Summary of last lesson Excellent review of techniques as long as pop gen Methods of ana
Burney, Jim, Music Director has reference to this Academic Journal, PHwiki organized this Journal Summary of last lesson Excellent review of techniques as long as 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 Frequency-, or density dependent, or balancing selection New alleles, if beneficial because linked to a trait linked to fitness will be positively selected as long as . Example: two races of pathogen are present, but only one resistant host variety, suggests second pathogen race has arrived recently Rapid generation time of pathogens. Reticulated evolution very likely. Pathogens will be selected as long as 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
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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 Life cycle: Reproduction Sexual Basidiocarps release spores (n) after karyogamy in addition to 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 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 in addition to then go on to show that this genotype likely represents in addition to individual – Myron Smith
Researchers 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 Approach 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 Methods in addition to Materials 1 1. Collecting samples Researcher collected samples over a 30 hectare area by baiting Armillaria with poplar stakes in addition to taking tissues in addition to spores They then grew the successfully colonized stakes in soil taken from the study site Each fungal colony cultured was called an isolate.
Methods in addition to Materials 2 2. Checking mating type – Somatic incompatibility For two fungal isolates to fuse, all somatic compatibility loci must be the same. Fusion means theyre clones Example (not Armillaria) Methods in addition to 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 in addition to B. (They have an incompatibility system) If fruit bodies had the same alleles at A in addition to B, in addition to were collected from the same area, they were assumed to be from the same clone Result 1 Somatic compatilbilty: isolates from vegetative mycelium from a large sampling area fused Mating alleles They had the same mating type
Result 1 Clone 1 was found to exceed 500 m in diameter Used previously collected mtDNA restriction fragment patterns Sensitivity of Approach Problem: These tests alone are not enough to distinguish a clone from closely related individuals 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.
Methods in addition to Materials 3 3. Molecular Testing – RFLP analysis at 5 polymorphic, heterozyg. loci of mtDNA from Clone 1 – RAPD analysis at 11 loci RAPDS vs. RFLPs 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 in addition to see how amplicon was cut Single locus is co-dominant Result 2 RFLP All 5 loci from Clone 1 were heterozygous in addition to identical (both alleles present at loci: 1,1) RAPD All 11 RAPD products were present in all vegetative isolates
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 as long as their results, not inbreeding More testing, just in case To be completely confident, they tested: 1) that nearby Clone 2 was different in addition to 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 Sensitivity of RAPDs Tested on subset of spores from same basidiocarp RAPDs differentiated among full sibs
Conclusions Somatic compatibility, mating allele loci, mtDNA, RFLP, in addition to RAPD tests all indicate that a single organism could indeed occupy a 15 hectare area Conclusions The larger individual, Clone 1 was estimated to weigh 9700 kg in addition to be over 1500 years old Implications Fungi are one of the oldest in addition to largest organisms on the planet Recycle nutrients very important! Armillaria bulbosa also a pathogen; its effects on as long as est above may be huge as well.
HOST-SPECIFICITY Biological species Reproductively isolated Measurable differential: size of structures Gene- as long as -gene defense model Sympatric speciation: Heterobasidion, Armillaria, Sphaeropsis, Phellinus, Fusarium as long as ma speciales Phylogenetic relationships within the Heterobasidion complex Fir-Spruce Pine Europe Pine N.Am.
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
From Garbelotto in addition to Chapela, Evolution in addition to 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)
Burney, Jim Music Director
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