maria eugenia d’amato. molecular genetics techniques types and properties of molecular makers...
TRANSCRIPT
Maria Eugenia D’Amato
• Molecular genetics techniques
• Types and properties of molecular makers
• Factors that determine the patterns of genetic variation
1.Southern blot
2.PCR
3.DNA sequencing
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
Sir Edwin Southern
1938-
Nobel Price
Restriction enzymes
molecular scissors
Southern blot steps
(GATA)4 (GGAT)4
Trout DNA digested with Hinf I
Multilocus
Unilocus
homozygote
heterozygote
PCR
500 bp
Restriction site
500250
mtDNA
Kary Mullis
1938-
Nobel Price 1993
Polymerase Chain Reaction
• In vitro replication of DNA
DNA Copies = 2n , n = number of cycles
After 30 cycles: 107 million copies
PCR machines
priming site
priming site
x ♂
♀
Pedigree analysis
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
RAPDs
(Random Amplified Polymorphic DNA)
AFLPs
(Amplified Random Length Polymorphism)
Dominant
multilocus
biallelic markers
A C G T
CTCCGGCTGTAACCTTCAC…
The old days….
Automatic sequencing
• 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
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
(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
Consequences of the model
• Allele frequencies remain constant, generation after generation
• Genotype frequencies can be determined from allele frequencies
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
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
• 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
Original population
Population
crash recovery
Cheetah:
Late Pleistocene bottleneck
American bison:
Over hunting bottleneck
Skin photo-sensitivity in a porphyria patient
1 couple carrying the allele immigrated SA in 1688
Today: 30 000 descendant South Africans are affected
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
• 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
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
Lineage: individuals or taxa related by
a common ancestor
Phylogenetic tree
h =n haplotypes
Total n individualsHaplotype diversity
= Σ xixjijn
n -1
Nucleotide diversity
Study of geographic
distribution of lineages
Population bottlenecks, expansions
Gene flow
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.
Crandall et al 2000 ecological exchangeability
genetic exchangeability
Reciprocal
monophyly
Phylogeographic reconstruction: Cytochrome b (540 bp)
16S rDNA (476 bp)
MP tree ML tree
Pachydactilus rugosus
Pronolagus rupestris
Vicariant event cycles of dry-humid period during
glacial –interglacial produced fragmentation of habitat
Hybrids in the wild?
O. aureus O. niloticus
Nuclear Mitochondrial
Haplotypes common
to both species
O.aureus*
*
Cluster of population
Cluster of haplotypesSenegal
Nile
Niger
Main results
• shallow mtDNA divergence between species in Sudano- Sahelian zone
• large divergence between Nile- western Africa
Retention of ancestral polymorphisms?
Secondary contact + introgression?
hypotheses
Kasinthula
O. mossambicus
O. andersoniiO. mortimeri O. karongae
Parsimony network of mtDNA control region
Cluster 1 O.urolepis-aureus
2a
Cluster 3 O . malagarasi
2b
Cluster 2 O. mossambicus
Cluster 4 O. karongae
Cluster 5 O. niloticus
Cluster 6 O. andersonii
O.urolepis
O. aureus
6a 6b
6c
2c
HaliotidaeFissurellidae
260 MY
350 MY paralogs
Orthologs ~ 65% identity
Duplication event
orthologs
orthologs
paralogs
65 % identity
80-95 %
identity
Japanese Chinese OverallMuroto Sukumo Shizuoka Nagasaki Shimane Mean Chinese1 Chinese2 Mean mean
Aver. no. of alleles/locus 8.8 11 9.3 10.8 9.8 9.9 9 11 10 10Ho 0.782 0.773 0.783 0.756 0.739 0.767 0.803 0.772 0.787 0.773He 0.777 0.756 0.76 0.76 0.753 0.761 0.762 0.768 0.765 0.762
Chinese
threeline grunt
Population structure analysis with 4 microsatellites lociJapanese samples
Test Japanese Chinese TotalFst 0 0.015 0.013P 0.399 0 0
significant
Pairwise Fst between populations
significant
LocationJapanese ChineseMuroto Sukumo Nagasaki Shizuoka Shimane Chinese1 Chinese2
MurotoSukumo -0.0005
Nagasaki -0.0044 0.0012
Shizuoka 0.0002 -0.0001 0.002
Shimane 0.0024 -0.0005 -0.0035 0.0027
Chinese1 0.0301a 0.0272 0.0272 0.0203 0.0249a
Chinese2 0.0216a 0.0129 0.0236 0.0147 0.0150a 0.0150a