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The Fungal Mating Game: A Simple Demonstration of the Genetic Regulation of Fungal Mating Compatibility

Vaillancourt, L.J. 2018. The Fungal Mating Game: A Simple Demonstration of the Genetic Regulation of Fungal Mating Compatibility. *The Plant Health Instructor*. DOI: 10.1094/PHI-T-2018-1001-01

### Bipolar Mating (Ascomycete model; *S. cerevisiae* and *N. crassa*).

### Tetrapolar Mating with two loci and multiple alleles (*U. maydis* model).

### Tetrapolar Mating with two pairs of linked loci and multiple alleles (*S. commune* model).

### RESULTS

### REFERENCES

**Lisa J. Vaillancourt**University of Kentucky, Lexington KY

vaillan@uky.edu

Some fungi have more than 20,000 “sexes” (Raper and Fowler, 2004). The genetic controls that underlie this extreme promiscuity have relevance to mechanisms of fungal development, evolution, and population genetics, and they provide an excellent model for other systems regulating self-compatibility, e.g. antigen recognition systems. I include an overview of the genetics of fungal compatibility systems as one topic in my Fungal Biology graduate class. In particular, I cover the mating behaviors of four model fungi: *Saccharomyces cerevisiae* and *Neurospora crassa* (both bipolar systems); and *Ustilago maydis* and *Schizophyllum commune* (both tetrapolar). These models illustrate the range of different compatibility systems in the Dikarya, and are reviewed in detail in (Brown and Casselton 2001; Dyer et al., 2016; Fraser and Heitman, 2003).

The genetics of mating compatibility are very complex, and can be difficult for the beginning mycology student to visualize or appreciate. This card game was developed as a simple and fun way to illustrate the regulation of mating compatibility in bipolar and tetrapolar systems, and also to demonstrate the effect on inbreeding and outcrossing potential of these various mating systems. Student feedback has indicated that the game helps to clarify the concepts in an enjoyable way. The game can be adapted for larger or smaller classes: in larger classes it can be played competitively in teams, and in smaller classes the game can be played individually and without competition. The only materials needed are several decks of standard playing cards, two for each participant. Jokers should be removed from the decks prior to beginning the exercise. An automated card shuffler will also be very helpful.

Give each student two decks of playing cards, and have them shuffle the decks together thoroughly. It is very important that the cards be well-shuffled. Instruct each student to draw one card from their deck. This card represents the mating type (MAT) genotype of a single individual. Red and black suits symbolize the two MAT alleles (aka. idiomorphs), and are compatible with one another. The students should continue to draw cards, one at a time, from their decks until they can make a compatible pair with the first card drawn. Incompatible cards should be returned to the stack (sampling with replacement).

Ask the student to count how many additional draws it takes until they get a card that is compatible with the first card drawn. Each student should play ten or more rounds of the game and keep track of the number of draws each time. After the additional rounds have been played, the instructor should poll the class for their results and combine them on the board, to come up with an average.

Evaluation of inbreeding potential: Ask the students to predict the progeny genotypes that would result from crossing the compatible pair of cards. The students should then calculate the inbreeding potential, ie. the proportion of the siblings resulting from the cross that can mate with one another.

Ask the students to remove all the face cards from the combined deck, leaving just the ace to nine of the four suits. They should combine the face cards into a second stack, and shuffle both stacks very thoroughly. The face card stack represents the “A” locus, with Red and Black as the two A idiomorphs. The number card stack is the “B” locus, which has nine idiomorphs represented by the ace through nine. A compatible mating will occur with a Red versus Black card from the A stack, AND any two different idiomorphs (number cards of any suit) from the B.

The students should simultaneously draw one card from the A stack and one from the B stack. This pair of cards represents the A and B MAT genotypes of a single individual. The students should then draw additional pairs of cards from the two stacks simultaneously, until they can make a compatible group of four cards, representing two individuals. Incompatible cards should be replaced in the decks.

Again, the students should count how many additional draws it takes until they can make a compatible mating. The students should play several rounds and then share their data so that the instructor can calculate an average.

Ask the students to determine the progeny types that will result if their compatible individuals (each represented by one pair of cards) are crossed. Remind the students that the A and B loci are unlinked and thus can recombine freely. The students should then calculate the inbreeding potential, ie. the proportion of the siblings resulting from the cross that can mate with one another.

The students should now be instructed to divide the number card stack into four stacks according to suit. Each of the four stacks should be shuffled very thoroughly. These will represent the four MAT loci, A alpha and A beta (Hearts and Diamonds) and B alpha, and B beta (Spades and Clubs). The different numbers (ace to nine) represent the different specificities at each locus. The students should draw one card simultaneously from each of the four stacks. The student should then draw additional groups of four cards, one drawn simultaneously from each of the four stacks, until they obtain a compatible group of eight cards. Compatible matings must differ in at least ONE red suit (A locus) and at least ONE black suit (B locus). Different at BOTH the red or black suits is also OK, because the alpha and beta loci are redundant.

Once again the student should count how many additional draws it takes until they can make a compatible mating of two individuals, represented by the eight cards. After playing several rounds, they should share their data with the instructor who can calculate an average on the board to compare with the averages generated during the previous games.

Ask the students to determine the progeny types that will result from the cross among the two genotypes represented by the eight cards. Remind the students that the A and B loci are unlinked, but that the alpha and beta loci are closely linked in each case and are unlikely to recombine. The students should then calculate the inbreeding potential, ie. the proportion of the siblings resulting from the cross that can mate with one another.

The results of the game should demonstrate the following. The inbreeding potential for the bipolar cross is 50%: each progeny is compatible with ½ of its siblings. For the two tetrapolar crosses, the inbreeding potential is only 25%, because each progeny is compatible with only ¼ of its siblings. After playing the game over several rounds, the students should discover that the bipolar cross and the tetrapolar cross with two A loci (*U. maydis*) average to close to 2 additional draws to obtain compatible matches with the first card: these systems generate ~50% outbreeding potential, so every draw has a 50% probability of producing a compatible pair. In *U. maydis* the second locus must also differ, which decreases the inbreeding potential. However since there are multiple (nine) specificities, it has a relatively small effect on the outbreeding potential. The first locus, with only two specificities, is the limiting factor. In *S. commune* they will find that the average will be close to 1, because the outbreeding potential is near 100%.

COMPETITIVE VERSIONS of the games for larger classes: Divide the class into pairs, and ask each pair to play against one another by drawing cards in turns, after deciding who goes first by drawing the high card. The first one to match their cards into a compatible mating will win the game. If the class is very large the students can compete in teams of two to four.

Brown, A. J., & Casselton, L. A. (2001). Mating in mushrooms: increasing the chances but prolonging the affair. *TRENDS in Genetics*, *17*(7), 393-400.

Dyer, P. S., Inderbitzin, P., & Debuchy, R. (2016). 14 Mating-Type Structure, Function, Regulation and Evolution in the Pezizomycotina. In *Growth, differentiation and sexuality* (pp. 351-385). Springer, Cham.

Fraser, J. A., & Heitman, J. (2003). Fungal mating-type loci. *Current Biology*, *13*(20), R792-R795.

Raper, C. A., & Fowler, T. J. (2004). Why Study Schizophyllum?. *Fungal Genetics Reports*, *51*(1), 30-36.