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 Phylogenetic relationships relationships within the within the HeterobasidionHeterobasidion complexcomplex

Het INSULARE

True Fir EUROPE

Spruce EUROPE

True Fir NAMERICA

Pine EUROPE

Pine NAMERICA

0.05 substitutions/site

NJ

Fir-SpruceFir-Spruce

Pine EuropePine Europe

Pine N.Am.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 Long branches in between groups between groups suggests no sex is suggests no sex is occurring in between occurring in between groupsgroups

Het INSULARE

True Fir EUROPE

Spruce EUROPE

True Fir NAMERICA

Pine EUROPE

Pine NAMERICA

0.05 substitutions/site

NJ

Fir-SpruceFir-Spruce

Pine EuropePine Europe

Pine N.Am.Pine N.Am.

Small branches within a clade Small branches within a clade indicate sexual reproduction is indicate sexual reproduction is

ongoing within that group of ongoing within that group of individualsindividuals

11.10 SISG CA

2.42 SISG CA

BBd SISG WA

F2 SISG MEX

BBg SISG WA

14a2y SISG CA

15a5y M6 SISG CA

6.11 SISG CA

9.4 SISG CA

AWR400 SPISG CA

9b4y SISG CA

15a1x M6 PISG CA

1M PISG MEX

9b2x PISG CA

A152R FISG EU

A62R SISG EU

A90R SISG EU

A93R SISG EU

J113 FISG EU

J14 SISG EU

J27 SISG EU

J29 SISG EU

0.0005 substitutions/site

NJ

890 bpCI>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 Phylogenetic relationships relationships within the within the HeterobasidionHeterobasidion complexcomplex

Het INSULARE

True Fir EUROPE

Spruce EUROPE

True Fir NAMERICA

Pine EUROPE

Pine NAMERICA

0.05 substitutions/site

NJ

Fir-SpruceFir-Spruce

Pine EuropePine Europe

Pine N.Am.Pine N.Am.

Geneaology of “S” DNA insertion into Geneaology of “S” DNA insertion into P ISG confirms horizontal transfer.P ISG confirms horizontal transfer.

Time of “cross-over” uncertainTime of “cross-over” uncertain

11.10 SISG CA

2.42 SISG CA

BBd SISG WA

F2 SISG MEX

BBg SISG WA

14a2y SISG CA

15a5y M6 SISG CA

6.11 SISG CA

9.4 SISG CA

AWR400 SPISG CA

9b4y SISG CA

15a1x M6 PISG CA

1M PISG MEX

9b2x PISG CA

A152R FISG EU

A62R SISG EU

A90R SISG EU

A93R SISG EU

J113 FISG EU

J14 SISG EU

J27 SISG EU

J29 SISG EU

0.0005 substitutions/site

NJ

890 bpCI>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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Thermalcycler

DNA template

Forward primer Reverse primer

Gel electrophoresis to visualize PCR product

Ladder (to sizeDNA 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)