Genetic recombination in sexual crosses of phycomyces

Sexual crosses between strains of Phycomyces blakesleeanus , involving three auxotrophic and one color marker and yielding a high proportion of zygospore germination, are described. Samples of 20–40 germ spores from 311 individual fertile germ sporangia originating from five two-factor and three three-factor crosses were characterized. The results show: (1) absence of any contribution of apogamic nuclei to the progeny, (2) confirmation of Burgeff's conjecture that the germ spores of any germ sporangium in most cases derive from one meiosis. In a cross involving two allelic markers the analysis of 175 pooled germ sporangia suggests an intragenic recombination frequency of 0.6%. All other factor combinations tested are unlinked. The bulk of the germ spores are homokaryotic. However, a small portion (4%) are heterokaryotic with respect to mating type.     

A New Genetic Linkage Map of the Zygomycete Fungus Phycomyces blakesleeanus

Phycomyces blakesleeanus is a member of the subphylum Mucoromycotina. A genetic map was constructed from 121 progeny of a cross between two wild type isolates of P. blakesleeanus with 134 markers. The markers were mostly PCR-RFLPs. Markers were located on 46 scaffolds of the genome sequence, covering more than 97% of the genome. Analysis of the alleles in the progeny revealed nine or 12 linkage groups, depending on the log of the odds (LOD) score, across 1583.4 cM at LOD 5. The linkage groups were overlaid on previous mapping data from crosses between mutants, aided by new identification of the mutations in primary metabolism mutant strains. The molecular marker map, the phenotype map and the genome sequence are overall congruent, with some exceptions. The new genetic map provides a genome-wide estimate for recombination, with the average of 33.2 kb per cM. This frequency is one piece of evidence for meiosis during zygospore development in Mucoromycotina species. At the same time as meiosis, transmission of non-recombinant chromosomes is also evident in the mating process in Phycomyces. The new map provides scaffold ordering for the genome sequence and a platform upon which to identify the genes in mutants that are affected in traits of interest, such as carotene biosynthesis, phototropism or gravitropism, using positional cloning.     

Sex Determination Directs Uniparental Mitochondrial Inheritance in Phycomyces

Uniparental inheritance (UPI) of mitochondria is common among eukaryotes. The underlying molecular basis by which the sexes of the parents control this non-Mendelian pattern of inheritance is yet to be fully understood. Two major factors have complicated the understanding of the role of sex-specific genes in the UPI phenomenon: in many cases (i) fusion occurs between cells of unequal size or (ii) mating requires a large region of the genome or chromosome that includes genes unrelated to sex determination. The fungus Phycomyces blakesleeanus is a member of the Mucoromycotina and has a simple mating type locus encoding only one high-mobility group (HMG) domain protein, and mating occurs by fusion of isogamous cells, thus providing a model system without the limitations mentioned above. Analysis of more than 250 progeny from a series of genetic crosses between wild-type strains of Phycomyces revealed a correlation between the individual genes in the mating type locus and UPI of mitochondria. Inheritance is from the plus (+) sex type and is associated with degradation of the mtDNA from the minus (−) parent. These findings suggest that UPI can be directly controlled by genes that determine sex identity, independent of cell size or the complexity of the genetic composition of a sex chromosome.     

A meiotically reproducible chromosome length polymorphism in the ascomycete fungus Ophiostoma ulmi (sensu lato)

We have followed the transmission of Ophiostoma ulmis.l. chromosome length polymorphisms (CLPs) into the F₂ generation to determine the reproducibility of a genome rearrangement culminating in the conversion of a 1.0 Mb chromosome into a 800 kb chromosome. The 1.0 Mb chromosome in strain CESS16K is thus far unique among O. ulmi s.l. wild-type strains, as no other wild-type strains have been observed with chromosomes smaller than 2.3 Mb. It has been previously shown that the 1.0 Mb chromosome is mitotically stable, carries at least one normally expressed gene, and is transmitted through meiosis. In this study, a series of crosses were performed to further elucidate the pattern of inheritance of the 1.0 Mb chromosome and the process of conversion of the 1.0 Mb species to 800 kb. In crosses where the 1.0 Mb chromosome was allowed to pair with itself or with the 800 kb chromosome, all progeny inherited a copy of the 1.0 Mb or 800 kb form, further demonstrating the A-type nature of these small chromosomes. When a cross was repeated between the strains CESS16K (1.0 Mb chromosome) and FG245B^r-O (no 1.0 Mb or 800 kb chromosome), the occurrence of a 800 kb chromosome was observed in 9% of the progeny. A reciprocal cross between an 800 kb strain and a strain with no 800 kb or 1.0 Mb chromosome was conducted, and a progeny strain containing a 1.0 Mb chromosome was recovered. The reproducibility and reciprocality of the 1.0 Mb to 800 kb chromosome conversion demonstrates that meiotic processes are responsible for this CLP, and that O. ulmi s.l. strains with various divergent genome architectures can remain sexually compatible.     

Genetic exchanges in the macrocysts of Dictyostelium discoideum.

Crosses were made between strains of Dictyostelium discoideum involving two drug resistance markers and the mating-type locus. Over 6000 progeny from 263 individual germinated macrocysts from four single-factor crosses, five two-factor crosses and one three-factor cross were characterized. In most cases the progeny from a single macrocyst were of one genotype, although in the population of macrocysts from any two-factor cross all possible parental and recombinant genotypes were recovered. There was no evidence of linkage between any of the markers examined. No selection against progeny carrying the methanol or the cycloheximide resistance markers was found in two-factor crosses, but selection against progeny carrying both resistance markers was found in the three-factor cross. Germination of macrocysts in all crosses was poor, only once exceeding 2.5% of the total macrocyst population. A variety of crosses and back-crosses with different parental strains indicated that germination might be influenced by both extrinsic (environmental) and multiple genetic factors. About 10% of the macrocysts yielded progeny spores that were ambivalent in their mating reactions. After extensive recloning these populations could be resolved to the normal matA (formerly A1) and mata (formerly A2) mating-types and might therefore have represented aneuploids. The results obtained with D. discoideum macrocysts differ from those obtained with other cellular slime moulds--Dictyostelium mucoroides, Dictyostelium giganteum and Polysphondylium pallidum--and are reminiscent of the results reported for germinated zygospores of Phycomyces blakesleeanus.     

Altered Mating-Type Identity in the Fungus Podospora Anserina Leads to Selfish Nuclei, Uniparental Progeny, and Haploid Meiosis

In wild-type crosses of the filamentous ascomycete Podospora anserina, after fertilization, only nuclei of opposite mating type can form dikaryons that undergo karyogamy and meiosis, producing biparental progeny. To determine the role played by the mating type in these steps, the four mat genes were mutagenized in vitro and introduced into a strain deleted for its mat locus. Genetic and cytological analyses of these mutant strains, crossed to each other and to wild type, showed that mating-type information is required for recognition of nuclear identity during the early steps of sexual reproduction. In crosses with strains carrying a mating-type mutation, two unusual developmental patterns were observed: monokaryotic cells, resulting in haploid meiosis, and uniparental dikaryotic cells providing, after karyogamy and meiosis, a uniparental progeny. Altered mating-type identity leads to selfish behavior of the mutant nucleus: it migrates alone or paired, ignoring its wild-type partner in all mutant X wild-type crosses. This behavior is nucleus-autonomous because, in the same cytoplasm, the wild-type nuclei form only biparental dikaryons. In P. anserina, mat genes are thus required to ensure a biparental dikaryotic state but appear dispensable for later stages, such as meiosis and sporulation.     

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.     

A meiotically reproducible chromosome length polymorphism in the ascomycete fungus Ophiostoma ulmi ( sensu lato )

We have followed the transmission of Ophiostoma ulmis.l. chromosome length polymorphisms (CLPs) into the F₂ generation to determine the reproducibility of a genome rearrangement culminating in the conversion of a 1.0 Mb chromosome into a 800 kb chromosome. The 1.0 Mb chromosome in strain CESS16K is thus far unique among O. ulmi s.l. wild-type strains, as no other wild-type strains have been observed with chromosomes smaller than 2.3 Mb. It has been previously shown that the 1.0 Mb chromosome is mitotically stable, carries at least one normally expressed gene, and is transmitted through meiosis. In this study, a series of crosses were performed to further elucidate the pattern of inheritance of the 1.0 Mb chromosome and the process of conversion of the 1.0 Mb species to 800 kb. In crosses where the 1.0 Mb chromosome was allowed to pair with itself or with the 800 kb chromosome, all progeny inherited a copy of the 1.0 Mb or 800 kb form, further demonstrating the A-type nature of these small chromosomes. When a cross was repeated between the strains CESS16K (1.0 Mb chromosome) and FG245B^r-O (no 1.0 Mb or 800 kb chromosome), the occurrence of a 800 kb chromosome was observed in 9% of the progeny. A reciprocal cross between an 800 kb strain and a strain with no 800 kb or 1.0 Mb chromosome was conducted, and a progeny strain containing a 1.0 Mb chromosome was recovered. The reproducibility and reciprocality of the 1.0 Mb to 800 kb chromosome conversion demonstrates that meiotic processes are responsible for this CLP, and that O. ulmi s.l. strains with various divergent genome architectures can remain sexually compatible. Received: 6 February 1996 / Accepted: 21 January 1997     

Complementation betweenPhycomyces mutants of mating type (+) with abnormal phototropism

Summary Twelve mutants ofPhycomyces blakesleeanus with defects in sporangiophore phototropism (genotypemad) were obtained from a wild type of the (+) mating type by mutagenesis with nitrosoguanidine. These mutants were tested for genetic complementation against standard (+)mad mutants derived from sexual crosses between the isogenic (+) strain and established (-)mad mutants (Ootaki et al., 1974; Eslava et al., 1976). Heterokaryons for complementation tests were obtained by grafting stage I sporangiophores. The (+) mutants were also investigated for their sensory responses such as photoinduction of sporangiophores and avoidance. The mutants were grouped into two classes, based on the phenotypic classification scheme of Bergman et al. (1973). There were eleven class 1.2 mutants and one class 2 mutant. Complementation tests revealed that all eleven class 1.2 mutants carry the genemadC and the class 2 mutant carriesmadD. There was no evidence that any were double mutants. These results are consistent with the phenotypic classification and with the complementation results of themad mutants of the (-) mating type.     

Intersexual partial diploids of phycomyces

Sexual interaction between strains of opposite sex in many fungi of the order Mucorales modifies hyphal morphology and increases the carotene content. The progeny of crosses of Phycomyces blakesleeanus usually include a small proportion of anomalous segregants that show these signs of sexual stimulation without a partner. We have analyzed the genetic constitution of such segregants from crosses that involved a carF mutation for overaccumulation of beta-carotene and other markers. The new strains were diploids or partial diploids heterozygous for the sex markers. Diploidy was unknown in this fungus and in the Zygomycetes. Random chromosome losses during the vegetative growth of the diploid led to heterokaryosis in the coenocytic mycelia and eventually to sectors of various tints and mating behavior. The changes in the nuclear composition of the mycelia could be followed by selecting for individual nuclei. The results impose a reinterpretation of the sexual cycle of Phycomyces. Some of the intersexual strains that carried the carF mutation contained 25 mg beta-carotene per gram of dry mass and were sufficiently stable for practical use in carotene production.     

Test of the isoallele hypothesis at the mouse first arch (far) locus

We tested the hypothesis that differences in wild-type (+) alleles (isoalleles) at the first arch (far) locus between mouse strains ICR/Bc and BALB/cGaBc are responsible for the partially dominant expression of far in the ICR/Bc genetic background, in contrast to its recessive expression in the BALB/cGaBc background. A similar hypothesis concerning isoalleles has been suggested to explain differences in heterozygote expression in some human genetic diseases but appears not to have been tested directly in mammals. First arch is lethal when homozygous. The dominant effects in ICR/Bc mice include defects in mystacial vibrissa pattern formation and cleft palate. To test the isoallele hypothesis, we made the four appropriate genetic crosses between +/far and +/+ mice between strains. The F1 progeny were scored on day 16 of gestation for defective mystacial vibrissa pattern formation and cleft palate. From all four crosses, approximately 30% of F1 fetuses (i.e., 60% of +/far fetuses) had disrupted mystacial vibrissa patterns, and only one fetus had cleft palate. The fact that all crosses produced approximately equal rates of defective progeny disproves the isoallele hypothesis for far. Therefore, differences between strains in alleles at other loci (modifier loci) must cause the differences in heterozygote (+/far) expression. This genetic design can be used for other mutations with strain differences in heterozygote expression to test the importance of isoalleles in mammals.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^? mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

Effect of differing degrees of genetic variability on the toxic effects of aflatoxin B1 among resistant strains of Drosophila melanogaster (Diptera)

Toxicity studies, using outbred lines with much genetic variability and isogenic lines with no genetic variability from two strains of Drosophila melanogaster, Lausanne-S and Oregon-R, are reported. In both of these wild-type strains, larval and pupal development are known to be relatively resistant to the toxic effects of media containing aflatoxin B₁, a mycotoxin with carcinogenic and mutagenic properties. To eliminate genetic variability, each strain was made “isogenic” by a standard chromosomal substitution technique. Each isogenic strain, in comparison to the appropriate outbred control strain, showed a significant decrease in egg-to-adult viability when offspring were allowed to develop from the egg stage on media containing 1.0 ppm aflatoxin B₁. However, the resistance levels shown by the offspring of crosses between the two isogenic strains were not significantly different in viability than those of the appropriate controls. The relationship of these results with the level of genetic variability prossessed by the outbred and isogenic lines is discussed.     

Mating interactions between coexisting dipoloid, triploid and tetraploid cytotypes ofHieracium Echioides (Asteraceae)

Experimental crosses between diploids, triploids and tetraploids ofHieracium echioides were made to examine mating interactions. Specifically, cytotype diversity in progeny from experimental crosses, intercytotype pollen competition as a reproductive barrier between diploids and tetraploids, and differences in seed set between intra- and intercytotype crosses were studied. Only diploids were found in progeny from 2x × 2x crosses. The other types of crosses yielded more than one cytotype in progeny, but one cytotype predominated in each cross type: diploids (92%) in 2x × 3x crosses, tetraploids (88%) in 3x × 2x crosses, triploids (96%) in 2x × 4x crosses, triploids (90%) in 4x × 2x crosses, tetraploids (60%) in 3x × 3x crosses, pentaploids (56%) in 3x × 4x crosses, triploids (80%) in 4x × 3x crosses and tetraploids (88%) in 4x × 4x crosses. No aneuploids have been detected among karyologically analyzed plants. Unreduced egg cell production was detected in triploids and tetraploids, but formation of unreduced pollen was recorded only in two cases in triploids. Triploid plants produced x, 2x and 3x gametes: in male gametes x (92%) gametes predominated whereas in female gametes 3x (88%) gametes predominated. Cytotype diversity in progeny from crosses where diploids and tetraploids were pollinated by mixture of pollen from diploid and tetraploid plants suggested intercytotype pollen competition to serve as a prezygotic reproductive barrier. No statistically significant difference in seed set obtained from intra- and intercytotype crosses between diploids and tetraploids was observed, suggesting the absence of postzygotic reproductive barriers among cytotypes.     

A Hybrid Dysgenesis Syndrome in Drosophila Virilis

A new example of ``hybrid dysgenesis' has been demonstrated in the F(1) progeny of crosses between two different strains of Drosophila virilis. The dysgenic traits were observed only in hybrids obtained when wild-type females (of the Batumi strain 9 from Georgia, USSR) were crossed to males from a marker strain (the long-established laboratory strain, strain 160, carrying recessive markers on all its autosomes). The phenomena observed include high frequencies of male and female sterility, male recombination, chromosomal nondisjunction, transmission ratio distortion and the appearance of numerous visible mutations at different loci in the progeny of dysgenic crosses. The sterility demonstrated in the present study is similar to that of P-M dysgenesis in Drosophila melanogaster and apparently results from underdevelopment of the gonads in both sexes, this phenomenon being sensitive to developmental temperature. However, in contrast to the P-M and I-R dysgenic systems in D. melanogaster, in D. virilis the highest level of sterility (95-98%) occurs at 23-25°. Several of the mutations isolated from the progeny of dysgenic crosses (e.g., singed) proved to be unstable and reverted to wild type. We hypothesize that a mobile element (``Ulysses') which we have recently isolated from a dysgenically induced white eye mutation may be responsible for the phenomena observed.     

The genetic architecture of Drosophila sensory bristle number

We have mapped quantitative trait loci (QTL) for Drosophila mechanosensory bristle number in six recombinant isogenic line (RIL) mapping populations, each of which was derived from an isogenic chromosome extracted from a line selected for high or low, sternopleural or abdominal bristle number and an isogenic wild-type chromosome. All RILs were evaluated as male and female F(1) progeny of crosses to both the selected and the wild-type parental chromosomes at three developmental temperatures (18 degrees, 25 degrees, and 28 degrees ). QTL for bristle number were mapped separately for each chromosome, trait, and environment by linkage to roo transposable element marker loci, using composite interval mapping. A total of 53 QTL were detected, of which 33 affected sternopleural bristle number, 31 affected abdominal bristle number, and 11 affected both traits. The effects of most QTL were conditional on sex (27%), temperature (14%), or both sex and temperature (30%). Epistatic interactions between QTL were also common. While many QTL mapped to the same location as candidate bristle development loci, several QTL regions did not encompass obvious candidate genes. These features are germane to evolutionary models for the maintenance of genetic variation for quantitative traits, but complicate efforts to understand the molecular genetic basis of variation for complex traits.     

Genetic and Molecular Analysis of a Long-Lived Strain of Podospora Anserina

A genetic and molecular analysis of a long-lived strain of Podospora anserina, Mn19, was undertaken to detect mutations in genes responsible for senescence. In crosses between Mn19 and wild type about 15% of the progeny were long-lived, regardless of the female parent. Molecular analysis of the long-lived progeny showed that none of the strains inherited a mtDNA rearrangement characteristic of the Mn19 parent. Instead, all long-lived strains initially inherited wild-type mtDNA. Over time the mtDNA of most long-lived strains underwent rearrangements, deletions and amplifications. The change over time in the presence of two previously characterized plasmids associated with either senescence or longevity was monitored. Crosses between Mn19 and its long-lived progeny also yielded only a small percent of individuals recovering from senescence. Analysis of mtDNA from crosses suggests that wild-type mtDNA from the paternal parent can be selected over mtDNA from the maternal parent. The life span phenotypes of progeny were not consistent with the hypothesis that mutations in a few nuclear genes were responsible for longevity.     

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus’s Role in Virulence

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F₉ MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.     

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.     

Identification of new pisatin demethylase genes (PDA5 and PDA7) in Nectria haematococca and non-Mendelian segregation of pisatin demethylating ability and virulence on pea due to loss of chromosomal elements

Previous studies have shown that high virulence on pea in Nectria haematococca Mating Population VI is linked to the ability to detoxify the pea phytoalexin, pisatin, via demethylation (Pda). To test this linkage further, a highly virulent Pda(+) isolate (34-18) was used as the recurrent parent in backcrosses to Pda(-) isolates, but most of the progeny were low in virulence on pea, and tetrad analysis gave conflicting ratios for the genetic control of Pda. Southern analysis of 34-18 and progeny showed that 34-18 carries a gene similar to PDA1 (PDA1-2), two new PDA genes, PDA5 and PDA7, and that all three genes can be lost during meiosis. Southern analysis of electrophoretic karyotypes showed that PDA1-2 is on a 1.5-Mb dispensable chromosome in 34-18 and that PDA5 and PDA7 are on a 4.9-Mb chromosome in 34-18 but are found on variably sized chromosomes in progeny. Loss of PDA5 or PDA7 in progeny was not generally associated with morphological phenotypes, except in progeny from some crosses between PDA5 parents. Loss of PDA5 was associated with growth abnormalities in these crosses, suggesting that in some genetic backgrounds at least a portion of the PDA5/PDA7 chromosome is essential for normal growth. All highly virulent progeny had PDA1-2 or a combination of PDA5 and PDA7 while isolates that lacked the three genes were low in virulence, supporting the hypothesis that Pda, or genes linked to PDA genes, are necessary for virulence on pea. However, low virulence isolates with PDA genes were also identified, suggesting that there are pathogenicity genes that can segregate independently of PDA genes.