Molecular Markers

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Molecular Markers

• DNA PROTEINS
• mtDNA often used in systematics in general, no
  recombination uniparental inheritance
• cpDNA often used in systematics in general, no
  recombination uniparental inheritance
• Microsatellites tandem repeats genotyping
  population structure
• Allozymes variations of proteins population
  structure
• RAPDs short segments of arbitrary sequences
  genotyping
• RFLPs variants in DNA exposed by cutting with
  restriction enzymes genotyping, population
  structure
• AFLPs after digest with restriction enzymes, a
  subset of DNA fragments are selected for PCR
  amplification genotyping

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Codominant Molecular Tools

• Allozymes different versions of proteins.
• One of the major first tools for analyzing
  population structure
• Advantages
• Inexpensive
• Easily Obtained
• Disadvantages
• Coding regions violate assumptions of
  analytical techniques
• Invariable in many fungi inadequate for looking
  at variation

• Microsatellites repetitive sequences in the DNA
  (e.g. AC)12
• Very popular for analyzing population structure
• Forensic applications
• Advantages
• Hypervariable
• Genotyping
• Population Structure
• Disadvantages
• High cost of Development

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Dominant Marker
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Levels of Analyses

• Individual
• identifying parents offspring very important
  in zoological circles identify patterns of
  mating between individuals (polyandry, etc.)
• In fungi, it is important to identify the
  "individual" -- determining clonal individuals
  from unique individuals that resulted from a
  single mating event.

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Levels of Analyses cont

• Families looking at relatedness within colonies
  (ants, bees, etc.)
• Population level of variation within a
  population.
• Dispersal indirectly estimate by calculating
  migration
• Conservation Management looking for founder
  effects (little allelic variation), bottlenecks
  (reduction in population size leads to little
  allelic variation)
• Species variation among species what are the
  relationship between species.
• Family, Order, ETC. higher level phylogenies

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Armillaria gallica Humongous Fungus
rhizomorphs
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What is Population Genetics?

• About microevolution (evolution of species)
• The study of the change of allele frequencies,
  genotype frequencies, and phenotype frequencies

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Goals of population genetics
Natural selection (adaptation) Chance (random
events) Mutations Climatic changes
(population expansions and contractions) To
provide an explanatory framework to describe the
evolution of species, organisms, and their
genome, due to Assumes that the same
evolutionary forces acting within
species (populations) should enable us to explain
the differences we see between species
evolution leads to change in gene frequencies
within populations
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Pathogen Population Genetics

• must constantly adapt to changing environmental
  conditions to survive
• High genetic diversity easily adapted
• Low genetic diversity difficult to adapt to
  changing environmental conditions
• important for determining evolutionary potential
  of a pathogen
• If we are to control a disease, must target a
  population rather than individual
• Exhibit a diverse array of reproductive
  strategies that impact population biology

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Analytical Techniques

• Hardy-Weinberg Equilibrium
• p2 2pq q2 1
• Departures from non-random mating
• F-Statistics
• measures of genetic differentiation in
  populations
• Genetic Distances degree of similarity between
  OTUs
• Neis
• Reynolds
• Jaccards
• Cavalli-Sforza
• Tree Algorithms visualization of similarity
• UPGMA
• Neighbor Joining

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Allele Frequencies

• Allele frequencies (gene frequencies)
  proportion of all alleles in an all individuals
  in the group in question which are a particular
  type
• Allele frequencies
• p q 1
• Expected genotype frequencies
• p2 2pq q2

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Evolutionary principles Factors causing changes
in genotype frequency

• Selection variation in fitness heritable
• Mutation change in DNA of genes
• Migration movement of genes across populations
• Vectors Pollen, Spores
• Recombination exchange of gene segments
• Non-random Mating mating between neighbors
  rather than by chance
• Random Genetic Drift if populations are small
  enough, by chance, sampling will result in a
  different allele frequency from one generation to
  the next.

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The smaller the sample, the greater the chance of
deviation from an ideal population. Genetic
drift at small population sizes often occurs as a
result of two situations the bottleneck effect
or the founder effect.
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Founder Effects

• Establishment of a population by a few
  individuals can profoundly affect genetic
  variation
• Consequences of Founder effects
• Fewer alleles
• Fixed alleles
• Modified allele frequencies compared to source
  pop
• Perhaps due to new environment

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Bottleneck Effect

• The bottleneck effect occurs when the numbers of
  individuals in a larger population are
  drastically reduced
• By chance, some alleles may be overrepresented
  and others underrepresented among the survivors
• Some alleles may be eliminated altogether
• Genetic drift will continue to impact the gene
  pool until the population is large enough

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Founder vs Bottleneck
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Northern Elephant Seal Example of Bottleneck
Hunted down to 20 individuals in
1890s Population has recovered to over
30,000 No genetic diversity at 20 loci
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Potato Blight

• Phytophthora infestans
• great Irish famine of 1845-1849
• 1,000,000 died
• Origin of P. infestans
• Mexico highest genetic diversity likely origin
• Ireland decreased genetic diversity due to
  founder effect
• Decreased genetic differentiation in other
  regions
• Europe, North America

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Hardy Weinberg Equilibriumand F-Stats

• In general, requires co-dominant marker system
• Codominant expression of heterozygote
  phenotypes that differ from either homozygote
  phenotype.
• AA, Aa, aa

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Hardy-Weinberg Equilibrium

• Null Model population is in HW Equilibrium
• Useful
• Often predicts genotype frequencies well

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Hardy-Weinberg Theorem
if only random mating occurs, then allele
frequencies remain unchanged over time. After one
generation of random-mating, genotype frequencies
are given by AA Aa aa p2 2pq q2 p freq
(A) q freq (a)
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Expected Genotype Frequencies

• The possible range for an allele frequency or
  genotype frequency therefore lies between ( 0
  1)
• with 0 meaning complete absence of that allele
  or genotype from the population (no individual in
  the population carries that allele or genotype)
• 1 means complete fixation of the allele or
  genotype (fixation means that every individual in
  the population is homozygous for the allele --
  i.e., has the same genotype at that locus).

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ASSUMPTIONS
1) diploid organism 2) sexual reproduction 3)
Discrete generations (no overlap) 4) mating
occurs at random 5) large population size
(infinite) 6) No migration (closed population) 7)
Mutations can be ignored 8) No selection on
alleles
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Locus Locus Locus
Sample 1 2 3
1 3,4 2,2 1,1
2 4,4 2,2 1,2
3 4,4 1,2 1,2
4 4,4 2,2 1,1
5 4,4 1,2 1,1
6 1,4 1,2 1,1
7 2,4 2,2 1,1
8 4,4 2,2 1,1
9 2,4 1,2 1,1
10 1,4 2,3 2,2
11 2,4 2,2 2,2
12 2,3 2,2 2,2
13 4,4 1,2 1,1
14 1,4 2,3 1,2
15 4,4 1,2 1,2
16 1,4 1,1 1,1
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Locus 1 Allele 1 4/32 0.125 Allele 2 4/32
0.125 Allele 3 2/32 0.0625 Allele 4
22/32 0.6875 Allele frequencies 0.125
0.125 0.00625 0.6875 1 Locus 2 Allele 1
8/32 0.2500 Allele 2 22/32 0.6875 Allele
3 2/32 0.0625 Locus 3 Allele 1 10/32
0.3125 Allele 2 22/32 0.6875
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EXP OBS (OBS-EXP)2/EXP
LOCUS 1 1,1 (0.1250)2 0.0156 0.0000 0.0156
1,2 (0.1250.125)2 0.0313 0.0000 0.0313
1,3 (0.1250.0625)2 0.0157 0.0000 0.0157
1,4 (0.1250.6875)2 0.1718 0.2500 0.0356
2,2 (0.125)2 0.0156 0.0000 0.0156
2,3 (0.1250.0625)2 0.0156 0.0625 0.1410
2,4 (0.1250.6875)2 0.1719 0.1875 0.0014
3,3 (0.0625)2 0.0039 0.0000 0.0039
3,4 (0.06250.6875)2 0.0859 0.0625 0.0064
4,4 (0.6875)2 0.4727 0.4375 0.0026



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EXP OBS (OBS-EXP)2/EXP
LOCUS 2 1,1 (0.2500)2 0.0625 0.0625 0.0000
1,2 (0.25000.6875)2 0.3438 0.3750 0.0028
1,3 (0.25000.0625)2 0.0313 0.0000 0.0313
2,2 (0.6875)2 0.4727 0.4375 0.0026
2,3 (0.68750.0625)2 0.0859 0.1250 0.0178
3,3 (0.0625)2 0.0038 0.0000 0.0038
LOCUS 3 1,1 (0.3125)2 0.0977 0.5625 2.2112
1,2 (0.31250.6875)2 0.4297 0.2500 0.0752
2,2 (0.6875)2 0.4726 0.1875 0.1720

CHI-SQUARED TEST 2.7858 CHI-SQUARED TEST 2.7858 CHI-SQUARED TEST 2.7858
P 0.999984

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IMPORTANCE OF HW THEOREM
If the only force acting on the population is
random mating, allele frequencies remain
unchanged and genotypic frequencies are
constant. Mendelian genetics implies that
genetic variability can persist indefinitely,
unless other evolutionary forces act to remove it
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Departures from HW Equilibrium

• Check Gene Diversity Heterozygosity
• If high gene diversity different genetic
  sources due to high levels of migration
• Inbreeding - mating system leaky or breaks down
  allowing mating between siblings
• Asexual reproduction check for clones
• Risk of over emphasizing particular individuals
• Restricted dispersal local differentiation
  leads to non-random mating

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Pop 3
Pop 2
Pop 1
Pop 4
FST 0.30
FST 0.02
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Pop1 Pop2 Pop3
Sample size 20 20 20
AA 10 5 0
Aa 4 10 8
aa 6 5 12
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Pop1 Pop2 Pop3
Freq
p (20 1/28)/40 0.60 (101/220)/40 .50 (01/216)/40 0.20
q (12 1/28)/40 0.40 (101/220)/40 .50 (241/216)/40 0.80
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Local Inbreeding Coefficient

• Calculate HOBS
• Pop1 4/20 0.20
• Pop2 10/20 0.50
• Pop3 8/20 0.40
• Calculate HEXP (2pq)
• Pop1 20.600.40 0.48
• Pop2 20.500.50 0.50
• Pop3 20.200.80 0.32
• Calculate F (HEXP HOBS)/ HEXP
• Pop1 (0.48 0.20)/(0.48) 0.583
• Pop2 (0.50 0.50)/(0.50) 0.000
• Pop3 (0.32 0.40)/(0.32) -0.250

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F StatsProportions of Variance

• FIS (HS HI)/(HS)
• FST (HT HS)/(HT)
• FIT (HT HI)/(HT)

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Pop Hs HI p q HT FIS FST FIT
1 0.48 0.20 0.60 0.40
2 0.50 0.50 0.50 0.50
3 0.32 0.40 0.20 0.80
Mean 0.43 0.37 0.43 0.57 0.49 -0.14 0.12 0.24

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Host islands within the California Northern
Channel Islands create fine-scale genetic
structure in two sympatric species of the
symbiotic ectomycorrhizal fungus Rhizopogon
Rhizopogon occidentalis
Rhizopogon vulgaris
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Rhizopogon sampling study area

• Santa Rosa, Santa Cruz
• R. occidentalis
• R. vulgaris
• Overlapping ranges
• Sympatric
• Independent evolutionary histories

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Sampling
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Bioassay Mycorrhizal pine roots
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Local Scale Population StructureRhizopogon
occidentalis
FST 0.26
8-19 km
N
E
FST 0.33
5 km
FST 0.24
W
B
T
FST 0.17
Populations are different
Populations are similar
Grubisha LC, Bergemann SE, Bruns TD Molecular
Ecology in press.
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Local Scale Population StructureRhizopogon
vulgaris
FST 0.21
N
E
FST 0.25
FST 0.20
W
Populations are different
Grubisha LC, Bergemann SE, Bruns TD Molecular
Ecology in press