Genetics of Sordaria fimicola. I. Ascospore Color Mutants
Author(s): Lindsay S. Olive
Source: American Journal of Botany, Vol. 43, No. 2 (Feb., 1956), pp. 97-107
Published by: Botanical Society of America
Stable URL: http://www.jstor.org/stable/2438817
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GENETICSOF SORDARIA FIMICOLA. I. ASCOSPORECOLORMUTANTS'
LindsayS. Olive2
Nineteen different cultures of S. fimicola were
obtained from various sources for the present study
(see table 1). Three of these cultures were the
sources of the three ascospore color mutants which
are the main subject of this report. All three mutant strains were obtained by subjecting Petri-dish
cultures to ultraviolet irradiation before perithecial
production. The first two (g and y) were found in
heterozvgous asci that appeared in a few perithecia
of irradiated cultures. The third (hy) appeared in
homozygous asci in a single perithecium. All
three appear to be the result of single-gene mutations which have no effect on the self-fertility of
the cultures. Each mutant strain may be readilv
crossed with the original wild-type culture from
which it was derived.
Crossing of cultures may be effected in several
ways. If a mutant and a wild-type culture are
placed on an agar plate a short distance apart they
may form where they come together a distinct line
of perithecia (fig. 1), some of which are hybrid
ones. Frequently such cultures inhibit each other
and do not form perithecial lines, in which case
they may cross better if started out as adjacent
inocula in plates or tubes. Heterokaryosisis often
involved and may have a direct bearing on the extent of hybridization.
Before any extensive crossing experiments were
attempted, single-spore cultures of all nineteen isolates were obtained. This was necessary for consistent results, since any of the original cultures
MATERIALS AND METHODS.-The culture medium
could have been, as some were actually found to
used throughout these experiments, except where be, heterokaryotic for various physiologic factors
otherwise indicated, was Difco corn meal agar including degree of fertility. These nineteen singleplus 0.1 per cent yeast extract. The minimal me- spore cultures will hereafter be referred to as
dium referred to later is the unbuffered medium strains.
of Westergaard and Mitchell (1947), only slightly
RESULTS. The nineteen strains are very similar
modified to contain 1 per cent sucrose and 8/,[g of morphologically. Some differences in spore meabiotin per 1. The initial pH of this medium is surements are found (table 1), and it is possible to
about 4.2.
detect microscopically that a strain such as Cl has
Bretzloff (1951, 1954) found that the ascospores slightly stubbier spores than the others. The overof S. fimicola (our Al strain), which ordinarily all spore measurements for our strains are 10.2germinate very poorly in the laboratory, will give 14.6 X 15.2-24.2 p- Probably the most closely
at least 60 per cent germination in solutions con- related species is So,rdaria humana, which has
taining 0.5-1.0 per cent sodium acetate. The spores that measure 16-19 X 22-28/A (Cain and
present writer finds that 95 per cent or more of the Groves, 1948). Sordaria macrosp!ora has still
spores will germinate on plain agar containing 0.7 larger spores. All attemptsto cross S. fimicola with
per cent sodium acetate.
these two species failed.
Table 1 shows that seven strains fruited abun1 Received for publication July 15, 1955.
dantly on minimal medium, the other twelve grow2 The author is grateful to Dr. Roy F. Cain and Dr. J. B.
ing on the medium, some at a reduced rate, without
Routien for most of the cultures used in this study and to
Dr. Seymour Hutner of the Haskins Laboratory and Miss
producing perithecia. The latter fruit well on miniJane Rhein for assistance with certain of the nutritional
mal medium to which thiamin has been added (2
studies.
mg./l). Thus the existence of distinct biotypes in
3 Heslot, H. 1953. Contribution a la gen6tique de
nature is indicated. Lilly and Barnett (1947), in a
l'ascomycete homothallique Sordaria macrospora (Auersw.).
Paper presented at 9th Int. Conr. Genetics, Bellagio, Italy.
study of five isolates of S. fimicola, found that all
SORDARIA FIMICOLA is a homothallic pyrenomy-
cete which, like 8-spored species of Neurospora,
produces asci, each with eight dark ascospores in a
single orderly series. No other type of spore is
produced by this species. Recently the writer
(1954) obtained a gray-sporedmutant by means of
ultraviolet irradiation. When the mutant culture
was paired with a wild-type culture, some perithecia were produced which contained asci with
four wild-type and four gray ascospores, thus making possible a direct analysis of segregation of the
spore color locus in the ascus. Zickler (1934)
studied a similar phenomenon in the asci of the
heterothallic pyrenomycete Bombardia lunata, and
Bistis and Olive (1954) reported on the segregation of two different loci affecting spore color in the
heterothallic discomycete Ascobolus stercorarzus.
During the past year the writer learned of another similar investigation being carried on independently by Professor H. Heslot on the homothallic species Sordaria macrospora at the Institut National Agronomique in Paris. In March of 1954,
Professor Heslot very kindly sent to the present
writer an outline of a paper3describing the characteristics of a considerable number of mutants, many
of which affect ascospore color and whose segregation patterns may be directly observed in the ascus.
Some of the writer's findings agree in several important details with those of Professor Heslot but
also include considerable data which were not a
part of the latter's report.
97
98
[Vol. 43
AMERICAN JOURNAL OF BOTANY
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February1956]
TABLE
Cult.
No.
Al
A2
A3
C
C2
C3
C4
C5
C6
C7
C8
C9
Co
Cl
RI
R2
R3
R4
R5
OLIVE-GENETICS
OF SORDARIA FIMICOLA.
99
I.
1. Data on the nineteen strains of Sordariafimicola. (MM = mimimalmedium)
Substrate
Locality
New York City?
New York City ---ATCC
-North Carolina -Ontario -10.9-13.0
Spinach seeds Pea seed Ontario -10.5-13.0
Radish seed Manitoba -11.5-13.0
Dung -Ontario
-11.2-12.7
-10.5-12.4
Dung -Ontario
-10.2-12.1
Dung -Ontario
-10.2-11.8
Leaf -Minnesota
-11.2-13.3
Dung -Michigan
S. A.? -10.5-12.1
-U.
-CBS
-11.5-13.3
Soil -Pilot
Knob, N. Y.-Dried leaves ---- Ithaca, N. Y.
Dried leaves ---- Ithaca, N. Y.N. Y.Dried leaves --Ithaca,
N. Y.
Soil -Ithaca,
Dung
Dung -
Ran,e,in sporesize (u)
11.2-13.6
10.9-12.4
11.2-14.0
11.8-13.6
11.2-13.6
11.8-14.3
11.8-14.6
10.5-12.4
10.9-13.6
required the presence of Fiotin in the medium for
adequate growth and for fruiting. Four, including
a culture from the Centraalbureau at Baarn, required only biotin, while one also required thiamin
for fruiting. The culture (C 11) which we received
from the Centraalbureaurequires both biotin and
thiamin for fruiting.
Origin of ascospore color mutants.-Wild-type
ascospores of S. fimicola during maturation pass
through the following series of colors: hyaline, yellow, greenish yellow, deep green, and finally dark
gray-brown. All pigments in these and in the spores
of all mutant strains are located in the cell wall,
while the cytoplasm is consistently hyaline.
The gray-spored mutant (g), which was derived
from strain Al, produces ascospores that are bluish
gray, then gray in color. They are conspicuously
lighter than mature wild-type spores (fig. 4-6).
Their germination approaches 100 per cent. Yellow
and green pigments have not been observed during
maturation of these spores. The gene g also has an
effect upon spore size. In both heterozygous and
homozygous asci g spores tend to be somewhat
larger than g+ spores (table 2). But in heterozygous asci the spores of both genotypes are larger
than they are in homozygous asci. Ascospores
carrying the g factor are inclined to germinate
earlier than wild-type spores.
The yellow-sporedmutant (y), which was derived
from strain Cl, has bright yellow ascospores that
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
18.6-21.7
17.4-21.7
16.7-22.9
18.0-21.3
17.4-21.1
18.0-22.9
18.8-22.9
18.0-21.7
19.2-22.9
16.7-19.8
16.7-19.5
17.4-22.3
16.7-19.8
17.4-21.4
16.1-21.7
18.0-23.6
15.5-22.9
16.1-24.2
15.2-22.3
Average
size (,u)
12.5 X
11.6 X
12.5 X
13.1 X
11.8 X
11.7 X
12.1 X
11.8 X
11.6 X
11.1 X
11.2 X
12.4 X
11.3 X
12.1 X
12.5 X
13.0 X
13.1 X
11.8 X
12.1 X
MM
2-0.3
19.5
20.0
19.8
19.3
20.6
20.7
20.1
21.2
18.2
18.4
20.4
17.9
19.9
19.3
21.6
21.0
20.5
19.0
Alg
Interfertility
Cly
C7hy
+
-+
_
-
-
-
-
-
+
+
+
-
-
-
-
-
+
+
+
-
-
-
-
+
-
-
-
-1-
-
-
-
-
+
+
+
+
-
-
-
-
+
-
+
-
-
-
+
-
-
+
+
-
become somewhat duller and darker with age. No
green pigments have been observed during spore
maturation in this mutant. The gene y appears to
have little or no effect upon spore size (fig. 10), but
the germination of y spores is greatly reduced,
about 36 per cent germination being obtained with
spores taken from pure y cultures. Strangely, the
percentage of germination is much more drastically
reduced in y spores produced in asci heterozygous
for the y locus, the proportion of viable spores from
such asci being about 3.3 per cent. Germinationof
y+ spores from these asci is unaffected.
The hyaline-spored mutant (hy) produces ascospores that are at first hyaline, then sub-hyaline
with a faint yellowish tint. The germination of these
spores is drastically reduced. Only six or eight
spores in the entire cluster of homozygous asci from
which the mutant was originally isolated were able
TABLE2. Size of wild-type (g+)
spores of the Al strain
and mutant (g) asco-
Genotype
Type of
Range in spore size
Average
spore size
of spores
ascus
(,u)
(,u)
g+
Homozygous
Heterozygous
Homozygous
Heterozygous
g
11.2-13.6 X
12.1-17.4 X
11.8-16.1 X
12.4-18.6 X
18.6-23.3
20.1-23.6
16.1-26.4
20.5-26.7
12.8 X
15.5 X
14.9 X
16.9 X
20.7
21.6
22.5
24.0
Fig. 1-7. Sordaria fimicola.-Fig. 1. Cross between self-fertile g and g+ cultures of the Al strain.-Fig. 2. Cross between self-fertile g and self-sterile g+ cultures of the Al strain.-Fig. 3. Cross between fertile wild-type (left) and selfsterile yellow-spored cultures of the Cl strain. Nuclear migration has converted the mutant mycelium into a fertile one.
-Fig. 4. Clusters of wild-type (g+) and gray-spored (g) asci of the Al strain.-Fig. 5. Heterozygous asci from the
cross g X g+.-Fig.
6. Homozygous wild-type and heterozygous (gg+) asci from the same perithecium.-Fig. 7. Possible translocation effects in a wild-type perithecium of the A2 strain.
AMERICAN JOURNAL OF BOTANY
100
to germinate. Also, the hy spores are conspicuously
larger than wild-type spores, as may be readily
observed in heterozygous asci (fig. 8. 11).
Single-spore isolates from original wild-type cultures not previously obtained in single-sporeculture
or which have been maintained for long periods in
the laboratory by mass transfer, may vary considerably in their crossing reactions with singlespore mutant cultures. When two such cultures are
paired some distance apart on an agar plate, a
distinct line of perithecia will often form where
they come together (fig. 1). Such a line may contain few to many crossed perithecia in addition to
mutant and wild-type perithecia. In some pairings
a double line of perithecia forms; in others perithecia fail to form at all at the line of contact.
Cultures of the latter type usually may be induced
to form some crossed perithecia by inoculation at
the same point on plates or in tubes of agar. The
degree of crossing appears to be genetically controlled but independent of the spore color loci. In
the pairing of two fully fertile cultures, it has never
been possible to demonstrate the "relative heterothallism" of Pontecorvo et al. (1953) ; i.e., the
number of homogeneous crossed perithecia in a
line does not exceed 50 per cent of the total, which
always includes pure wild-type and mutant perithecia. In crosses involving spore color mutants and
wild-type cultures of S. fimicola, quite a few mixed
perithecia are also produced. These are perithecia
that contain homozygous as well as heterozygous
asci (fig. 6), and their occurrencedemonstratesthat
the asci in a single perithecium may originate from
more than one nuclear pair. There is no evidence
that more than two different nuclear pairs may be
involved in ascus production in the same perithecium, for no peritheciumhas been found to contain
heterozygous asci and homozygous asci of both
parental genotypes. In a cross between g and g+
cultures of the Al strain, it was found that approximately 25 per cent of the perithecia containing
heterozygous asci were of the mixed type.
Inter-crossing of strains.-It
has been suggested
that ascospore color mutants of a homothallic
species might prove of value in assisting with the
classification of other isolates suspected of belonging to the same species (Olive, 1954). To test this
hypothesis each of the three spore-color mutants
was paired with all other strains. Table 1 shows
that Alg may be crossed only with A3, C4, C9, and
ClI; Cly crosses only with C4, C9, and R2; and
C7hy crosses only with RI, R3, and R5. In no case
was crossing between different strains extensive, but
the resultant heterozygous asci were normal in
development. It will be noted that eight strains
failed to cross with any of the mutant strains.
Although R3 and R4 were isolated from the same
soil sample only R3 could be crossed with C7hy.
It is also clear that spore size may not be used as a
primary criterion to indicate which strains may be
crossed.
[Vol. 43
From the cross Alg X Cllg+ four mutant (g)
progeny were isolated from heterozygousasci. When
attempts were made to backcross these with the
parental wild-type strains Alg+ and C11g+ by pairing the cultu-reson agar plates, the only crossing
which occurred was between one of the g progeny
and the C11g+ parent. Only a single hybrid perithecium was found. When four of the g progeny
from this perithecium were paired with Alg+ and
Cllg+, respectively, one of the four crossed only
with Alg+- and the other three crossed very poorly
with both. Later one of these failed to cross with
either of the wild-type cultures. It is therefore concluded that, whereas crossing among different
isolates of a homothallic fungus strongly indicates
that they are correctly classified as the same species,
the failure of such crossing to occur does tiot necessarily indicate that the isolates do not belong to
the same species. It appears likely that the existence
of intersterility factors may prevent strains of a
homothallic species from crossing. There is some
evidence that mutations causing intersterility may
occur spontaneously without affecting self-fertility.
Effects of temperatureupon segregation. Crosses
of each spore-color mutant with its wild-tvpe strain
were allowed to mature at four different controlled
temperatures: 7?C., 13?C., 230C., and 31'C., and at
a variable laboratory temperature ranging, from
230 to 28?C. Because so many asci of the Cl strain
abort at 310C., the upper temperaturelimit for the
cross y X y+ was set at 29.50C. Most of the controlled temperatures varied plus or minus 0.5?,
while at the 130 level the temperature at times
dropped to 120C. At 70C. the cultures matured so
slowly that it was necessary to allow the crosses to
grow for three days at room temperature,by which
time protoperithecia have appeared, before placing
them in the cold chamber. Even so, mature asci do
not begin to appear until about four or five weeks
later, and it was therefore necessary at the end of
this period to maintain the cultures at room temperature for one or two days longer in order to
permit maturation of the spores already present.
The data on temperature effects, representing a
total analysis of 10,666 asci, are given in table 3
and fig. 15.
The use of shorter temperature intervals undoubtedly would have made possible a more accurate graph. Nevertheless, the results demonstrate
that different chromosomes or segments of chromosomes may behave quite differently under the
same set of environmental influences. No explanation is available for the unexpected data obtained
for the y locus at the variable laboratory temperature of 230-28?C.
In the absence of interference, the extent of
second division segregation is a measure of the
amount of crossing-over between the centromere
and the locus of the mutant factor. If interference
is absent here, the results would indicate that very
low temperatures tend to inhibit crossing-over
February1956]
OLIVE-GENETICS
OF SORDARIA FIMICOLA.
101
I.
*~~~~~~~~~~~~~~~~~~~~~~~~~~~~
^
8
s.
w
:~~~~~~qw.
. 9
-
:
A~~~
Fig. 8-12. Sordaria fimicola.-Fig. 8. Segregation in asci of the C7 strain heterozygous for the hy locus. Fig. 9. Cluster of asci frim hybrid perithecium resulting from dihybrid cross, g+y X gy+. Note the presence of three main
types of asci.-Fig. 10. Segregation in asci heterozygous for the y locus. Fig. 11. Segregation in asci heterozygous for
the hy locus. Note larger size of mutant spores.-Fig. 12. Segregation in asci from dihybrid cross, g+y X gy+. Note
the presence of both ditype and tetratype asci.
102
[Vol. 43
AMERICAN JOURNAL OF BOTANY
TABLE 3.
Relationshipof segregationto temperatureat the
three spore color loci
Temperature
70C.
MI
asci
PercentMuI
age of
asci Total MI asci
420
301
382
316
61
365
g X g+
yXy+
319
533
298 309
607 50.9%
hy X hy+
360
726
g X g+
y X y+
hy X hy+
269 573
842 68.1%
315
314
255
421
570
735
44.7%
57.3%
Cross
g X g+
y X y+
hy X hy+
130C.
230C.
366
pa
736
362
747
42.9% <0.01
16.9%o <0.01
49.0% <0.01
852
62.6%
50.4%
0.016
0.038
<0.01
230-280C.
gXg+
(Variable)
Y X y+
hy X hy+
303
669
408
600
337
537
903
1006
945
66.4%
33.5%
56.8%
0.49
<0.01
0.83
y X y+
335
256
591
43.3%
0.64
gXg+
hy X hy+
116
255
235
438
351
693
67.0%
63.2%
0.78
0.023
29.50C.
310C.
a Computations based on comparisons
with results obtained at 23?C.
between centromere and spore color locus for all
three mutants, while the optimum temperature for
crossing-over varies with all three loci, and high
temperaturesmay reduce or increase crossing-over.
Another explanation of the data might be that
chiasma interference occurs and is variously
affected by different temperatures. For example,
the high percentage of second division segregation of the y locus at 13?C. may have resulted from
an increase in single crossovers at the expense of
multiple crossovers, the reduction of the latter
being due to chiasma interference (Barratt et al.,
1954). This theory does not appear to apply as well
to the g locus, for if chiasma interference were
operative here, one would expect at some point in
the temperature range to exceed the theoretical
limit of 66.7 per cent for the number of Mll asci
produced, but this was not the case. It is not likely
that a great deal more light can be shed upon this
subject until other loci linked to the spore color loci
have been obtained.
Dihybrid crosses.-The most obvious method of
testing for linkage among the three loci is to cross
the mutant cultures with one another. However, as
table 1 shows, none of the mutant cultures can be
crossed with any other mutant culture. But both
Alg and Cly may be crossed with wild type C4
and C9. Yellow-spored isolates (g+y) from the
cross Clg+y X C9g+y+ were crossed with grayspored isolates (gy+) from the cross Algy+ X
C9g+y+. This cross (g+y X gy+) resulted in the
production of hybrid perithecia containing the expected three types of asci (fig. 9, 12): (1) parental
ditype (4g+y :4gy+), (2) recombinant ditype
(4g+y+ : 4gy), and (3) tetratype (2g+y : 2gy :
2g+y+
2gy). The gy spores were of a genotype
not previously observed. They are light tan in color,
being lighter than either g+y or gy+ spores, and
unlike g+y spores, they give a high percentage of
germination (90 per cent or more). The gy cultures
were back-crossedto wild-type Cl and C9 cultures.
Hybrid perithecia appeared in both crosses, but
better results were obtained with the C9 strain.
Hybrid perithecia produced by this cross (gy X
g+y+) contained the same three types of asci resulting from the foregoing cross. Table 4 gives the
proportions of the three types of asci produced by
these crosses at 13?C.,23?C., and room temperature
(230-280C.). The fact that at all temperaturesthe
non-parentalditypes are essentially equal in number
to the parental ditypes demonstratesthat the g and
y loci segregate independently. The number of
tetratype asci obtained at 13?C. and at 230--280C.
closely approximates the 66.7 per cent expected if
the two loci were on separate chromosomes. It is
not clear why at 230C. the tetratypescomprise only
about 60 per cent of the total in spite of a relatively
large sample. Even if the two loci were on different arms of the same chromosome, one would expect, in view of the segregation data obtained
separatelyfor these loci (table 3), that at all temperatures the tetratype asci would comprise two-thirds
of the total (Lindegren, 1949). While the evidence
clearly shows that the g and y loci cannot be linked
on the same chromosome arm, it has not been
possible to demonstrateconclusively, in the absence
of other gene markers, that these two genes are not
on different arms of the same chromosome (Barratt et al., 1954).
For some unknown reason, mixed perithecia did
not appear in the dihybrid crosses described above.
However, when the heterokaryon, Clg+y+ (carrying a sterility factor) + Clg+y was paired with the
heterokaryon C9g+y+ + gy, two perithecia were
found to contain, in addition to the three types of
asci expected from the cross g+y+ X gy, a number
of asci which segregated in a 4:4 ratio for g+y and
gy, or yellow and light tan.
It was noted that in dihybrid crosses grown at
130C. one or both of the gy+ spores in tetratvpeasci
TABLE
Temp.
?C.
130
230
23?280
4. Analysis of asci from dihybrid crosses
Cross
gy X g+y+
g+y X gy+
gy X g+y+
g+y X gy+
gy X g+y+
g+y X gy+
NonPer cent
Parental parental Tetratetraditypes ditypes types Total type
64
84
351
97
104
19
62
83
333
114
90
26
251
324
1052
313
383
110a
377
491
1736
524
577
155
66.6%
66.0%
60.6%
59.7%
66.49%
71.0%
a
The small size of this sample resulted from poor crossing and is probably responsible for the somewhat higher
percentage of tetratypes than expected.
February1956]
OLIVE
GENETICS OF SORDARIA FIMICOLA.
frequently developed little or none of the characteristic gray color, in which case they were difficult or
impossible to distinguish from gy spores. The relative position of the aberrant spores in the ascus did
not appear to be the controlling factor in their
appearance, and no explanation is available at this
time for this unexpected behavior.
Test for biased segregation.-Segregation at the
g and y loci was studied to determine whether there
is polarized segregation as reported by Catcheside
(1944) in an analysis of Zickler's data on Bombardia lunata. Of 581 MI asci from the cross g X
g+, 294 had the four wild-tvpe spores in the upper
half of the ascus, while 287 had them in the lower
half. The difference in the two figures is obviously
not significant. From the cross y X y+ a total of
978 MI asci were examined, and 502 had the wildtype spores in the upper half of the ascus, 476 in
the lower half. Again the difference in the two
figures is not significant (X2 0.7; P -0.41).
Whitehouse and Haldane (1946), in an analysis
of Zickler's data on Bombardia lunata as well as
more limited data on Neurospora sitophila, concluded that there was a significant difference in the
number of asci showing asymmetrical (AaAa and
aAaA) and symmetrical (AaaA and aAAa) postreduction in favor of the former. Thus far the
writer has subjected only the y locus to a similar
analysis. Out of a total of 662 Mll asci from the
cross y X y+, 347 were asymmetrical and 315
symmetrical.The difference between the two figures
is not considered significant (X2 -1.5; P - 0.24).
Thus it may be stated, with respect to the loci
analyzed, that there is no significant evidence for
any kind of biased segregation in Sordaria fimicola.
Meiotic aberrations.-The results of what appear
at first to be the segregation of ascospore color
factors, but which are possibly due to translocations, may frequently be observed in perithecia of
strains (untreated) such as A2, Cl, C6, and CIO
(fig. 7). Other cultures show this rarely if at all.
No detailed explanation of the translocation
phenomenon is attempted here, since this has
already been adequately explained by McClintock
(1945) for Neurospora. In asci such as those of
the A2 strain shown in fig. 7, ascospores of various
shades of color are found, frequently two or three
types in the same ascus. Probably the most common
asci in such a cluster are those which contain four
wild-type, two yellow, and two hyaline spores. Some
asci in these clusters contain eight wild-type spores,
while others contain no wild-type spores. Only
the wild-type spores are germinable. Some aberrant
spores abort in the ascus. Frequently several perithecia in the same area of a culture contain asci
with these characteristics. If a translocation were
involved this would indicate that it had occurred
earlier in the mycelium followed by mitotic divisions of the translocation nuclei and the eventual
association of some of these nuclei with normal
ones in the ascogonia of several perithecia. As
I.
103
previously mentioned, certain strains are more
susceptible to this behavior than others. Successive
single-spore cultures of these strains do not appear
to be less susceptible to these aberrations.
Obviously more than one type of such aberrant
behavior is involved in the various strains or even
in the same strain. For example the A2 strain may
produce perithecia whose asci contain aberrant
spores that are most commonly olive-green and
yellow or tan, rather than yellow and hyaline as
described above. And still different effects may be
observed in other strains. Mutant and heterozygous
asci may show similar effects. Wheneverthis occurs
in hybrid asci it is generally impossible to use
them in genetic analysis.
What appear to be reverse mutations have been
observed rarely for all three spore color loci, but
only in heterozygous asci. In one or two out of
hundreds of these asci five spores are wild-type in
appearance and three mutant, indicating a reverse
mutation from mutant to wild-type. As yet no further analysis has been made of these asci. Although
several thousands of homozygous mutant asci of all
three types have been examined from both untreated and ultra-violet irradiated cultures, no reverse
mutation has ever been found in these cultures.
Factors affecting grouwthand fertility.-The Al
and Cl strains, which were the only ones studied
from this standpoint, were apparently heterokaryotic for various physiological factors at the time
these studies began. Some single-spore wild-type
isolates derived from the original cultures before
single-spore stock cultures had been obtained
showed marked reduction in perithecial production,
a few being completely self-sterile. Many of these
isolates also showed differences in growth rate and
general appearanceon agar plates. When y and y+
cultures differing in growth characters were
crossed, the four wild-type spores in each ascus
gave rise to two pairs of colonies which were
usually distinguishable with respect to their growth
characters (fig. 13). Because of their very low
germinability the yellow spores could not be tested
further. When a self-sterile culture is crossed with
a self-fertile one, the cross being heterozygous at
the y locus for purposes of identifying hybrid asci,
the four wild-type spores in each ascus germinate
to produce four fertile, four sterile, or two sterile
and two fertile cultures (fig. 14). The sterile
cultures usually have a denser, whiter appearance.
There is more than one locus involved in the
sterility phenomenon, for the progeny of these
crosses may be completely self-sterile or show varying degrees of fertility. Many that are completely
self-sterile on plates are partially fertile in test
tubes. Apparently the substance inhibiting fertility
becomes more diluted by diffusing through the
deeper agar. Other cultures remain completely selfsterile in both plates and tubes.
Greis (1942) reported the crossing of self-sterile
("male" and "female") X-ray induced mutants of
104
[Vol. 43
AMERICAN JOURNAL OF BOTANY
Heterokar-yosis and nuclear migration. - The
showing complete or partial sterility has, in com- widespread occurrence of heterokaryosis and nubination with any other such mutant culture, shown clear migration in S. fimicola is best demonstrated
any evidence of a stimulation in production of with the use of spore color mutants. Single-hyphal
hybrid perithecia. Crosses between sterile and isolates from groups of germinating ascospores of
fertile cultures are usually quite successful in plates different genotypes or from transfers taken from
or in tubes, some of the most extensive crossing the line of contact between two genetically different
occurring with such combinations. When a mutant cultures most often will contain mixed nuclei of the
spore-color factor is used as a marker, the results genotypes involved. Heterokaryosis occurs much
may be readily interpreted. For example, when a more freely on highly nutrient agars rather than on
fertile g culture is crossed with the self-sterile wild- agars weak in nutrients, since the former induce
type of the Al strain, the perithecia formed in the much more hyphal proliferation and anastomosis.
line where the two come together will be hybrid In a study of g + g+ heterokaryons, it was found
(and mixed), mutant, and wild-type, the latter often that heterokaryosis is much more readily mainbeing fairly common (fig. 2). Obviously the fertile tained during serial transfers on richer media rather
mutant culture in some way stimulates the sterile than on media poorly supplied with nutrients. On
wild-type culture to produce perithecia of its own. the latter the cultures tend to become homokaryotic,
Even more striking is the cross between a fertile usually with only the g genotype surviving.
When paired cultures come together across the
wild-type culture y+ ) and a completely sterile
mutant-sporedculture (y) of the Cl strain (fig. 3). middle of an agar plate, the hyphae pass one
The sterile culture suppresses perithecial production another only for a short distance, generally only
within a broad band on the y+ side of the line of a few millimeters, after which they taper off and
contact, but on the y side a broad dense band of cease growing. But hyphal anastomoses occur
perithecia develops and gradually extends further freely at the line of contact, and it is through these
back on that side, often reaching the periphery of anastomoses that the nuclei become intermixed.
the dish and converting the entire originally sterile Twenty different paired combinations of fertile
mycelium into a fertile one. This is the result of mutant (g) and wild-type (g+) isolates of the Al
unilateral nuclear migration; i.e., a migration of strain were studied on agar plates to determine the
y+ nuclei through the y mycelium by miieansof frequenCyand direction of nuclear migration. This
hyphal anastomoses at the line of contact. The was done by transferring to tubes of media small
perithecia on the y (sterile) side are mainly wAild- blocks of agar and mycelium from a certain distype and crossed or mixed, with very few or none tance back in this case one centimeter 0on each
of the y genotype. In crosses between fully fertile side of the line of contact between paired cultures.
and partially fertile cultures, the latter being self- If nuclear migration has occurred from one side
sterile on agar plates, a similar phenomenon may to the other, than the transfer from the latter side
occur, but the proportion of perithecia of the less will be heterokaryotic and will give rise to crossed
fertile type is much greater than in the previous as well as mutant and wild-type perithecia. Seven
cross.
of the twenty pairings gave no evidence of nuclear
Since the sterility-fertility mechanism is one of migration in either direction, although crossing
the latest phases of this study to be investigated, occurred at the line of contact. In five of the comthere is not yet a sufficient amount of evidence on binations nuclear migration occurred from the g
the genetic factors involved to permit a fuller dis- into the g+ mycelium, and in the remaining eight
cussion of the subject at this time.
the direction was reversed. In no combination did
Sordlaria fimicola, but none of our mutant cultures
x
--
-~~~~~~~~~~~~~P~
x
Fig. 13, 14. Sordaria fimicola.-Fig. 13. Segregation of growth factors in wild-type (y+) spores of two asci from the
cross y X y+, in which the parental cultures showed differences in growth characters. Fig. 14. Two sterile white cultures and two wild-type cultures from the four dark spores of a 4:4 ascus heterozygous for the y locus and sterility factors.
February 1956]
OLIVE
GENETICS OF SORDARIA FIMICOLA.
70
/
60
7
oei
z
h~~~~~~~y
/
0
w 40
z
0
530
0
z
20
w
I0-
5
10
20
15
TEMPERATUREOC
25
30
Fig. 15. Segregation patterns of the three loci affecting
color at different temperatures (70, 13?, 230, and
310C.).
ascospore
the nuclei migrate in both directions. Furthermore,
a culture which is the recipient of nuclei in one
combination may be the donor in another. Thus it
seems likely that a number of genetic factors control
nuclear migration and that there is a kind of
valence among the cultures with respect to their
behavior towards nuclear migration.
Heterokaryosis and nuclear migration have also
been investigated in combinations of self-fertile
cultures with cultures var)ying from partly fertile
to fully sterile. Test transfers were taken from each
paired culture at two centimeters from the line of
contact and grown in agar tubes. Fifty different
combinations of cultures were studied in this manner, and in all cases nuclear migration was found to
occur in the direction of the fertility mutant, never
in the reverse direction (fig. 2, 3). The rate of
nuclear migration has not been determined, but
hybrid perithecia may be found at the extreme
outer periphery of the fertility mutant within about
seven to nine days following contact of the two
cultures. This is particularly evident when completely sterile cultures are paired with fertile ones.
In the Cl strain, as previously mentioned, nuclear
migration from a fertile culture into a completely
self-sterile one may eventually convert the latter
into a fertile mycelium with numerous perithecia
(fig. 3). At the same time a broad zone of perithecial suppression is apparent opposite the line of
contact on the side of the self-fertile culture. It
appears that the sterile culture produces some substance which passes into the medium and causes
sterility not only of this culture but of any adjacent
culttire for some distance from the line of contact.
I.
105
The completely self-sterile mutant produces no
distinct protoperithecia when grown alone. However, it has no difficultyin forming them after wildtype nuclei have migrated into the mycelium. There
must be some interaction between the two genotypes in a common cytoplasm which overcomes the
sterility originally imposed upon the mycelium by
the mutant nuclei.
The partially fertile mutant cultures of the Cl
strain, which usually remain sterile on plates but
are partly fertile in test tubes or glass jars in
which the agar is deep, behave much like the
completely sterile cultures when paired with fertile
cultures on plates. Here, also, the fertility mutant
exerts a depressing effect upon perithecial production by the fertile culture in an area adjacent to
the line of contact. Nuclear migration into the
mutant mycelium frequently does not proceed as
rapidly or as extensively as in the completely
sterile mycelium. Also, in crosses involving a
partially fertile mutant and a self-fertile culture,
perithecia of the mutant genotype appear on the
mutant mycelium in nearly equal proportions with
hybrid perithecia and perithecia of the self-fertile
genotype. On the other hand, when the completely
sterile mutant culture is used very few or no pure
mutant perithecia appear, while hybrid perithecia
and perithecia of the self-fertile genotype tend to
be about equally common on the mutant mycelium.
The degree of sterility or fertility does not necessarily determine the direction of nuclear migration.
A certain partially fertile y+ culture was paired on
a plate with a less fertile y culture and on another
plate with a completely sterile y culture. Transfers
taken from each culture at two centimetersfrom the
line of contact were then tested for heterokaryosis
by pairing them with self-fertile y and y+ cultures.
These tests showed that in both cases nuclei had
migrated from the less fertile mycelium into the
more fertile one. While it is probable that one (or
more) of the genes causing reduced fertility in the
Cl strain also controls the direction of nuclear
migration, the degree of sterility is not an adequate
index to the direction of nuclear migration.
DISCUSSION.-The
behavior of the three spore
color loci at various temperaturesdemonstratesthat
different chromosomes or segments of chromosomes
may behave quite differently under the same set
of environmentalconditions. The various crossover
values obtained for each locus at different temperatures are interpreted as being due to the effects of
temperature upon crossing-over between the locus
and its centromere. The fact that all three loci
differ in their behavior at the various temperatures
may be indicative of differences in chromosome
structure with reference to such features as size
and distribution of heterochromaticregions, length
of chromosome, etc. Chiasma interferencemay also
be involved, as discussed earlier in this paper.
Bistis (1955) found in Ascobolus stercorarius a
considerable increase in MII asci with respect to a
106
[Vol. 43
AMERICAN JOURNAL OF BOTANY
spore color factor (t) as the temperatureis reduced
from 25?C. to 14?-16C., the results being due at
least in part to the effects of temperature upon
crossing over. He found that another locus (1) on
a different chromosome which gave approximately
67 per cent second division segregation at room
temperaturewas unaffected by lower temperatures.
The g locus of S. fimicola segregates at second division in about 67 per cent of the asci at room
temperature as well as at 31?C. Like Bistis, the
present writer found that the 67 per cent theoretical
limit could not be exceded by varying the temperature. However, the percentage of MII asci from the
cross g X g+ dropped to 62.6 per cent at 13?C.
and to 42.9 per cent at 70C. It seems likely, therefore, that the g locus is not far beyond the detectable limit of 33.3 crossover units from its centromere.
Plough (1917, 1921) studied the effects of
temperature upon crossing-over in Drosophila and
found that maxima of crossing-over in the second
chromosome were reached at 13?C. and at 31?C. A
minimum was observed at 220-270C., and a drop
occurred below 130C. Plough further observed that
crossing-over is unaffected by temperature in certain chromosomes. In the third chromosome of
Drosophila he found one region sensitive and the
remainder insensitive to temperatureeffects. White
(1934) studied cytologically three different insects
at various temperatures. He made actual counts
of chiasmata at meiosis, and much of the data
which he obtained agree with the findings of
Plough. For example, in Stenobothrus parallelus
studied at 20, 100, 230, and 37?C., he found that
maximum numbers of chiasmata were obtained at
1OC. and at 370C. White discusses the possibility
that temperature governs chiasma frequency by
interference, which in return is related to viscosity
of the chromosome. But as White points out, little
is known about the temperature coefficient of
chromatin viscosity.
One point which is made obvious by these studies
is that crossover data should always be carefully
correlated with the temperatureat which they were
obtained. An examination of fig. 15 will quickly
show that an investigator working at a laboratory
temperature around 18?C. might obtain crossover
data for a particular locus which would be quite
at variance with data obtained for the same locus
by an investigator working at a laboratory temperature of about 28?C
SUMMARY
With the use of ultraviolet irradiation, three
ascospore color mutants (g, y, hy) were obtained
in the homothallic pyrenomyceteSordaria fimicola.
Nineteen different wild-type strains of the fungus
were studied, seven of them fruiting on a minimal
medium containing biotin as the only growth substance and the remaining twelve requiring both
biotin and thiamin for fruiting. With the use of
the ascospore color mutants, which make possible
the direct identification of hybrid perithecia and
heterozygous asci crosses were attemptedamong the
nineteen wild-type strains. It was found that certain
strains could be crossed and others could not. In
the latter cases it is believed that intersterility
factors operate against crossing. Segregation of the
three independent spore color loci was studied at
four different temperatures. Different patterns of
segregation were obtained for all three loci. It is
concluded that the results are due to the effects of
temperature upon crossing-over and that chiasma
interference in conjunction with differences in
chromosome structure may account for the three
varied patterns of segregation. No significant evidence for biased segregation of any kind was
obtained, nor was there any convincing evidence
of linkage among the spore color loci. Dihybrid
crosses involving g and y loci were obtained, and
these yielded the three expected types of heterozygous asci in the same hybrid perithecium. What
appear to be reverse mutations have been found to
occur rarely at all three loci, but only in heterozygous asci and never in pure mutant cultures. Factors
affecting growth and fertility also segregate at
meiosis. Cultures ranging from partially fertile to
completely sterile may be crossed readily with
completely fertile cultures. Nuclear migration and
heterokaryosis are common in this fungus and
appear to be partly under the control of genetic
factors. Nuclear migration is always unilateral
when it occurs in paired cultures.
DEPARTMENTOF BOTANY,
COLUMBIA UNIVERSITY,
NEW YORK 27, N. Y.
LITERATURE CITED
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LAURA GARNJOBST. 1954. Map construction in Neuro-
spora crassa.. Advances in Genetics 6: 1-93.
BISTIS, G. 1955. Studies on the genetics of Ascobolus
stercorarius (Bull.) Schrot. Thesis. Columbia Univ.
New York.
, AND L. S. OLIVE. 1954. Ascomycete spore mutants
and their use in genetic studies. Science 120: 105-106.
BRETZLOFF, C. W. 1951. Fungus fruiting in submerged
culture. Science 114: 418-419.
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CAIN, R. F., AND J. W. GROVES., 1948. Notes on seed-borne
fungi. VI. Sordaria. Canadian Jour. Res. C. 26: 486-495.
CATCHESIDE, D. G. 1944. Polarized segregation in an
ascomyCete. Ann. Bot. N.S. 8: 119-130.
GREIS, H. 1942. Mutations- und Isolationsversuche zur
Beeinflussung des Geschlechtes von Sordaria fimicola
(Rob.). (Ein Beitrag zur Frage nach der Stabilitat
und Labilitat der Sexualreaktionen der Mon6zisten und
Dizisten.) . Zeitschr. Bot. 37: 1-116.
February1956]
ZALOKAR AND COCHRANE-DIPHOSPHOPYRIDINE
V. G., AND H. L. BARNETT. 1947. The influence of
pH and certain growth factors on mycelial growth and
perithecial formation by Sordaria fimicola. Amer. Jour.
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LINDEGREN, C. C. 1949. Chromosome maps of Saccharomyces.
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of chromosomes in bivalents and multivalents. Jour.
Genetics 30: 53-78.
MCCLINTOCK, BARBARA. 1945. Neurospora.. I. Preliminary
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DIPHOSPHOPYRIDINENUCLEOTIDASEIN THE LIFE CYCLE OF
NEUROSPORACRASSA1
MarkoZalokarand VincentW. Cochrane
IN THE COURSEOF
an investigation of respiratory
enzymes in Neurospora crassca it was found that
cell-free extracts made from conidia fail to show
triose phosphate dehydrogenase activity, although
the enzyme is easily demonstrablein extracts made
from mycelium. It was found further that very
small amounts of the conidial extract completely inhibit the reduction of diphosphopyridinenucleotide
(DPN) by the mycelial extract in the presence of
an oxidizable substrate. These phenomena were
traced to the occurrence in the conidial extract of a
diphosphopyridinenucleotidase (DPN-ase) in high
concentration. The DPN-ase of Neurospora crassa
has been described by Kaplan et al. (1951), who
also devised a method for the estimation of the relative activity of the enzyme. Using this method, we
have followed the changes of activity of DPN-ase
in the various stages of growth of the organism.
MATERIALS ANID METHODS.-Neurospora
crassa,
wild type, obtained from crosses of E-5256A and
E-5297a, was used in all experiments. Conidia were
obtained from cultures grown on a niedium containing sucrose, 20 g. per 1., agar 15 g. per 1, and the
mineral solution of Horowitz (1947), which consists of (per 1.): K Na tartrate, 5.0 g.; NaNO3, 3.0
g., KH2PO4, 3.0 g.; MgSO4-7 H20, 0.5 g.; NaCl
0.1 g.; CaCl2,0.1 g., and minor elements. The agar
medium, in Erlenmeyer flasks, was inoculated and
held for two days at 35?C. and six additional days
at room temperature. Eight-day-old cultures were
used because conidia of this age have the highest
germinability (IRyan,1948).
1 Received for publication August 8, 1955.
This investigation was supported by a research grant
(G3154) from the Division of Research Grants of the National Institutes of Health, and by a grant-in-aid (MET18A) from the American Cancer Society on recommendation of the Committee on Growth of the National Research
Council.
The mycelium was grown in Fries minimal medium with sucrose (20 g. per 1.) as the carbon
source. For most experiments the mycelium was
grown in 100 ml. of medium in a 500-ml. flask on
a reciprocating shaker at 25?C. In experiments in
which it was desired to suppress sporulation the
mycelium was grown in still culture, with Tween 80
(sorbitan monooleate) in the medium, in a 125-ml.
flask with 25 ml. of medium. A dense suspension of
conidia was used for all inoculations.
Cell-free extracts of mycelium were prepared by
grinding filtered and washed material with quartz
sand (Reagent grade, 3 g. per g. fresh weight of
mycelium) and 0.6 per cent cysteine hydrochloride
in 0.1 M KHCO3 (3 ml. per g. fresh weight of mycelium), after which the suspension was centrifuged at 3000 r.p.m.for 10 min. and the supernatant,
containing the enzyme, was decanted off. A slightly different procedure was used for the preparation
of extracts from conidia, which are more difficultto
disintegrate than is mycelium. Conidia were suspended in water, filtered through cotton to remove
mycelial fragments, and collected on filter paper on
a Buchner funnel. The mass of conidia so collected was moistened with cysteine solution and
ground with three times its fresh weight of powdered Pyrex glass (40 mesh) in a chilled mortar.
The slurry was then diluted with twice its weight
of cysteine solution, and the extract was collected
by centrifugation as above.
For the determination of DPN-ase activity, the
procedure of Kaplan et al. (1951) was used: 0.1 ml.
of DPN solution (4 mg. per ml., 90 per cent purity) was incubated at 37?C. with 0.3 ml. of 0.1 M
KH2PO4 and 0.1 ml. of suitably diluted extract.
After incubation for 7.5 min., 3.0 ml. of 1.0 M
KCN was added to the reaction mixture and the
optical density at 340 my was measured. A unit of