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
Summary of seventh lesson SEX; the great homogenizing force, and also ability to create new alleles INTERSTERILITY/ MATING> SOMATIC COMPATIBILITY NEED TO USE MULTIPLE MARKERS; SC does that, otherwise go to molecular markers PCR/ RAPDS
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)
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
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
AFLPs are read like RAPDs (0/1)
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
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 –Discriminant, canonical analysis
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
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 –Discriminant, canonical analysis –Frequency: does allele frequency match expected (hardy weinberg), F or Wright’s statistsis
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)---
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
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
White mangroves: Corioloposis caperata
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
Pacifico Caribe
From Garbelotto and Chapela, Evolution and biogeography of matsutakes Biodiversity within species as significant as between species