Lab 9 Sordaria XO frequency

Lab Topic 9:
Meiosis and Determining Cross Over Frequency

Meiosis in Ascaris eggs

Cross section of an Oviduct in Ascaris. Ascaris is a nematode worm that has a diploid number of chromosomes equal to 4. Meiosis is studied in the eggs of these worms. To the right is a crossection of a female's reproductive system. You can see the outer wall of the tube and the eggs in tne tube. These eggs are shown at higher magnifications in the following photos so that you can see meiotic stages.
Sperm (angular) and mature fertilized egg A photo of an egg and a sperm taken from a cross section of the oviduct at the upper end of the reproductive track. The sperm of Ascaris are not flagellated as they are in many other animal species. They are amoeboid. The sperm here appears triangular and stains darkly. The egg is starting to undergo meiosis I. Sperm must fuse with teh egg cytoplasm before the shell form because they can not penetrate the thick shell. When the eggs are shed by the female, it protects the developing embryo from dessiccation and physical damage.
Metaphase I of meiosis Sperm has penetrated the egg and its nucleus is the darkly staining structure in the center of the cytoplasm. An egg shell surrounds the egg cytoplasm. The egg is in metapase I of meiosis and the spindle forms off center. When the cell divides in telophase I one daughter cells will be very small and will become a polar body. The other will be large and udergo meiosis II. Note the four chromosomes in the egg cell; each consists of two chromatids which are clsely pressed together and can not be seen distinctly.
Metaphase II A cell in metaphase II of meiosis. Note the darl staining first polar body just inside the shell.
Just before sperm and egg nuclei fuse A great picture showing haploid sperm nucleus and haploid egg nucleus before they fuse (syngamy). Two polar bodies are visible under the egg shell as dark structures. After these nuclei fuse, the fertilized egg will divide by mitosis.
First mitosis in fertilized egg Mitosis in a fertilized egg. Chromosomes are aligned at center of cell in metaphase.
First two cells of new generation Two cell stage resulting from mitosis of fertilized egg. These cells will divide and so on to produce an embryonic worm.

Sordaria System

Sordaria fimicolais an ascomycete fungus, a division of fungi, that reproduce by producing haploid spores that are contained in an ascus. It is this property that makes it excellent material in which to study the results of crossing over. The lab manual (Biological Investigations, Dolphin, 2001) describes the the reasons for this and outlines chromsomal behavior during meiosis.

Life Cycle

Strains of Sordaria that produce black and tan spores can be purchased. The strains can be used to inoculate a Petri plate containing an agar. At first, the strain grows as a mycelial mat and then it produces fruiting (reproductive) bodies.

Hyphae growing from the points of the inoculation are seen in the magnified view of the agar surface to the right.

If a strain producing tan spores is inoculated on one half of the plate and a strain producing black spores is placed on the other half, hyphae grow from both points and eventually meet at the center of the plate where they fuse in the equivalent of mating. Since the hyphae of both strains are haploid, the fusion product is diploid. The diploid hyphae start to differentiate into a fruiting body called a perithecium as seen to the right. You can see the perithecia forming in first picture of the Petri plate; they are the dark line down the center of the plate.

Experimental Procedure

In the perithecium, diploid cells divide first by meiosis and then by mitosis to produce 8 haploid spores. The spores are contained in a translucent saclike structure called an ascus (pl. asci). To the right , you can see a ruptured mature perithecium releasing several asci. Normally the asci would break open and release haploid spores (seen below) which would be air-carried to new locations where they would germinate and divide by mitosis to produce new hyphae.

You can view the asci by taking a perithecium from a culture and placing it in a drop of water on a slide. If you gently press on a coverslip covering the drop, the perithecium will burst open and release the asci. Too much pressure and you will burst the asci as well, ruining the preparation.

Experimental Results

To the right is a photo of asci that resulted from a cross between two black strains. All of the spores are black.

To the right is a photo of asci that resulted from a cross between two tan strains. All of the spores are tan.

To the right is a photo of asci that resulted from a cross between black and tan strains. Look at a single ascus and note that it contains both black and tan spores. Those on which the pattern of spore distribution in the ascus is 4 tan to 4 black were produced from cells in which no crossing over occurred. Such asci are called non-recombinants. Other asci contain black and tan spores that are distributed in 2:4:2 patterns or 2:2:2:2 patterns. These asci only result from cells in which crossing over has occurred and are called recombinants. Because the recombinant patterns result only from crossing over, the frequency of occurrence of recombinants is a measure of how often crossing over occurs.

Your task is to look at the asci in the following photos and to count number of asci that show the recombinant and non-recombinant patterns. You should count 100 asci and then calculate the percentage of crossing over that occurs.

To the right are asci that have been expelled from a perithecium. View it as clock face and starting at 12 o'clock, count the asci that are the non-recombinant or recombinant type.

To the right are asci that have been expelled from a perithecium. View it as clock face and starting at 12 o'clock, count the asci that are the non-recombinant or recombinant type.

To the right are asci that have been expelled from a perithecium. View it as clock face and starting at 12 o'clock, count the asci that are the non-recombinant or recombinant type.

Analysis

Read the lab manual analysis section. You are to use a statistical Chi square test to determine if the cross over frequency you calculated matches that which has been reported in the scientific literature.

General and Comparative Essay Questions

1. Draw metaphase I and metaphase II as they would appear in a cell that had a diploid number of chromosomes equal to 6.
2. Can a haploid cell undergo meiosis? Can it divide by mitosis?
3. Why do you expect the diploid number of chromosomes always to be an even number and never an odd number?
4. How does crossing over contribute to gentic variability? Does this have any evolutionary significance?

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Credits
Photos by Maria Oehler and Warren Dolphin
Layout and text by W. D. Dolphin

To the right is a photo of asci that resulted from a cross between two black strains. All of the spores are black.
To the right is a photo of asci that resulted from a cross between two tan strains. All of the spores are tan.
To the right is a photo of asci that resulted from a cross between black and tan strains. Look at a single ascus and note that it contains both black and tan spores. Those on which the pattern of spore distribution in the ascus is 4 tan to 4 black were produced from cells in which no crossing over occurred. Such asci are called non-recombinants. Other asci contain black and tan spores that are distributed in 2:4:2 patterns or 2:2:2:2 patterns. These asci only result from cells in which crossing over has occurred and are called recombinants. Because the recombinant patterns result only from crossing over, the frequency of occurrence of recombinants is a measure of how often crossing over occurs. Your task is to look at the asci in the following photos and to count number of asci that show the recombinant and non-recombinant patterns. You should count 100 asci and then calculate the percentage of crossing over that occurs.

To the right are asci that have been expelled from a perithecium. View it as clock face and starting at 12 o'clock, count the asci that are the non-recombinant or recombinant type.
To the right are asci that have been expelled from a perithecium. View it as clock face and starting at 12 o'clock, count the asci that are the non-recombinant or recombinant type.
To the right are asci that have been expelled from a perithecium. View it as clock face and starting at 12 o'clock, count the asci that are the non-recombinant or recombinant type.