The biology of the organism drives an epidemic

1. The biology of the organism drives an epidemic • Autoinfection vs. alloinfection • Primary spread=by spores • Secondary spread=vegetative, clonal spread, same genotype . Completely different scales (from small to gigantic) Coriolus Heterobasidion Armillaria Phellinus

3. OUR ABILITY TO: • Differentiate among different individuals (genotypes) • Determine gene flow among different areas • Determine allelic distribution in an area

4. WILL ALLOW US TO DETERMINE: • How often primary infection occurs or is disease mostly chronic • How far can the pathogen move on its own • Is the organism reproducing sexually? is the source of infection local or does it need input from the outside

5. IN ORDER TO UNDERSTAND PATTERNS OF INFECTION • If John gave directly Mary an infection, and Mary gave it to Tom, they should all have the same strain, or GENOTYPE (comparison=secondary spread among forest trees) • If the pathogen is airborne and sexually reproducing, Mary John and Tom will be infected by different genotypes. But if the source is the same, the genotypes will be sibs, thus related

6. Recognition of self vs. non self • Intersterility genes: maintain species gene pool. Homogenic system • Mating genes: recognition of “other” to allow for recombination. Heterogenic system • Somatic compatibility: protection of the individual.

7. INTERSTERILITY • If a species has arisen, it must have some adaptive advantages that should not be watered down by mixing with other species • Will allow mating to happen only if individuals recognized as belonging to the same species • Plus alleles at one of 5 loci (S P V1 V2 V3)

8. MATING • Two haploids need to fuse to form n+n • Sex needs to increase diversity: need different alleles for mating to occur • Selection for equal representation of many different mating alleles

9. MATING ALLELES • All heterokaryons will have two mating allelels, for instance a, b • There is an advantage in having more mating alleles (easier mating, higher chances of finding a mate) • Mating allele that is rare, may be of migrant just arrived • If a parent is important source, genotypes should all be of one or two mating types

10. A, A, B, C, D, D, E, H, I, L A, A, A,B, B, A, A Two scenarios:

11. A, A, B, C, D, D, E, H, I, L Multiple source of infections (at least 4 genotypes) A, A, A,B, B, A, A Sible source of infection (1 genotype) Two scenarios:

12. SEX • Ability to recombine and adapt • Definition of population and metapopulation • Different evolutionary model • Why sex? Clonal reproductive approach can be very effective among pathogens

13. Long branches in between groups suggests no sex is occurring in between groups Fir-Spruce Pine Europe Pine N.Am.

14. Small branches within a clade indicate sexual reproduction is ongoing within that group of individuals NA S NA P EU S 890 bp CI>0.9 EU F

15. Index of association Ia= if same alleles are associated too much as opposed to random, it means sex is not occurring Association among alleles calculated and compared to simulated random distribution

16. SOMATIC COMPATIBILITY • Fungi are territorial for two reasons • Selfish • Do not want to become infected • If haploids it is a benefit to mate with other, but then the n+n wants to keep all other genotypes out • Only if all alleles are the same there will be fusion of hyphae • If most alleles are the same, but not all, fusion only temporary

17. SOMATIC COMPATIBILITY • SC can be used to identify genotypes • SC is regulated by multiple loci • Individual that are compatible (recognize one another as self, are within the same SC group) • SC group is used as a proxy for genotype, but in reality, you may have some different genotypes that by chance fall in the same SC group • Happens often among sibs, but can happen by chance too among unrelated individuals

18. Recognition of self vs. non self • What are the chances two different individuals will have the same set of VC alleles? • Probability calculation (multiply frequency of each allele) • More powerful the larger the number of loci • …and the larger the number of alleles per locus

19. Recognition of self vs. non self:probability of identity (PID) • 4 loci • 3 biallelelic • 1 penta-allelic • P= 0.5x0.5x0.5x0.2=0.025 • In humans 99.9%, 1000, 1 in one million

20. Evolution and Population genetics • Positively selected genes:…… • Negatively selected genes…… • Neutral genes: normally population genetics demands loci used are neutral • Loci under balancing selection…..

21. Evolution and Population genetics • Positively selected genes:…… • Negatively selected genes…… • Neutral genes: normally population genetics demands loci used are neutral • Loci under balancing selection…..

22. Evolutionary history • Darwininan vertical evolutionary models • Horizontal, reticulated models..

23. Phylogenetic relationships within the Heterobasidioncomplex Fir-Spruce Pine Europe Pine N.Am.

24. Geneaology of “S” DNA insertion into P ISG confirms horizontal transfer.Time of “cross-over” uncertain NA S NA P EU S 890 bp CI>0.9 EU F

25. Because of complications such as: • Reticulation • Gene homogeneization…(Gene duplication) • Need to make inferences based on multiple genes • Multilocus analysis also makes it possible to differentiate between sex and lack of sex (Ia=index of association), and to identify genotypes, and to study gene flow

26. Basic definitions again • Locus • Allele • Dominant vs. codominant marker • RAPDS • AFLPs

27. How to get multiple loci? • Random genomic markers: • RAPDS • Total genome RFLPS (mostly dominant) • AFLPS • Microsatellites • SNPs • Multiple specific loci • SSCP • RFLP • Sequence information Watch out for linked alleles (basically you are looking at the same thing!)

28. Sequence information • Codominant • Molecules have different rates of mutation, different molecules may be more appropriate for different questions • 3rd base mutation • Intron vs. exon • Secondary tertiary structure limits • Homoplasy

29. Sequence information • Multiple gene genealogies=definitive phylogeny • Need to ensure gene histories are comparable” partition of homogeneity test • Need to use unlinked loci

30. DNA template Reverse primer Forward primer Thermalcycler

31. Gel electrophoresis to visualize PCR product Ladder (to size DNA product)

32. From DNA to genetic information (alleles are distinct DNA sequences) • Presence or absence of a specific PCR amplicon (size based/ specificity of primers) • Differerentiate through: • Sequencing • Restriction endonuclease • Single strand conformation polymorphism

33. Presence absence of amplicon • AAAGGGTTTCCCNNNNNNNNN • CCCGGGTTTAAANNNNNNNNN AAAGGGTTTCCC (primer)

34. Presence absence of amplicon • AAAGGGTTTCCCNNNNNNNNN • CCCGGGTTTAAANNNNNNNNN AAAGGGTTTCCC (primer)

35. RAPDS use short primers but not too short • Need to scan the genome • Need to be “readable” • 10mers do the job (unfortunately annealing temperature is pretty low and a lot of priming errors cause variability in data)

36. RAPDS use short primers but not too short • Need to scan the genome • Need to be “readable” • 10mers do the job (unfortunately annealing temperature is pretty low and a lot of priming errors cause variability in data)

37. RAPDS can also be obtained with Arbitrary Primed PCR • Use longer primers • Use less stringent annealing conditions • Less variability in results

38. Result: series of bands that are present or absent (1/0)

40. Root disease center in true fir caused by H. annosum

41. Ponderosa pine Incense cedar

42. Yosemite Lodge 1975 Root disease centers outlined

43. Yosemite Lodge 1997 Root disease centers outlined

50. Are my haplotypes sensitive enough? • To validate power of tool used, one needs to be able to differentiate among closely related individual • Generate progeny • Make sure each meiospore has different haplotype • Calculate P