Sexual Crosses Research Articles

Mitogen-Activated Protein Kinase Cascade Required for Regulation of Development and Secondary Metabolism in Neurospora crassa

Mitogen-activated protein kinase (MAPK) signaling cascades are composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In this study, we characterize components of a MAPK cascade in Neurospora crassa (mik-1, MAPKKK; mek-1, MAPKK; and mak-1, MAPK) homologous to that controlling cell wall integrity in Saccharomyces cerevisiae. Growth of basal hyphae is significantly reduced in mik-1, mek-1, and mak-1 deletion mutants on solid medium. All three mutants formed short aerial hyphae and the formation of asexual macroconidia was reduced in Deltamik-1 mutants and almost abolished in Deltamek-1 and Deltamak-1 strains. In contrast, the normally rare asexual spores, arthroconidia, were abundant in cultures of the three mutants. Deltamik-1, Deltamek-1, and Deltamak-1 mutants were unable to form protoperithecia or perithecia when used as females in a sexual cross. The MAK-1 MAPK was not phosphorylated in Deltamik-1 and Deltamek-1 mutants, consistent with the involvement of MIK-1, MEK-1, and MAK-1 in the same signaling cascade. Interestingly, we observed increased levels of mRNA and protein for tyrosinase in the mutants under nitrogen starvation, a condition favoring sexual differentiation. Tyrosinase is an enzyme that catalyzes production of the secondary metabolite l-DOPA melanin. These results implicate the MAK-1 pathway in regulation of development and secondary metabolism in filamentous fungi.

Methods for detecting chromosome rearrangements inGibberella zeae

Chromosome rearrangements between fungal strains may reduce fertility in sexual crosses through the production of genetically inviable recombinant progeny. As such, rearrangements can be important postzygotic reproductive barriers that contribute to the speciation process. The presence of chromosome rearrangements in crosses with Gibberella zeae was tested by counting asci with 8, 6, 4, or 2 viable ascospores. Counts were made by observing rosettes of asci extruded from crushed perithecia and by observing unordered ascospore tetrads ejected onto agar slabs from mature perithecia. The two methods gave similar results. Self-fertilized cultures served as controls and produced the normal eight ascospores per ascus in >98% of cases. Crosses with strains known to carry chromosome rearrangements produced significant frequencies of asci with 6, 4, or 2 ascospores, as expected. These results suggest that these methods will be useful to survey populations of G. zeae for chromosome rearrangements.

A New and Improved Automated Technology for Early Sex Determination of Ginkgo biloba

Random amplified polymorphic DNA (RAPD) technique with male associated decamer primer S1478 was used to amplify DNA from 72 leaf samples collected from Ginkgo biloba trees with known sexual determinism in the canton of Geneva, Switzerland. This marker was found to be male-specific and was lacking in all female plants. Automated random polymorphic DNA analysis (ARPA), a new automated technology developed in the frame of this work, proved highly effective in distinguishing males and females with 100% efficiency and successful in male and female discrimination from a collection of young seedlings derived from a sexual cross. Our findings provide unambiguous evidence that ARPA combined with the male-associated decamer primer S1478 could be considered an efficient, rapid, and easy method to make an early sex determination in the dioecious tree Ginkgo biloba.

In search of new tractable diatoms for experimental biology

Diatoms are a species-rich group of photosynthetic eukaryotes, with enormous ecological significance and great potential for biotechnology. During the last decade, diatoms have begun to be studied intensively using modern molecular techniques and the genomes of four diatoms have been wholly or partially sequenced. Although new insights into the biology and evolution of diatoms are accumulating rapidly due to the availability of reverse genetic tools, the full potential of these molecular biological approaches can only be fully realized if experimental control of sexual crosses becomes firmly established and widely accessible to experimental biologists. Here we discuss the issue of choosing new models for diatom research, by taking into account the broader context of diatom mating systems and the place of sex in relation to the intricate cycle of cell size reduction and restitution that is characteristic of most diatoms. We illustrate the results of our efforts to select and develop experimental systems in diatoms, using species with typical life cycle attributes, which could be used as future model organisms to complement existing ones.

The Evolution of the Pheromonal Signal System and Its Potential Role for Reproductive Isolation in Heterothallic Neurospora

Comparative sequencing studies among a wide range of taxonomic groups, including fungi, provide the overall pattern that reproductive genes evolve more rapidly than other genes, and this divergence is believed to be important in the establishment of reproductive barriers between species. In this study, we investigated the molecular evolution of the pheromone receptor genes pre-1 and pre-2 of strains belonging to 12 and 13 heterothallic taxa, respectively, of the model genus Neurospora. Furthermore, we examined the regulatory pattern of both pheromone precursor and receptor genes during sexual crosses of Neurospora crassa and Neurospora intermedia, for which reinforcement of interspecific reproductive barriers in sympatry previously has been documented. We conclude that the part encoding the C-terminal intracellular domain of pre-1 and pre-2 genes evolves rapidly. Both stochastic and directional processes drive this divergence; both genes contain neutrally evolving codons, and in addition, pre-1 contains codons evolving under positive selection, whereas in pre-2 we found highly variable regions with numerous repeats encoding glycine, threonine, or aspartic acid. In addition, we found regulatory changes of the pheromone and receptor genes during crosses between N. crassa and N. intermedia with different reproductive success. Gene expression levels are higher in the interspecific sympatric crosses with low reproductive success than in their intraspecific and/or allopatric equivalents, both at the stage of initial communication and contact and later at postfertilization stages. Taken together, our data indicate that pheromones and receptors are important key players during reproductive isolation between Neurospora species, and this study provides a general framework for future studies on the role of reproductive proteins for reproductive isolation.

Meiotic Parthenogenesis in a Root-Knot Nematode Results in Rapid Genomic Homozygosity

Many isolates of the plant-parasitic nematode Meloidogyne hapla reproduce by facultative meiotic parthenogenesis. Sexual crosses can occur, but, in the absence of males, the diploid state appears to be restored by reuniting sister chromosomes of a single meiosis. We have crossed inbred strains of M. hapla that differ in DNA markers and produced hybrids and F(2) lines. Here we show that heterozygous M. hapla females, upon parthenogenetic reproduction, produce progeny that segregate 1:1 for the presence or absence of dominant DNA markers, as would be expected if sister chromosomes are rejoined, rather than the 3:1 ratio typical of a Mendelian cross. Codominant markers also segregate 1:1 and heterozygotes are present at low frequency (<3%). Segregation patterns and recombinant analysis indicate that a homozygous condition is prevalent for markers flanking recombination events, suggesting that recombination occurs preferentially as four-strand exchanges at similar locations between both pairs of non-sister chromatids. With this mechanism, meiotic parthenogenesis would be expected to result in rapid genomic homozygosity. This type of high negative crossover interference coupled with positive chromatid interference has not been observed in fungal or other animal systems in which it is possible to examine the sister products of a single meiosis and may indicate that meiotic recombination in this nematode has novel features.

Zeolin is a recombinant storage protein that can be used to produce value-added proteins in alfalfa (Medicago sativa L.)

The specific features of plants make them particularly suitable for the production of recombinant proteins and alfalfa is one of the recommended plant production systems. We have transformed alfalfa with a gene coding for a chimaeric protein made previously by fusing phaseolin to the N-terminal region of γ-zein and have analyzed the accumulation of this fusion protein, named zeolin. Zeolin was expressed both in T0 Regen SY alfalfa plants and in the progeny resulting from the sexual cross between Regen SY transformants and alfalfa cv. Adriana plants. In some alfalfa plants a 95 kDa zeolin glycosylated polypeptide is the most abundant polypeptide detected by Western-blot analysis, whereas in tobacco the most abundant zeolin polypeptide has a molecular mass around 60 kDa, expected for intact zeolin. Zeolin has been stably accumulated in alfalfa leaves because it forms endoplasmic reticulum-located protein bodies in the cell. As regards zeolin quantisation, in the progeny alfalfa plants a value of about 0.22–0.28 mg of zeolin / g of fresh leaf weight has been estimated.

RAPD-based genetic analysis of offsprings from the sexual cross using allotetraploid citrus somatic hybrid as pollen parent

Thirty-one polymorphic decamer primers were selected to genotype 92 progenies from the cross between Yiben No.4, a monoembryonic diploid F1 hybrid of Citrus reticulata Blanco cv Huanongbendizao tangerine and C. ichangensis Swingle, and [Hamlin sweet orange + Rough lemon], an allotetraploid somatic hybrid of Citrus sinensis Osbeck cv. Hamlin and C. jambhiri Lush cv. Rough Lemon. chi2 (Chi-square) analysis of RAPD markers in the progenies indicated they were randomly transmitted from the four donor parents, without significant difference between the diploids and triploids. However, these progenies were clustered into three major groups using dendrogram constructed by UPGMA, skewed to three parents in certain degrees, 15 (13 triploids and 2 diploids) to Hamlin, 16 (9 and 7) to Yiben No. 4, and 61 (57 and 4) to [Hamlin sweet orange + Rough Lemon] from which genomic contribution was predominant in progenies, respectively.

Cloning and characterization of Fpmtr1, an amino acid transporter gene of Fusarium proliferatum (Gibberella intermedia)

Fpmtr1, an amino acid transporter gene from Fusarium proliferatum was strongly expressed during conidial germination and repressed in late stationary phase. To identify the specific function of this gene, DeltaFpmtr1 knock-out mutants were generated by gene replacement. Vegetative growth of the DeltaFpmtr1 mutants was normal both in liquid and on solid media, but conidial germination was delayed. The DeltaFpmtr1 mutants and the wild type were equally fertile when used as males in sexual crosses, however if the mutants were used as the female parent then the fertility of the cross decreased dramatically. Inactivation of Fpmtr1 abolished vegetative self-incompatibility in strain ITEM 2287 of F. proliferatum, but the DeltaFpmtr1 mutants were still vegetatively incompatible with the other strains of the fungus. Endophytic colonization capability of the mutants, assessed on maize seedlings also was adversely affected. These data suggest that Fpmtr1 is involved in multiple developmental processes related to both sexual and parasexual events in F. proliferatum. Furthermore, the fungus might have problems in adapting to a less than optimal environment if this otherwise dispensable transporter has been inactivated.

Asexual Recombination in a uvsH Mutant of Aspergillus nidulans

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.

Sexual reproduction in the soybean sudden death syndrome pathogen Fusarium tucumaniae

We investigated the sexual reproductive mode of the two most important etiological agents of soybean sudden death syndrome, Fusarium tucumaniae and Fusarium virguliforme. F. tucumaniae sexual crosses often were highly fertile, making it possible to assign mating type and assess female fertility in 24 South American isolates. These crosses produced red perithecia and oblong-elliptical ascospores, as is typical for sexual members of the F. solani species complex. Genotyping of progeny from three F. tucumaniae crosses confirmed that sexual recombination had occurred. In contrast, pairings among 17 U.S. F. virguliforme isolates never produced perithecia. Inter-species crosses between F. tucumaniae and F. virguliforme, in which infertile perithecia were induced only in one of the two F. tucumaniae mating types, suggest that all U.S. F. virguliforme isolates are of a single mating type. We conclude that the F. tucumaniae life cycle in S. America includes a sexual reproductive mode, and thus this species has greater potential for rapid evolution than the F. virguliforme population in the U.S., which may be exclusively asexual.

The molecular biology of cytoplasmically inherited male sterility and prospects for its engineering

Nucleocytoplasmic male sterilities are binary genetic systems driven by mitochondrial, maternally inherited genes that induce male sterility and a female phenotype and which are overcome by nuclear restorers of fertility. They contribute to the reproductive biology and evolution of natural populations and are valuable tools for the commercial production of hybrid seeds in crops. For species with no natural form of cytoplasmic male sterility, such sterility can in some cases be introduced from different, but related, species through sexual crosses or somatic hybridisation. Somatic hybridisation is the only technique currently available for manipulating plant mitochondrial genomes. Recent successes in plastid transformation have opened up entirely new perspectives for the engineering of cytoplasmic male sterilities in transplastomic plants.

Evidence of Genetic Exchange by Parasexual Recombination and Genetic Analysis of Pathogenicity and Mating Type of Parasexual Recombinants in Rice Blast Fungus, Magnaporthe oryzae

ABSTRACT A selectable marker gene conferring resistance to bialaphos (BI) was introduced into rice blast isolate Y90-71BI and another conferring resistance to blasticidin S (BS) into isolate 3514-R-2BS of Magnaporthe oryzae to demonstrate exchange of DNA. Colonies obtained from co-cultures of these two isolates were resistant to both BI and BS and had both resistance genes as shown by Southern blot analysis of their genomic DNA. Conidia from these BI-BS-resistant isolates had only one nucleus per cell after staining with 4',6-diamidino-2-phenylindole (DAPI). Using flow cytometry, however, these BI-BS-resistant isolates were found to be haploid. Segregation of BI-BS-resistant isolates for pathogenicity (avirulence to virulence) on rice line K59-1 was consistent with a 1:1 ratio, as was segregation for mating type. These BI-BS-resistant isolates were thus apparently derived from parasexual exchange of DNA and the segregation of pathogenicity and of mating type of the parasexual recombinants might correspond to that of the progeny of the offspring of the sexual cross.

Breeding strategies for combining fermentative qualities and reducing off-flavor production in a wine yeast model

In agricultural sciences, breeding strategies have historically been used to select new, optimized plant varieties or animal breeds. Similar strategies are possible for genetic improvement of wine yeasts. We optimized 11 relevant enological traits in a single clone using successive hybridization and segregation steps. A hybrid obtained by crossing two parent strains derived from commercial wine yeasts showed that some of the traits were readily optimized. Dominance/recessivity, heterosis and transgression were observed among 51 segregating progeny. On the basis of this information, all the optimal characters from both parents were combined in a single strain following two targeted sexual crosses. This article presents a powerful methodology for obtaining a single wine strain with numerous fermentative qualities that does not produce off-flavors.

Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives

The major constrains for practical exploitation of the somatic hybrids between eggplant and its wild relatives have been their sterility and tetraploidy which prevented their incorporation into breeding programs. Here we demonstrate that anther culture was successfully utilized to bring back the ploidy level to the diploid status in tetraploid interspecific hybrids between eggplant and the allied species S. integrifolium and S. aethiopicum gr. gilo. Both the relative species are resistant to Fusarium oxysporum f. sp. melongenae and to some strains of bacterial wilt (Ralstonia solanacearum) which are very destructive diseases of eggplant. Dihaploid androgenetic plants were obtained from the somatic hybrids, from the “double somatic hybrid” obtained by sexual cross of the two somatic hybrids [(eggplant + S. aethiopicum) × (eggplant + S. integrifolium)], and from tetraploid backcrossed plants between the somatic hybrid with S. aethiopicum and eggplant. Phenotypical, molecular, biological and biochemical characterization, and also artificial inoculation with Fusarium oxysporum are consistent with a recombination between the genomes of the species involved in the hybridizations. Dihaploids resistant to Fusarium were successfully backcrossed with eggplant. Besides their utility as potential valuable breeding materials, the introgressed lines obtained may be utilized in genetic and molecular studies about the resistance to Fusarium from S. integrifolium and S. aethiopicum gr. gilo.

Natural variation of ascospore and conidial germination by Fusarium verticillioides and other Fusarium species

Fusarium verticillioides and other Fusarium species were examined for their spore germination phenotypes. In general, germinating spores of F. verticillioides formed germ tubes that immediately penetrated into agar. Such invasive germination was the predominant growth phenotype among 22 examined field isolates of F. verticillioides from a broad range hosts and locations. However, two of the field isolates were unique in that they formed conidial germ tubes and hyphae that grew along the surface of agar before penetration eventually occurred. Conidia of 22 other Fusarium species were assessed for their germination phenotypes, and only some strains of F. annulatum, F. fujikuroi, F. globosum, F. nygamai, and F. pseudoanthophilum had the surface germination phenotype (21 % of the strains assessed). Sexual crosses and segregation analyses involving one of the F. verticillioides surface germination strains, NRRL 25059, indicated a single locus, designated SIG1 (surface vs. invasive germination), controlled the germ tube growth phenotypes exhibited by both conidia and ascospores. Perfect correlation was observed between an ascospore germination phenotype and the germination phenotype of the conidia produced from the resulting ascospore-derived colony. Recombination data suggested SIG1 was linked (∼7 % recombination frequency) to FPH1, a recently described locus necessary for enteroblastic conidiogenesis. Corn seedling blight assays indicated surface germinating strains of F. verticillioides were less virulent than invasively germinating strains. Assays also indicated pathogenicity segregated independently of the FPH1 locus. Invasive germination is proposed as the dominant form of spore germination among Fusarium species. Furthermore, conidia were not necessary for corn seedling disease development, but invasive germination may have enhanced the virulence of conidiating strains.

Heterothallic mating observed between Mexican isolates of Glomerella lindemuthiana

Although several reports have described the occurrence of the teleomorphic state of Glomerella lindemuthiana (anamorph, Colletotrichum lindemuthianum), there has been a lack of continuity in this research. To identify G. lindemuthiana isolates capable of developing the teleomorphic state, 19 Mexican isolates were analyzed. Three types of response were observed: (i) negative, where only mycelial growth with or without acervuli was observed; (ii) potential, where in addition to the above, spherical perithecia-like structures were observed; (iii) positive, where perithecia containing asci and ascospores were observed. All strains were self-sterile and only one combination of strains produced fertile perithecia. From this fertile combination 168 individual ascospore cultures were isolated, including five from a single ascus. Forty-four monoascospore cultures were characterized with AFLP, confirming that these individuals were progeny from a sexual cross between the original two G. lindemuthiana isolates and that sexual reproduction in G. lindemuthiana is heterothallic in nature. Analysis of the parental strains with degenerate PCR primers indicated that sequences homologous to the HMG box of the MAT1-2 idiomorph are present in both parental isolates. This supports previous observations in other Glomerella species where the standard ascomycete configuration of distinct idiomorphs at the MAT locus does not hold true. The significance of these results is discussed.

Heterothallic mating observed between Mexican isolates of Glomerella lindemuthiana

Although several reports have described the occurrence of the teleomorphic state of Glomerella lindemuthiana (anamorph, Colletotrichum lindemuthianum), there has been a lack of continuity in this research. To identify G. lindemuthiana isolates capable of developing the teleomorphic state, 19 Mexican isolates were analyzed. Three types of response were observed: (i) negative, where only mycelial growth with or without acervuli was observed; (ii) potential, where in addition to the above, spherical perithecia-like structures were observed; (iii) positive, where perithecia containing asci and ascospores were observed. All strains were self-sterile and only one combination of strains produced fertile perithecia. From this fertile combination 168 individual ascospore cultures were isolated, including five from a single ascus. Forty-four monoascospore cultures were characterized with AFLP, confirming that these individuals were progeny from a sexual cross between the original two G. lindemuthiana isolates and that sexual reproduction in G. lindemuthiana is heterothallic in nature. Analysis of the parental strains with degenerate PCR primers indicated that sequences homologous to the HMG box of the MAT1-2 idiomorph are present in both parental isolates. This supports previous observations in other Glomerella species where the standard ascomycete configuration of distinct idiomorphs at the MAT locus does not hold true. The significance of these results is discussed.

Description of Gibberella sacchari and neotypification of its anamorph Fusarium sacchari

We described the teleomorph of Fusarium sacchari as Gibberella sacchari, sp. nov. This species can be separated from other species of Gibberella on the basis of the longer, narrower ascospores found in G. sacchari and by sexual cross fertility. Female-fertile mating type tester strains were developed that can be used for making sexual crosses with this heterothallic fungus under laboratory conditions. The anamorph, Fusarium sacchari, was neotypified.

Description of Gibberella sacchari and neotypification of its anamorph Fusarium sacchari

We described the teleomorph of Fusarium sacchari as Gibberella sacchari, sp. nov. This species can be separated from other species of Gibberella on the basis of the longer, narrower ascospores found in G. sacchari and by sexual cross fertility. Female-fertile mating type tester strains were developed that can be used for making sexual crosses with this heterothallic fungus under laboratory conditions. The anamorph, Fusarium sacchari, was neotypified.