Genetic analysis of the polyauxotrophy of the early mitotic progeny of Saccharomyces cerevisiae zygotes. III. The behavior of the chromosome-III markers in polyauxotrophic clones and their mitotic and meiotic segregants

Cloning and segregation analysis of polyauxotrophic (PA) progeny, as well as the study of diploid segregants, revealed an unusual state of the diploid genome in these strains. Analysis of linkage markers of chromosome III in all crosses may be expected to illuminate relationships between the homologous chromosomes. All PA strains were assigned to two major classes. In the class of PA strains with recombinant chromosome III, the effect of approachment of third linkage markers was noted. In other strains, transposition of genetic marker metA1 to chromosome III and its linkage to leu2 were discovered. The present study has demonstrated that the unusual state of the diploid genome of PA strains induces multiple nonspecific alterations in chromosome relationship and structure. It is likely that these alterations are the consequence of disturbance in mitotic apparatus of cell.     

Systems and results of tests for chemical induction of mitotic malsegregation and aneuploidy in Aspergillus nidulans

In Aspergillus several types of test systems have been developed for detection of chemicals which induce aneuploidy and/or malsegregation of chromosomes. Results from 23 papers were reviewed in which numerical data for 42 chemicals had been reported. The test systems fall into two groups. One group includes all purely genetic tests that detect euploid mitotic segregants from heterozygous diploids and identify these either as products of malsegregation of chromosomes or as products of crossing-over (13 papers, several reviewed in detail previously; K?fer et al. (1982) and Scott et al. (1982)). The other group includes tests that treat haploid or diploid strains and detect aneuploids as unstable abnormally growing segregants which can be identified as specific disomics or trisomics by their characteristic phenotypes. In addition, such tests characterize abnormal segregants from heterozygous diploids by correlating phenotypes with patterns of genetic segregation in spontaneous euploid sectors. This analysis makes it possible to distinguish between induced primary aneuploidy of whole chromosomes and partial tri- or monosomy resulting from chromosome breakage and secondary spontaneous malsegregation (10 papers). Based on results of both types of tests, it is postulated that chemicals which cause increases of euploid malsegregants, but not of crossovers, normally induce aneuploids as primary products (as shown for 7 of the 14 cases). These include compounds which damage spindles or membranes (especially the well-known haploidizing agents) and generally are effective only when growing cells are exposed. (8 chemicals that may belong in this category could not be classified for certain, because information was insufficient.) On the other hand, chemicals which cause increases of all types of euploid segregants (11 cases), mostly induce drastic mutations and aberrations as primary effects and cause spontaneous malsegregation or crossing-over only as secondary events (as demonstrated for radiation-induced abnormals). In addition, a few chemicals were negative, because they increased only crossing-over or showed no increased segregation at all at concentrations which reduced survival or growth rate (9 cases). Recommendations are made for standardization of methods and protocols. New tester strains and specific procedures are outlined which should be useful for conclusive tests of chemicals that may induce aneuploidy.     

Regulation of Mating and Meiosis in Yeast by the Mating-Type Region

A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rme enables the diploids a*/α, a/a*, and α/α to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains.     

Induced somatic segregation inArmillaria mellea diploids

Armillaria mellea is a bifactorially heterothallic fungus with a fertile, diploid, vegetative phase. While diploids of this fungus are readily recovered by nutritional selection from compatible and incompatible pairings of haploids, genetic analyses have been limited both because this organism does not produce fruiting bodies reliably in the laboratory and because somatic segregation occurs infrequently in diploid cultures. In this study, diploids ofA. mellea heterozygous at nutritional and mating-type marker loci were treated with formaldehyde, para-fluorophenylalanine, benomyl, and ultraviolet light in order to recover somatic segregants. Of these agents, only benomyl dramatically increased the frequency of somatic segregation under the conditions used. Auxotrophic segregants were recovered from macerates of prototrophic, diploid mycelia that had been grown in the presence of benomyl. Many of these segregants differed from their diploid progenitors in mating behavior as well as in nutritional phenotype. The development of a reliable method for the recovery of somatic segregants from diploids ofA. mellea permits parasexual analysis and reduces the need forin vitro production of fruiting bodies in future genetic studies.     

Inheritance of extrachromosomal rDNA in Physarum polycephalum.

In the acellular slime mold Physarum polycephalum, the several hundred genes coding for rRNA are located on linear extrachromosomal DNA molecules of a discrete size, 60 kilobases. Each molecule contains two genes that are arranged in a palindromic fashion and separated by a central spacer region. We investigated how rDNA is inherited after meiosis. Two Physarum amoebal strains, each with an rDNA recognizable by its restriction endonuclease cleavage pattern, were mated, the resulting diploid plasmodium was induced to sporulate, and haploid progeny clones were isolated from the germinated spores. The type of rDNA in each was analyzed by blotting hybridization, with cloned rDNA sequences used as probes. This analysis showed that rDNA was inherited in an all-or-nothing fashion; that is, progeny clones contained one or the other parental rDNA type, but not both. However, the rDNA did not segregate in a simple Mendelian way; one rDNA type was inherited more frequently than the other. The same rDNA type was also in excess in the diploid plasmodium before meiosis, and the relative proportions of the two rDNAs changed after continued plasmodial growth. The proportion of the two rDNA types in the population of progeny clones reflected the proportion in the parent plasmodium before meoisis. The rDNAs in many of the progeny clones contained specific deletions of some of the inverted repeat sequences at the central palindromic symmetry axis. To explain the pattern of inheritance of Physarum rDNA, we postulate that a single copy of rDNA is inserted into each spore or is selectively replicated after meiosis.     

Induction and Characterization of Artificial Diploids from the Haploid Yeast Torulaspora delbrueckii

The yeast Torulaspora delbrueckii, which propagates as a haploid, was made into a diploid by treatment with dimethyl sulfoxide (DMSO) on the regeneration of protoplasts. The diploid state was stably inherited; the cell volume was three times that of the parent strain and the cellular DNA content was two times that of the parental strain. No essential difference was found between diploids induced by DMSO and those formed through intraspecific protoplast fusion. The diploid strains sporulated fairly well, with their cells converting directly into asci. Random spore analysis revealed that diploids induced through protoplast fusion gave rise to auxotrophic segregants (haploids) with the parental genetic marker or to segregants formed by recombination, while diploids induced by DMSO from a doubly auxotrophic parent gave rise to no recombinant, indicating that it was chromosomally homoallelic in nature. The magnesium level in the protoplast regeneration medium was found to be an important factor for inducing diploid formation. At 0.2 mM magnesium diploids appeared even in the absence of DMSO, while at 2 mM magnesium diploids never appeared unless DMSO was added to the regeneration medium. Evidence is provided that the diploids induced by DMSO or a low magnesium level are due to direct diploidization but not protoplast fusion. UV light irradiation of intact cells (without protoplasts), 10% of which survived, also produced diploids among this surviving population. From these results we conclude that the perturbation of protoplast regeneration or of cell division by the treatments mentioned above somehow induced direct diploidization of T. delbrueckii.     

Single cross alfalfa (Medicago sativa L.) hybrids produced via 2n gametes and somatic chromosome doubling: experimental and theoretical comparisons

Summary The potential breeding value of 2n gametes from diploid alfalfa (2n = 2x = 16) was tested by comparing single cross alfalfa hybrids produced via 2n = 2x gametes from diploids versus n = 2x gametes from somatic-chromosome-doubled, tetraploid counterparts. Three diploid clones, designated 2x-(rprp), homozygous for the gene rp (conditions 2n gamete formation by a first division restitution mechanism) were colchicine-doubled to produce their tetraploid counterparts, designated 4x-(SCD). These six clones were crossed as males to the same cytoplasmic male sterile clone. Yield comparisons of progeny from the six clones demonstrated a significant yield increase of the hybrid progeny from 2n = 2x gametes from the diploids over the hybrid progeny from n = 2x gametes from the chromosome doubled tetraploid counterparts. The yield gain ranged from a 12% increase to a 32% increase. Theoretical comparisons indicated the 2n = 2x gametes from diploids would have 12.5 to 50% more heterozygous loci, on average, than the n = 2x gametes derived from somatic doubling. These results confirm the importance of heterozygosity on alfalfa yield, and the results demonstrate that 2n gametes formed by first division restitution offer a unique method for producing highly heterotic alfalfa hybrids.     

Genetic-cytoplasmic male sterility in progeny of 4x-2x crosses in cultivated potatoes

Summary Forty-three percent of the progeny from 4x × 2x crosses [Group Tuberosum cultivar x diploid hybrid (Groups Phureja or Stenotomum x Group Tuberosum haploid)] were male sterile. In contrast only four percent of the progeny from the reciprocal crosses were male sterile. Male sterility among the former progeny is presumed to result from the interaction of Tuberosum cytoplasm with dominant genes from the cultivated diploids, Groups Phureja and Stenotomum, an interaction known to occur in crosses of Tuberosum haploid x cultivated diploid species (2n = 2x = 24). The frequency of fertile progeny from the 4x × 2x crosses (57%) was significantly higher than that from the 2x × 2x crosses (Tuberosum haploid x cultivated diploid), (28%). The frequency of male fertility among progeny from different cultivars in 4x × 2x crosses varied from 31–82 percent. The difference between cultivars strongly suggests that some cultivars may have dominant male fertility restorer genes.     

STABLE DIPLOIDS FROM INTRAGROUP ZYGOSPORES OF CLOSTERIUM EHRENBERGII MENEGH. (CONJUGATOPHYCEAE)1

Two pairs of stable diploid clones were obtained as aberrant forms among F₁ progeny of an intragroup (intraspecific) cross between R-11-4 (mating type +) and M-16-4b (mating type -) of Group A of Closterium ehrenbergii Menegh. Each pair was derived from the two germination products of a single zygospore, and both clones were mating type minus. The cell size range of these four diploid minus clones was considerably above that of normal (haploid) Group A clones. Chromosome counts at the second meiotic metaphase indicated that these clones were diploid with approximately 200 chromosomes, which was double the number for normal Group A clones. Diploid minus clones conjugated normally with any haploid Group A plus clones, and yielded many triploid zygospores. Triploid zygospores germinated normally as did intragroup diploid zygospores. In metaphase I preparations, only bivalents were observed except on a few occasions where some uni- and multivalents were also detected. Viability of F₁ progeny from triploid zygospores (55–74%) was somewhat lower than from diploid zygospores of Japanese Group A populations (65–90%), but higher than intergroup (interspecific) hybrid zygospores from Groups A, B and H (0–12%). In addition to lower viability, some F₁ progeny from triploid zygospores exhibited slow vegetative growth. Almost all pairs of F₁ clones from single triploid zygospores were of opposite mating type, similar to normal diploid zygospores of the intragroup cross. Morphological variability of F₁ progeny of triploid zygospores was great. The apparently normal meiosis of triploid zygospores and the high viability of F₁ progeny suggested that the genome of Group A contains several sets of chromosome complements with mechanisms by which bivalents are regularly formed in the first meiotic division.     

Studies of the inheritance of virulence in the entomopathogenic fungus Metarhizium anisopliae

A forced heterocaryon was established between two auxotrophic conidial color mutants of Metarhizium anisopliae. From the heterocaryon, a prototrophic somatic diploid was selected which, in turn, yielded somatic segregants. The virulence of the original mutants, the somatic diploid, and the somatic segregants was evaluated on three species of mosquitoes as well as on Ostrinia nubilalis larvae. The virulence of the somatic diploid was comparable to that of the wild-type parental strain while the auxotrophic somatic segregants exhibited virulence approximately equal to that of the auxotrophic components of the heterocaryon. Putative somatic diploids were obtained between morphological mutants of the two species varieties (M. anisopliae var. minor and var. major). The presumptive diploids were avirulent for the insect species to which the parental strains exhibited virulence.     

Inter-embryo competition in the progeny of autotriploid Aloineae (Liliaceae)

In reciprocal crosses between diploid and triploid Aloineae the progeny are largely diploid or diploid plus one or two chromosomes, but in reciprocal crosses between triploids and tetraploids they are tetraploid or nearly so. Thus the triploids contribute circa haploid gametes to the progeny when crossed with diploids but circa diploid gametes when crossed with tetraploids. These results are compared with those of a number of earlier workers. It is concluded that the bias in the frequency of progeny types towards diploidy or tetraploidy, depending on the ploidy level of the plant which is crossed with the triploid, is caused by inter-embryo competition. Those embryos with an endosperm/embryo factor of 1.5, the value found in normal diploid/diploid crosses having triploid endosperms, are selected in preference to those with factors higher or lower than 1.5.Inter-gamete competition also occurs among the euploid and aneuploid gametes produced by the triploids. This is more pronounced on the male side, because the degree of survival of aneuploid pollen from the triploids into the next generation is much lower than that of aneuploid egg nuclei.Non-reduction in the triploids gives rise to occasional pentaploid progeny in crosses with tetraploids, but it is more probable that in diploid/triploid crosses tetraploid progeny are the products of non-reduction in the diploid.     

Genetic Analysis of the Reciprocal Translocation T2(I;VIII) of Aspergillus Using the Technique of Mitotic Mapping in Homozygous Translocation Diploids

A UV-induced sulphite-requiring mutant (sD50) consistently shows mitotic linkage to groups I and VIII in haploids from heterozygous mapping diploids. This linkage was found to be due to a reciprocal translocation T2(I;VIII) which could not be separated from the sulphite requirement in about 100 tested progeny from heterozygous crosses, and both may well have been induced by the same mutational event. T2(I;VIII) is the first case of a reciprocal translocation in Aspergillus which showed meiotic linkages between markers of different linkage groups, and, in addition, involved chromosome arms containing markers suitable for complete mapping by the technique of mitotic recombination in homozygous translocation diploids.-Using various selective markers, haploid segregants and diploid crossovers of all possible types were isolated from homozygous translocation diploids. (1) Haploid segregants showed new linkage relationships in T/T diploids: all available markers of VIII now segregated as a group with the majority of the markers of I, except for the markers of the left tip of I. These formed a separate linkage group and are presumably translocated to VIII. (2) Diploid mitotic crossovers confirmed this information and showed that the orientation of the translocated segments was unchanged. These findings conclusively demonstrate that T2(I;VIII) is a reciprocal translocation due to an exchange of the left tip of group I with the long right arm of group VIII.-Since the position of the break on VIIIR was found to be at sD50 this marker could be used to map the break on IL by meiotic recombination in heterozygous crosses. In addition, such crosses showed reduced recombination around the breaks, so that it was possible to sequence markers which normally are barely linked.     

Genetic diversity among Plantagos VII. Nature of aneuploids in the progeny of aneutriploid x diploid plants ofPlantago lagopus L.

Although half of the progeny of a cross between aneutriploid (3x + 1) and diploid individuals ofP. lagopus are diploids (2n = 12), hybrids with 2n = 14, 15 & 22 also occur. These have been screened for mode of chromosome pairing and phenotype. Data thus raised have been employed for evaluating their status and possible mode of origin.     

The Genetic System Controlling Homothallism in Saccharomyces Yeasts

There are four types of life cycles in Saccharomyces cerevisiae and its related species. A perfect homothallic life cycle (the Ho type) is observed in the classic D strain. Two other types show semi-homothallism; one of them shows a 2-homothallic diploid:2alpha heterothallic haploid segregation (the Hp type) and another, a 2-homothallic:2a segregation (the Hq type). In the segregants from these Ho, Hp, and Hq diploids, each homothallic segregant shows the same segregation pattern as its parental diploid. The fourth type has a heterothallic life cycle showing a 2a:2alpha segregation and the diploids are produced by the fusion of two haploid cells of opposite mating types. The diploids prepared by the crosses of alpha Hp (an alpha haploid segregant from the Hp diploid) to a Hq (an a haploid from the Hq diploid) segregated two types (Type I and II) of the Ho type homothallic clone among their meiotic segregants. Genetic analyses were performed to investigate this phenomenon and the genotypes of the Ho type homothallic clones of Type I and Type II. Results of these genetic analyses have been most adequately explained by postulating three kinds of homothallic genes, each consisting of a single pair of alleles, HO/ho, HMalpha/hmalpha, and HMa/hma, respectively. One of them, the HMalpha locus, was proved to be loosely linked (64 stranes) to the mating-type locus. A spore having the HO hmalpha hma genotype gives rise to an Ho type homothallic diploid (Type I), the same as in the case of the D strain which has the HO HMalpha HMa genotype (Type II). A spore having the a HO hmalpha HMa or alpha HO HMalpha hma genotype will produce an Hp or Hq type homothallic diploid culture, respectively. The other genotypes, a HO HMalpha hma, alpha HO hmalpha HMa, and the genotypes combined with the ho allele give a heterothallic character to the spore culture. A possible molecular hypothesis for the mating-type differentiation with the controlling elements produced by the HMalpha and HMa genes is proposed.     

Hybridization studies in the aster occidentalis (Asteraceae) polyploid complex of western North America

Hybridization experiments were carried out with 12 of the approximately 15 species in the complex. The crosses involved plants at various ploidy levels from diploid (n=8) to duodecaploid (n=48). Viable F₁ progeny were obtained from 26% of the crosses involving diploids, and from 66% of the crosses between polyploids. All but a few of the progeny had a high degree of pollen fertility, and all of those examined had regular pairing of chromosomes at meiosis. Even at the diploid level few reproductive barriers isolate the taxa of this complex, although morphologically distinct entities can be recognized.     

Life history divergence of sympatric diploid and polyploid populations of brine shrimp Artemia parthenogenetica

In order to study how polyploidy affects life history patterns in animals, we have examined sympatric diploid and polyploid brine shrimp (Artemia parthenogenetica) from China, Italy and Spain under laboratory conditions. At optimal temperature and salinity (25°C and 90 ppt), diploids from the three populations had much higher intrinsic rates of increase, higher fecundity, faster developmental rates, and larger brood sizes than their sympatric polyploids. The Chinese and Italian populations were selected for further analysis to determine the life history responses of diploids and polyploids to temperature and salinity changes. Under intermediate and high salinities, Chinese and Italian polyploids produced most of their offspring as dormant cysts while their sympatric diploids produced most of their offspring as nauplii. This relationship is reversed in the Spanish diploid-polyploid complex. For the Chinese population at 25° C, pentaploid clones had higher developmental rates than diploid clones at 35 ppt; at 90 ppt, diploid clones had higher developmental rates than the pentaploids. Italian diploids and tetraploids had different responses to variation in both temperature (25° C and 31° C) and salinity (30 ppt and 180 ppt). Our results demonstrate that relative fitness of the two cytotypes is a function of environmental conditions and that sympatric diploids and polyploids respond differently to environmental changes. Chinese and Italian polyploids are expected to have lower fitness than their sympatric diploids when the physical environment is not stressful and when intraspecific competition is important. However, polyploids may have advantages over sympatric diploids in stressful habitats or when they encounter short-term lethal temperatures. These results suggest that polyploid Artemia have evolved a suite of life-history characteristics adapting them to environments that contrast to those of their sympatric diploids.     

Genetics of CHLAMYDOMONAS REINHARDTII Diploids I. Isolation and Characterization and Meiotic Segregation Pattern of a Homozygous Diploid

A strain of Chlamydomonas reinhardtii has been investigated which, when mated with known wild-types, produces very few viable germination products and transmits its Mendelian markers to more than half of those products. Cytogenetic observations, fluorometric measurements of DNA and genetic data all suggest that the strain, d mt^-ery-M3a sr-u-1 is a stable homozygous diploid. This strain has twice as many nuclear chromatin bodies at metaphase and twice as much DNA as its haploid progenitor, and the phenotypes of its meiotic progeny are consistent with predictions based on triploid meiosis. Data from crosses involving d mt^-ery-M3a sr-u-1 and from crosses involving hybrid diploids indicate that the frequency of second division segregation increases in triploid zygotes and that mitotic segregation following triploid meiosis is a frequent event which may more often result from mitotic recombination than from chromosome loss.     

The Significance of Agamospermous Triploid Pollen Donors in the Sexual Relationships between Diploids and Triploids in Taraxacum (Compositae)

Abstract We review in this article the investigations of the significance of agamospermous triploid pollen donors in the sexual relationships between diploids and triploids in Taraxacum . Crossing experiments between diploid sexual mother plants and agamospermous polyploid pollen donors and recent isozyme analyses of the progeny have exhibited the following results: 1) Pollen from Agamospermous polyploid pollen donors have the potential to give rise to the polyploid agamospermous offspring when fertilizing diploid sexual plants. Ploidy level of the progeny is usually the same or higher, but occasionally lower, compared to the pollen donor. 2) Diploid progeny also occur from diploid (♀)-polyploid (♂) crosses, however, these diploids were in our results not hybrids but the results of self-fertilization of the diploids. The self-fertilization is regarded as a cosequence of the breakdown of the self-incompatibility barrier through the sterile triploid's pollen. This breakdown is in all probability a common phenomenon in diploid (♀)-polyploid (♂) crosses. Some examples suggest that agamospermous polyploids can increase their genetic diversity through obtaining genes from coexisting diploids. The evolutionary implications of this phenomenon and the reduction mechanism of chromosome number through agamospermous pollengenesis are discussed.     

A cryptic clonal line of the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) evidenced by induced gynogenesis,interspecific hybridization,microsatellite genotyping and multilocus DNA fingerprinting

In Memanbetsu town, Hokkaido island, Japan, a high frequency of natural triploid loaches Misgurnus anguillicaudatus (7.4% on average) was detected by flow cytometry for relative DNA content. Among sympatric diploid females (n=6) from a single population, we found two unique females that laid unreduced diploid eggs. They gave normal diploid progeny even after induction of gynogenesis with genetically inert UV-irradiated sperm. When fertilized with normal loach sperm, some unreduced eggs developed into triploids, but the rest into diploids. Hybridization using goldfish Carassius auratus sperm gave both normal diploid loaches and inviable allotriploid hybrids possessing the diploid loach genome and the haploid goldfish genome. Microsatellite genotyping and DNA fingerprinting demonstrated that the diploid progeny developing from the unreduced eggs were genetically identical to the mother, while the triploids had some of the paternal DNA. These results indicate that the diploid eggs reproduced unisexually as a diploid clone and in other cases developed into triploids after accidental incorporation of sperm nucleus. The presence of at least one clonal line in this area was shown by the identical DNA fingerprint detected in five out of 17 diploid loaches examined.     

Characterization of the Heterokaryotic and Vegetative Diploid Phases of MAGNAPORTHE GRISEA

The heterokaryotic and vegetative diploid phases of Magnaporthe grisea, a fungal pathogen of grasses, have been characterized. Prototrophic heterokaryons form when complementary auxotrophs are paired on minimal medium. Hyphal tip cells and conidia (vegetative spores) taken from these heterokaryons are auxotrophs with phenotypes identical to one or the other of the parents. M. grisea heterokaryons thus resemble those of other fungi that have completely septate hyphae with a single nucleus per cell. Heterokaryons have been utilized for complementation and dominance testing of mutations that affect nutritional characteristics of the fungus. Heterokaryons growing on minimal medium spontaneously give rise to fast-growing sectors that have the genetic properties expected of unstable heterozygous diploids. In fast-growing sectors, most hyphal tip cells are unstable prototrophs. The conidia collected from fast-growing sectors include stable and unstable prototrophs, as well as auxotrophs that exhibit a wide range of phenotypes, including many recombinant classes. Genetic linkage in meiosis has been detected between two auxotrophic mutations that recombine in vegetatively growing unstable diploids. The appearance of recombinants suggests that homologous recombination occurs during vegetative growth of M. grisea. No interstrain barriers to heterokaryosis and diploid formation have been detected. The mating type of the strains that are paired does not influence the formation of heterokaryons or diploids.