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