RECESSIVE MUTATIONS FROM NATURAL POPULATIONS OF NEUROSPORA CRASSA THE

Copyright 0 1985 by the Genetics Society of America

RECESSIVE MUTATIONS FROM NATURAL POPULATIONS OF NEUROSPORA CRASSA T H A T ARE EXPRESSED IN T H E

SEXUAL DIPLOPHASE

JOHN F. LESLIE’.’ AND NAMBOORl B. RAJU

Department of Biological Sciences, Stanford University, Stanford, Calijornia 94305 and *Department OJ Plant Pathology, Kansas State University, Manhattan, Kansas 66506

Manuscript received June 7 , 1985 Revised copy accepted August 3, 1985

ABSTRACT

Wild-collected isolates of Neurospora crassa Shear and Dodge were systematically examined for recessive mutations affecting the sexual phase of the life cycle, which is essentially diploid. Seventy-four of 99 wild-collected isolates from 26 populations in the United States, India and Pakistan carried one or more recessive mutations that reduced fertility significantly when homozygous; mutations affecting spore morphology were also detected. Limited complementation tests indicate that most of the 106 recovered mutations are unique.- T h e recessive diplophase (= sexual phase) mutations were uncovered by crossing each wild-collected isolate to a marked two-chromosome double-reciprocal translocation strain as “balancer.” Surviving progeny receive approximately 60% of their genome from the wild parent, but receive the mating-type allele from the “balancer” parent. These progeny were backcrossed to the wild parent and were also crossed with a standard laboratory strain ( f l ) . Reduced fertility in the backcross us. normal fertility in the cross with the laboratory standard signals the presence of a recessive mutation in the wild-collected isolate.-Most of the mutants (95 of 106) fall into two major classes: those producing barren perithecia with no or few viable ascospores (51) and those with spore maturation defects (44). Most of the recessive barrens result either from an early block in meiosis o r ascus development (25) o r from a late disturbance in post- meiotic ascus behavior (1 8).-These recessive mutations are formally equivalent to recessive lethals in higher eukaryotes and may be important in determining the breeding structure of natural Neurospora populations.

EUROSPORA crassa Shear and Dodge is genetically and biochemically one of the most intensively studied eukaryotic microbes, but little was known

of natural Neurospora populations until systematic collections were begun in 1969 (PERKINS, TURNER and BARRY 1976). Wild-collected Neurospora have since been examined for electrophoretic enzyme polymorphisms (SPIETH 1975), Spore killers (a meiotic drive phenomenon, TURNER and PERKINS 1979), vegetative (heterokaryon) incompatibility (MYLYK 1976), variations in ribosomal DNA organization (RUSSELL et al. 1984), DNA restriction fragment length polymorphisms (METZENBERG et al. 1984) and mitochondrial DNA composition

’ To whom correspondence should be addressed.

Genetics 111: 759-777 December, 1985

760 J. F. LESLIE AND N. B. RAJU

((~OLLINS and LAMROWITZ 1983). Mitochondrial plasmids have been isolated from wild-collected strains (COLLINS et al. 1981; STOHL et al. 1982) and used as the basis for recombinant DNA cloning vectors (STOHL and LAMROWITZ 1983). It is clear from these studies that the heterothallic Neurospora species N. crassa and N. intermedia are both highly polymorphic outbreeders. This determination is based on data from both local and extended populations.

The present work focuses on the isolation of recessive mutants that effect qualitative differences between wild-collected isolates of N. crassa and are expressed during the transient diplophase portion of the life cycle. To identify vlich mutants in a heterothallic fungal species like N. crassa, the mutation must be ~ecombined into a strain carrying the opposite mating type of the wild- collected parent. This recombined strain is then backcrossed to the wild parent to permit detection and characterization of the recessive mutation. To mini- mize the number of progeny from such crosses that must be examined to ensure that at least one will carry the recessive mutation, we have used a mal-ked two-chromosome double-reciprocal translocation (LESLIE 1985) as “balancer” to increase the proportion of the wild parent’s genome in the progeny of a cross of wild-type X balancer. In addition to receiving much of their genome from the wild parent, these isolates also receive the mating-type allele of the balancer parent. DELANCE and GRIFFITHS (1980a) designed a disomic complementation method for the recovery of laboratory-induced, recessive diplophase mutants and isolated 46 mutants on linkage groups 11-VII. Their method is not suitable for the recovery of mutants from natural populations however.

In this study over 100 niutants were isolated by screening 99 wild-collected strains from 26 geographically different natural populations. Many of these mutants have also been studied cytologically and used for elucidating the genetic control of ascus development (N. B. RAJU and J. F. LESLIE, unpublished results). Although we have termed these mutants “diplophase-specific,” the loci we have detected may affect haploid, diploid and dikaryotic portions of the sex0 (a1 phase. We have termed these mutants “diplophase-specific” because we can detect their effects only when they are homozygous and not when they are heterozygous. We cannot yet rule out interactions between new combina- tions of alleles at more than one locus (4 LESLIE and LEONARD 197913) as an explanation for some of our mutants.

I n nature, genes expressed during the diplophase portion of the life cycle would not necessarily be subject to selection during the extended vegetative portion of the life cycle. These mutations are formally equivalent to the recessive lethal mutations of higher organisms. They thus provide a means, using conventional population genetic measures, to estimate both genetic load and mutation rate in natural Neurospora populations. They also provide informa- tion on the mating structure of the population and may be useful in determining the degree of genetic relationship between individuals. Portions of this work were presented previously in abstract form (LESLIE and RAJU 1981).

MATERIAL3 AND MEIHODS

Strains: M’ild-collrc.tetl strains used i n this study are from the collection of 1). D. PERKINS at St;inford (we ‘I‘able I ) . All of these strains werc c.ollected fresh from burned substrate in nature

RECESSIVES IN N . CRASSA POPULATIONS 76 1

TABLE 1

Geographical origin of strains examined for recessive diplophase mutations

Location Strain no. (number of mutants identified)

Everglades, Florida

Florida City, Florida

Groveland, Florida

Homestead-2, Florida

Homestead-3, Florida

Okeechobee, Florida Sweetwater-2, Florida

Yeehaw Junction, Florida

Bayou Chicot, Louisiana

Coon, Louisiana

Elizabeth, Louisiana

Houma, Louisiana

Iowa, Louisiana

Marrero, Louisiana

Ravenswood, Louisiana

Roanoke, Louisiana

Sugartown, Louisiana

Welsh, Louisiana

Fred, Texas

Mauriceville, Texas

Saratoga, Texas

Spurger, Texas

Bombay-Aarey, (Maharashtra),

Dagguluru, (A.P.), India Lankala Koderu, (A.P.), India

Lahore, Pakistan

India

PI441 (I): P1443 (2), PI445 (1)" P1453 (0)

P438 [FGSC 19451 (l) , P441 ( I ) , P445 (2)

PI410 (0), P1417 (1)

PI460 (l), PI462 (I): PI463 (l) , P1465 ( I ) , PI466 ( I ) ,

P1352 (1) P1476 (0)

P1365 (0)

P872 ( I ) , P873 [FGSC 32271 (1) P879 [FGSC 32001 ( I ) , P880 (2); P881 (2), P882 [FGSC

P861 (0), P864 [FGSC 32231 (0), P866 (0), P867 ( l ) , P868

P491* [FGSC 22211 ( l ) , P492 (l), P493 (l) , P496 (0), P497b

P1470 (1)

31991 ( I ) , P884 (1)

[FGSC 32241 (2), P869 (2), P870 (0)

(2): P498 (1): P499 (l), P501b [FGSC 39431 (l), P502 (2), P503* [FGSC 22201 (1)

( I ) , P532 [FGSC 22231 (1) P527 [FGSC 22221 ( I ) , P528 (2), P529 (2), P530 (l) , P531

P474 [FGSC 22241 (1)

P888 (0)

P516* [FGSC 22281 (0), P517 (0), P518' (l), P519 (l) , P520 (0), P521* (l) , P522 (2), P523 (2), P524* (l) , P525 (1)

P852 [FGSC 32101 (0), P853 (2), P854 (0), P858 [FGSC 321 11 ( 1 )

P504 (2): P506' (l), P5076 [FGSC 22301 (0), P508 [FGSC 22291 (0), P509* (0), P512* (1); P513 (l), P515* (1)

P828 [FGSC 32251 (2), P829 (0), P830 (a), P832 (0), P833

P538 [FGSC 22251 (l), P539 [FGSC 22261 (0)

P822 (l) , P825 [FGSC 33261 (1) P836 (2)," P838 [FGSC 32011 ( I ) , P839 (4). P840 (0), P841

(2), P842 [FGSC 32021 (l) , P843 (a), P844 (0), P845 (1)" P676 [FGSC 25001 (2)," P680 [FGSC 24991 ( I ) , P682 [FGSC

27121 (4) P1120 [FGSC 33601 (2), PI121 [FGSC 33611 (3) P1105 [FGSC 33581 (2), P1117 [FGSC 33591 (2)

(2)

P349 [FGSC 18241 (2), P350 [FGSC 18251 (2)

Strains with an FGSC number are also available from the Fungal Genetics Stock Center, Kansas

Several progeny showed a similar, but not identical, phenotype; the possibility of additional City, Kansas.

mutants has not been excluded. ' Strains studied by MYLYK (1976).


(PERKINS, TURNER and BARRY 1976) and were preserved in suspended animation on silica gel sliortly aftrr collecrioii (PERKINS 1977).

The wild-collected strains examined for recessive mutations were limited in two ways. First, only N. crassa isolates were examined. The availability of the BLNC-I balancer strains to speed the analysis and the capability to compare the mutations uncovered in this study with the numerous nieiotic and other recessive diplophase mutants already known to exist in N. crassa were the priniary reasons for thih limitation. Second, t i e emphasis of the study was placed on isolates from North America (all of the available isolates were tested) with only a few isolates from the Indian subcontinent included for comparison (see Table 1). The best regions for collecting N. crassa so far have been in the Gulf Coast states in the southern United States, even though other species, p.g. , N . tetrasperma, A'. intermedia and N. sitophila have much wider distributions (PERKINS, TURNER and BARRY 1976). Most laboratory stocks o f iV. crassa are based 011 strains originally isolated from this geographic region, and we presumed that differences in genetic background between the wild- collected isolates from this region and the laboratory standards would be less than the differences between the wild-collected isolates from other regions, e.g., India or Pakistan, and the laboratory standa rds.

The LILSC-1 b;il;uncer strains contain selective markers and two reciprocal chromosome translo- cations involving opposite arms of linkage groups I 1 and V. BLNC-I strains will be referred to as Ixilancer sti-aim. When these strains are crossed to Normal sequence strains and appropriate selection pressure is applied, viable progeny rrceive the mating-type region from the balancer parent and receive 80% of linkage groups I1 and V and 60% of the entire genome from the Normal sequence parrnt (LESLIE 1985). Thus, i t is necessary to test only ten viable prototrophic progeny in order to detect a recessive diplophase gene with 99% confidence, rather than the 24 progeny required if the balancer strains were not used. The study could have proceeded without the use of niarked balancer strains, but vvould have required the testing of more progeny. A description of the syntliesis and detailed properties of the T(1IL;VL;IIR;VR) BLIVC-I arg-5 j l ilv; acr-3 A / a

Mutant stocks originating from crosses of wild-collected isolates X BLNC-1 are available from J.F.L..; only strains with the mating-type opposite to that of the wild-collected parent are available. Some of these mutant stocks may also carry the T(llL;VLL)AK30 chromosome rearrangement resulting from the breakdown of BLNC-1 into its component rearrangenietits (LESLIE 1982, 1985). l'tie original wild-collected isolates (which carry the opposite mating-type of the F I progeny) are available from the Fungal Genetics Stock Center (FGSC) or from DAVII) PERKINS at Stanford.

Design o j crosscs and backcrosses to detect recessives: The general procedure used to detect recessives is outlined i n Figure I . Wild-collected isolates were crossed to a balancer strain and prototrophic progeny were isolated from random spores growing on a minimal sorbose medium (BROCK- MAN and DESERRES 1963). 'l'hese progeny were crossed to a standard laboratory strain and were backcrossed to the wild-collected parent. I f the two crosses differed, then they were examined in more detail. Some of the progeny differ morphologically in nrinor ways from the standard wild type; the genetic basis for these differences has not been investigated.

Detailed protocol for mutant identzjcation: Each wild-collected isolate was first crossed with BLNC- 1 arg-5 j l ilv or B L N G I arg-5 j l ilv; acr-? on appropriately supplemented Synthetic Crossing nicdium (M'ESTERGAARD and MI?'CHEI.I. 1947); the balancer strain was used as the female parent. (:I-osses were incubated at 25", with a 12 hr-12 hr dark-light cycle. Ten days after fertilization, crosses werr .;cored for the percentage of black spores ejected, and progeny were isolated either t o a niinirnal medium containing sorbose (BROCKMAN and DESERRES 1963) or to a minimal medium containing sorbose plus 50 nig/liter acriflavin. I n crosses without acr-3, 30 arg-5+ iZv+ progeny were isolated, arid in crosses with acr-3, 20 arg-5+ ilv' progeny were isolated. These numbers of isolates provide for sampling error and some nonviable progeny and still give >99% certainty of i.ecovering any recrssive diplophase mutants located on linkage groups 11-VI1 and on the portions of linkage group I not tightly linked to mating type. Mutants near mating type, e.g., mei-3, will usually not be detected by this technique, because only progeny that receive the mating-type allele o f the balancer parent can be trackcrossed to the wild parent.

All of the progeny were c-rossed with jl' standard strains of the same mating type as the wild- collrc-red pretnt; the jl' standard was used as the feniale parent. These highly fertile, aconidiate

4633 and 4634) is given elsewhere (LESLIE 1985).

RECESSIVES

Balancer x N N . CRASSA POPULATIONS

Wild-Collected Isolate

763

minimal sorbose medium + acrif lavin

Pick survivors

Laboratory Standard

Wild Parent

X

r - 7 Note discrepancies such as:

<90% black spores Few perithecia Partially or totally barren perithecia Abnormal spore morphology

FIGURE 1 .-Procedure for identifying recessive genes in wild-collected isolates. Each isolate is crossed to the balancer. Enough ascospores are isolated to provide 10 or 20 acriflavin-resistant prototrophic progeny. (Since acr-3 is closely linked to mating type, these progeny are mostly of opposite mating type to the wild parent.) Each of the F1 progeny is backcrossed to the wild parent and to a standard laboratory tester. Abnormalities in the backcross, as compared to the cross with the laboratory standard, signal a recessive mutant that affects the sexual phase.

strains are isogenic with the standard Oak Ridge laboratory wild-type strains (PERKINS et al. 1982). Compatible progeny were backcrossed to the wild-collected parent, which was used as the female parent. A difference between the cross to the j‘ standard and the cross to the wild-collected parent indicated the presence of the same recessive diplophase mutation(s) in both the wild- collected parent and in the progeny. Both of the crosses with each of the tested progeny were scored initially by J.F.L. for abnormal spore morphology and for the percentage of black spores ejected. When the crosses were both fertile and identical, no further examination was made. When the crosses were different, N.B.R. made preliminary cytological examinations and provisionally assigned the isolates to one of the classes listed below. All of the isolates except those classified as “male-barren” and “ascospore maturation” were reexamined cytologically before final class assign- ments were made. In isolates where more than one recessive diplophase mutation was recovered, the mutants were distinguished on the basis of their cytological phenotypes (see Table 2). This procedure will underestimate the number of mutants in the population since different mutations in a strain may be classed as a single mutation, either because of their similar cytological phenotypes or because the late effects of one mutation cannot be observed against the early effects of another.

Complementation tests: Complementation tests were made with progeny from the crosses of BLNC- 1 X wild-collected isolate. Crosses for complementation tests were made using the protocol described above on unsupplemented Synthetic Crossing medium. N o preference was shown for which isolate served as the male parent and which as the female parent.

RESULTS

Phenotype and frequency of mutants: The wild-collected isolates and the mutants recovered from them are listed in Table 2. The 106 mutants which we

764 J. F. LESLIE AND N . B. RAJU

TABLE 2

Recessive diplophase mutants isolated from wild-collected isolates of Neurospora crassa

~~

Mutant class (number of mutants)

Early ascus abortion (25) Defect prior to the end of meiosis 11; may delimit a few spores of ir- regular shape or size

Late ascus abortion (18) Usually delimits eight spores per ascus, but most of the spores abort shortly after delimitation

Ascospore maturation (44) Deliniits eight spores per ascus but 50% or less blacken normally, no rearrangement-associated ascus patterns

Ascospore color (2) Delimits eight spores, 4 black: 4 white; no other ascus patterns

Perithecial development (8) The perithecium cannot release the available ascospores; frequently perithecia are not full size and have no beaks or abnormal beaks; the defect may be caused by in- complete ascus development or by a gene(s) unrelated to ascus formation and maturation

Male barren ( 3 ) Conidia do not function as fertiliz- ing parent; female fertility is not affected

Mutant and origin

JL104(P498), JLI 5YtC(P499), JLI 53"(P501), JL155(P502), JL161(P506), JL164(P515), JL175(P527), JLI 79(P529), JL182(P532), JL187(P350), JL193(P1121), J1,200(P512), JL202(P522), JL204(P525), JL208(P828), JL209(P538), JL213(P349), JL216M(P1105), JL217'(P833), JL2 18'(P833), JL223(P841), JL226(P839), JL234(P858), JL23gb(P869), JL245'(P880)

JL157(P503), JL163(P513), JL177(P528), JL184'(P682), JL185(P682), JL188'(P350), JL190a(P676), JL192(P676), JL206'(P445), JL214"(P822), JL2 15'(P825), JL224'(P841), JL225(P842), JL233'(P853), JL235(P867), JL250(P 1352), JL26 1 (P 1463), JL262(P1465)

JL146(P497), JL147(P497), JL149(P474), JL150(P491), JLI 56(P502), JL158'(P504), JLl65(P518), JL166(P519), JL 169(P52 I ) , JL 17 l"(P523), JL 176(P528), JL 180(P53 l), JLI 8l(P530), JL183'(P682), JL194(P112 l), JLI95"(PI 121), JL196(P1117), JL198(P441), JL205'(P524), JL206M(P445), JL207'(P828), JL210(P1120), JL211(P680), JL212(P349), JL219(P836), JL220(P836), JL227(P839), JL228(P839), JL229(P839), JL23O(P845), JL236(P872), JL237(P873), JL238(P879), JL242(P868), JL244(P880), JL246MC(P881), JL248'(P882), JL249"(P884), JL25 I(P1441), JL256(P1445), JL258(PI417), JL259(P1462), JL263(P1466), JL264(P1470)

JL240(P869), JL253(P1443)

JL186"(P682), JL189(P438), JL197(PI 117), JL203(P522), JL210M(P1120), JL216(P1105), JL232"(P853), JL241(P868)

JL15lC(P492), JL.16OC(P504), JL1 70'(P523)

RECESSIVES IN N . CRASSA POPULATIONS 765

TABLE 2-Continued

Mutant class (number of mutants) Mutant and origin

Ascospore size (3) JL144(P493), JL178(P529), JL254(P1443) Delimits more or less than eight spores per ascus; some spores may be 2-4 times larger or smaller than normal; the larger spores are usually black

Ascospore shape ( 3 ) JL222(P838), JL246(P881), JL257(P1460) Spores are round or roundish, with less pointed ends than normal

a Partial or complete dominant. “Synthetic”-two or more genes from different parents required for the expression of this trait.

Precrozier block. Also displays a minor morphological phenotype whose genetic basis has not been determined.

recovered were grouped into eight major classes-early ascus abortion, late ascus abortion, ascospore maturation, ascospore color, perithecial development, male-barren, ascospore size and ascospore shape. Brief descriptions of the mutant classes are given in Table 2. (For an illustrated description of normal meiosis and ascospore genesis see RAJU 1980.) The first six classes were detected on the basis of low fertility or differences in the percentage of black spores ejected between crosses of the tested isolate with the wild-collected parent and the laboratory standard. The last two classes were identified on the basis of abnormal spore morphology. The early ascus abortion and late ascus abortion classes have been examined cytologically and the results will be reported elsewhere (N. B. RAJU and J. F. LESLIE, unpublished results). One leaky thiamine auxotroph was detected in strain P1105 from Lankala Koderu, India.

Some of the mutants (e.g., JL152 and JL213) are denoted as dominants or partial dominants. These mutants had similar significant effects in crosses with both the laboratory standard strain and their wild-collected parent. Since both of the parents of these strains were fertile, we assume that these phenotypes are due to the recombination of genes in the original cross of wild-collected parent with balancer and that these phenotypes are the result of the interactions between alleles at two or more loci and are not due to single dominant genes. These results suggest that the laboratory standard strains carry alleles at some loci that can reduce fertility when these alleles are placed in the proper genetic background or combined with a suitable allele(s) at the same or other loci. The male-barren class of mutants may also be viewed as synthetic because all of the wild-collected isolates will function as male parents.

T o test for cytoplasmic effects, 15 mutants (five early ascus abortion, five late ascus abortion and five spore maturation) were crossed reciprocally with their wild-type parents. Each strain served as the female parent in one of the two crosses and as the male parent in the other cross. In all 15 cases, no significant differences in phenotypes between the two crosses were detected.

766 J. F. LESLIE ANI) N. R . RAJU

Complementation tests: Two different criteria for complementation may be employed in this context. T h e first compares the fertility of strain 1 “selfed” (wild-isolate crossed with F1 progeny from BLNC-1 X wild isolate) and the fertility of strain 2 “selfed” with the fertility of the intercross of strains 1 and 2. If the intercross proved more fertile than either strain was when selfed, then the two strains were said to complement one another. T h e second criterion compares the fertility of strain 1 crossed with a laboratory standard, and similarly that of strain 2, with the fertility of the intercross of strains 1 and 2. I f the intercross was less fertile than either of the crosses with the laboratory standards, then the two strains were said to not complement one another.

Of the 4005 possible complementation tests (excluding dominants, partial dominants, male-barrens and ascospore color, shape and size mutants), approximately 30% ( I 206) were made (see Figure 2). T h e intercrosses made using mutants in the early ascus abortion, late ascus abortion and perithecial development classes were all fertile. Even progeny with very similar cytological phenotypes, e.g., JL 157 and JL16 1, produced fertile perithecia when they were intercrossed. Thus, using the first criterion for complementation, all of the tested pairs of strains were complementary.

Using the second criterion for complementation given above, 182 of the 1206 tested pairs were noncomplementary; in Figure 2 these pairs are denoted as displaying partial complementation. This partial complementation is not a randomly distributed phenomenon, however. Four strains-JL185, JL189, JL 192, and JL 193-participated in 103 of the 182 partially complementary pairings.

Partial complementation between strains as described here could result from several different unrelated phenomena: (1) Interactions between allelic mutations which are not completely recessive; such interactions are what is usually meant by the term “partial complementation.” (2) Inter- and intra-locus interactions between minor genes in the genetic backgrounds of the two mutant strains which affect fertility in a quantitative rather than a qualitative manner. T h e partial complementation of the strains originating in India is probably best explained by this type of minor gene interaction. (3) Partial complementation could result if a chromosome rearrangement from the balancer went undetected in one of the strains being tested, resulting in the production of fewer viable black spores than would normally be expected. This type of partial complementation, an artifact of the mutant recovery process, could be elimi- nated by reisolating the mutants in question from crosses where chromosomal rearrangements are not involved.

DISCUSSION

I n this study w e have systematically examined natural populations of N. crassa for recessive diplophase mutations. We detected a large number of such mutations with diverse phenotypes at a large number of genetically distinct loci. These results will first be discussed in relation to previous work with fungi and other organisms. Then some of the implications of the work will be examined as they relate to the strategy for collecting N . crassa from natural


JL 1 4 6 j1147 j1155 JL 1 5 6 j1157 JL 1 5 8 j1161 j1164 j1165 j1166 j1169 j1171 j1175 j1209 JL2 1 0 JL21OM j1211 JL2 1 2 j1214 j1215 JL2 16 JL2 16M JL2 1 9 j1220 j1223 j1224 j1226 j1227 j1228 j1229 j1230 j1232 j1233 j1236 j1237 j1239 j1248 j1249 j1250 JL25 1 j1254 j1256 j1258 j1259 JL26 1 j1262 j1263

5 1 6 0 ~ ~ 0 h 0 ~ ~ ~ Q 0 0 ~ ~ 6 0 ~ 6 ~ 0 0 Q ~ Q 6 ~ 6 ~ 0 0 0 ~ ~ 0 h h h Q Q ~ Q Q 0 0 0 0 0 0 0 0 0 0 0 ~ ~ ~ ~ 6 ~ 6 6 r r r r r r r r r r r r r r r r ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

7 ~ ~ ~ 7 ~ 7 ~ ~ ~ 7 ~ 7 ~ ~ ~ ~ ~ 7 7 ~ 7 7 ~ ~ ~ ~ 7 7 ~ 7

FIGURE 2.-Cotnplementation tests of recessive diplophase mutants isolated from wild-collected Neurospora crassa. Of a total of 4005 possible tests, 1024 showed complete complementation H by both criteria 1 and 2, as defined in the text, and 182 showed partial complementation 0, i .e . , by criteria 1 only. 0 represents the tests not done. All of the tested pairs complemented as assessed by criteria 1. a isolates, listed vertically, were crossed with A isolates listed horizontally. Numbers identify JI. mutants listed in Table 2.


sources, to the demographics and evolution of natural Neurospora populations and to the general development of the field of fungal population genetics.

Relationship of this work to previous work

Many organisms have loci which are phase-specific in their expression. In the present case (and in most fungi), some genes are expressed in the diplophase but not in the haplophase. These genes should be subject to the same types of selection pressure as are genes in organisms without a protracted haplophase. In both ferns and the Hymenoptera, some or all of the members of the population will spend a portion of the life cycle as haploids. It is of interest, then, to compare representative data from these quite different systems to see what similarities exist as regards the diplophase-limited genes, even though meiotic mutants are known in numerous other organisms (BAKER et al. 1976).

Strict comparisons of the Neurospora system with diploid systems, such as Drosophila, are difficult because viability of the diploid and fertility of the diploid can be relatively easily separated in Drosophila but not in Neurospora. The mutants described in this study reduce the fertility of the strains which carry them to the extent that homozygotes produce fewer viable ascospores than do heterozygotes. If this reduction in ascospore production is not complete, as in the case of the spore maturation mutants, then the changes caused by these mutants in N. crassa are similar to the decreases in male and female fertility reported by TEMIN (1966) in D. melanogaster and by MARINKOVIC (1967) in D. pseudoobscura. If the reduction in fertility is complete and no viable ascospores are formed, then the distinction between reduced fertility and nonviability is very difficult to make. For example, consider a mutant which disrupts meiosis and prevents the division of the diploid nucleus. Should this mutant be considered a mutant blocking fertility, which it certainly would in any diploid organism, or should it instead be considered a mutant blocking viability since the diploid nucleus in Neurospora will divide only meiotically and not mitotically? We have treated these mutants as recessive lethals for theoretical purposes (see below), but great caution should be exercised in any attempt to draw strict comparisons in these areas between these fungi and many higher organisms.

Fungi: Wild-collected strains unable to complete the diplophase portion of the life cycle have been previously documented in several ascomycetes by different workers and have been noted because of their effects on ascus development and fertility. The observations were incidental to the main thrust of the studies, and the data were not used to study the genetic structure of the populations from which the isolates were obtained (e.g., HARPER 1905; DODGE 1934; DODGE and SEAVER 1938; WILFRED, FREDERICK and AUSTIN 1985).

NELSON and his colleagues studied wild-collected Cochliobolus strains with both qualitative and quantitative differences in fertility. They found strains that differed qualitatively in their ability to form perithecia (NELSON 1959d, 1964, 1970; WEBSTER and NELSON 1968), mutants that affect perithecial development (NELSON 1 9 5 9 ~ ) and mutants that resulted in numerous perithecia

RECESSIVES I N N . CRASSA POPULATIONS 769

with sterile asci (NELSON 1959b, 19’70; WEBSTER and NELSON 1968). Quanti- tative differences were also observed in the number of perithecia with viable ascospores and in the number of viable ascospores per perithecium (NELSON 1959a; NELSON and KLINE 1964a).

We were surprised by the relatively large number of recessive mutations affecting the diplophase portion of the Neurospora life cycle that our survey uncovered, because the work by NELSON and his colleagues suggested that most of the genetic variability was of a quantitative (multiple minor loci) and not a qualitative (major locus) nature. That quantitative effects are probably important in determining Neurospora fertility is supported by our “partial complementation” results. These multiple locus effects have been muted, however, by crossing the wild-collected isolates with the balancer and then looking for effects in backcrosses with the wild-collected isolates and their progeny rather than simply intercrossing the wild-collected isolates as NELSON and his co-workers did. Mutations of the type we detected in this study would be detected only infrequently if we had simply intercrossed the wild-collected isolates with one another, because most of the mutant alleles are present only once in our sample. Our results indicate that the intercrossing procedure is not a good technique to use to detect recessive lethals in natural fungal populations.

Studies with Neurospora have identified many loci that affect UV-sensitivity or chemical mutagen-sensitivity and also impair meiosis ($ RAJU and PERKINS 1978; NEWMEYER and GALEAZZI 1978; DELANCE and GRIFFITHS 1980a,b; KA- FER and PERLMUTTER 1980; KAFER 1981; DELANCE and MISHRA 1981, 1982; PERKINS et al. 1982; SCHROEDER and OLSON 1983; INOUE and ISHII 1984). The relationship of these laboratory-induced mutations to the mutants recovered from the wild-collected isolates has not been established, but should be most interesting. The “dominant” and “partially dominant” mutants detected in the present study could result from a phenomenon similar to hybrid dysgenesis in Drosophila (SVED 1979). Estimation of the number of minor loci affecting the diplophase may also be possible (CHOVNICK and Fox 1953).

Ferns: In the fern genus Osmusda, KLEKOWSKI and his colleagues have identified numerous recessive diplophase mutants (KLEKOWSKI 1973, 1976a,b, 1982). They have related the genetic load generated by these mutations to the life-style and ecological limits of the organism. As in Neurospora (see below), numerous recessive diplophase mutations are present in natural populations, even though these ferns, unlike N. crassa, are self-fertile. In the fern genus Bommeria, self-fertile isolates are rare and outbreeding is usually required for the production of the sporophyte (HAUFLER and GASTONY 1978). One means by which such a situation could arise would occur if the population had ac- quired so many recessive diplophase mutations that virtually every isolate carried at least one such mutation and was, thus, self-sterile.

KLEKOWSKI and his colleagues have also used changes in the frequencies of recessive diplophase mutations to calculate the genetic load imposed on some fern populations by chemical pollutants (KLEKOWSKI 1982; KLEKOWSKI and KLEKOWSKI 1982; KLEKOWSKI and POPPEL 1976). Similar studies with N. crassa


should be possible in the southern United States, Hawaii, Puerto Rico and other moist tropical and semitropical regions.

Hymenoptera: In these insects, females are normally diploid and males are normally haploid. Both naturally occurring and laboratory-induced sex-limited mutations have been described in these insects (cJ KERR 1974, 1976; SAUL et al. 1965). In Nasonia (also cited in the literature as Mormoniella), there are many genes that cause sterility in homozygous females but do not affect fertility in hemizygous males (SAUL et al. 1965). In Apis mellifera, 20-40% of the total number of genes have been identified as female sex-limited genes (KERR 1974), although at least some of these genes also affect artificially produced diploid drones (WOYKE 1963, 1969; KERR 1974). Although not strictly analogous to the fern or fungal systems, these results do suggest that the accumulation of mutations with minimal effects in the haplophase but with substantial delete- rious effects in the diplophase is common whenever both the haplophase and the diplophase are each a significant portion of the organism’s life cycle.

Strategy for collection of additional N. crassa isolates The distribution of recessive diplophase mutants in the available samples

suggests that the strategy for collecting wild Neurospora should be altered for at least a few local populations. D. D. PERKINS’ present collection procedure has been to sample seven to ten well-isolated colonies from each local collection site. This procedure works well for the initial characterization of populations from a large number of geographic locations when the detection of rare alleles is not a primary objective. In the case of the recessive diplophase mutants examined here, however, mutations derived from the available isolates suggest great diversity both within and between local populations. It appears that most of the nonfunctional alleles at these loci are rare. Further studies of these mutants could best be made by the collection of a relatively large sample (about 100) from each of several sites in Texas and Louisiana, where N . crassa is abundant and recessive diplophase mutants are common, and might provide the best base from which to study the relative frequency of these mutants and their maintenance in a natural population.

Mutation rate, genetic load and mating structure in natural N. crassa populations The relatively large number of recessive diplophase mutants detected in this

survey provides an opportunity to use conventional population genetic techniques to estimate the mutation rate and genetic load in natural N . crassa populations. Since the wild-collected isolates we examined were collected fresh from burned substrate in nature and were preserved in suspended animation on silica gel shortly after collection, the mutations we have detected are pre- sumably present in the natural population and are not artifacts that accumu- lated during laboratory storage.

The frequency of the recessive diplophase mutations can be used to estimate the number of loci affecting the diplophase. If the frequency of recessive alleles at the ith locus is q. then the probability of a tested strain carrying a recessive allele at that locus is simply q, and the probability for not carrying the recessive

RECESSIVES IN N . CRASSA POPULATIONS 77 1

allele is 1 - qt. Assuming that the recessive alleles at the different loci are scattered at random throughout the genome, then the probability, P, that a tested strain carries no recessive diplophase mutations is P = Il (1 - ql), or ln(P) = Cln(1 - ql ) 5 - Est. Of the 99 strains listed in Table 1, 25 carried no detectable recessive allele at a locus affecting the diplophase, so P = 0.2525 and ln(P) = -1.3763. Assuming that mating is random and the mutation rate, p, is the same for each of the i loci, then qz = 4~ and N J ~ = Cqt, where N is the total number of loci. Assuming p = per locus per sexual generation (AUERBACH 1976), then N = 435 loci. This number is a lower estimate for the number of loci involved since the balancer will not render the portion of the genome near the mating-type locus homozygous in a backcross with the wild- collected parent, because only loci with easily recognized phenotypes will be identified and because the calculations assume that each recessive allele is a complete lethal, which many of the isolated mutants, e.g., spore maturation mutants, are not. The number of loci estimated in this manner is consistent with the number of loci (1000) estimated by TIMBERLAKE et al. (1985) to be involved in conidiation in Aspergallus nidulans.

The Ala mating-type system requires a disassortative mating structure, i .e. , individuals must cross with unlike partners, in Neurospora populations. The recessive diplophase mutations reduce the number of cross-fertile available partners even further. The closest mathematical homologs to such a system are the pollen incompatibility systems in higher plants (FINNEY 1952; MORAN 1962) and the tetrapolar mating systems of higher basidiomycetes (BURNETT 1975). When compared with either of these systems however, two differences can be noted: (1) the N. crassa system (composed of a mating-type locus and the recessive diplophase loci) involves many more loci (>50) than the other systems (one to four) and (2) there is no evidence (from limited data) for more than two alleles at the N. crassa loci, whereas the other systems have many alleles at each locus. The A l a Neurospora mating types (and the tetrapolar basidiomycete mating systems) act similarly to the pollen eliminator genes of higher plants and impose a mating structure on the population, i.e., fertile diploids are heterozygous at the locus (loci) in question. The recessive diplophase mutants will act as zygote eliminators, since fertility is unaffected until after the diploid has been formed, and will impose a load in the sense that for each infertile perithecium the organism has wasted the resources normally used for the production of a fertile perithecium.

The mathematical analysis of disassortively mating populations is complex (FINNEY 1952; MORAN 1962; CROW and KIMURA 1970), but some generaliza- tions can be made. First a multiple-locus system with both mating-type and recessive diplophase loci will generate many more types of progeny (2N, where N = the number of loci segregating) from a single cross than will a mating- type system alone [four or perhaps as many as 16 with recombination in a system such as Schizophyllum commune (RAPER 1966)l. Thus, the possible number of types of progeny from a Neurospora cross is potentially quite large. In a typical Neurospora cross, however, only mating-type and two or three recessive diplophase loci will be heterozygous, and eight to 16 classes of progeny


with respect to both mating-type and the recessive diplophase loci will be produced. This typical set of progeny classes is very similar in inbreeding potential to the progeny classes produced from a single cross of a tetrapolar basidiomycete. The analog; to a tetrapolar system is weakened whenever a population rather than a single cross is considered, because mating-type loci are important in every cross, whereas recessive diplophase loci are important only when one of these loci is homozygous for the recessive allele. Thus, any single locus’s importance in a population framework is related to the frequency of the nonfunctional recessive allele. The accumulation of recessive diplophase mutations within natural N. crassa populations suggests that the “realized load” of these mutations is low and that outbreeding is the rule for this species in nature. If placed in a situation in which inbreeding was necessary, however, we would expect that a large fraction of the matings would have reduced fertility until the system was purged of the detrimental recessive diplophase alleles.

Implications f o r fungal population genetics

Work in fungal population genetics is spotty (CJ: BURNETT 1975 for a general discussion), but has many uses. Mating types and vegetative incompatibility loci are typically well-studied (e.g., RAPER 1966; FINCHAM, DAY and RADFORD 1979; CROFT and JINKS 1977; MYLYK 1976; PUHALLA and SPIETH 1985) and detection techniques are readily available (CJ: RAPER 1966; MYLYK 1975; MEIN- HARDT and LESLIE 1982; DALES and CROFT 1983). Isozyme studies have been used to trace the origin and follow the evolution of pathogen populations (e.g., BURDON et al. 1982) and to examine general population polymorphisms (e.g., SPIETH 1975); results from other haploid populations are similar (e.g., liver- worts, KRZAKOWA and SZWEYKOWSKI 1979). Naturally occurring heterogeneity in fungi has been documented for auxotrophs (e.g., WHELAN and MAGEE 198 l) , developmental abnormalities (e.g., LESLIE and LEONARD 1980), fungicide re- sistance (e.g., GRINDLE 198 l), pathogenicity (e.g., NELSON and KLINE I964b; MICHELMORE and INGRAM 1981; HILL and NELSON 1983; GROTH and ROELFS 1982), recombination frequency (e.g., FROST 1961; SIMCHEN and CONNOLLY 1968), restriction fragment length polymorphisms (e.g., METZENBERC et aE. 1984) and growth rate (e.g., JINKS et al. 1966; SIMCHEN 1966; MCDONALD and ANDREWS 1982).

Fungal populations may serve as a source of alleles not readily generated in the laboratory. For example, GRINDLE (1 979) reported a difference between mutants segregating from natural isolates of Botrytis cinerea and those he could induce in the laboratory. The existence of the partially and totally dominant synthetic mutants detected in this study suggests that the concept of multiple loci affecting a given trait such as fertility in a cooperative but qualitative manner [similar to the complicated system with haploid fruiting in Schizophyl- lum commune (LESLIE and LEONARD 1979a,b; ESSER, SALEH and MEINHARDT 1979)] is more common than was originally supposed. Detection of such gene sets is difficult, however, and their analysis is laborious and time-consuming.


Natural populations of S. commune are highly polymorphic for such traits (LES- LIE and LEONARD 1980).

The present work unequivocally documents the existence of “recessive lethal” genes in fungal populations and provides a class of mutations which can be used to determine important population parameters, such as genetic load and mating structure. The rarity of individual mutations in combination with other naturally occurring markers, such as isozyme polymorphisms, DNA restriction fragment length polymorphisms and heterokaryon incompatibility genotypes, should permit the identification of individual fungal isolates. Such identification could be useful for the study of the survival and dispersal of fungal vegetative clones and in estimating the importance of sexual reproduction in the organism’s life cycle. Application of such identification techniques to an economically important fungus, e.g., a plant pathogen, could aid in the study of disease epidemiology and in the detection of new fungal races.

This work was supported by United States Public Health Service Research Grant AI-01462. We thank D. D. PERKINS, P. T. SPIETH, M. SIMMONS, and J. B. ANDERSON for critically reading the manuscript. This is contribution 85-459-5 from the Department of Plant Pathology, Kansas Agricultural Experiment Station, Kansas State University, Manhattan.

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Communicating editor: R. L. METZENBERC

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