Ex4 Genetic analysis in Aspergillus nidulans.
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We have been studying fungal growth and response to changing environments, and mold
isolation and identification. With few exceptions, none of the organisms in these lab exercises
were characterized beyond genus. In contrast, when fungi are used as experimental tools they
are generally very precisely identified and characterized.
The matings for this lab were done between strains with different spore colours and nutritional
auxotrophies (see table below). In order to test whether individual cleistothecia resulted from
an outcross, that is, between parental strains, a small volume of ascospore suspension is spread
on rich medium and incubated at 28°C for two to three days. The ascospore suspension is stored
at 4°C, where the spores will remain viable for months to years.
Today you’ll be cleaning Aspergillus cleistothecia, suspending the ascospores in sterile distilled
water, and plating a sample of spores to test whether they came from a cross between the two
parents. Aspergillus can mate with itself, so this is not a trivial test. However, we will pick
cleistothecia that are likely to be outcrossed by choosing large ones formed close to conidia that
have both parental spore colours. The conidia will be plated on medium that will support most of
the auxotrophies, enough that we will be able to tell outcrosses for next time.
Analyzing the progeny of an outcrossed cleistothecium
Just learning that you have an outcrossed cleistothecium doesn’t get you very far. Generally you
are trying to do two things – learn about a genetic interaction which sometimes requires isolating
strains that have particular combinations of mutations, and determining the nutritional
requirements of those strains. To accomplish these aims, you need single-spore progeny. The
original spread of your newly isolated ascospores was done at high spore density. All you needed
then was to distinguish between outcrossed and self-crossed cleistothecia.
The next objective requires precision: you will be spreading a diluted spore sample on
more CM+ plates, and next time you will be picking those progeny for analysis.
Preparation: make a glass spreader. Get a 5 1/4
inch Pasteur pipette. Hold the narrow end in a
Bunsen burner flame until it seals and droops
slightly. Pull the pipette out of the flame, and
rotate it about 90° until the droopy tip is pointing
towards you. Then, flame again so that the pipette
bends about an inch from the tip. Remove and
place in an Eppendorf rack to cool.
Ex4 Genetic analysis in Aspergillus nidulans.
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Examine the Eppendorf tubes that have your ascospore suspension: you should be able to
see a dark spot at the bottom, which is the mass of ascospores originally in the cleistothecium.
Obviously, these spores are heavier than water. To spread plates for isolated colonies (likely to
have been produced from a single spore), thoroughly mix your original ascospore suspension and
make a 1:100 dilution in sterile distilled water. Use 2µl of ascospores in 200 µl of water. Mix
again, and them pipette 30µl, 60µl and 90µl of this suspension onto three labeled plates of CM+
agar. Spread the suspension evenly over the surface of the plate using a sterile spreader. Dip your
spreader in alcohol, flame briefly, dip again in alcohol, flame to dry and let cool. Test the
coolness by touching the agar. Leave the plates right side up for 30-60 min to allow the water in
the suspension to absorb into the agar. Tape shut and invert. Store at room temperature for two to
three days. A. nidulans ascospores take longer to germinate than do conidia.
We will be analyzing individual ascospore progeny by random ascospore analysis. Colonies
from single ascospores are sampled by probing gently with a sterile toothpick that was first by
touching to a clean part of the agar, and then point inoculated by touching to a master plate that
has a fixed position with respect to the locator grid. Each toothpick is used for only one colony,
but several replicate plates can be inolculated from the same toothpick.
This whole process must be done with minimal air disturbance. The plates are opened once
only for the entire process, you should avoid waving your hands over the source or recipient
plates, and you should not breathe over the plates. Done properly, you will have 50 progeny plus
2 parental colonies growing well separated on a single plate. Otherwise, you will have many
small colonies as well as the ones you inoculated intentionally.
By inoculating replicate series of colonies onto media of different compositions, or grown under
different conditions, you will be able to deduce the genotype of each progeny strain. It is
important to inoculate the two parental strains onto each plate for cross reference purposes. The
parental strains have been well characterized, but some of the phenotypes of the alleles can be
difficult to score. Generally the strains mated for this cross have easy to score phenotypes.
Scoring phenotypes used sheets designed for convenient tallying of characteristics.
An interesting analysis is to compare inheritance amongst pairs of genes. You might find that if
the parental cross is AB :: ab, that these combinations occur far more frequently than then
recombinant Ab and aB. This can be tested statistically to determine if the genes are linked, that
is, whether they are physically close on the same chromosome. Regardless of linkage (which is
only relevant when genes are compared pairwise), a/A and b/B should be inherited by 50% of the
progeny.
Your tasks for this exercise will be to
 identify and isolate strains that have interesting and useful genetic marker comparisons
for future research
 to study gene linkage
 to study gene epistasis, using colour markers