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

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

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

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

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

Two scenarios: 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)

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

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

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

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

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

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

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 evolutionary 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), and to identify genotypes, and to study gene flow

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)

Root disease center in true fir caused by H. annosum

Ponderosa pineIncense cedar

Yosemite Lodge 1975 Root disease centers outlined

Yosemite Lodge 1997 Root disease centers outlined

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

RAPD combination

Conclusions Only one RAPD combo is sensitive enough to differentiate 4 half-sibs (in white) Mendelian inheritance? By analysis of all haplotypes it is apparent that two markers are always cosegregating, one of the two should be removed

If we have codominant markers how many do I need IDENTITY tests = probability calculation based on allele frequency… Multiplication of frequencies of alleles 10 alleles at locus 1 P1=0.1 5 alleles at locus 2 P2=0,2 Total P= P1*P2=0.02

AFLP Amplified Fragment Length Polymorphisms Dominant marker Scans the entire genome like RAPDs More reliable because it uses longer PCR primers less likely to mismatch Priming sites are a construct of the sequence in the organism and a piece of synthesized DNA

How are AFLPs generated? AGGTCGCTAAAATTTT (restriction site in red) AGGTCG CTAAATTT Synthetic DNA piece ligated –NNNNNNNNNNNNNNCTAAATTTTT Created a new PCR priming site –NNNNNNNNNNNNNNCTAAATTTTT Every time two PCR priming sitea are within bp you obtain amplification

Have we sampled enough? Resampling approaches Saturation curves –A total of 30 polymorphic alleles –Our sample is either 10 or 20 –Calculate whether each new sample is characterized by new alleles

Saturation curves No Of New alleles

Dealing with dominant anonymous multilocus markers Need to use large numbers (linkage) Repeatability Graph distribution of distances Calculate distance using Jaccard’s similarity index

Jaccard’s Only 1-1 and 1-0 count, 0-0 do not count

Jaccard’s Only 1-1 and 1-0 count, 0-0 do not count A: AB= (1-AB) B: BC= C: AC=0.20.8

Now that we have distances…. Plot their distribution (clonal vs. sexual)

Now that we have distances…. Plot their distribution (clonal vs. sexual) Analysis: –Similarity (cluster analysis); a variety of algorithms. Most common are NJ and UPGMA

Now that we have distances…. Plot their distribution (clonal vs. sexual) Analysis: –Similarity (cluster analysis); a variety of algorithms. Most common are NJ and UPGMA –AMOVA; requires a priori grouping

AMOVA groupings Individual Population Region AMOVA: partitions molecular variance amongst a priori defined groupings

Results: Jaccard similarity coefficients Coefficient Frequency P. nemorosa P. pseudosyringae: U.S. and E.U. 0.3 Coefficient Frequency

Pp U.S. Pp E.U Jaccard coefficient of similarity 0.7 P. pseudosyringae genetic similarity patterns are different in U.S. and E.U.

P. nemorosa P. ilicis P. pseudosyringae Results: P. nemorosa

Results: P. pseudosyringae P. nemorosa P. ilicis P. pseudosyringae = E.U. isolate

The “scale” of disease Dispersal gradients dependent on propagule size, resilience, ability to dessicate, NOTE: not linear Important interaction with environment, habitat, and niche availability. Examples: Heterobasidion in Western Alps, Matsutake mushrooms that offer example of habitat tracking Scale of dispersal (implicitely correlated to metapopulation structure)---

White mangroves: Corioloposis caperata Distances between study sites

Coriolopsis caperata on Laguncularia racemosa Forest fragmentation can lead to loss of gene flow among previously contiguous populations. The negative repercussions of such genetic isolation should most severely affect highly specialized organisms such as some plant- parasitic fungi. AFLP study on single spores

Using DNA sequences Obtain sequence Align sequences, number of parsimony informative sites Gap handling Picking sequences (order) Analyze sequences (similarity/parsimony/exhaustive/bayesian Analyze output; CI, HI Bootstrap/decay indices

Using DNA sequences Testing alternative trees: kashino hasegawa Molecular clock Outgroup Spatial correlation (Mantel) Networks and coalescence approaches

From Garbelotto and Chapela, Evolution and biogeography of matsutakes Biodiversity within species as significant as between species

Microsatellites or SSRs AGTTTCATGCGTAGGT CG CG CG CG CG AAAATTTTAGGTAAATTT Number of CG is variable Design primers on FLANKING region, amplify DNA Electrophoresis on gel, or capillary Size the allele (different by one or more repeats; if number does not match there may be polimorphisms in flanking region) Stepwise mutational process (2 to 3 to 4 to 3 to2 repeats)