Plant Pathology 526
Advanced Fungal Biology
Spring 2014
Laboratory #1 – Genetic Crosses of Sordaria fimicola
Setting up the Matings:
1. Find the stock plates of the wild type, gray spore mutant and tan spore mutant strains. The gray and tan
strains are color mutants and have gray and tan ascospores, respectively. The wild-type ascospores are
black. Depending on the maturity of the cultures, you may be able to see color differences among the
three strains on the plates. Obtain 2 plates of each isolate per group.
2. Obtain 22 plates of Sordaria crossing medium per group and label each with your group name.
3. Cut 4 small cubes (approx. 3mm x 3mm x 3mm) of the mycelium plus agar from the plate with the
wild-type strain and place them in 4 positions (90 degrees equidistant from each other) on a fresh plate of
crossing agar as shown below. Place the cubes on the plate with the fungal mycelium side facing down, in
contact with the surface of the fresh plate (i.e, turn the cubes upside-down).
4. Cut 4 cubes from the tan mutant stock plate and place them 2-4 mm to the right of the wild-type cubes.
Repeat the process on another plate to give you 5 replicate plates for each cross. All plates should look
like this:
Wild-type
Tan mutant
5. Repeat the above procedure crossing the wild-type with the grey mutant (5 replicate plates).
6. Repeat the above procedure crossing the tan mutant with the grey mutant (12 replicate plates).
7. Wrap the plates with parafilm and incubate at 25C for 7 to 10 days on the lighted growth shelves in
Peever’s lab until perithecia are evident in the zones of interaction between the strains.
As the fungus grows out over the surface, the two strains will meet along a boundary between the two
strains. This is where hybrid matings will occur. Sordaria fimicola is a homothallic fungus and will
undergo selfing if only a single strain (ie. single genotype) is placed on a plate. The fungus can be forced
to outcross if paired with a strain of different genotype. We are most interested in these outcrossed or
hybrid perithecia as these will be the only perithecia which contain asci segregating for spore color.
1
Plant Pathology 526
Advanced Fungal Biology
Spring 2014
Seven to Ten Days Later:
It is difficult to predict exactly when the asci will be mature but it generally takes 7-10 days with S.
fimicola. We will need to be somewhat flexible in terms of when we score the tetrads to allow for
differences in rate of maturation. If the crosses are started on a Tuesday, they should be ready by the next
Tuesday and/or Thursday. Students will need to start observing the crossing plates carefully at 7 days to
monitor maturity. If they start to mature and shoot ascospores onto the lids of the Petri dishes, refrigerate
the plates to slow them down.
Scoring the Crosses
Ascospores of Sordaria fimicola are small so in order to see them you will need to prepare a wet mount
slide and observe them under a compound microscope. Prepare the mounts carefully and then look
patiently to find intact asci to score. You may need to prepare several slides before you get one in which
the asci are well spread out but which are still intact.
1. Pick up the plates that you set up last week containing the paired strains. Examine the plate. You
should be able to clearly see a boundary between the sectors of the plate containing the two strains. You
should attempt to collect perithecia from this boundary. These are the regions most likely to contain
hybrid perithecia. Check several of the replicate plates for each cross.
2. Use a toothpick to scrape the surface of the plate along one of the boundary regions and pick up some
of the perithecia that are located there. The perithecia are the dark bodies visible on the crossing plates.
3. Transfer approximately 10-15 perithecia to a drop of water on a microscope slide and add a coverslip.
Then gently tap the coverslip and apply gentle pressure. The ascospores are contained in long sacs called
asci (singular ascus). The spores in one ascus all arose from a single meiotic division. A group of asci are
contained in a large round structure called the perithecium. You need to break open the perithecia to
release the asci, but you must avoid applying too much pressure and breaking open the asci and releasing
the ascospores. It is essential that the spores that you score are contained within unbroken asci. In fact,
what you will attempt to score are not single ascospores but the arrangement of the ascospores in each
ascus.
Wild-type X Mutant Crosses
Collect asci from the zone of contact between the mutant and wild-type strains. Crush and mount
these asci and look for those that contain ascospores of two colors. The dark spores are the wild-type ones
and the lighter-colored ones are the mutants. You may be able to detect a grayish hue to the ascospores
carrying the mutant allele from the gray strain and a tan hue to the spores carrying the mutant allele from
the tan strain. You will only score asci that contain spores of both colors. Any asci that contain all dark,
all tan or all grey spores did not result from a cross between two different strains and are selfs.
The asci will be of two types: non-recombinant and recombinant. These are classified by the
arrangement of the ascospores within the ascus. The non-recombinant types contain a series of four tan or
grey ascospores at one end and four dark ascospores at the other. These patterns are called "first division
segregation patterns" or "MI" because alleles segregate into different nuclei after the first meiotic
division. The ascospores will be arranged in two patterns that should occur in equal frequency.
2
Plant Pathology 526
Advanced Fungal Biology
Spring 2014
Below are two possible arrangements of non-recombinant asci resulting from a tan mutant x wild type
cross:
T = tan ascospore, B = black, wild-type ascospore
TTTTBBBB
BBBBTTTT
You should see the same pattern for the grey mutant X wild-type cross.
Recombinant asci contain ascospores that are arranged in groups of two. These are called "second
division segregation patterns" or "MII" because alleles do not segregate until the second meiotic
division. There are four possible arrangements for the spores in a recombinant ascus:
TTBBTTBB
TTBBBBTT
BBTTBBTT
BBTTTTBB
You should see the same patterns for the grey mutant X wild-type cross.
3
Plant Pathology 526
Advanced Fungal Biology
Spring 2014
Count at least 50 asci and score them as either non-recombinant or recombinant. Record the results. Pool
your results with the rest of the students on the blackboard.
Questions:
1) Calculate the recombination frequency and the map the distance between the tan locus
and the centromere.
2) Calculate the recombination frequency and the map the distance between the grey locus
and the centromere.
3) Why is it equally likely that you will see asci with TTTTBBBB patterns and
BBBBTTTT patterns from ascus tip to base?
4) Why is the map distance equal to the recombination percentage divided by 2? In other
organisms (like Drosophila, humans and peas) the map distance is equal to the
recombination percentage.
4
Plant Pathology 526
Advanced Fungal Biology
Spring 2014
Grey Mutant x Tan Mutant Crosses
In this cross you should see a total of 7 different ascospore patterns rather than the 6 you observed
with the wild-type x mutant crosses. In addition to black, gray and tan ascospores you observed above,
you will also observe colorless ascospores in this cross. The 7 patterns can be classified into 3 basic types:
a) parental ditypes (PD) - no recombination - 2 spores of each parental type (no crossovers OR a 2strand double crossover)
b) non-parental ditypes (NPD) - 4 recombinant spores (4-strand double crossovers OR
independent assortment with no recombination)
c) tetratype (T) - 2 parental types, 2 reciprocal recombinants (single crossover OR 3-strand double
crossover)
These patterns are the result of crossovers and MI and MII segregation. Tetratype (T) patterns can
only result when a crossover has occurred between at least one locus and the centromere. For two linked
genes, a T ascus can only be generated at the 4-strand stage. If crossing over occurred at the 2-strand
stage prior to chromosome replication, it would create an NPD ascus. The existence of T asci proves that
crossing over occurs after DNA replication.
Count at least 50 asci and place them into the 7 classes. Record the results. Pool your results with the rest
of the students on the blackboard.
Questions:
5) estimate the distance of the tan locus from the centromere. Does this estimate agree with the one
you estimated above?
6) estimate the distance of the grey locus from the centromere. Does this estimate agree with thoe
one you estimated above?
7) are the tan and grey loci linked? Why or why not? Justify your answer.
8) why is this sort of analysis referred to as tetrad (meaning "four") analysis if there are eight
ascospores in each ascus?
5
Plant Pathology 526
Advanced Fungal Biology
Spring 2014
6