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DNA variation in Ecology and
Evolution
II- Technical approach and concepts
Maria Eugenia D’Amato
Available at http://planet.uwc.ac.za/nisl
BCB 703:
Scientific Methodology
Methodological approaches to
the study of genetic diversity
•
Molecular genetics techniques
• Types and properties of molecular
makers
• Factors that determine the patterns of
genetic variation
Molecular techniques
1.Southern blot
2.PCR
3.DNA sequencing
Southern blot (1977)
1. Fragmentation of genomic
DNA in a reproducible way
2. Separation of the fragments
in an electric field
Sir Edwin Southern
3. Transfer of the fragments from gel to
a membrane
1938Nobel Price
4. Probing of the membrane with known DNA
5. Detection of the probe
Southern blot
Restriction enzymes
molecular scissors
Southern blot steps
DNA fingerprinting
Multilocus
Trout DNA digested with Hinf I
(GATA)4
Unilocus
(GGAT)4
homozygote
heterozygote
RFLPs
Restriction Fragment Length
Polymorphism.
mtDNA
PCR
500
250
500 bp
Restriction site
PCR (1981)
Polymerase Chain Reaction
• In vitro replication of DNA
Kary Mullis
1938Nobel Price 1993
PCR
 DNA Copies = 2n ,
n = number of cycles
 After 30 cycles: 107 million
copies
PCR machines
Applications of PCR:
microsatellite genotyping
priming site
x 

♂
♀
priming site
Pedigree analysis
Applications of PCR
microsatellites for mating strategies
Polyembryony in
bryozoans?
Loci
Mother Chamber N
A
A1
10
A2
5
A3
5
A4
5
A5
4
A6
5
A7
9
A8
5
BA
Incubating chamber
BA1
BA2
BA3
6
6
5
Cd 4b
149159
159159
159159
149159
149159
149159
149159
159159
149149
Cd 5
180180
180180
180180
180180
180180
180188
180180
180180
180180
Cd 6
168168
168168
168168
168168
168168
168176
168168
168168
168168
Cd 7-1
147171
145147
147171
147147
145171
145171
171171
147147
147171
Cd 17-3
233233
237237
233233
229229
233233
229229
229229
233233
237237
149159
149149
149149
149159
182182
180182
182188
180182
170170
168170
170176
168170
145149
145167
145167
145149
237253
237253
229237
253253
Applications of PCR.
Anonymous loci
RAPDs
AFLPs
(Random Amplified
Polymorphic DNA)
(Amplified Random
Length Polymorphism)
Dominant
multilocus
biallelic markers
DNA sequencing
The old days….
ACGT
CTCCGGCTGTAACCTTCAC…
Automatic sequencing
Molecular Markers
• Physical location in a genome whose inheritance can be monitored
• polymorphic
Parentage,
1. Individual identification
relatedness,
mating systems
2. Genic variation
Gene flow, drift
Phylogeography,
3. Gene genealogies
speciation,
deeper phylogenies
Genes in populations
N
N
A
a
A
A
a
p = 0.6
p = 0.4
a
AA
p2
A
p = 0.6
a
p = 0.4
Aa
pq
0.36
0.24
Aa
pq
aa
q2
0.24
0.16
Genes in populations:
equilibrium of Hardy Weinberg
(p +
q) 2 = p2
+ 2pq +
q2
p = freq A
q = freq a
the organism is diploid
with sexual reproduction
generations are non overlapping
Assumptions
loci are biallelic
allele frequencies are identical in males and females
random mating
population size is infinite
no migration, no mutation, no selection
Hardy Weinberg Equilibrium
Consequences of the model
• Allele frequencies remain constant,
generation after generation
• Genotype frequencies can be determined from
allele frequencies
HWEMathematical example of deviation from equilibrium
Genotypes
pop
I
II
II
IV
AA
0.2
0.36
0.5
0.6
Aa
0.8
0.48
0.2
0
Allele freqs Expected genotype freqs
aa
0
0.16
0.3
0.4
p
0.6
0.6
0.6
0.6
q
0.4
0.4
0.4
0.4
AA Aa
aa
0.36 0.48 0.16
0.36 0.48 0.16
0.36 0.48 0.16
0.36 0.48 0.16
Expected genotype freqs
In pop I:
(0.6 + 0.4)2 = 0.62 + 2 x 0.6 x 0.4 + 0.42
= 0.36 + 0.48 + 0.16
2 = ∑ (O – E)2
2 = 44.4
d.f. = (R-1) x (C-1) = 2
2 d.f =2 = 5.99 highly significant
Charles Darwin
Departures from HWE:
Selection
Differential survival and
reproductive success of genotypes
Balancing selection
Frequency dependent
Directional selection

0.5







Normal and sickling
forms of erythrocytes
selection

1 2 3 4 5 6 7 8 9
sites
Heliconius erato
Deviations from HWE:
Genetic drift
•
Random variation of allele frequencies
generation after generation
• Generated by the random sampling process
of drawing gametes to form the next generation
dq = q1 – q0
2 dq
=
p0 q0
2N
Variance in 1 generation
•Alleles become fixed (freq = 1) or lost (freq = 0)
•The effect is more pronounced
in small populations
• Genetic diversity decreases
Genetic drift:
Bottlenecks




 






 
Original population
Cheetah:
Late
Pleistocene
bottleneck



Population
crash
recovery
 








American
bison:
Over hunting
bottleneck
Genetic drift:
Founder effect









 
Skin photosensitivity in a
porphyria
patient








 
1 couple carrying the allele
immigrated SA in 1688
Today: 30 000 descendant South
Africans are affected
HWE departure/restoration
Migration
Migration = Gene flow
transfer of alleles from one gene pool to another
After m,
80% of the island is A1A1
m
and 20% A2A2
Genotypes out of HWE
A1A1 = 1
After 1 generation
genotypes are in HWE
A2A2 = 1
non random mating- drift – no gene flow
Population structure
• Differential allele frequencies between subpopulations
• inbreeding coefficients : measure of H deficiency at
different hierarchical levels
• Wahlund effect: H deficiency due to subdivision, drift
and inbreeding
FIS = (Hs – Ho) / Ho within a subpopulation
FIT = (HT – H0) / HT among individuals overall populations
FST = (HS – HT) / HT between subpopulations
Ho = aver. observed H within a subpopulation over loci
Hs = aver. expected H within subpopulation over loci
Ht = aver. expected H overall
Examples of population
structure
1
Out of HWE
2
In HWE
subpop A1A1 A1A2 A2A2
1 0.25
2 0.35
0.5
0.3
1
2
0.5
0.42
0.25
0.49
0.5
0.35
0.25
0.09
fA1
0.5
0.5
0.5
0.7
fA2
0.5
0.5
Fis =
Fit =
Fst =
0.5 Fis =
0.3 Fit =
0.2
0.2
0
0
0.0625
Fst = 0.0625
Gene genealogies:
a historical perspective
Lineage: individuals or taxa related by
a common ancestor
Phylogenetic tree
Diversity with
uniparental markers
h
=
p =
n haplotypes
Total n individuals
Haplotype diversity
n
Σ xixjpij
n -1
Nucleotide diversity
Phylogeography
Study of geographic
distribution of lineages
Population bottlenecks,
expansions
Gene flow
ESUs
Waples 1991: populations that are reproductively separate from
other populations and have unique or different adaptations.
Moritz 1994: populations that are reciprocally monophyletic for
mtDNA alleles and show significant divergence of allele
frequencies at nuclear loci.
Reciprocal
monophyly
Crandall et al 2000
ecological exchangeability
genetic exchangeability