Ascus development in two temperature-sensitive four-spore mutants of Neurospora crassa

Two nonallelic Four-spore mutants are known in which ascospore walls enclose the four immediate products of meiosis rather than the normal eight products of a postmeiotic mitosis. Expression depends on temperature. The Four-spore phenotype is expressed when the developing asci are subjected either to high temperatures (25-30 degrees C) for Fsp-1 or to low temperatures (15-20 degrees C) for Fsp-2. Heterozygous Fsp-1 X Fsp-1+ crosses make eight-spored asci at 15-20 degrees C but produce many four-spored asci at 25 degrees C and mostly four-spored asci at 30 degrees C. Homozygous Fsp-1 X Fsp-1 crosses respond similarly to increasing temperature but make 40-50% four-spored asci even at 20 degrees C. Heterozygous Fsp-2 X Fsp-2+ crosses produce almost exclusively four-spored asci at 15 degrees C but a mixture of four- and eight-spored asci at 20, 25, and 30 degrees C. Homozygous Fsp-2 X Fsp-2 crosses make all four-spored asci at 15 and 20 degrees C and a mixture of four- and eight-spored asci at 25 and 30 degrees C. When both Fsp-1 and Fsp-2 are present in a cross, either homozygous or heterozygous, no asci contain more than four ascospores at any temperature. Limited temperature-shift experiments with Fsp-1 and Fsp-2 show that the sensitive period for Four-spore expression is sometime after meiotic prophase, possibly at interphase II.     

An insertional translocation in neurospora that generates duplications heterozygous for mating type

In strain T(I-->II)39311 a long interstitial segment is transposed from IL to IIR, where it is inserted in reversed order with respect to the centromere. In crosses of T x T essentially all asci have eight viable, black spores, and all progeny are phenotypically normal. When T(I-->II)39311 is crossed by Normal sequence (N), the expected duplication class is viable while the corresponding deficiency is lethal; 44% of the asci have 8 Black (viable) spores and 0 White (inviable) spores, 41% have 4 Black: 4 White, and 10% have 6 Black: 2 White. These are the ascus types expected from normal centromere disjunction without crossing over (8B:0W and 4B:4W equally probable), and with crossing over between centromere and break point (6B:2W). On germination, 8B:0W asci give rise to only parental types-4 T and 4 N; 4B:4W asci usually give four duplication (Dup) progeny; and 6B:2W asci usually give 2 T, 2 N, 2 Dup. Thus one third of all viable, black ascospores contain duplications.-Recessive markers in the donor chromosome which contributes the translocated segment can be mapped by duplication coverage. Ratios of 2 Dominant: 1 Recessive vs. 1 Dominant: 2 Recessive distinguish location in or outside the transposed segment. Eleven loci including mating type have been shown to lie within the segment, and markers at four loci have been transferred into the segment by meiotic recombination. The frequency of marker transfer indicates that the inserted segment usually pairs with its homologue. Ascus types that would result from single exchanges within the insertion are infrequent, as expected if asci containing dicentric bridges usually do not survive.-Duplication ascospores germinate to produce distinctive inhibited colonies. Later these "escape" to grow like wild type, and genes that were initially heterozygous in the duplication segregate when escape occurs. As with duplications from pericentric inversion In(IL-->IR)H4250 (Newmeyer and Taylor 1967), the initial inhibition is attributed to mating-type heterozygosity, and escape to a somatic event that makes mating type homoor hemizygous.-Twenty additional duplication-generating Neurospora rearrangements are listed and described briefly in an Appendix.     

Most Uv-Induced Reciprocal Translocations in SORDARIA MACROSPORA Occur in or near Centromere Regions

In fungi, translocations can be identified and classified by the patterns of ascospore abortion in asci from crosses of rearrangement x normal sequence. Previous studies of UV-induced rearrangements in Sordaria macrospora revealed that a major class (called type III) appeared to be reciprocal translocations that were anomalous in producing an unexpected class of asci with four aborted ascospores in bbbbaaaa linear sequence (b = black; a = abortive). The present study shows that the anomalous type III rearrangements are, in fact, reciprocal translocations having both breakpoints within or adjacent to centromeres and that bbbbaaaa asci result from 3:1 disjunction from the translocation quadrivalent.-Electron microscopic observations of synaptonemal complexes enable centromeres to be visualized. Lengths of synaptonemal complexes lateral elements in translocation quadrivalents accurately reflect chromosome arm lengths, enabling breakpoints to be located reliably in centromere regions. All genetic data are consistent with the behavior expected of translocations with breakpoints at centromeres.-Two-thirds of the UV-induced reciprocal translocations are of this type. Certain centromere regions are involved preferentially. Among 73 type-III translocations, there were but 13 of the 21 possible chromosome combinations and 20 of the 42 possible combinations of chromosome arms.     

Preferential Ascus Discharge during Cross Maturation in SORDARIA BREVICOLLIS

Crosses involving spore color mutants of Sordaria brevicollis all showed a decline in the frequency of second division asymmetric asci (2:2:2:2's) as the cross matured. This decline was due to the preferential maturation and/or discharge of these asci. The proportion of spindle overlap and recombinational asci within the group did not change as shown by ascus dissection. The preferential discharge was also found to occur in two-point crosses where the asci did not contain wild-type spores.     

A Putative Rhamnogalacturonase Required for Sexual Development of Neurospora Crassa

In previous work, the asd-1 (ascus development) gene of the filamentous fungus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-1 gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-1 protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-1 crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-1 asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-1 protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.     

New CMS types in Plantago lanceolata and their relatedness

 Mitochondrial variation in Plantago lanceolata was used to detect new CMS types. Directional reciprocal crosses were made between plants which differed in mtDNA restriction patterns. Differential segregation of male steriles in reciprocal crosses indicated that the parents differed in CMS type. MtDNA variation revealed nine RFLP patterns, which could be categorised according to the sex phenotype of the plants as MS1 (brown-anther type), MS2 (petaloid flower type) and MS3 (more yellow anthers than MS1). A single mtDNA pattern was found within MS1, six mtDNA patterns were found within the MS2 group, and two mtDNA patterns were found within hermaphrodites which segregated MS3 in the crosses. MS1 and MS2 are known to represent different CMS types, CMSI and CMSII. In reciprocal crosses between plants with different mtDNA patterns within the MS2 group, different ratios of male steriles segregated in the crosses, indicating that the parents differed in CMS type. Within the MS2 group two CMS types were found, designated CMSIIa and b. Finally, the sex phenotype H/MS3 turned out to be a different type. From previous studies it was known that the MS3 phenotype can also occur in CMSI and CMSII types, hence it is unclear whether MS3 is diagnostic for CMSIII. Since the data in this study cannot distinguish between the new type being a fully restored new CMS type or a ‘Normal’ cytoplasm, it was denoted as CMSIII. In total, four CMS types were found in the material studied. CtDNA variation was screened and three chloroplast haplotypes were identified. Two haplotypes were associated with CMSI plants, and one haplotype with the other CMS types. The ctDNA variation indicated that the CMSI type is widespread within the species, due to migration rather than to recurrent mutation. This may lead to the conclusion that only a limited number of CMS types are successful. Received: 9 August 1996 / Accepted: 6 September 1996     

Genetics of rough seed coat texture in cowpea

Seed coat texture is an important trait in determining the acceptability of cowpea varieties in different regions. A rough seed coat is preferred in western and central Africa, since it permits easy removal of the seed coat which is essential for indigenous food preparations. On the other hand, a smooth seed coat is preferred in eastern and southern Africa as well as in parts of South America where cowpea is consumed as boiled beans without removing the seed coats. This study was undertaken to elucidate the inheritance of seed coat texture so that cowpea breeders may adopt appropriate breeding strategy to develop cowpea varieties with preferred seed types for different regions. The F1 plants between smooth- and rough-seeded parents as well as between rough- and rough-seeded parents produced smooth seeds, indicating a complementary gene action and dominance for smooth seed coat. The F2 plants from the smooth x rough cross segregated into a 3 smooth:1 rough seed coat ratio, but the F2 plants from rough x rough crosses segregated into a 9 smooth:7 rough seed coat ratio. The F1 plants from backcross to the smooth parent were all smooth, while the F1 plants from backcross to rough parent segregated in a 1 smooth:1 rough seed coat ratio. However, both the backcross populations in rough x rough crosses segregated into 1 smooth:1 rough seed coat ratio. These results indicate that two pairs of independent recessive genes confer rough seed coat texture in cowpea and the presence of at least one dominant gene at each of the two loci results into smooth seed coat. The gene symbols rt1rt1 and rt2rt2 are being assigned for rough seed coat texture in cowpea.     

Genetic analysis of spore killing in the filamentous ascomycete Podospora anserina

In the present study, we analysed different Podospora anserina strains for their ability to induce spore killing and identified three new killer strains. Test crosses of killer strains with different sensitive strains revealed different second division segregation ratios suggesting an influence of the sensitive strain on the crossing-over frequency. In crosses of killer strain O with a sensitive strain, the frequency of two-spored asci was found to vary extremely from perithecium to perithecium. Furthermore, crosses of strain O with sensitive strain Us5 led to a significant proportion of asci containing an unexpected high number of surviving spores as the result of gene conversion. Finally, for the first time, we present data demonstrating that in a number of ascospores the killer and the corresponding sensitive allele is located in one individual nucleus. Mycelia derived from such ascospores display a "sensitive killer" phenotype. Crosses of these mycelia with a killer strain as well as with a sensitive strain result in spore killing. Strikingly, heterokaryotic spores containing the recombined "sensitive/killer" allele and a nucleus with a killer allele give rise to mycelia protected against spore killing during selfing.     

江苏省稻瘟病菌有性态的研究*

用标准菌株对１９９７～１９９９年在江苏省吴江市,宜兴市、通州市、高邮市和赣榆县采集的３２５个猪瘟病菌单孢分离菌株的可育性和交配型进行了测定,结果表明江苏省稻瘟病菌菌株的育性较低,可交配率为２２．７７％,可育率仅为７．０８％。不同年份、不同地区采集的猪瘟病菌茵株的性亲和力和交配型有较大的差异,三年的交配率分别为２６．６１％、８．２６％和３３．６４％;温州地区和赣榆地区菌株的交配率相对较高,分别为２６．１５％和２５．４２％,宜兴地区菌株的交配率较低,只有１５．３８％。江苏省稻瘟病菌菌株的交配型在不同年份亦出现很大差别,１９９７年２９个可交配菌株中有２１个菌株表现为ＭＡＴ１－２ 交配型,而１９９９年３６个可交配茵株均为ＭＡＴ１－１交配型。用江苏省稻瘟病菌的可育菌株进行互交,２５个组合中只有６个组合能产生子囊壳和子囊,但均不产生子囊孢子,提示江苏省稻瘟病菌在田间产生健康有性后代的几率不大。对杂交后代的遗传学分析表明,菌株的交配型是受单基因控制的。     

The intergradation, genetic interchangeability and interpretation of gene conversion spectrum types

In the Pasadena strains of Ascobolus immersus, the gene conversion propperties of 29 induced (nine UV, nine NG, and 11 ICR-170) and nine spontaneous white-ascospore mutations have been studied. Each mutant was crossed to three types of derived wild-type strains; single mutants often gave very different conversion results in the three types of crosses, with any or all of the following changes in: percentage with post-meiotic segregation among aberrant-ratio asci; percentage with conversion to wild type among aberrant-ratio asci; and in total conversion frequency. - These results are compared with those of Leblon (1972 a, b) from Ascobolus immersus and Yu-Sun, Wickramaratne and Whitehouse (1977) from Sordaria brevicollis. It is shown that conversion spectrum types are not necessarily distinct, but can completely intergrade, on the criteria of both post-meiotic segregation frequency and direction of correction. Genetic differences between strains in the present work resulted in much interchangeability of spectrum types for the same mutation in different crosses; e.g., from type C in one cross to type B/D type in another cross, although the mutation is presumably of the same molecular type (addition or deletion frame shift, or base substitution) in each cross. These changes of conversion properties for a given mutation in different crosses mean that previous interpretations of spectrum types in terms of specific conversion properties for various molecular types of mutation are inapplicable, or inadequate on their own, to explain the present data. Other factors, such as heterozygous cryptic mutations or conversion control genes, are probably involved. Because of asymmetric hybrid DNA formation, correction properties may differ from observed conversion properties.     

Gene conversion: point-mutation heterozygosities lower heteroduplex formation.

The effects of heterozygosity on meiotic gene conversion characteristics have been studied in the fungus Ascobolus immersus. The non-Mendelian segregation patterns of seven white ascospore mutants of the b2 gene were established in the presence or the absence of additional neighbouring allelic mutations. These correspond to nine different double mutants with wild-type or pseudo-wild-type phenotypes, constituted by two +1, -1 frameshift mutations of complementary phases. When heterozygous, these double point mutations decrease, by an average of one third, the gene conversion frequencies of the mutants located on their right, toward the low conversion end of the gene. The decrease corresponds either to a reduction in all classes of non-Mendelian segregation (6:2, 5:3 and aberrant 4:4 asci) or to a reduction restricted to the single class of aberrant 4:4 asci. These modifications are explained by changes in hybrid DNA parameter values: frequencies of formation and modalities of distribution (asymmetric versus symmetric ratio). Besides the nature of the non-homology, point mutation versus deletion, which leads to quantitative differential effects, the region where the non-homology is located within the gene also appears to play an important role.     

Expression of Meiotic Drive Elements Spore Killer-2 and Spore Killer-3 in Asci of Neurospora Tetrasperma

It was shown previously that when a chromosomal Spore killer factor is heterozygous in Neurospora species with eight-spored asci, the four sensitive ascospores in each ascus die and the four survivors are all killers. Sk-2K and Sk-3K are nonrecombining haplotypes that segregate with the centromere of linkage group III. No killing occurs when either one of these killers is homozygous, but each is sensitive to killing by the other in crosses of Sk-2K x Sk-3K. In the present study, Sk-2K and Sk-3K were transferred by recurrent backcrosses from the eight-spored species Neurospora crassa into Neurospora tetrasperma, a pseudohomothallic species which normally makes asci with four large spores, each heterokaryotic for mating type and for any other centromere-linked genes that are heterozygous in the cross. The action of Sk-2K and Sk-3K in N. tetrasperma is that predicted from their behavior in eight-spored species. A sensitive nucleus is protected from killing if it is enclosed in the same ascospore with a killer nucleus. Crosses of Sk-2K x Sk-2S, Sk-3K x Sk-3S, and Sk-sK x Sk-3K all produce four-spored asci that are wild type in appearance, with the ascospores heterokaryotic and viable. The Eight-spore gene E, which shows variable penetrance, was used to obtain N. tetrasperma asci in which two to eight spores are small and homokaryotic. When killer and sensitive alleles are segregating in the presence of E, only those ascospores that contain a killer allele survive. Half of the small ascospores are killed. In crosses of Sk-2K x Sk-3K (with E heterozygous), effectively all small ascospores are killed. The ability of N. tetrasperma to carry killer elements in cryptic condition suggests a possible role for Spore killers in the origin of pseudohomothallism, with adoption of the four-spored mode restoring ascospore viability of crosses in which killing would otherwise occur.     

Ascospore Dimorphism and Mating Type in Chromocrea spinulosa (Fuckel) Petch n. comb

The asci contain four large and four small ascospores, eachtwo-celled, arranged in the six patterns expected if spore sizewere controlled by a pair of allelic genes, the locus showing65 per cent, second-division segregation. The small ascosporesproduce sterile colonies, the large ones moderately fertilecolonies whose asci again show segregation for spore size. Fertilityis stimulated where colonies from large and small spores meet.In crosses of various mutants with the wild type, all asci inperithecia developed along the line of junction show segregationfor the mutant as well as for spore size. Evidently Chromocreais heterothallic, the spore-size difference being a pleiotropicexpression of mating type. One allele, that governing largespores, occasionally mutates to the other allele, resultingin fertility of colonies from large spores.     

Inheritance of a hair character in <Emphasis Type="Italic">Helianthemum oelandicum</Emphasis> var. <Emphasis Type="Italic">canescens</Emphasis> and allele frequencies in natural populations

Helianthemum oelandicum var. canescens (Hartm.) Fr. is an endemic taxon with a restricted distribution (less than 10 km²) in the southernmost part of the Baltic island of Öland, SE, Sweden. The taxon varies with respect to stellate hairs. Most plants can be classified into two morphs, the stellated morph (with a dense carpet of stellate hairs on the abaxial surface of the leaves) and the bristled morph (without a carpet of stellate hairs). In crosses between plants assumed to be homozygous for the trait that characterises the phenotypes of the two morphs, F1 offspring was indistinguishable from the bristled morph. Segregation in F2 did not deviate from the expected 3:1 ratio (bristled morph/stellated morph), indicating one major gene with a dominant allele for the phenotype of the bristled morph and a recessive allele for the phenotype of the stellated morph. Besides the Mendelian inheritance of the presence/absence of a whitish cover of stellate hairs, the density of hairs appeared to be further modified by quantitative genes. The frequency of the recessive allele for the phenotype of the stellated morph varied among populations and showed a geographical structure. Possible mechanisms behind the spatial variation in indumentum are discussed.     

Genetic recombination at the buff spore colour locus in Sordaria brevicollis

Summary Asci showing aberrant segregation at the buff spore colour locus in Sordaria brevicollis were selected from crosses between buff mutants and wild type in the presence of closely-linked flanking markers. The frequency of crossing-over associated with aberrant segregations was calculated and corrected to allow for crossovers between the flanking markers incidental to the aberrant segregation. The average frequency of crossing over was found to be related to the class of aberrant ascus studied. 5+:3m and 3+:5m asci showed 16% associated marker recombination while 6+:2m and 2+:6m asci showed 27% recombination. The frequency of tritype and tetratype postmeiotic segregation asci was calculated. Only 3% tetratypes were found and this is thought to indicate a low frequency of symmetric hybrid DNA formation.     

Randomness tests on the sequence of ascal segregation classes in Neurospora crassa.

Various statistical tests for randomness were made on the order of ascal classes in groups of asci from wild-type X asco crosses. There was no significant nonrandom clustering of asci of the same segregation class, nor a regular twinning of similar asci. Any apparent observed clustering of similar ascal classes is probably an artefact or due to chance. 2 x n X2 tests showed that frequencies of individual ascus classes from different perithecia were generally homogeneous, as were second division segregation frequencies. The tests described here for randomness in sequences of occurrences could be of general use in other areas of genetics.     

Mutations at the smo genetic locus affect the shape of diverse cell types in the rice blast fungus

Teflon film surfaces are highly conducive to the formation of infection structures (appressoria) in the plant pathogenic fungus, Magnaporthe grisea. We have utilized Teflon films to screen and select for mutants of M. grisea that are defective in appressorium formation. This approach and several others yielded a group of 14 mutants with a similar phenotype. All the mutant strains make abnormally shaped conidia and appressoria. When two mutant strains are crossed, abnormally shaped asci are formed. Ascus shape is normal when a mutant strain is crossed with a wild-type strain. Despite dramatic alterations in cell shape these strains otherwise grow, form conidia, undergo meiosis, and infect plants normally. This mutant phenotype, which we have termed Smo(-), for abnormal spore morphology, segregates in simple Mendelian fashion in crosses with wild-type strains. Some ascospore lethality is associated with smo mutations. In genetic crosses between mutants, smo mutations fail to recombine and do not demonstrate complementation of the abnormal ascus shape phenotype. We conclude that the smo mutations are alleles of a single genetic locus and are recessive with regard to the the ascus shape defect. Mutations at the SMO locus also permit germinating M. grisea conidia to differentiate appressoria on surfaces that are not normally conducive to infection structure formation. A number of spontaneous smo mutations have been recovered. The frequent occurrence of this mutation suggests that the SMO locus may be highly mutable.     

Premeiotic and Meiotic Instability Generates Numerous b2 Mutation Derivatives in Ascobolus

We have studied the genetic characteristics of an unstable mutation located in the central region of the b2 gene of the fungus Ascobolus. In crosses to wild type, this spontaneous white ascospore mutation (G0 ) gives rise to a stable white spored derivative (G1) at a frequency of 5 x 10(-3). G1 is a frameshift mutation and differs from G0 by its gene conversion pattern. In self crosses, G0 gives asci with colored spore derivatives at a frequency of 1 x 10(-3). We isolated and analyzed genetically 97 independent colored derivatives ("G2" series). All but one are pseudorevertants. By the criteria of phenotype and gene conversion pattern with wild type and with G1, the pseudorevertants represent at least 13 distinct classes. Two of them are large silent deletion mutations. In crosses with wild type, some G2 derivatives, represented by G21, continue to exhibit instability, G21 yields white spored b2 mutant derivatives at a frequency of 2.6 x 10(-3). In turn, some of these "G3" mutants are themselves unstable. All the derivatives lie at the same site within the b2 locus as the parental mutation G0 . Different mutations in the G series manifest their instability at different times in the Ascobolus life cycle. Derivatives of G0 arise premeiotically (leading to two derivative meiotic products among the four), while those of G21 arise during meiosis (leading to only one derivative out of four products). The characteristics of the G instability system are similar to those of unstable mutations in other eukaryotes which are due to insertion of mobile elements.     

Gene conversion spectrum of 15 mutants giving post-meiotic segregation in the b2 locus of Ascobolus immersus

Summary The conversion spectrum of fifteen mutants giving post-meiotic segregations and located in the b2 locus of Ascobolus immersus was studied in 77 mutant x wild-type crosses. These mutants all yield aberrant 4:4 asci, mutants located in the right portion of the locus yielding more aberrant 4:4 than left mutants. The basic frequency of conversion is higher in the left portion. The frequency of hybrid DNA, its symmetrical or asymmetrical distribution and the frequency of correction of the mismatch in hybrid DNA were estimated. The left region shows a higher frequency of hybrid DNA formation than the right region. The fraction of hybrid DNA with a symmetrical distribution tends to increase from left to right in the locus. The frequencies of mismatch correction show considerable variation from one mutant to another and have no relationship to their location. The implications of these observations on the molecular models of genetic recombination are discussed.     

Ribosomal DNA sequence polymorphism and the delineation of two ascosporic yeast species: Metschnikowia agaves and Starmerella bombicola

The relationship between mating success and sequence divergence in the internal transcribed spacer (ITS)/5.8S-D1/D2 rDNA region was examined in isolates tentatively assigned to Metschnikowia agaves and Starmerella bombicola. Both species are haplontic and heterothallic, such that the formation of mature asci can be used as a measure of genetic compatibility. Parsimony haplotype network analysis and mating success confirmed that all known isolates of M. agaves are conspecific. The previously reported D1/D2 polymorphism of five substitutions was not corroborated; the maximum divergence observed between any two strains was three substitutions, four with ITS. Of 39 putative S. bombicola strains, 36 formed an ITS-D1/D2 haplotype network using the 95% criterion. Thirty-five strains could mate with one or more compatible partner. The excluded strains did not mate. Mature asci arose from crosses between individuals differing by as many as five, but not six or seven substitutions in the D1/D2 domain. All strains capable of mating formed mature asci with at least one partner and all network members could be linked to another member by three or fewer substitutions. These results support the use of sequence divergence as a criterion for species delineation, but caution against describing poorly sampled species solely on the basis of that criterion.