MARKERS and CENTROMERE DISTANCES in NEUROSPORA Tetraspermal

MARKERS AND CENTROMERE DISTANCES IN NEUROSPORA TETRASPERMAl

H. BRANCH HOWE, JR.

Department of Bacteriology, University of Georgia, Athens, Georgia Received September 24, 1962

ENETIC studies with Neurospora tetrasperma were begun by the late B. 0. GD~~~~( 1928) soon after the genus Neurospora was described (SHEARand DODGE1927). DODGE(1927) showed that the linear ascus of N. tetrasperm contains four binucleate ascospores and that events during ascus formation usually provide that the two nuclei in each ascospore will differ in mating type. The four ascospores from a single ascus, therefore, usually yield four self-fertile mycelia, a phenomenon variously known as facultative, secondary, or pseudo- homothallism. Genetic differences studied include mating type (DODGE1928; LINDEGREN 1932) ; conidial variation (DODGE1930,1935) ;lethals ( UBERand GODDARD1934; DODGE1934, 1935; SEAVER1937; DODGE,SINGLETON and ROLNICK1950); substrate color (TAI1936); dwarfism (DODGE,SCHMIDT and APPEL1945); and other morphological traits ( GOODSPEED1942; MALLOCH1942). Until recently (HOWE1961) mainly visible differences were employed. Genetic work in N. tetrasperma clearly lags behind that in N. crmsa both in numbers of mutants produced and in linkage analysis. Linkage analysis in N. tetrmperma is compli- cated by nuclear passing during ascus formation (DODGE1927) and by the dicaryotic condition of the ascospores. Single ascospore isolation does not neces- sarily insure homokaryosis for every allele, as in N.crassa. The present investigation involves the recovery of both morphological and nutritional mutants as well as a preliminary determination of gene-centromere distances using both tetrads and random ascospores. The genetic analyses are based upon current understanding of ascus formation and the relationship between crossing over and nuclear content of the ascospores. These concepts are briefly traced below and outlined in Figures 1 and 2. DODGE(1 927) was the first to describe nuclear events in the developing ascus. His description may be summarized as follows. First meiotic division spindle orientation is longitudinal (with respect to the long axis of the ascus), and the two daughter nuclei come to rest one somewhat above the other. Second meiotic division spindle orientations occur in two ways, but both produce the same result. In one the two spindles are oriented longitudinally, with a spindle near each end of the ascus. In the other the two spindles are oriented obliquely and lie nearly parallel to each other near the center of the

1 Work supported by National Science Foundation Grant G-13267.

Genetics 48: 121-131 January 1963. 1 22 H. B. HOWE, JR.

MV DIV DIV FIGURE1.-Diagrammatic representation of ascus development in Neurospora tetraperma with respect to an allelic pair. Based upon several authors (see text). First division segregation results if a crossover does not occur between locus and centromere; second division segregation, if a single crossover does occur in this interval. Multiple exchanges are not considered. Nuclear passing (denoted by curved lines) occurs at both the second and third divisions. Sister nuclei shown by heavy and light circles, respectively. Upper Row: First division segregation resulting in heterokaryotic ascospores (Type I ascus). Middle Row: Second division segregation. Tetrad nuclei with nonidentical alleles at both ends of the ascus resulting in heterokaryotic ascospores (Type I ascus). Bottom Row: Second division segregation. Tetrad nuclei with identical alleles at both ends of the ascus resulting in homokaryotic ascospores (Type I1 ascus). ascus. Both, however, result in a pair of nonsister nuclei becoming situated near each end of the ascus. The four spindles in the third (mitotic) division are obliquely or nearly trans- versely oriented. The resulting eight nuclei assume nonsister pairwise arrangements, after which a pair of nonsister nuclei is cut off in each ascospore. COLSON ( 1934) verified DODGE’Scytological findings. Clearly, nuclear passing normally occws at both the second and third divisions. This is quite different from ascus development in N. crassa, where nuclear Neurospora tetrasperma MARKERS 123

TYPE I ASCUS TYPE U ASCUS

ASCOSPORES ASCOSPORES HETEROKARYOTIC HOMOKARYOTIC

ORIGIN: 1ST. DIV. SEC. ORIGIN: IR ZND. DIV. YG.

+ IR 2ND. DIV. SEC. FIGURE2.-Determination of gene-centromere distance in N. tetrasperma. Based upon several authors (see text). A. Tetrad analysis: Percent 2nd. div. seg. = 2 x percent Type I1 asci. percent 2nd. div. seg. Centromere distance = =percent Type I1 asci. B. Random, binucleate 2 ascospore analysis: Percent Type I1 asci = 2 x percent homokaryotic-mutant ascospores. Cen- tromere distance = 2 x percent homokaryotic-mutant ascospores. Centromere distance for mating type = percent self-sterile ascospores.

passing is very rare, requiring special techniques for detection ( MCCLINTOCK 1945; HOWE1956), and ascospores are initially uninucleate. LINDEGREN(1933) showed that second division segregation of a marker in the N. crassa ascus manifests crossing over between marker and centromere. COLSON (1934) suggested from her cytological studies that homokaryosis for a marker in the N. tetrasperma ascus manifests crossing over between marker and centromere. Homokaryosis for mating type had already been observed in N. tetrasperma by DODGE(1927) and LINDEGREN(1932) and mentioned in a review by KNIEP ( 1929). More recently SANSOME( 1946) and CATCHESIDE( 195 1 ) again discussed the role of homokaryotic ascospores in linkage studies in this species.

MATERIALS AND METHODS Stocks were derived from the DODGEN. tetrasperma wild type strain 87 (Columbia University stock Number 163) obtained from DR.L. S. OLIVE.Strain 87 is a typical secondarily homothallic strain, but reference to it has not yet been found in DODGE’Spapers. Strain 87 was inbred to increase isogenicity by twelve successive generations of single dicaryotic-ascospore isolations, a procedure equivalent to repetitive 124 H. B. HOWE, JR. intra-ascus crossing. Following inbreeding the heterothallic A and a mating type components were extracted by conidial plating and used in all subsequent work. These inbreds are referred to as 85A and 85a to distinguish them from the original strain 87. Prior to completion of inbreeding, however, some mutants were induced in strain 87. Standard N. crassa media were found satisfactory for growth of N. tetrasperma. WESTERGAARD-MITCHELL( 1947) crossing medium, however, was used almost exclusively as the routine culture medium so that heterokaryosis for mating type could be more easily detected. VOGEL’SMedium N (1956) was used when en- hancement of conidiation was desired. All mutants were of ultraviolet origin and were obtained by means of the filtration-concentration technique (WOODWARD,DEZEEUW and SRB 1954). Be- cause the heterothallic A and a components of N. tetrasperma usually conidiate poorly as homokaryons, 85A and 85a were grown in mixed culture (85A i- 85a) to increase conidial yield for irradiation purposes. Formation of unwanted perithecia in the mixed cultures was avoided by incubation at 35°C. Use of the mixed cultures actually aided in screening isolates from the post-irradiation sorbose plates, since isolates which fruited could not have originated from uninucleate conidia and therefore would probably not be homokaryotic for a mutation. Mutant strains were crossed to wild type. Both ascus (unordered) and random- ascospore isolations were made directly from dissection plates to slants, followed by heat shocking in a 60°C water bath for 4.0 minutes to induce germination. Sorbose colonizing medium ( NEWMEYER1954) was not employed in the original ascospore isolations. First division segregations and half of second division segregations both produce asci having ascospores heterokaryotic with respect to a marker-gene (Type I ascus). Asci having homokaryotic ascospores (Type I1 ascus), however, uniquely represent the other half of the second division segregations (Figure 1) . Second division segregations, therefore, are detected by observing Type I1 asci. Second division segregation of a marker results from crossing over between the marker and the centromere. Following second division segregation, the tetrad nuclei assume by chance either one of two kinds of arrangements (Figure 1) . One arrangement results in Type I asci; the other in Type I1 asci. Since these two ascus types should be equally frequent, second division segregation frequency is found by doubling the observed frequency of Type I1 asci (Figure 2). The centromere distance in standard map units, being half the percentage of second division segregation (LINDEGREN1933), is therefore simply the observed percentage frequency of Type I1 asci. Random ascospore analysis utilizes the fact that half the ascospores which originate in Type I1 asci are homokaryotic for the mutant allele. Doubling the observed percentage of random mutant ascospores, therefore, gives the percentage of Type I1 asci that produce them and hence the centromere distance (Figure 2). Neurospora tetrasperna MARKERS 125 Occasionally only one nucleus is cut out in a N. tetrasperma ascospore (DODGE 1927). Such ascospores are usually dwarf and were purposely avoided in these studies. Even in normal-sized, binucleate ascospores, however, only one nucleus may survive and produce homokaryosis interpreted as crossing over. This anomaly was minimized in these studies by scoring all isolates for self-fertility and hence for the presence of two initially viable nuclei. Self-sterile isolates were analyzed separately (Table 6).

RESULTS Mutants: Mutants obtained by UV irradiation of DODGEstrain 87 or of inbred strain 85 are shown in Table 1. Some of the nutritional mutants have only partial deficiencies necessitating use of small inocula and careful rechecking of growth tests during scoring. Mutant 101 shows a clear adenine requirement and is also scorable by sterility when homokaryotic for the requirement but heterokaryotic for mating type; demonstration of mating-type heterokaryosis for 101 necessi- tates crossing tests. Mutant 105 is also sterile when selfed, but incipient perithecia develop sufficiently to show mating type heterokaryosis without necessitating crossing tests. The nutritional requirement of 108 is not known, but shows tem- perature sensitivity. Ascus classification: An ascus may be completely scored if at least three of the four ascospores germinate. To avoid bias, therefore, only asci with at least three viable ascospores are considered. These constituted 57 percent of all asci isolated. Five different classes of asci with at least three viable ascospores are obtainable when a single recessive marker is segregating (Table 2). The five classes are then scored as either Type I or Type 11, providing the basis for finding centromere distance without requiring conidial plating to determine all eight nuclear components of each ascus.

TABLE 1

Mutant strains

Reaction of mutant Origin when selfed -_____Mutant number (strain) (mA X ma) Other characteristics 101 87A Sterile Initial growth and conidiation slower than wild type; adenine-requiring 102 87A No recomb. Albino conidia; no pigmentation with mating type. after prolonged illumination on Hence not tested enriched media 103 87a Fertile Stimulated by arginine 104 87A Fertile Stimulated by arginine 105 85a Incipient perithecia Colonial growth but no ascospores 106 85a . Fertile Stimulated by lysine 107 87A Fairly fertile Stimulated by methionine 108 85a Fertile Unknown requirement at 35°C; slow growth on minimal at 25°C 126 H. B. HOWE, JR.

TABLE 2

Fiue unordered ascus classes obtainable in crosses of a recessive mutant x uild type (upper half of table) and A x a mating type alleles (lower half of table). Only asci with at least three viable ascospores are considered. m = recessiue mutant allele; + = wild type allele; A, a = mating type alleles; mut = mutant; w.t.= wild type; s- f = self-fertile; s -s = self-sterile; = inuinble ascospore

Ascus Ascus class type Genotype Phenotype 1 i-Jn +,m +,m +,m w.t. w.t. w.t. w.t. I 2 +,m +,m +,m . . . . w.t. w.t. w.t. .... 3 m,m m,m +,+ +,+ mut. mut. w.t. w.t. 4 I1 m,m m,m +,+ . .. . mut. mut. w.t. .... 5 m,m +,+ +,+ . . . mut. w.t. w.t. .... 1 A,a A,a A,a A,a s-f s-f s-f s-f I 2 A,a A,a A,a ..., s-f s-f s-f .... 3 A,A A,A a,a a,a s-s s-s s-s s-s 4* I1 A,A A,A a,a .... s-s s-s s-s .... 5* A,A a,a a,a . .. s-s s-s s-s . ..

* Distinguishable by crossing tests.

Tetrad analysis: Crosses of the mutants to wild type are shown in Table 3. Centromere distances of the various mutants range from zero to 46 units. Mutant 102 (albino) has not shown recombination with the centromere in a total of 76 asci. Conidial plating and isolation of the eight nuclear components in 14 of these

TABLE 3

Classification of 442 unordered asci with at least three uiable ascospores from crosses of mutant x wild type. All ascospores self-fertile except those from the three Type II asci in mating type data

Ascus type and class I I1 Type totals Percent type I1 (centromere Cross 1 2 3 4 5 I I1 distance) ZOla X 85A 13 6 12 3 1 19 16 46 102A x 85a 56 20 0 0 0 76 0 0 103A X 85a 24 5 6 0 0 29 6 103a X 85A U) 3 3 0 3 23 6 19 104A X 85a 19 9 1 0 2 28 3 6 104a X 85A 12 9 0 0 0 21 0 105a X 85A 35 12 14 0 2 47 16 25 106a X 85A 4 2 0 0 1 6 1 14 107A X 85a 43 10 5 0 0 53 5 9 108, X 85A 58 21 3 0 2 79 5 6 mating type (from above crosses) 328 111 2 I* 439 3 0.7

* Crossing tests to distinguish between Classes 4 and 5 were not made. Neurospora tetrasperma MARKERS 127 76 asci selected at random (Table 5) verified the Type I heterokaryotic classification; this particular heterokaryon, moreover, is usually discernible by pink instead of bright orange conidia. Mutant 101, on the other hand, shows a very high incidence of second division segregation, about 92 percent. Random ascospore analysis: Table 4 shows data obtained from randomly isolated ascospores. Only ascospores of normal size were selected and all were checked for mating type heterokaryosis on the basis of mycelial self-fertility. Mating-type heterokaryosis indicates that each ascospore initially contained two viable nuclei. Table 4 also shows that centromere distances determined from random ascospores were not always homogeneous with distances obtained by tetrad analysis. A major portion of the nonhomogeneity is believed to be the result of inaccuracies in scoring the randoms. Scoring of markers having only partial nutritional deficiencies is likely to be more accurate in whole asci, where one has the other members of the tetrad available for comparison. A secondary cause of nonhomogeneity is believed to be nuclear lethality (see below). No random ascospores were analyzed from crosses of mutant 106. Gene-geminteruals: Finding distances between marker-genes requires conidial plating to determine genotypes of the component nuclei of the segregants. Visible mutants which may be scored directly on the sorbose plates, such as 105 (colonial), or soon after isolation into tubes, such as 102 (albino), are more readily handled by the plating procedure. So far only these two have been so analyzed. Conidia of all four members of 14 of the 76 asci from the 102A x 85a cross (Table 3) were plated. The nuclei of all 14 asci had parental combinations with respect to the albino and mating-type markers (Table 5), indicating linkage of 102 to mating type. Conidia of the 95 phenotypically wild type isolates from the

TABLE 4

Classification of 1629 randomly isolated ascospores. All ascospores self-fertile except as noted in mating type data

Ascospores mutant wild type Centromere Cross (m,m) (m,+or +,+) Percent mutant distance

lOlA x +a 61 212 21.5 43 lOla X +A 54 209 102A x +a 0 96 0 0 103A x +a 3 30 6.8 14 103a X +A 8 1 22 104A X +a 20 146 12.1 24+ 105a X +A 17 95 15.2 30 107A X +a I2 127 8.6 17* 108a X +A 13 128 9.2 18'

~~ ~~ Ascospores Percent Centromere Cross self-sterile self-fertile self-sterile distance +A X +a (mating type) 1 275 0.4 0.4

~ * Not homogeneous with centromere distances obtained from tetrads. 12s H. B. HOWE, JR.

TABLE 5

Evidence for linkage of mutants 102 and 105 to mating type. Mutant 102 data are frasm unordered asci; mutant 105 data, from random, self-fertile ascospores

~ ~ ~~~~~~~ ~ Deviation from No am Recombination 1 1 ratio Cross PD NPD T with mating type (PD NPD)

102A X 85a 14 0 0 none x2 = 14, P < .01

Mating types of 04 ni nuclei Ascospore No. randomly selected from the Deviation from Cross genotypes randoms 79 m,+ heterokaryons 1:l ratio (aA) m,m 17 105a X +A m,+ 79 18a:6A x2 = 6, P < .05 +,+ 16 PDzparental dltype, NPD=nonparentdl ditype, T= tetratype.

105a x 4- A cross (Table 4) were plated. Seventy-nine of the isolates proved to be heterokaryotic, with the two homokaryons equally frequent, as expected (Table 5).When colonial cultures isolated from 24 of the 79 heterokaryons were sex-tested, a nonrandom 1Sa: 6A ratio was obtained, indicating linkage of 105 to mating type. Nuclear lethality and aberrant asci: If events other than crossing over should produce homokaryotic ascospores, estimates of crossing over would be biased. One such source of bias is the loss of one of the two nuclei subsequent to being cut out in heterokaryotic ascospores. Nuclear lethality can usually be detected in whole asci, because the unaffected ascospores are available for comparison, but random ascospore analyses are especially vulnerable to this source of error ( SEAVER1937). Since normal-size ascospores are nearly always heterokaryotic for mating type, self-sterility in normal-size ascospores suggests probable nuclear lethality, especially when found in some but not all ascospores of any one ascus. Self-sterility in all ascospores of an ascus, on the other hand, may be alternatively interpreted as second division segregation for mating type. A second independent centromere marker would increase the probability of detecting nuclear lethality, especially in randomly isolated ascospores, although mating type is the best single marker for the purpose, because loss of either allele may be detected. Thirty-one asci were found which contained self-sterile ascospores interpreted as nuclear lethality (Table 6). Three additional asci which had all self-sterile ascospores are interpreted as second division segregations for mating type and are used to determine centromere distance for this locus (Table 3). Alternatively, these three asci could have resulted from nuclear lethality occurring in each ascospore simultaneously, although this seems unlikely in view of the observed frequency of the event in individual asci. All three asci had phenotypically wild- type ascospores. Conidial platings were unfortunately not made to check for via- bility of the recessive markers (107 and 108) that were segregating. A fourth ascus showed self-sterility in all three ascospores that were viable but homo- Neurospora tetrasperma MARKERS 129

TABLE 6

Normal and aberrant ratios for mating type alleles in 473 unordered asci with at least three viable ascospores

No. viable ascospores Ratio self-fertile: No. asci with self-sterile ascospores noma1 ratio aberrant ratio per ascus __ 4:0 328 .. 0:4 2* .. 4 3: 1 .. 17 2:2 .. 7 1:3 .. 0 3:O 111 .. .. 3 0:3 I* 2: 1 .. 6 1:2 .. 1 Totals 4.1.2 31

* Interpreted as second division segregation for mating type. karyosis for the nutritional marker (103) as well. This ascus is considered too ambiguous for inclusion in the data.

DISCUSSION Mutants for nutritional deficiencies and for morphological differences, as well as the mating-type locus, have been mapped with respect to their centromeres. The mapping method used depends upon homokaryotic ascospore frequency, the principle of which is based upon cytological observations of nuclear behavior by previous investigators. Mutants 202 (albino) and 105 (colonial) are both linked to mating type and therefore to each other. The fact that 202 and mating type consistently segregate to give Type I asci, whereas 105 consistently segregates to give 25 percent Type I1 asci, suggests normal prereduction of the centromere, at least for this linkage group. The segregational behavior of 102 is interesting. The only albino mutants so far found in N. crassa are very loosely linked to mating type (over 30 units) and over 25 units from centromere (BARRATT,NEWMEYER, PERKINS and GARNJOBST, 1954). Mutant 202, on the other hand, satisfies present criteria for very close linkage to both mating type and centromere. The simplest interpretation is that 102 is not allelic to the N. crmsa albinos but rather is a new locus close to centromere, although, on this hypothesis, it seems surprising that such a locus would not have been detected during the numerous mutation experiments that have been conducted with N. crmsa. The possibility also exists that the two species differ by one or more rearrangements in linkage group I. This view is supported by the fact that not only 102 but also the mating type locus appears to map in a different position from the albino and mating type loci in N. crmsa. 130 H. B. HOWE, JR. Mutant 101 shows about 92 percent second division segregation. Another in- stance of high second division segregation frequency in N. tetrasperma was reported by DODGE,SCHMIDT and APPEL (1945) who found 67 percent second division segregation for the U locus. Values in excess of 67 percent indicate the occurrence of positive chiasma interference (PERKINS1955). Nothing is known about the cause of the apparent loss of mating types alleles interpreted as nuclear lethality in 31 asci. Concomitant allelic loss at the other marked loci can be inferred in only a portion of the 31 asci, so it is not possible to determine total relative loss of mutant and wild type alleles. Allelic loss occurs with sufficient frequency, however, to warrant experimental control by use of one or more centromere markers. DODGE(1931) found ascospores homokaryotic for mating type about one percent of the time (centromere distance of one unit). The values of 0.4 to 0.7 unit reported here are in good agreement with DODGE’Sfinding in view of the fact that in the present experiments nuclear lethality was at least partially controlled. It is striking that these low values are similar to the frequency of abnormal meiotic nuclear passing detected in N. crassa (HOWE1956). If normal meiotic nuclear passing failed to occur with similar frequency in N. tetrasperma, centromere distances for mating type such as those reported here could be obtained without crossing over. SUMMARY Gene-centromere intervals for eight mutant strains of Neurospora tetrasperma, as well as the mating type locus, range from zero to 46 units in length, based upon homokaryotic ascospore frequencies obtained from both whole asci and random ascospores. Single-strand analyses of two visible mutants revealed that both are linked to mating type. Nuclear lethality in heterokaryotic ascospores mimicking legitimate homokaryosis was found to be sufficiently common to recommend the continued use of an independent centromere marker for its detection.

ACKNOWLEDGMENTS The writer wishes to express appreciation to DR. D. D. PERKINSfor his critical reading of the manuscript. The technical assistance of MRS. M. V. MINCHEYand MRS.T. M. MULLMANNis also gratefully acknowledged.

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