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 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 mtDNA

Kary Mullis Nobel Price 1993 Polymerase Chain Reaction In vitro replication of DNA

 DNA Copies = 2 n, n = number of cycles  After 30 cycles: 107 million copies PCR machines

priming site xx ♂  ♀  Pedigree analysis

Polyembryony in bryozoans? Incubating chamber

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 monitored polymorphic polymorphic 1.Individual identification 2.Genic variation 3.Gene genealogies Parentage, relatedness, mating systems Gene flow, drift Phylogeography, speciation, deeper phylogenies

Aa Aa NN A p = 0.6 A p = 0.6 a p = 0.4 a p = 0.4 AA Aa aa p2p2 q2q2 pq

(p + q) 2 = p 2 + 2pq + q 2 p = 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

Expected genotype freqs In pop I: ( ) 2 = x 0.6 x =  2 = ∑ (O – E) 2  2 = 44.4 d.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 sites 0.5 Charles Darwin Balancing selection Directional selection f ACE 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 p 0 q 0 2N = q = q 1 – q 0 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: descendant South Africans are affected

Migration = Gene flow transfer of alleles from one gene pool to another A 1 A 1 = 1 A 2 A 2 = 1 After m, 80% of the island is A 1 A 1 and 20% A 2 A 2 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 inbreeding F IS = (Hs – Ho) / Ho within a subpopulation F IT = (H T – H 0 ) / H T among individuals overall populations F ST = (H S – H T ) / H T between subpopulations Ho = aver. observed H within a subpopulation over loci Hs = aver. expected H within subpopulation over loci Ht = aver. expected H overall

Out of HWE In HWE 1 2

Lineage: individuals or taxa related by a common ancestor Phylogenetic tree

h = n haplotypes Total n individuals Haplotype diversity  = Σ x i x j  ij n 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 treeML 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 haplotypes Senegal 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

O. mossambicus O. andersonii O. mortimeriO. karongae Parsimony network of mtDNA control region

Haliotidae Fissurellidae 260 MY 350 MY paralogs Orthologs ~ 65% identity Duplication event

orthologs paralogs 65 % identity % identity

Chinese threeline grunt Population structure analysis with 4 microsatellites loci Japanese samples significant

Pairwise Fst between populations significant