MITOSIS AND MEIOSIS LAB Sordaria / Crossing-Over

Sordaria fimicola is 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. 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 below/right.  You can see the perithecia forming in the first picture of the Petri plate; they are the dark line down the center of the plate.  Perithecia  Black Tan Hyphae   Hyphae 2n “n” “n”

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 which would be air-carried to new locations where they would germinate and divide by mitosis to produce new hyphae.  Sordaria sp. Perithecium

ANALYSIS OF SINGLE MEIOSES meiosis & post-meiotic mitosis in linear tetrad / octad

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.

Four black ascospores in a row next to four tan ascospores in a row indicates that crossing over has NOT occurred. Any other arrangement of ascospores indicates that crossing over has taken place.

Below 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. 

Possible Arrangements of Ascospores Ascus (pl. asci)  Ascospore  4:4 2:2:2:2 2:4:2

Your task is to look at the asci in the following photos and to count the 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. 

Below 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. Photo 1

Below 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. Photo 2

Below 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.  Photo 3

Other Slides That Might Help

Orderd Tertrads and Gene Mapping in Fungi 6/8/2018 Establishing Map Distance Between the Centromere and a Linked Locus Genetic linkage maps are give distances between loci based on how often recombination occurs between linked genes. One map unit (mu or cM) is equal to the distance between loci that causes 1% of gametes to have a recombinant chromosome. G. Podgorski, Biol. 1610 Lab

Orderd Tertrads and Gene Mapping in Fungi 6/8/2018 Establishing Map Distance Between the Centromere and a Linked Locus An example: You observe 150 octads and discover 30 of these are recombinant. Map distance between the centromere and the locus (spore color locus in our case) = mu = (½ x 30) x 100 = 15 x 100 = 10 150 150 G. Podgorski, Biol. 1610 Lab

From the crossing over data you gather for Sordaria, you will be able to calculate the map distance between the gene for spore color and the centromere. To find the number of map units, you divide the percent of crossovers by 2. (In an actual lab, you should count at least 50 asci before calculating map units.)

Why divide by two? Each crossover produces two spores like the parents and two spores that are a result of the crossover. Thus, to determine the number of crossovers, you must divide the number of asci counted by two since only half the spores in each ascus result from crossing over.

Calculate the Map Units Calculate the percent of crossovers by dividing the number of crossover asci (2:2:2:2 or 2:4:2) by the total number of asci x 100. Calculate the map distance, divide the percent of crossover asci by 2. % of crossover asci is divided by 2 because only half of the spores in each ascus are the result of crossing over.