)mating type A2 (US)

• MS39a (GA)11 129 bp
• MS39b (GA)4(GATA)31 234, 242, 246, 250, 254 bp
• MS43a (CAGA)69 309, 329, 349, 353, 357, 361,
  365, 369, 373, 377, 381 bp
• MS43b (CAGA)75()(CAGA)16 416, 420, 466, 470,
  474, 476, 478, 482, 486, 490, 494, 498 bp
• MS45 (TCCG)11 167, 183, 187 bp
• MS18 (AC)39 218, 220, 222, 254, 264, 272, 274,
  276, 278, 282 bp
• MS64 (CT)16 342, 374, 376, 378 bp

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• Ind. MS39a MS39b MS43a MS43b MS45 MS18 MS64 Matin
  g type
• 1 129-129 246-246 369-369 486-486 167-187 220-278
  342-374 A1
• 2 129-129 246-246 369-369 486-486 167-187 220-278
  342-374 A1
• 3 129-129 246-246 373-373 486-486 167-187 220-274
  342-374 A1
• 4 129-129 246-246 373-373 486-486 167-187 220-27
  4 342-378 A1
• 5 129-129 246-246 373-373 486-486 167-187 220-27
  4 342-378 A1
• 6 129-129 246-246 373-373 486-486 167-187 220-27
  4 342-378 A1
• 7 129-129 246-246 373-373 486-486 167-187 220-27
  8 342-378 A1
• 8 129-129 246-246 373-373 486-486 167-187 220-27
  8 342-374 A1
• 9 129-129 250-250 369-369 486-486 167-187 220-278
  342-374 A1
• 10 129-129 250-250 369-369 486-486 167-187 220-2
  78 342-374 A1
• 11 129-129 250-250 369-369 486-486 167-187 220-27
  8 342-374 A1
• 12 129-129 250-250 377-377 490-490 167-187 220-2
  78 342-374 A1
• 13 129-129 250-250 377-377 490-490
  167-187 220-278 342-381 A1
• 14 129-129 250-250 377-377 490-490
  167-187 220-278 342-381 A1
• 15 129-129 250-250 377-377 490-490
  167-187 220-278 342-381 A1
• 16 129-129 246-246 377-377 490-490
  167-187 220-278 342-374 A1
• 17 129-129 246-246 377-377 486-486 167-187 220-2
  78 342-374 A1

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Ind. MS39a MS39b MS43a MS43b MS45 MS18 MS64 Matin
g type 1 11-11 32-32 69-69 17-17 11-16 39-68 18-
29 A1 2 11-11 32-32 69-69 17-17 11-16 39-68
18-29 A1 3 11-11 32-32 70-70 17-17 11-16
39-66 18-29 A1 4 11-11 32-32 70-70 17-17
11-16 39-66 18-30 A1 5 11-11 32-32 70-70
17-17 11-16 39-66 18-30 A1 6 11-11 32-32
70-70 17-17 11-16 39-66 18-30 A1 7 11-11
32-32 70-70 17-17 11-16 39-68 18-30
A1 8 11-11 32-32 70-70 17-17 11-16 39-68
18-29 A1 9 11-11 33-33 69-69 17-17 11-16
39-68 18-29 A1 10 11-11 33-33 69-69 17-17
11-16 39-68 18-29 A1 11 11-11 33-33 69-69
17-17 11-16 39-68 18-29 A1 12 11-11 33-33
71-71 18-18 11-16 39-68 18-29 A1 13 11-11
33-33 71-71 18-18 11-16 39-68
18-31 A1 14 11-11 33-33 71-71 18-18 11-16
39-68 18-31 A1 15 11-11 33-33 71-71 18-18
11-16 39-68 18-31 A1 16 11-11 32-32 71-71
18-18 11-16 39-68 18-29 A1 17 11-11
32-32 71-71 17-17 11-16 39-68 18-29
A1 18 11-11 32-32 69-69 17-17 11-16 39-68
18-29 A1 19 11-11 32-32 72-72 17-17 11-16
40-null 18-29 A2 20 11-11 9 32-32 72-72
19-19 11-16 40-null 18-29 A2
132
133

• China Camp State Park (Marin) n24
• Bean Creek Forest (Santa Cruz) n24
• Nurseries n14

133