Fungi are considered an ideal model organism as a result of their easily

manipulatable nature and their eukaroytic properties, including genetic recombination as

a result of meiosis and fusion of gametes, as opposed to partial, unidirectional transfers
seen in prokaryotes. The fungi, especially ascomycetes that produce isolated, ordered
tetrads in ascii spores have been fundamentally important to the teaching of genetics.1
Microscopic studies originally done with Drosophila melanogaster showed that the
exchange of genetic information, called crossing over, takes place during prophase I
when dichromatid homologous chromosomes matched in synapsis. During crossing over,
breakage points called chiasmata develop between synapsed chromosomes. These
chiasmata result from pieces of the two homologues being switched, usually in an equal
and reciprocal fashion. Crossing over combines genetic material that had previously been
on separate homologues and produces progeny with increased genetic variation as a result
of the combination of genetic material from two parents. Geneticists also realized that the
visualization of crossing over could be used as an important tool for learning more about
the distance between genes on chromosomes. They reasoned that if chiasmata can form at
any point between two homologous chromosomes, then the frequency of crossing over in
the region between two different genes on a chromosome should vary directly with the
physical distance between the genes. The higher the frequency of crossing over, the larger
the distance between the genes in question.
In certain fungi, such as the fecal resident Sordaria fimicola, meiosis occurs
within a reproductive structure called an ascus which isolates each tetrad, the four
products of mitosis. In tetrad analysis, the genetic make-up of each cell of a tetrad can be
studied as a result of observable phenotypes, and this information can be related to the
meiotic division that produced the tetrad.
Tetrad analysis makes it possible to
determine when crossing over has
occurred, and to use this information to
determine if genes are linked and to map
the locations of genes on chromosomes.
First, plasmogamy occurs in which fusion
of the contents of two cells forms one cell
with two haploid nuclei. Second, in
karyogany there is a fusion of the haploid
nuclei in which a zygote with a diploid
nucleus is formed. In most organisms,
karyogamy immediately follows
plasmogamy. In the fungi, dikaryotic cells
remain for extended periods of time, with
karyogamy occuring at seperate distant
period of time.2 In Sordaria fimicola
(Figure 1.1), the multicellular fungal body
1. Mertens, Thomas R. and Cassell, Peggy. “A Laboratory Exercise on the Genetics of Ascospore Color in Sordaria fimicola.” The
American Biology Teacher Vol. 30.No. 5 (1968): 367-372. Web.
is composed of haploid cells arranged in long filaments called hyphae, the whole of
which is called a mycelium. If conditions are right, the fungus forms sex organs
containing numerous haploid nuclei. Each zygote immediately undergoes meiosis,
producing four haploid nuclei which each divide once, mitotically, yielding a total of
eight haploid cells. Each cell develops a resistant cell wall and is called an ascospore. All
of these events occur within an elongate sac called an ascus, so that at maturity an ascus
contains eight ascospores arranged in a precise manner. Clusters of asci develop within a
special spore dispersal structure called a perithecium, which ruptures as a result of
hydrostatic force, shooting the ascii in a phototrophic manner. Karyogamy occurs within
an ascus primordium to form a diploid zygote.
The spore color of wild type Sordaria is black, a phenotype derived from the
production of the pigment melanin and its deposition in the cell walls. Several different
genes are involved in the control of the melanin biosynthetic pathway and each gene has
two possible allelic forms. The gray spore gene has two allelic forms: the wild type allele
(g+) and a mutant allele (g). The tan spore gene also has two forms: a wild type allele (t+)
and a mutant allele (t). Normal black spores are produced only if both wild type alleles
are present at the loci of both genes. Thus, black ascospores have the genotype g+t+.
Those with the genotype g t+ are gray, while g+ t ascospores are tan. Ascospores that are
g t show a cumulative effect of the two mutations and are colorless.
Two petri dishes were obtained containing cornmeal-glucose agar supplemented
with 0.1% yeast extract. With a marking pen, the bottoms of the petri dishes were divided
into fourths. Each quadrant was labeled with a g+t or g t+. For aseptic technique, an
inoculating loop was flamed with an alcohol burner. The lid of one of the mutant stock
culture plates was lifted slightly and the loop was used to cut a small piece of agar
containing abundant mycelial growth. This inoculum was transferred to the center of the
appropriate quadrant of one of the petri plates. The plates were placed in an incubator at
25°C. The perithecia that result from these genetic crosses are listed as being mature after
8 to ten days, while checked seven days later the level of mitotic events was considered
insufficient therefore the dishes were incubated for another week. After incubation, 10 to
15 perithecia were removed by scraping the loop’s tip back and forth over the surface of
the agar and placed into the drop on the slide and uniformly distributed. A step vital to
success incuded the gentle push on the cover slip to induce perithicia ejection of ascii.
The segregation of alleles from the two parents occurs during anaphase I of
meiosis. If crossing over occurs, however, the alleles rearranged by the crossover are not
segregated until anaphase II of meiosis, thus itt is said that crossing over leads to second
division segregation of the alleles involved in the crossover. Gene mapping became
possible when it was realized that the frequency of second division segregation was
related to the physical distance separating the genes involved. In Figure 1.2, notice that
only four of the eight ascospores are genetically changed due to crossover; the two tan
spores with copies of chromatid 1 and the two gray spores with copies of chromatid 3.
These spores are called recombinant ascospores because they contain chromosomes that
have been changed by the crossover, and are now a combination of the parental
chromosomes. The other four spores that bear chromosomes not affected by the crossover
and are still like the parental chromosomes, are called non-recombinant ascospores.
2. Glase, J. C. A study of gene linkage and mapping using tetrad analysis in the fungus Sordaria fimicola. Tested studies for laboratory
teaching, Volume 16 (C. A. Goldman, Editor). 1–24.Proceedings of the 16th Workshop/Conference of the Association for Biology
Laboratory Education (ABLE), 273 pages. 1995.
Sordaria Mapping Data:
Grey: g t+
Parental Ditype 4:4 246
Tetratype 2:2:2:2 238
Tetratype 2:4:2 210
TOTAL: 694
(238+210)/694*100=64.55 map units

Tan: g+ t
Parental Ditype 4:4 190
Tetratype 2:2:2:2 235
Tetratype 2:4:2 160
TOTAL: 585
(160+235)/585*100= 67.52 map units

Ascii Tan: g+ t Grey: g t+

Observed Crossover 395 448
Observed Non-crossover 190 246
Expected Crossover 0.54*585=315.9 0.66*694=458.04
Expected Non-crossover 0.46*585=269.1 0.34*694=235.96
(395-315)+(190-269.1)+(448-458.04)+(246-235.96)=0=x2

Published gene-centromere map locations for the two genes are approximately 27
map units for the tan spore gene and 33 map units for the gray spore gene.3 If the distance
between the tan locus and its centromere is 27 map units, then the frequency of meiotic
divisions with a single crossover between the tan locus and its centromere is equal to
0.54. Therefore, the frequency of meiotic divisions with no crossover for that gene will be
0.46 (1.00 – 0.54). If the distance between the gray locus and its centromere is 33 map
units, then the frequency of meiotic divisions with a single crossover between the gray
locus and its centromere is equal to 0.66.
The increase of crossover tan with the decrease of crossover grey from expected
quantities shows that while the map units derived from the data shows a lack of linkage
between the genes, with the grey gene appearing to have a distance of 64 map units from
the centromere and the tan gene appearing 67 map units from the centromere, the
difference between the distances of these two genes is similar to the map units observed
by L.S. Olive in 1956. Through chi-square analysis, it has been determined that the
observed quantity of crossover events is in accordance with the quantity obtained through
data analysis with previously stated gene distance.

3. Olive, L. S. Genetics of Sordaria fimicola. I. Ascospore color mutants. American Journal of Botany, 43:97–107. 1956.