A Laboratory Exercise on the Genetics of Ascospore Color in Sordaria

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A LaboratoryExercise on the Genetics of Ascospore Color in Sordariafimicola

* Peggy Cassell and Thomas R. Mertens, Biology Department, Ball State University, Muncie, Indiana 47306

An exercise that has application to both secondary school and college instruction in helping with the understanding of the details of the genetic behavior of Sordaria.

introduction ic Continuity, p. 53), in that Sordaria is Soraaria fimicola has become a popular shown to reproduce by conidia, when actual- organism for use in the laboratory exercise ly it reproduces only by ascospores. on segregation of ascospore color mutants The present paper will describe and dis- described in this paper. One of the biological cuss in detail a laboratory exercise on the supply companies now maintains and sup- genetics of ascospore color in Sordaria fimi- plies the necessary cultures for this exercise cola. Although included in some laboratory and several laboratory manuals, such as manuals, as indicated above, a complete and Genetics Laboratory Exercises by Gardner detailed explanation of the exercise is not (1964) and Principles of Genetics by Hart- readily available to most biology teachers. man, Suskind and Wright (1965), include Sordaria is a suitable organism for this study this exercise. Perhaps the most widely known because most of its life cycle is spent in a account of the exercise is that given by Bent- haploid state and analysis of the ascospores ley Glass (1965) in the BSCS Laboratory which are produced by meiosis and main- Block, Genetic Continuity. Unfortunately, it tained in a linear order in the asci enables appears that Dr. Glass's account is marred the detection of the behavior of chromosomes by an error in the description of the life during meiosis. The exercise has been devel- cycle of Sordaria fimicola (See Glass: Genet- oped so as to utilize the simplest techniques

367 and the minimum amount of equipment several ascomycetesform over a wide range necessary to provide the opportunity for of pH values, but asci matureonly at neutral students to learn basic concepts of fungal or slightly alkaline reactions (Cochrane, genetics and to observe the segregation of a 1958). Specifically, Sordaria needs a near- single gene pair in Sordaria. maximummycelial growth and a pH of six for perithecial development and abundant Literature Review fruiting (Lilly and Barnett,1947). The pH is, Sordariafimicola belongs to the family Sor- however, a limiting factor only, while the dariaceae,which includes the widely studied amount of biotin present is the major con- genus Neurospora.In nature Sordarialeads trolling factor in perithecial production and a saprophytic life on dung or on decaying in the formation of mature asci (Lilly and plant material. Sordaria fimicola produces Barnett, 1947). Sordariafimicola requires an neither conidia nor microconidiaand repro- exogenous source of biotin for growth; duces solely by ascosporesborne in a linear studies have been made showing a direct Downloaded from http://online.ucpress.edu/abt/article-pdf/30/5/367/24399/4442092.pdf by guest on 26 September 2021 order of eight and containedin a tight-fitting correlationbetween available supply of bio- ascus (Alexopoulos, 1962). This gives it a tin and percentage of mature ascospores distinct advantage over Neurosporafor lab- produced (Barnett and Lilly, 1947). oratory investigations. Neurospora can re- Other studies on the physiology of growth produce asexually by vegetatively formed in ascomycetes, lead to the conclusion that conidia. These conidia are producedin great suppression, relative or absolute, of repro- abundance,germinate readily, and enable the ductive stages occurredat higher concentra- mold to spread widely through the air to tions of carbohydrates.The production of any available medium that will support its perithecia occurred sooner and more abun- growth. Since Sordaria does not reproduce dantly when the supply of carbohydrateswas by conidia, it cannot become the laboratory unfavorable for vegetative growth (Bretz- pest that Neurosporacan. loff, 1954). Sordariafimicola is homothallic, and tra- ditionallythe entire genus Sordariawas con- Materialsand Methods sideredto be sexuallyself-fertile. But in 1961 When doing this experiment,the medium the discovery of a new heterothallicspecies, to be used is Difco corn meal agar with dex- S. brevicollis, was reported and since that trose (19 grams/1000 ml of distilled water) time other new heterothallic species have to which is added 0.1%Difco yeast extract, been found (Fields and Maniotis,1963). The i.e., one gram of extractper liter of medium. details of perithecial development and how The agar and yeast extract are rehydrated plasmogamytakes place in Sordariahave not in distilled water and boiled to finish sus- been completely elucidated (Alexopoulos, pending the medium. The medium is then 1932). However, various species have been dispensed into test tubes and autoclaved at extensively studied and much is known con- 15 poundspressure and 121?Cfor 15 minutes cerning the physiology and genetics of this and stored in the refrigerator. ascomycete. The stock cultures of Sordariafimicola Dr. L. S. Olive first investigated Sordaria used in the present study were 58 with wild- ascosporecolor mutants, including the gray- type or black ascospores and 5A with gray spored strain, by inducing mutations in a ascospores obtained from Dr. Ralph Baker wild-type strainwith ultravioletlight (Olive, at Colorado State University, Fort Collins. 1956). Ascospore color is autonomouslyde- These strains,unlike most S. fimicola,are not terminedby the genotype of the spore itself, self-fertile. Test tube slants of the medium so segregationof alleles affectingspore color were inoculated with either strain 5A or 58, can be observed directly in the ascus (Fin- incubated for one or two days at 25?C, and cham and Day, 1963). then stored in a refrigerator.Stock cultures A prerequisitefor normalsexual reproduc- then remained viable for a few months or tion in Sordaria is the proper balance be- until the medium began to dehydrate, at tween the amount of biotin, the supply of which time it was necessary to transfer the nutrients, pH and possibly other factors stocks again. (Barnett and Lilly, 1947). The perithecia of Before inoculating a petri dish containing

368 The AmericanBiology Teacher, May 1968 sterile medium, a wax pencil was used to ascospore pattern were obtained (+ repre- mark the bottom of the dish into fourths, and the quadrants were labeled 5A or 58 in a manner that would place 5A and 58 in adja- cent quadrants. An arrowhead needle was flamed, cooled in 70%ethyl alcohol, and then flamed slightly to remove the excess alcohol. A small piece of agar approximately 3mm square containing mycelium was cut from the stock culture and transferred to the center of the appropriate section of the petri dish using sterile technique. The procedure IM was repeatedly until two pieces each of 58

and SA mycelium were placed in the labeled Downloaded from http://online.ucpress.edu/abt/article-pdf/30/5/367/24399/4442092.pdf by guest on 26 September 2021 quadrants of the petri dish. The dish was then placed in a darkened incubator at 25?C.

ExperimentalObservations 4 Following the cross of the two strains, protoperithecia were formed within three days. Within seven days perithecia contain-

.0::; ff 70 li; ing mature ascospores could be recognized ~~~~~~~~~~~~~~...... jf as black dots on the mycelium. The line of perithecia was formed in the area where the mycelia of the two strains grew together. The perithecia formed more closely around the 5A inoculum because of its slower, less vigorous growth in comparison to the 58 strain (Fig. 1). From approximately seven to twelve days after crossing or until the spores were forci- bly discharged from the asci, the perithecia contained mature ascospores from which data for first and second division segregation could be obtained. The wild-type ascospores passed through the following series of colors during maturation: hyaline, yellow, and finally dark brown or black. The gray mutant ascospores passed from hyaline to light gray to dark gray with all the pigment located in the cell wall. In order to obtain accurate data, it is essential that mature ascospores be counted. From the line of hybrid perithecia, wet mounts of mature perithecia were made The black with little (Fig. 2). perithecia, as Fig. 2.IWet ;:ountof a mature perithecium in the centet adhering agar as possible, were removed Fig. 3. Petri dish showing the crossed strains 58 and 5A from the plate with an arrowhead needle mendthe inthe ofhybrid perithecia, represented by the using sterile technique. The perithecia were black dots on the mycelia. Note that the perithecia formed more closely around the SA inoculum. placed in a drop of water on a clean slide. Fig. 2. Wet mount of a mature perithecium in the center Gentle pressure was then applied to the and a small, immature perithecium to the left. Magni. fication: 200X. coverslip with the eraser of a pencil to force Fig. 3. Wet mount showing the various ascospore arrange- the asci to be ejected from the perithecia ments in the asci wh:ch have been ejected from the (Fig. 3). The linear order of the ascospores perithecia. Note the various ascospore arrangements which are also shown diagrammatically in Fig. 4. was observed and the following data for Magnification: 200X.

A LaboratoryExercise on the Genetics of AscosporeColor 369 sents a wild-type ascospore; g represents a Map distance. Percent of crossing over or mutant gray ascospore). map distance between the gene and the centromere is estimated on the basis of the fre- Ascospore DistributionPattern in Sordaria Fimicolaat 250C quency of second division segregation. The First Division Segregation Number Counted frequency of second division segregation, 4+:4g 621 60.1%,was calculated using the data obtained Second Division Segregation from crosses incubated at 25?C. Second divi- 2+:2g:2+:2g 443 sion asci result from a single crossover be- 2+:4g:2+ 266 tween the g locus and the centromere. Since 2g:4+:2g 226 crossing over involves only two of the four chromatids, one half of the strands are there- Total 1556 fore recombinant and half are parental. The measure of the distance between the gene The percentage of second division segre- locus and the centromere is thus one half the gation was calculated from the above data Downloaded from http://online.ucpress.edu/abt/article-pdf/30/5/367/24399/4442092.pdf by guest on 26 September 2021 percentage of second division segregation or in the following manner: the total number 30.05 map units (one percent recombination of second division segregation asci, 935, was represents one map unit). divided by the total number of asci counted, 1556, and multiplied by 100 to yield 60.1% Sordariafimicola grows and fruits under a second division segregation. wide range of temperatures from 7-31?C. The percentage of second division segrega- Discussion tion varies with the temperature. Olive in- Ascospore patterns. The production of an vestigated this variation in crossing over at ascus containing eight ascospores from a various temperatures and obtained the fol- diploid zygote involves four nuclear divi- lowing frequencies of second division segre- sions. The first two divisions are meiotic and gation for the g locus: 7?C-42.9%; 13?C- produce four haploid nuclei. The third divi- 62.6%;23?C-68.1%; and 31?C-67.1% (Olive, sion is mitotic and results in the four haploid 1956). From the above data it was concluded nuclei giving rise to eight ascospore nuclei. that the g locus is approximately 33.3 cross- The fourth division is also mitotic and occurs over units from the centromere (Olive, 1956). after ascospore delimitation so that the mature spore is binucleate (Carr and Olive, Some petri dishes containing strains 58 and 1958). 5A were incubated at 31?C instead of 25?C. The first three nuclear divisions that pro- At the higher temperature, data for the fre- duce the ascus and the various possible asco- quency of the various ascospore patterns spore patterns of hybrid asci are depicted in were more difficult to obtain because many Fig. 4. When no crossing over occurs between ascospores did not mature and those that did from the gene and the centromere, segregation of mature were ejected the ascus within the alleles at the first meiotic division (i.e., approximately two days after maturation. when homologous centromeres segregate at Also, unusual or aberrant ascospore patterns 1 anaphase I) results in the sequence 4+:4g such as 3+: Ig: 1+:-3g, 2g: +: lg: 1+: lg:2+, or 4g:4+. When crossing over has occurred, 2+:3g:1+:lg:1+, etc. occurred with much 25C. The the other ascospore arrangements are ob- greater frequency at 31?C than at tained following second division segregation total number of second division segregation (i.e., when the centromere duplicates at asci, 140, was divided by the total number 100 anaphase II and the sister chromatids sep- of asci counted, 243, and multiplied by arate). The 2+:2g:2+:2g arrangement is to yield 57.6%second division segregation at the result of crossing over between chroma- 31?C. It would appear that 25?C was a much tids two and three (Fig. 4), while if chroma- more desirable temperature at which to incu- bate the crosses because of the greater abun- tids one and four are involved, a 2g:22+ :2g:2+ sequence results. The pattern of dance of mature ascospores formed and be- in 2+ :4g:2+ results from crossing over in- cause of the longer period of time which volving strands two and four while crossing to obtain data. over between chromatids one and three gives Variationsin the medium. In addition to the pattern 2g:4+:2g. the one gram of yeast extract per liter of

370 The American Biology Teacher, May 1968 medium,other concentrationsof extractwere units between the g locus and the centro- tried. At five grams of yeast extract per liter mere. The method of obtaining the various of medium,vegetative growthwas abundant, ascosporepatterns was discussed, and perti- but few perithecia were formed, and they nent literaturewas reviewed. contained no asci. With one-half of a gram Two factors are especially implicated in of yeast extractper liter of medium,mycelial the successful completion of this laboratory growthwas good, and many peritheciawhich exercise: (1) using the correct concentra- contained mature ascospores were formed. tions of nutrients and yeast extract in the The ascosporesalso matured slightly sooner medium for adequate sporulation and (2) than on the medium containing 0.1%yeast collecting data at the proper time between extract. ascospore maturationand ejection from the Summary ascus. A temperatureof approximately250C This paper describes and discusses a lab- and a concentration of one gram of yeast oratory exercise on the genetics of an asco- extract per liter of medium give the best spore color mutant in Sordaria fimicola. Two results in terms of the number of perithecia Downloaded from http://online.ucpress.edu/abt/article-pdf/30/5/367/24399/4442092.pdf by guest on 26 September 2021 strains of Sordaria,58 with wild-type asco- formed containing mature asci. Ascospore spores and 5A with gray ascospores, were arrangementdata are then obtainable from crossed, and the hybrid asci containing or- approximately7 to 12 days after crossingthe dered ascosporeswere analyzed.The number two strains. of first and second division segregationasco- spore arrangementswere determined, and Additional Related Exercises the percent of second division segregation Other exercisescan be suggested using the was used to calculate the number of map basic techniques described in this paper.

FIRST DIVISION SEGREGATION

Fl RST SECOND MITOTIC MEIOTIC MEIOTIC DJVISION DIVISION I DIVISION 4 0 4 2--:ZZZ4 -- zzoz4 --- 0--- 7

4--- _::Q 4 I 3----- __m-9------9- --4

SECOND DIVISION SEGREGAT ION

=, FIRST SECOND MITOTIC 2-G MEIOTIC MEIOTIC DIVISION ~ DIVISION DIVISION +

5 ---0-t~~~~~~~~~0-2

- 3 -0------~~~~~~~~~~~~~~~~~~-- ~ -0- -

4 ~ ~ -

5 --o------i-~~~~~~~0 --

I + ~ ~ ~ ~ ~ ~ ~ ~~~~~-+ ---O----q

3-- _j %__-0- _------

+ + 0~~~~+ jgzii

PATTERN Fig. 4. Illustration of the first three nuclear divisions thot produce the various possible arrongements of ascospores in the ascs flloingfist nd ecod iviionsegegaio. Se txt ordetoiled explanaton.

A Laboratory Exercise on the Genetics of Ascospore Color 371 Ascospores can be germinated on the medium all biology students-and their teachers. used in this exercise to which has been added seven grams of sodium acetate per liter. The Literature Cited sodium acetate stimulates spore germination Alexopoulos, Constantine J. 1962. Introductory my- without heat treatment. After the ascospores cology. 2nd Ed., John Wiley and Sons, Inc. New are allowed to germinate for 8 to 12 hours, York. Alexopoulos, ConstantineJ. and E. S. Beneke. 1962. observations of the growth of the germ tube Laboratory manual for introductory mycology. and its branches can be made during a two Burgess Publishing Co. Minneapolis. hour laboratory period (Alexopoulos and Bamett, H. L. and Virgil Greene Lilly. 1947. The Beneke, 1962). The chromosomes of Sordaria effects of biotin upon the formation and development of perithecia, asci, and ascospores by Sor- fimicola have been shown to be suitable for daria fimicola. American Journal of Botany 34: cytological studies (Carr and Olive, 1958) 196-204. and interesting laboratory exercises involving Bretzloff, Carl W. Jr. 1954. The growth and fruiting in could be de- of Sordaria fimicola. American Journal of Botany mitosis and meiosis Sordaria Downloaded from http://online.ucpress.edu/abt/article-pdf/30/5/367/24399/4442092.pdf by guest on 26 September 2021 veloped. Various physiological studies can be 41:58-67. Carr, A. J. H. and Lindsay S. Olive. 1958. Genetics made by varying the pH and the concentra- of Sordariafimicola II. Cytology. American Jour- tion of biotin and carbohydrates, and some of nal of Botany 45:142-150. the results would be readily visible by the Cochrane, Vincent W. 1958. Physiology of fungi. presence or absence of perithecia and asco- John Wiley and Sons, Inc. New York. spores. Fields, William G. and James Maniotis. 1963. Some cultural and genetic aspects of a new heterothallic The temperature at which crosses are in- Sordaria. American Journal of Botany 50:80-85. cubated can be varied in order to determine Fincham, J. R. S. and P. R. Day. 1963. Fungal genetics. 2nd Ed., F. A. Davis Co. Philadelphia. the effect upon crossing over and second Gardner, Eldon J. 1964. Genetics laboratory exer- division segregation. In the data obtained by cises. 4th Ed., Burgess Publishing Co., Minneapo- Olive (1956), the effect of temperature upon lis. the percent of second division asci produced Glass, Bentley. 1965. Genetic continuity. D. C. was slight except at 7?C, at which tempera- Heath and Company. Boston. Hartman, P. E., S. R. Suskind and J. R. F. Wright. ture data were obtained with some difficulty 1965. Principles of genetics laboratory manual. since the reproductive cycle required several Wm. C. Brown Co. Dubuque, Iowa. weeks in which to be completed. At this Lilly, Virgil Greene and H. S. Barnett. 1947. The temperature the frequency of crossing over influence of pH and certain growth factors on was markedly depressed. mycelial growth and perithecial formation by Sordaria fimicola. American Joumal of Botany From these comments it would appear that 34:131-138. Sordaria fimicola is sufficiently versatile to Olive, Lindsay S. 1956. Genetics of Sordaria fimi- provide a wide array of genetic, cytological, cola. I. Ascospore color mutants. American and physiological experiments to challenge Joumal of Botany 43:97-107.

Student Summer Program in Academic Director, Explorer Science Camp, Animal Physiology Berkshire School, Sheffield, Massachusetts Two summer programs will be offered in 01257. animal physiology this year at the Explorer Science Camp in Wingdale, New York. An BSCSSecond-Level Institute eight-week basic program will stress en- A summer institute to follow the BSCS docrinology in animal physiology, while a Second-Level course in molecular biology six-week advanced course will examine the will be held July 7-August 17 under the biochemical aspects of physiology. Both pro- direction of Dr. Gjerding Olsen, Brandeis grams are open to boys who have completed University, Waltham, Massachusetts 02154. high school biology and show promise of being able to work on individual research The NSF this year was ordered by Congress projects during the program session. not to cut funds for supplementary education Further information and application forms of high school science and mathematics may be obtained from: James V. Ekstrom, teachers.

372 The American Biology Teacher, May 1968