Mapping the location of genes on a chromosome can be accomplished with a diploid organism by following the percentage of crossover events much like the problems we have done in class.  But what happens when you have a haploid organism like fungi?  In this case, there are no "dominant" or "recessive" traits (since the organism is haploid).  However, the events of recombination and crossing over can be observed by looking at the spore patterns in the ascus (spore case of an Ascomycetes).  This process is called tetrad analysis since the outcome of meiosis in Ascomycetes results in a linear tetrad of haploid spores.  By analyzing spore patterns one can observe crossover events that occurred during meiosis when an allelic marker is located on each chromatid of a synapsed tetrad.  Such an analysis reveals two important pieces of information about the crossover event.  First, one can determine which two of the four chromatids participated in the cross over event.  Secondly, the gene can be mapped relative to an observable cytological marker such as the centromere.  By determining the location of several genes, one could eventually determine linkage groups and chromosome locations for all genes.

Sordaria fimicola is a common species of ascomycete found on dung.  Sordaria is haploid and spends most of its life cycle in the vegetative state.  Under favorable environmental conditions, different matting types of  Sordaria can undergo sexual reproduction. This occurs by the creation of the a binucleate hypha, and the eventual fusion of the two nuclei within a developing ascus fuse to produce a diploid (2n) zygote.  This zygote then undergoes meiosis to form a linear array of haploid ascospores contained in  the ascus (pl., asci).  In the case of Sordaria the meiotic division is followed by a mitotic division to produce eight ascospores (Figure 1).  The asci (about 20) are grouped together within a structure called the perithecium. It is the dark brown perithecium on the agar plate that you can observe with the naked eye.
 
When you observe the Sordaria in this lab, you will note that the ascospores are of two different colors. The one most often found in nature is called the wild type (+) and produces a dark spore.  The mutant form of this gene called "tan" (t) produces a light spore.  By observing the order of the ascospores in the ascus one can determine the order in which the chromosomes are segregated (separated) during meiosis.  If no crossover events occur, the two genes will segregate during meiosis I and produced a 4:4 arrangement of ascospores.  If a  crossover event does occur, the two genes will not segregate until meiosis II which will result in a 2:2:2:2 or 2:4:2 sequence of ascospores (Figure 2).
  

PROCEDURE:
Making the Cross:
Each student should obtain a starch agar petri plate from the instructor.  This plate will be designated the cross plate, and will be used to cross the wild type (dark) ascospore Sordaria with the mutant type (tan) ascospore Sordaria.  On the bottom of the cross plate write your initials.  With the cross plate upside down, center the petri plate over Figure 3 below.  Mark the "+" and "t" on the bottom of the cross plate.  Now turn the plate right side up.  There are two stock plates of Sordaria that will be circulating round the lab room.  One is the wild type (dark) Sordaria and one is the  mutant type (tan) Sordaria.  When the plates come to you, slightly lift the cover of the stock petri plate and, using a sterile toothpick, transfer a block of the fungi culture to your starch plate.  Place the block upside down over the "+" or "t" marks, depending on which culture you are using.  You will need two blocks of each type of Sordaria culture.

Leave the cross plate in the drawer indicated by the instructor.  It will take 8 to 10 days for the perithecia to mature.
 
Counting Hybrid Asci:
During the laboratory period when the Sordaria  cultures have matured, you will observe the asci under the compound microscope.   Obtain a microscope slide.  Place one drop of water in the center of the microscope slide.  Obtain the Sordaria cross plate that you set-up about a week and a half ago.  Using a toothpick, scrape across the top of the agar gently to remove several perithecia from the areas designated by arrows indicated in Figure 4.

Atypical Segregation Ratios and Gene Conversion:  As you are working with the asci you will occasionally observe unusual tetrad arrangements.  The possible arrangements are  3:1:1:3, 5:3, and 6:2 which are actually aberrant 4:4 ascospore patterns.  Changes of ascospore genes from wild type to mutant or mutant to wild type during meiosis can account for these ascospore patterns. But, the  frequency of these type changes are much too great to be accounted for by spontaneous mutation.  Instead, the genes in these asci are undergoing gene conversion.   The asci having 3:1:1:3 or 5:3 ascospore patterns contain two ascospores derived from a "hybrid" DNA chromatid.  Remember that DNA is a complementary double stranded macromolecule.  A hybrid DNA has strands that are noncomplementary in their nucleotide sequence for the gene in question (i.e. they do not have the same sequence).  Thus when the DNA strands segregate in Mitosis, the resulting two ascospores end up with different sequences in the genes affected.  The asci ratio 6:2 results from a complete chromatid change, which could have resulted from a DNA repair system.  Studies of aberrant ascospore patterns and the processes involved in gene conversion have provided researchers with important information on the molecular mechanisms of recombination.  Read the section in your lecture textbook about the mechanism of gene recombination and the Holliday model.

Mechanism of Crossing Over:  Two theories have been proposed to describe the involvement of chiasmata in crossing over.  The classical theory maintains that the chiasmata cause physical strains on the chromosomes and that the location and presence of the chiasmata area completely random.  A chiasmata may or may not induce breakage and rejoining at the cross over point.  This theory states that the chiasmata are responsible for crossover events and clearly precede them.  Since chiasmata appear during the diplotene stage of meiosis I, the classical theory holds that the actual crossover event occurs after diplotene but before the chromosomes separate at anaphase I.  The chiasmatype theory predicts that crossing over precedes chiasma formation and occurs in pachytene.  As a result, in this theory the chiasmata are formed at points of genetic exchange.  Therefore, the chiasmata seen in the diplotene stage are evidence of crossover events.  Some supporting evidence for the chiasmatype theory comes from the finding that DNA synthesis occurs in the zygotene stage of meiotic prophase I.  The actual role of this late-replicating DNA is not known.  However, it amounts to 0.3% of the total nuclear DNA and it is distributed randomly among chromosomes.  If this late DNA synthesis is inhibited, chromosomal synapsis is also inhibited and the process of  meiosis is stopped.  This suggests that this  DNA synthesis is somehow important for chromosome alignment during meiosis.

1. How many different arrangements did you find? __________________
2. What were the different arrangements?  To answer this question list the black spores as + and the tan (mutant) spores as t.  For example, no crossing over would be recorded as ++++tttt.
3. Which allele is dominant:  the + allele or the t allele?  Explain.
4. How do you explain the perithecia in which the asci contained either eight black or eight tan ascospores?
5. Draw a schematic map of the telemeric chromosome with the location of the gene.
6. What is the definition of a "map unit"?
7. How does this procedure, using the haploid Sordaria, differ from the mapping exercises we have done with genes on Drosophila chromosomes?
8. What does the aberrant ratio 3:1:1:3 (aberrant 4:4) represent?  If you encountered this arrangement, what frequency did you observe?  If you did not encounter this ratio, what do you know about its frequency?
9. Using drawings like to show how the 3:1:1:3 arrangement develops in the ascus.
10. How might you account for such ratios as 5:3 or 3:5?  Again diagram the possible crossing pattern that would give rise to these arrangements.
11. How might you account for such ratios as 6:2 or 2:6? ?  Again diagram the possible crossing pattern that would give rise to these arrangements.
12. Were any other ascospore arrangements observed other than the ones previously mentioned?  If so, how might these be explained?
13. By following the incorporation of tritiated thymidine, it has been demonstrated that a small amount of DNA synthesis occurs during pachynema.  What function might this DNA synthesis serve?  Relate this to what happens in the Holliday (or Chi) structure.