1. Maria Eugenia D’Amato

2. Molecular genetics techniques Types and properties of molecular makers Factors that determine the patterns of genetic variation

3. 1.Southern blot 2.PCR 3.DNA sequencing

4. 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

5. Restriction enzymes molecular scissors Southern blot steps

6. (GATA) 4 (GGAT) 4 Trout DNA digested with Hinf I Multilocus Unilocus homozygote heterozygote

7. PCR 500 bp Restriction site 500 250 mtDNA

8. Kary Mullis 1938- Nobel Price 1993 Polymerase Chain Reaction In vitro replication of DNA

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

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

11. Polyembryony in bryozoans? Incubating chamber

12. RAPDs (Random Amplified Polymorphic DNA) AFLPs (Amplified Random Length Polymorphism) Dominant multilocus biallelic markers

13. A C G T CTCCGGCTGTAACCTTCAC… The old days…. Automatic sequencing

14. 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

15. 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 0.36 0.24 0.16

16. (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

17. Consequences of the model Allele frequencies remain constant, generation after generation Genotype frequencies can be determined from allele frequencies

18. Expected genotype freqs In pop I: (0.6 + 0.4) 2 = 0.6 2 + 2 x 0.6 x 0.4 + 0.4 2 = 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

19. 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 ACE R Heliconius erato Frequency dependent selection

20. 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

21.                                         Original population Population crash recovery Cheetah: Late Pleistocene bottleneck American bison: Over hunting bottleneck

22.                                   Skin photo- sensitivity in a porphyria patient 1 couple carrying the allele immigrated SA in 1688 Today: 30 000 descendant South Africans are affected

23. 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

24. 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

25. Out of HWE In HWE 1 2

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

27. h = n haplotypes Total n individuals Haplotype diversity  = Σ x i x j  ij n n -1 Nucleotide diversity

28. Study of geographic distribution of lineages Population bottlenecks, expansions Gene flow

29. 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

30. Phylogeographic reconstruction: Cytochrome b (540 bp) 16S rDNA (476 bp)

31. MP treeML tree

32. Pachydactilus rugosus Pronolagus rupestris Vicariant event cycles of dry-humid period during glacial –interglacial produced fragmentation of habitat

33. Hybrids in the wild? O. aureus O. niloticus

34. Nuclear Mitochondrial Haplotypes common to both species O.aureus * * Cluster of population Cluster of haplotypes Senegal Nile Niger

35. 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

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

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

38. orthologs paralogs 65 % identity 80-95 % identity

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

40. Pairwise Fst between populations significant