Materials Included In Kit

Additional Materials Required

Safety Precautions

This laboratory activity is considered nonhazardous.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period. The prelaboratory assignment may be completed before coming to lab, and the data analysis and calculations may be completed the day after the lab.
• For the purpose of this simulation, map units for this gene will vary from actual. Published sources cite between 26 and 28 map units as the distance between the tan mutant gene and the centromere.

Teacher Tips

• Use this activity as a substitute for the Advanced Placement^® Biology Laboratory 3B.2 or as an introduction to the laboratory itself.

Answers to Prelab Questions

1. In order to calculate the number of map units between the centromere and the gene at least 100 hybrid or heterozygous asci are counted. Why is it necessary to count so many asci?

   A representative number of asci from several perithecium must be counted in order to reduce the likelihood of skewed data.

2. The drawings in Figures 3a–c show only heterozygous asci. What would homozygous asci look like?
   {10976_PreLabAnswers_Figure_4}

Sample Data

Answers to Questions

1. Take the sum of the tally marks for each genotype. Record each result in the Total column.

   See data table for an idealized answer. Student answers will vary.

2. Determine the total number of noncrossover asci counted.

   Based on the sample data in the table the number is 74. Student answers will vary.

3. Determine the total number of crossover asci counted.

   Based on the sample data in the table the number is 26. Student answers will vary.

4. Determine the total number of hybrid asci counted.

   Based on the sample data in the table the number is 100. Student answers will vary.

5. Determine the map distance between the gene for spore color and the centromere using Equation 1. Report the result in map units. However, keep in mind that each ascus contains 8 spores because the four haploid spores underwent an additional mitotic event after meiosis. To account for this, the map distance found in Equation 1 needs to be halved (Equation 2).
   {10976_Answers_Equation_1}
   13/50 x 100 = 26 map units
   {10976_Answers_Equation_2}
   26/2 = 13 map units
6. Was the number of each type of crossover phenotype observed relatively constant or equal? Explain why you would expect these numbers to be constant.

   Yes, each crossover phenotype occurred in similar numbers. This is due to the random segregation of each allele during meiosis.

7. A similar technique can be used to determine the distance between two genes on a single chromosome. In this laboratory a color mutation was used as the gene of interest. What is the benefit for using a color mutant gene for learning about map units.

   The absence of color is easily observed using common laboratory equipment (the microscope). A change in size or shape would also be easily observed.

Teacher Handouts

10976_Teacher1.pdf

References

Biology: Lab Manual; College Entrance Examination Board: 2001.

Cassell, P., Mertens, T. R., A Laboratory Exercise on the Genetics of Ascospore Color in Sordaria fimicola; The American Biology Teacher, 1968, 30, 367–372. 

Olive, L. S. Genetics of Sordaria fimicola; American Journal of Botany, 1956, 43, 97–107. 

The Tree of Life Web project. http://tolweb.org/Ascomycota (accessed August 2010). 

Introduction

Ascomycota are a diverse group of fungi containing the familiar single-celled baker’s yeast, the complex morel mushroom, and the deadly Aspergillus flavus. In fact, 75% of all fungi are grouped as Ascomycota. Geneticists have altered one particular species of Ascomycota, Sordaria fimicola, for use in studying crossing over during meiosis.

Concepts

• Centromere
• Genetics
• Crossing over
• Meiosis

Background

Crossing over occurs during prometaphase I of meiosis. During crossing over, homologous pairs of chromosomes exchange sections of DNA that contain the same genes. Therefore, the exact genotype of the new offspring will vary from that of its parents (see Figure 1). It is important to note that crossing over does not have to occur during each generation, nor does it always take place at the same point of exchange. Over time, however, crossing over leads to a greater variety of genes in a population and contributes to a diversity of characteristics and an overall stronger population. This strength is then reflected in the ability of the population to adapt to changes in the environment and also to evolve.

Meiosis involves two cellular divisions, meiosis I and meiosis II. In meiosis I the chromosomes condense, replicate, crossover, and divide in two. In meiosis II, the chromosomes do not replicate again. Instead each chromosome is split in half through the centromere leaving one copy of each gene in each haploid cell. In the fungi kingdom, meiosis occurs in specialized fruiting bodies. In the group Ascomycota this specialized fruiting body is called an ascocarp or perithecium. The frequency of crossing over is studied genetics because it allows scientists to map genes and estimate the distance between two genes or between a gene and the centromere of the chromosome. The daughter cells are called ascospores or, in more general terms, spores. The daughter cells are all contained within a single tube-like structure called an ascus (plural = asci). The structure and properties of the ascus make Sordaria fimicola useful for studying crossing over.

Many Ascomycota, like S. fimicola, spend most of their time as haploid cells. Numerous clone copies of each haploid cell unite to form thread-like hyphae. Small holes between cell walls allow the sharing of nutrients and water between the cells of each hypha. Masses of hyphae intertwine to form mats of fungi. One of the reasons that fungi spread so easily is that these haploid hyphae are able to break off and generate a new organism anywhere nutrients are available. S. fimicola grows on rotting vegetation or dung in the wild making it a common mold in the environment. If no nutrients are available, the fungus is able to form haploid spores using asexual reproduction or sexual reproduction. The spores can be dispersed in the wind or settle into the soil until conditions improve. In the lab, S. fimicola is easily cultured on agar plates.

During sexual reproduction hyphae of different haploid S. fimicola come into contact allowing cells in the hyphae to fuse and form a single cell with two nuclei, one from each individual. This fused cell is called a dikaryon. The dikaryon is not considered diploid since the two nuclei are from separate fungi and the nuclei are not fused together. The dikaryon cells undergo multiple rounds of mitosis to form a mass of cells. This mass of cells can exist for years without undergoing fusion of the nuclei. Sexual reproduction occurs when some of the dikaryon nuclei fuse. After fusion the fruiting body forms and meiosis occurs, creating the asci and ascospores of the next set of haploid cells.

The ascospores form inside the tight confines of the tube-like asci. The ascospores actually line up in order based upon which cell produced that particular ascospore. In 1956, a geneticist named Lindsay S. Olive (1917–1988) published an article about crossing over in S. fimicola. Dr. Olive used ultraviolet light to cause mutations in the genes of S. fimicola. After numerous trials Dr. Olive produced a mutation in the gene that produces the pigment in the ascospore. The production of the black pigment is either greatly reduced or completely repressed in the mutated strain of S. fimicola. A reduction in the amount of black pigment results in gray spores. An absence of black pigment results in tan ascospores. By collecting the gray or tan ascospores Dr. Olive was able to produce true breeding fungi much like Mendel’s peas.

Collecting the ascospores is easy because the fruiting body produced by S. fimicola is shaped like a vase (see Figure 2). The vase-shaped perithecium is produced on a dikaryon stalk above the dikaryon mass of cells. Within the perithecium each ascus lines up with the top opening of the perithecium. The ascospores are ejected out of the opening into the wind for dispersal. Wet-mount microscope slide preparations of the perithecium result in the asci spreading out like spokes on a wheel, lining up for analysis. The distance between the centromere and the gene that codes for the black pigment can be determined by counting the ascospores within a population of asci. This distance is called the map distance and is reported or measured in terms of map units. A map unit is an arbitrary unit of measurement where one map unit corresponds to 1% crossover. The likelihood of crossover occurring between two genes on the same chromosome increases as the distance between the genes increases. Similarly, a gene is more likely to crossover if the gene is not adjacent to the centromere of the chromosome. By definition, the number of map units between two genes or between the gene and the centromere is equivalent to the percent of genes that undergo crossover. In order to count the number of crossing over events a culture of wild type (black) S. fimicola and a culture of tan mutant S. fimicola are grown adjacent to each other in a culture dish. Recall that each ascospore can be tracked back to the parent chromosome. The pattern of black and tan ascospores shows whether crossing over occurred during meiosis. Look at Figure 3. Note that the diagram of the asci indicates eight ascospores in each ascus, not the expected four cells. With S. fimicola each of the four haploid daughter cells undergoes a single mitosis after the end of meiosis II. So each daughter cell produces a clone of itself. These clones reside next to each other within the ascus. If the cells come from parents with identical pigment genes the ascus will contain eight spores that are the same color whether black or tan. If the cells come from parents with each pigment type but crossing over did not occur the spores will appear as four black wild-type and four tan mutant spores (4b:4t) (see Figure 3a). If crossing over between a black wild-type and a tan mutant occurred during meiosis I the four spores will have one of two possible patterns. Patterns of 2:2:2:2 and 2:4:2 are possible. Each of the numbers can be either tan or black. This is written out as 2b:2t:2b:2t or 2t:2b:2t:2b (see Figure 3b) and 2b:4t:2b or 2t:4b:2t (see Figure 3c).

Experiment Overview

The purpose of this genetics simulation activity is to identify the phenotype for each ascospore within a hybrid ascus and to count the number of asci corresponding to each type of crossover and noncrossover genotype.

Materials

Prelab Questions

1. In order to calculate the number of map units between the centromere and the gene, at least 100 heterozygous asci are counted. Why is it necessary to count so many asci?
2. The drawings in Figures 3a–c show only heterozygous asci. What would homozygous asci look like?

Safety Precautions

Although this activity is considered nonhazardous, please follow all laboratory safety guidelines.

Procedure

1. Begin with one of the five Sordaria cards assigned. Categorize each heterozygous ascus beginning at the 12 o’clock position. Note: Do not categorize any homozygous asci. Homozygous asci are not the result of sexual reproduction between a tan mutant and a black wild type S. fimicola and will not be used to calculate the frequency of crossing over or the map distance.
2. Place the tally mark on the Counting Crossing Over Worksheet corresponding to the correct genotype for each ascus. The genotypes may reflect both crossover and noncrossover asci. Note: Refer to Figures 3a–c for schematics of possible hybrid asci.
3. Categorize all of the hybrid asci on the first card.
4. Repeat steps 1–3 with the four remaining cards.
5. Complete the calculations and data analysis on the Counting Crossing Over Worksheet.

Student Worksheet PDF

10976_Student1.pdf