available at bcb 703: scientific methodology maria eugenia d’amato dna variation in ecology and...
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Available at http://planet.uwc.ac.za/nislBCB 703:Scientific Methodology
Maria Eugenia D’Amato
DNA variation in Ecology and EvolutionII- Technical approach and concepts
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
1. Fragmentation of genomic DNA in a reproducible way
2. Separation of the fragments in an electric field
3. Transfer of the fragments from gel to a membrane
4. Probing of the membrane with known DNA
5. Detection of the probe
Southern blot (1977)
Sir Edwin Southern
1938-
Nobel Price
DNA fingerprinting
(GATA)4 (GGAT)4
Trout DNA digested with Hinf I
Multilocus
Unilocus
homozygote
heterozygote
PCR (1981)
Kary Mullis
1938-
Nobel Price 1993
Polymerase Chain Reaction
• In vitro replication of DNA
Applications of PCRmicrosatellites for mating strategies
Polyembryony in
bryozoans?
Incubating chamber
Loci
Mother Chamber N Cd 4b Cd 5 Cd 6 Cd 7-1 Cd 17-3 A 149159 180180 168168 147171 233233 A1 10 159159 180180 168168 145147 237237 A2 5 159159 180180 168168 147171 233233 A3 5 149159 180180 168168 147147 229229 A4 5 149159 180180 168168 145171 233233 A5 4 149159 180188 168176 145171 229229 A6 5 149159 180180 168168 171171 229229 A7 9 159159 180180 168168 147147 233233 A8 5 149149 180180 168168 147171 237237
BA 149159 182182 170170 145149 237253 BA1 6 149149 180182 168170 145167 237253 BA2 6 149149 182188 170176 145167 229237 BA3 5 149159 180182 168170 145149 253253
Applications of PCR. Anonymous loci
RAPDs
(Random Amplified Polymorphic DNA)
AFLPs
(Amplified Random Length Polymorphism)
Dominant
multilocus
biallelic markers
Molecular Markers• Physical location in a genome whose inheritance can be monitoredPhysical location in a genome whose inheritance can be monitored
• polymorphicpolymorphic
1. Individual identification
2. Genic variation
3. Gene genealogies
Parentage,
relatedness,
mating systems
Gene flow, drift
Phylogeography,
speciation,
deeper phylogenies
Genes in populations
A a
A a
N N
A
p = 0.6
A
p = 0.6
a
p = 0.4
a
p = 0.4
AA Aa
Aa aa
p2
q2pq
pq0.36 0.24
0.24 0.16
Genes in populations:equilibrium of Hardy Weinberg
(p + q) 2 = p2 + 2pq + q2p = freq A
q = freq a
the organism is diploid
with sexual reproduction
generations are non overlapping
loci are biallelic
allele frequencies are identical in males and females
random mating
population size is infinite
no migration, no mutation, no selection
Assumptions
Hardy Weinberg EquilibriumConsequences of the model
• Allele frequencies remain constant, generation after generation
• Genotype frequencies can be determined from allele frequencies
HWE- Mathematical example of deviation from equilibrium
Genotypes Allele freqs Expected genotype freqs
pop AA Aa aa p q AA Aa aaI 0.2 0.8 0 0.6 0.4 0.36 0.48 0.16II 0.36 0.48 0.16 0.6 0.4 0.36 0.48 0.16II 0.5 0.2 0.3 0.6 0.4 0.36 0.48 0.16IV 0.6 0 0.4 0.6 0.4 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 = 44.4d.f. = (R-1) x (C-1) = 2
2 d.f =2 = 5.99 highly significant
Departures from HWE: Selection
Differential survival and
reproductive success of genotypes
Normal and sickling
forms of erythrocytes
1 2 3 4 5 6 7 8 9
sites
0.5
Charles Darwin
Balancing selection
Directional selection
f A
CE
R
Heliconius erato
Frequency dependent
selection
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
q p0 q0
2N =
q = q1 – q0
•Alleles become fixed (freq = 1) or lost (freq = 0)
•The effect is more pronounced
in small populations
• Genetic diversity decreases
Variance in 1 generation
Genetic drift: Bottlenecks
Original population
Population
crash recovery
Cheetah:
Late Pleistocene bottleneck
American bison:
Over hunting bottleneck
Genetic drift: Founder effect
Skin photo-sensitivity 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
A1A1 = 1
A2A2 = 1
After m,
80% of the island is A1A1
and 20% A2A2
After 1 generation
genotypes are in HWE
Genotypes out of HWE
m
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 inbreedingFIS = (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
subpop A1A1 A1A2 A2A2 fA1 fA2 Fis = 0.2
1 0.25 0.5 0.5 0.5 0.5 Fit = 0.2
2 0.35 0.3 0.35 0.5 0.5 Fst = 0
1 0.25 0.5 0.25 0.5 0.5 Fis = 0
2 0.49 0.42 0.09 0.7 0.3 Fit = 0.0625
Fst = 0.0625
Out of HWE
In HWE
1
2
Gene genealogies: a historical perspective
Lineage: individuals or taxa related by
a common ancestor
Phylogenetic tree
Diversity with uniparental markers
h =n haplotypes
Total n individualsHaplotype diversity
= Σ xixjijn
n -1
Nucleotide diversity
Phylogeography
Study of geographic
distribution of lineages
Population bottlenecks, expansions
Gene flow
ESUsWaples 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.
Crandall et al 2000 ecological exchangeability
genetic exchangeability
Reciprocal
monophyly