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  1. Summary of last lesson • Excellent review of techniques for pop gen • Methods of analysis • Previous lesson: density dependence/janzen connel/red queen hypothesis/type of markers • humnogous fungus • Testing the marker/testing sample size

  2. Frequency-, or density dependent, or balancing selection • New alleles, if beneficial because linked to a trait linked to fitness will be positively selected for. • Example: two races of pathogen are present, but only one resistant host variety, suggests second pathogen race has arrived recently

  3. Rapid generation time of pathogens. Reticulated evolution very likely. Pathogens will be selected for INCREASED virulence • In the short/medium term with long lived trees a pathogen is likely to increase its virulence • In long term, selection pressure should result in widespread resistance among the host

  4. Overview Armillaria bulbosa (gallica) • Known as the Humungous Fungus, or honey mushroom • Form rhizomorphs, which make up much of the “humungous” part • Basidiocarp: cap 6 cm in diameter, stem is 5-10 cm tall • Facultative tree root pathogen

  5. Life cycle: Reproduction • Sexual • Basidiocarps release spores (n) after karyogamy and meiosis • 2 mating-type loci, each with multiple alleles in the population • Isolates (n) must have different alleles at two mating type loci to be sexually compatible • Asexual • vegetative spreading of rhizomorph • The large mass of rhizomorph that is genetically isolated is called a clone

  6. Building up the question… • “By extending the areas sampled in subsequent years, we were finally able to delimit the large area occupied by this genotype and then go on to show that this genotype likely represents and ‘individual’” - Myron Smith

  7. Researcher’s Question • The clonal “individual” is especially difficult to define because the network of hyphae is underground • How do you unambiguously identify an individual fungi within a local population?

  8. 1. Collect samples 2. Check mating type - Somatic compatibility test - Distrubution of mating-type alleles 3. Molecular testing - RFLP - RAPD 4. Statistics 5. More testing Approach

  9. Methods and Materials 1 1. Collecting samples • Researcher collected samples over a 30 hectare area by baiting Armillaria with poplar stakes and taking tissues and spores • They then grew the successfully colonized stakes in soil taken from the study site • Each fungal colony cultured was called an isolate.

  10. Methods and Materials 2 Example (not Armillaria) 2. Checking mating type - Somatic incompatibilityFor two fungal isolates to fuse, all somatic compatibility loci must be the same. Fusion means they’re clones 

  11. Methods and Materials 2 • 2. Checking mating type - Distrubution of mating alleles • Mating occurs only when coupled isolates have different alleles at two unlinked, multiallelic loci: A and B. (They have an incompatibility system) • If fruit bodies had the same alleles at A and B, and were collected from the same area, they were assumed to be from the same clone

  12. Result 1 • Somatic compatilbilty: • isolates from vegetative mycelium from a large sampling area fused • Mating alleles • They had the same mating type

  13. Result 1 • “Clone 1” was found to exceed 500 m in diameter • Used previously collected mtDNA restriction fragment patterns

  14. Sensitivity of Approach • Problem: These tests alone are not enough to distinguish a clone from closely related individuals

  15. Why? • Q: The first two tests were not sensitive enough to tell a clone from a close relative…Why? • A: Spores from same point source have the same mating-type alleles, but the offspring they produce after inbreeding are genetically distinct.

  16. Methods and Materials 3 3. Molecular Testing - RFLP analysis at 5 polymorphic, heterozyg. loci of mtDNA from “Clone 1” - RAPD analysis at 11 loci

  17. Use 1 short PCR primer When it finds match on template at a distance that can be amplified (primer binds twice within 50 to 2000 bp) RAPD amplicon Dominant, annoymous Total genomic, vs single locus Use endonuclease to digest DNA at specific restriction site Run digest and see how amplicon was cut Single locus is co-dominant RAPDS vs. RFLPs

  18. Result 2 • RFLP • All 5 loci from Clone 1 were heterozygous and identical (both alleles present at loci: 1,1) • RAPD • All 11 RAPD products were present in all vegetative isolates”

  19. Statistical Analysis • The probability of retaining heterozygosity at each parental locus in an individual produced by mating of sibling monospore isolates… = 0.0013 • So they were pretty confident that cloning was responsible for their results, not inbreeding

  20. More testing, just in case • To be completely confident, they tested: • 1) that nearby Clone 2 was different and lacked 5 of the Clone 1 heterozyg. RAPD fragments, • 2) more loci, totaling • 20 RAPD fragments • 27 nuclear DNA RFLP fragments ** all were identical in Clone 1

  21. Sensitivity of RAPDs • Tested on subset of spores from same basidiocarp • RAPDs differentiated among full sibs

  22. Conclusions • Somatic compatibility, mating allele loci, mtDNA, RFLP, and RAPD tests all indicate that a single organism could indeed occupy a 15 hectare area

  23. Conclusions • The larger individual, Clone 1 was estimated to weigh 9700 kg and be over 1500 years old

  24. Implications • ????? • Fungi are one of the oldest and largest organisms on the planet • Recycle nutrients…very important! • Armillaria bulbosa also a pathogen; its effects on forest above may be huge as well.

  25. HOST-SPECIFICITY • Biological species • Reproductively isolated • Measurable differential: size of structures • Gene-for-gene defense model • Sympatric speciation: Heterobasidion, Armillaria, Sphaeropsis, Phellinus, Fusarium forma speciales

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

  27. 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

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

  29. 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

  30. 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

  31. 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.

  32. 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

  33. Recognition of self vs. non self • It is possible to have different genotypes with the same vc alleles • VC grouping and genotyping is not the same • It allows for genotyping without genetic tests • Reasons behing VC system: protection of resources/avoidance of viral contagion

  34. Somatic incompatibility

  35. More on somatic compatibility • Perform calculation on power of approach • Temporary compatibility allows for cytoplasmic contact that then is interrupted: this temporary contact may be enough for viral contagion

  36. 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

  37. 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

  38. 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

  39. 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

  40. 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)

  41. INTERSTERILITY • Basis for speciation • These alleles are selected for more strongly in sympatry • You can have different species in allopatry that have not been selected for different IS alleles

  42. 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

  43. MATING • If one individuals is source of inoculum, then the same 2 mating alleles will be found in local population • If inoculum is of broad provenance then multiple mating alleles should be found

  44. MATING • How do you test for mating? • Place two homokaryons in same plate and check for formation of dikaryon (microscopic clamp connections at septa)

  45. Clamp connections

  46. 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

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

  48. 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 Siblings as source of infection (1 genotype) Two scenarios: