Summary of sixth lesson Janzen-Connol hypothesis; explanation of why diseases lead to spatial heterogeneity Diseases also lead to heterogeneity or changes through time –Driving succession –The Red Queen Hypothesis: selection pressure will increase number of resistant plant genotypes Co-evolution: pathogen increase virulence in short term, but in long term balance between host and pathogen Complexity of forest diseases: primary vs. secondaruy, modes of dispersal etc

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

Phylogenetic relationships within the Heterobasidion complex Fir-Spruce Pine Europe Pine N.Am.

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.

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)

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

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

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

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

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

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

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

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

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

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

Evolutionary history Darwininan vertical evolutionray models Horizontal, reticulated models..

Phylogenetic relationships within the Heterobasidion complex Fir-Spruce Pine Europe Pine N.Am.

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

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)

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

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!)

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

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

Thermalcycler DNA template Forward primer Reverse primer

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

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

Presence absence of amplicon AAAGGGTTTCCCNNNNNNNNN CCCGGGTTTAAANNNNNNNNN AAAGGGTTTCCC (primer)

Presence absence of amplicon AAAGGGTTTCCCNNNNNNNNN CCCGGGTTTAAANNNNNNNNN AAAGGGTTTCCC (primer)

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)

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)

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

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