Molecular Markers

1. Molecular Markers • DNA & PROTEINS • mtDNA = often used in systematics; in general, no recombination = uniparental inheritance • cpDNA = often used in systematics; in general, no recombination = uniparental inheritance • Microsatellites = tandem repeats; genotyping & population structure • Allozymes = variations of proteins; population structure • RAPDs = short segments of arbitrary sequences; genotyping • RFLPs = variants in DNA exposed by cutting with restriction enzymes; genotyping, population structure • AFLPs = after digest with restriction enzymes, a subset of DNA fragments are selected for PCR amplification; genotyping

2. Allozymes = different versions of proteins. One of the major first tools for analyzing population structure Advantages: Inexpensive Easily Obtained Disadvantages: Coding regions = violate assumptions of analytical techniques Invariable in many fungi = inadequate for looking at variation Microsatellites = repetitive sequences in the DNA (e.g. AC)12 Very popular for analyzing population structure Forensic applications Advantages: Hypervariable Genotyping Population Structure Disadvantages: High cost of Development Codominant Molecular Tools

4. Dominant Marker

5. Levels of Analyses • Individual • identifying parents & offspring– very important in zoological circles – identify patterns of mating between individuals (polyandry, etc.) • In fungi, it is important to identify the "individual" -- determining clonal individuals from unique individuals that resulted from a single mating event.

6. Levels of Analyses cont… • Families – looking at relatedness within colonies (ants, bees, etc.) • Population – level of variation within a population. • Dispersal = indirectly estimate by calculating migration • Conservation & Management = looking for founder effects (little allelic variation), bottlenecks (reduction in population size leads to little allelic variation) • Species – variation among species = what are the relationship between species. • Family, Order, ETC. = higher level phylogenies

7. Armillaria gallica “Humongous Fungus” rhizomorphs

9. What is Population Genetics? • About microevolution (evolution of species) • The study of the change of allele frequencies, genotype frequencies, and phenotype frequencies

10. Goals of population genetics • Natural selection (adaptation) • Chance (random events) • Mutations • Climatic changes (population expansions and contractions) • … To provide an explanatory framework to describe the evolution of species, organisms, and their genome, due to: Assumes that: • the same evolutionary forces acting within species (populations) should enable us to explain the differences we see between species • evolution leads to change in gene frequencies within populations

11. Pathogen Population Genetics • must constantly adapt to changing environmental conditions to survive • High genetic diversity = easily adapted • Low genetic diversity = difficult to adapt to changing environmental conditions • important for determining evolutionary potential of a pathogen • If we are to control a disease, must target a population rather than individual • Exhibit a diverse array of reproductive strategies that impact population biology

12. Analytical Techniques • Hardy-Weinberg Equilibrium • p2 + 2pq + q2 = 1 • Departures from non-random mating • F-Statistics • measures of genetic differentiation in populations • Genetic Distances – degree of similarity between OTUs • Nei’s • Reynolds • Jaccards • Cavalli-Sforza • Tree Algorithms – visualization of similarity • UPGMA • Neighbor Joining

13. Allele Frequencies • Allele frequencies (gene frequencies) = proportion of all alleles in an all individuals in the group in question which are a particular type • Allele frequencies: • p + q = 1 • Expected genotype frequencies: • p2 + 2pq + q2

14. Evolutionary principles: Factors causing changes in genotype frequency • Selection = variation in fitness; heritable • Mutation = change in DNA of genes • Migration = movement of genes across populations • Vectors = Pollen, Spores • Recombination = exchange of gene segments • Non-random Mating =mating between neighbors rather than by chance • Random Genetic Drift = if populations are small enough, by chance, sampling will result in a different allele frequency from one generation to the next.

15. The smaller the sample, the greater the chance of deviation from an ideal population. • Genetic drift at small population sizes often occurs as a result of two situations: the bottleneck effect or the founder effect.

17. Founder Effects • Establishment of a population by a few individuals can profoundly affect genetic variation • Consequences of Founder effects • Fewer alleles • Fixed alleles • Modified allele frequencies compared to source pop • Perhaps due to “new environment”

19. Bottleneck Effect • The bottleneck effect occurs when the numbers of individuals in a larger population are drastically reduced • By chance, some alleles may be overrepresented and others underrepresented among the survivors • Some alleles may be eliminated altogether • Genetic drift will continue to impact the gene pool until the population is large enough

20. Founder vs Bottleneck

21. Northern Elephant Seal: Example of Bottleneck Hunted down to 20 individuals in 1890’s Population has recovered to over 30,000 No genetic diversity at 20 loci

23. Potato Blight • Phytophthora infestans • great Irish famine of 1845-1849 • 1,000,000 died • Origin of P. infestans • Mexico = highest genetic diversity; likely origin • Ireland = decreased genetic diversity due to founder effect • Decreased genetic differentiation in other regions • Europe, North America

24. Hardy Weinberg Equilibriumand F-Stats • In general, requires co-dominant marker system • Codominant = expression of heterozygote phenotypes that differ from either homozygote phenotype. • AA, Aa, aa

25. Hardy-Weinberg Equilibrium • Null Model = population is in HW Equilibrium • Useful • Often predicts genotype frequencies well

26. Hardy-Weinberg Theorem if only random mating occurs, then allele frequencies remain unchanged over time. After one generation of random-mating, genotype frequencies are given by AA Aa aa p2 2pq q2 p = freq (A) q = freq (a)

27. Expected Genotype Frequencies • The possible range for an allele frequency or genotype frequency therefore lies between ( 0 – 1) • with 0 meaning complete absence of that allele or genotype from the population (no individual in the population carries that allele or genotype) • 1 means complete fixation of the allele or genotype (fixation means that every individual in the population is homozygous for the allele -- i.e., has the same genotype at that locus).

28. ASSUMPTIONS 1) diploid organism 2) sexual reproduction 3) Discrete generations (no overlap) 4) mating occurs at random 5) large population size (infinite) 6) No migration (closed population) 7) Mutations can be ignored 8) No selection on alleles

30. Locus 1 Allele 1 = 4/32 = 0.125 Allele 2 = 4/32 = 0.125 Allele 3 = 2/32 = 0.0625 Allele 4 = 22/32 = 0.6875 Allele frequencies = 0.125 + 0.125 + 0.00625 + 0.6875 = 1 Locus 2 Allele 1 = 8/32 = 0.2500 Allele 2 = 22/32 = 0.6875 Allele 3 = 2/32 = 0.0625 Locus 3 Allele 1 = 10/32 = 0.3125 Allele 2 = 22/32 = 0.6875

33. IMPORTANCE OF HW THEOREM If the only force acting on the population is random mating, allele frequencies remain unchanged and genotypic frequencies are constant. Mendelian genetics implies that genetic variability can persist indefinitely, unless other evolutionary forces act to remove it

34. Departures from HW Equilibrium • Check Gene Diversity = Heterozygosity • If high gene diversity = different genetic sources due to high levels of migration • Inbreeding - mating system “leaky” or breaks down allowing mating between siblings • Asexual reproduction = check for clones • Risk of over emphasizing particular individuals • Restricted dispersal = local differentiation leads to non-random mating

35. Pop 3 Pop 2 Pop 1 Pop 4 FST = 0.30 FST = 0.02

38. Local Inbreeding Coefficient • Calculate HOBS • Pop1: 4/20 = 0.20 • Pop2: 10/20 = 0.50 • Pop3: 8/20 = 0.40 • Calculate HEXP(2pq) • Pop1: 2*0.60*0.40 = 0.48 • Pop2: 2*0.50*0.50 = 0.50 • Pop3: 2*0.20*0.80 = 0.32 • Calculate F = (HEXP – HOBS)/ HEXP • Pop1 = (0.48 – 0.20)/(0.48) = 0.583 • Pop2 = (0.50 – 0.50)/(0.50) = 0.000 • Pop3 = (0.32 – 0.40)/(0.32) = -0.250

39. F StatsProportions of Variance • FIS = (HS – HI)/(HS) • FST = (HT – HS)/(HT) • FIT = (HT – HI)/(HT)

41. Host islands within the California Northern Channel Islands create fine-scale genetic structure in two sympatric species of the symbiotic ectomycorrhizal fungus Rhizopogon Rhizopogon occidentalis Rhizopogon vulgaris

42. Rhizopogon sampling & study area • Santa Rosa, Santa Cruz • R. occidentalis • R. vulgaris • Overlapping ranges • Sympatric • Independent evolutionary histories

43. Sampling

44. Bioassay – Mycorrhizal pine roots

45. Local Scale Population StructureRhizopogon occidentalis FST = 0.26 8-19 km N E 5 km FST = 0.33 FST = 0.24 W B T FST = 0.17 Populations are similar Populations are different Grubisha LC, Bergemann SE, Bruns TD Molecular Ecology in press.

46. Local Scale Population StructureRhizopogon vulgaris FST = 0.21 N E FST = 0.25 FST = 0.20 W Populations are different Grubisha LC, Bergemann SE, Bruns TD Molecular Ecology in press