Functional analysis of the mating type genes in Verticillium dahliae.

BACKGROUND: Populations of the plant pathogenic fungus Verticillium dahliae display a complex and rich genetic diversity, yet the existence of sexual reproduction in the fungus remains contested. As pivotal genes, MAT genes play a crucial role in regulating cell differentiation, morphological development, and mating of compatible cells. However, the functions of the two mating type genes in V. dahliae, VdMAT1-1-1, and VdMAT1-2-1, remain poorly understood. RESULTS: In this study, we confirmed that the MAT loci in V. dahliae are highly conserved, including both VdMAT1-1-1 and VdMAT1-2-1 which share high collinearity. The conserved core transcription factor encoded by the two MAT loci may facilitate the regulation of pheromone precursor and pheromone receptor genes by directly binding to their promoter regions. Additionally, peptide activity assays demonstrated that the signal peptide of the pheromone VdPpg1 possessed secretory activity, while VdPpg2, lacked a predicted signal peptide. Chemotactic growth assays revealed that V. dahliae senses and grows towards the pheromones FO-a and FO-α of Fusarium oxysporum, as well as towards VdPpg2 of V. dahliae, but not in response to VdPpg1. The findings herein also revealed that VdMAT1-1-1 and VdMAT1-2-1 regulate vegetative growth, carbon source utilization, and resistance to stressors in V. dahliae, while negatively regulating virulence. CONCLUSIONS: These findings underscore the potential roles of VdMAT1-1-1 and VdMAT1-2-1 in sexual reproduction and confirm their involvement in various asexual processes of V. dahliae, offering novel insights into the functions of mating type genes in this species.

Mating type and microsatellite genotyping indicate that the Tunisian population of Phyllosticta citricarpa is clonal and thrives only asexually.

Citrus black spot (CBS) caused by Phyllosticta citricarpa was reported for the first time in Tunisia in 2019. This was also the first reported occurrence of the disease in a Mediterranean climate. In Tunisia, CBS is mainly found in lemon (Citrus limon) orchards, and is seldom observed on sweet orange (Citrus × sinensis). This recent finding in North Africa raises questions about how the disease has been able to spread under Mediterranean climatic conditions. In this work, 216 Phyllosticta strains collected from lemon orchards in 2021, 2022 and 2023 throughout the country's main citrus-growing provinces were characterised by species morphological and molecular identification, mating type and Simple Sequence Repeats (SSR) microsatellite genotyping (MLG). P. citricarpa was the only species found to be associated with CBS in Tunisia. Although P. citricarpa is a heterothallic fungal species, potentially able to reproduce both sexually and asexually, a single mating type (MAT 1-1-1) idiomorph was found in the population. In addition, three MLGs were observed, across ten microsatellite loci, one of which was massively represented (93 %), indicating a clonal population. The clonality observed suggests a single recent introduction of the pathogen into the country. These findings support the idea that in Tunisia, P. citricarpa only reproduces asexually by pycniospores, with a relatively limited dispersal potential. This is consistent with the absence of pseudothecia on the leaf litter. These results show that CBS is able to thrive under Mediterranean conditions, even in the absence of sexual reproduction. This should be taken into consideration for CBS risk assessment and management.

Biological characteristics of Cordyceps militaris single mating-type strains.

Cordyceps militaris has been extensively cultivated as a model cordyceps species for commercial purposes. Nevertheless, the problems related to strain degeneration and breeding technologies remain unresolved. This study assessed the physiology and fertility traits of six C. militaris strains with distinct origins and characteristics, focusing on single mating-type strains. The results demonstrated that the three identified strains (CMDB01, CMSY01, and CMJB02) were single mating-type possessing only one mating-type gene (MAT1-1). In contrast, the other three strains (CMXF07, CMXF09, and CMMS05) were the dual mating type. The MAT1-1 strains sourced from CMDB01, CMSY01, and CMJB02 consistently produced sporocarps but failed to generate ascospores. However, when paired with MAT1-2 strains, the MAT1-1 strains with slender fruiting bodies and normal morphology were fertile. The hyphal growth rate of single mating-type strains (CMDB01, CMSY01, and CMJB02) typically surpassed that of dual mating-type strains (CMXF07, CMXF09, and CMMS05). The growth rates of MAT1-2 and MAT1-1 strains were proportional to their ratios, such that a single mating-type strain with a higher ratio exhibited an increased growth rate. As C. militaris matured, the adenosine content decreased. In summary, the C. militaris strains that consistently produce sporocarps and have a single mating type are highly promising for production and breeding.

Chromosome-level genome assembly of the yeast Lodderomyces beijingensis reveals the genetic nature of metabolic adaptations and identifies subtelomeres as hotspots for amplification of mating type loci.

Lodderomyces beijingensis is an ascosporic ascomycetous yeast. In contrast to related species Lodderomyces elongisporus, which is a recently emerging human pathogen, L. beijingensis is associated with insects. To provide an insight into its genetic makeup, we investigated the genome of its type strain, CBS 14171. We demonstrate that this yeast is diploid and describe the high contiguity nuclear genome assembly consisting of eight chromosome-sized contigs with a total size of about 15.1 Mbp. We find that the genome sequence contains multiple copies of the mating type loci and codes for essential components of the mating pheromone response pathway, however, the missing orthologs of several genes involved in the meiotic program raise questions about the mode of sexual reproduction. We also show that L. beijingensis genome codes for the 3-oxoadipate pathway enzymes, which allow the assimilation of protocatechuate. In contrast, the GAL gene cluster underwent a decay resulting in an inability of L. beijingensis to utilize galactose. Moreover, we find that the 56.5 kbp long mitochondrial DNA is structurally similar to known linear mitochondrial genomes terminating on both sides with covalently closed single-stranded hairpins. Finally, we discovered a new double-stranded RNA mycovirus from the Totiviridae family and characterized its genome sequence.

Yeast sexes: mating types do not determine the sexes in Metschnikowia species.

Although filamentous Ascomycetes may produce structures that are interpreted as male and female gametangia, ascomycetous yeasts are generally not considered to possess male and female sexes. In haplontic yeasts of the genus Metschnikowia, the sexual cycle begins with the fusion of two morphologically identical cells of complementary mating types. Soon after conjugation, a protuberance emerges from one of the conjugants, eventually maturing into an ascus. The originating cell can be regarded as an ascus mother cell, hence as female. We tested the hypothesis that the sexes, female or male, are determined by the mating types. There were good reasons to hypothesize further that mating type α cells are male. In a conceptually simple experiment, we observed the early stages of the mating reaction of mating types differentially labeled with fluorescent concanavalin A conjugates. Three large-spored Metschnikowia species, M. amazonensis, M. continentalis, and M. matae, were examined. In all three, the sexes were found to be independent of mating type, cautioning that the two terms should not be used interchangeably.

Discovery of New Genomic Configuration of Mating-Type Loci in the Largest Lineage of Lichen-Forming Fungi.

The genetic architecture of mating-type loci in lichen-forming fungi has been characterized in very few taxa. Despite the limited data, and in contrast to all other major fungal lineages, arrangements that have both mating-type alleles in a single haploid genome have been hypothesized to be absent from the largest lineage of lichen-forming fungi, the Lecanoromycetes. We report the discovery of both mating-type alleles from the haploid genomes of three species within this group. Our results demonstrate that Lecanoromycetes are not an outlier among Ascomycetes.

Homeodomain 1 Genes of the Different HD Subloci of Flammulina velutipes Can Activate the HD Pathway and Are Involved in Mating, Clamp Cell Formation, and Upregulation of FvClp1.

Flammulina velutipes has two independent and functional mating type factors, HD and PR. The HD locus contains two separate subloci: HD-a and HD-b. In this study, we investigated the roles of Hd1 genes of the HD-a and HD-b subloci in the process of mating, clamp cell formation, and regulation of FvClp1 (F. velutipes clampless1 gene) gene expression in F. velutipes. To this end, we introduced Hd1 genes from mating compatible strains into F. velutipes monokaryon L11. Overexpression of Hd1 gene FvHd-a1-1 of the HD-a sublocus resulted in the formation of pseudoclamps in L11 monokaryons. L11 mutants overexpressing the Hd1 gene FvHd-b1-2 of the HD-b sublocus also similarly developed pseudoclamps in the L11 monokaryons. Moreover, these mutant L11 monokaryons produced complete clamps when crossed with monokaryotic strains that differed at the PR loci, i.e., when selective activation of the PR pathway was obtained through crossing. Thus, Hd1 genes of the two different HD subloci in F. velutipes can activate the HD mating type pathway and induce clamp cell formation. In addition, activation of the HD pathway resulted in upregulation of the FvClp1 gene. Finally, to complete clamp cell formation, activation of the PR pathway appears to be essential. Overall, these findings were beneficial for deepening our understanding of sexual reproduction and fruiting body development of edible fungi.

Cip1, a CDK regulator, determines heterothallic mating or homothallic selfing in a protist.

Mating type (sex) plays a crucial role in regulating sexual reproduction in most extant eukaryotes. One of the functions of mating types is ensuring self-incompatibility to some extent, thereby promoting genetic diversity. However, heterothallic mating is not always the best mating strategy. For example, in low-density populations or specific environments, such as parasitic ones, species may need to increase the ratio of potential mating partners. Consequently, many species allow homothallic selfing (i.e., self-fertility or intraclonal mating). Throughout the extensive evolutionary history of species, changes in environmental conditions have influenced mating strategies back and forth. However, the mechanisms through which mating-type recognition regulates sexual reproduction and the dynamics of mating strategy throughout evolution remain poorly understood. In this study, we show that the Cip1 protein is responsible for coupling sexual reproduction initiation to mating-type recognition in the protozoal eukaryote Tetrahymena thermophila. Deletion of the Cip1 protein leads to the loss of the selfing-avoidance function of mating-type recognition, resulting in selfing without mating-type recognition. Further experiments revealed that Cip1 is a regulatory subunit of the Cdk19-Cyc9 complex, which controls the initiation of sexual reproduction. These results reveal a mechanism that regulates the choice between mating and selfing. This mechanism also contributes to the debate about the ancestral state of sexual reproduction.

The MAT1 locus is required for microconidia-mediated sexual fertility in the rice blast fungus.

Rice blast fungus (Pyricularia oryzae) is a heterothallic ascomycete that causes the most destructive disease in cultivated rice worldwide. This fungus reproduces sexually and asexually, and its mating type is determined by the MAT1 locus, MAT1-1 or MAT1-2. Interestingly, most rice-infecting field isolates show a loss of female fertility, but the MAT1 locus is highly conserved in female-sterile isolates. In this study, we performed a functional analysis of MAT1 using the CRISPR/Cas9 system in female- and male-fertile isolates and female-sterile (male-fertile) isolates. Consistent with a previous report, MAT1 was essential for sexual reproduction but not for asexual reproduction. Meanwhile, deletion mutants of MAT1-1-1, MAT1-1-2, and MAT1-1-3 exhibited phenotypes different from those of other previously described isolates, suggesting that the function of MAT1-1 genes and/or their target genes in sexual reproduction differs among strains or isolates. The MAT1 genes, excluding MAT1-2-6, retained their functions even in female-sterile isolates, and deletion mutants lead to loss or reduction of male fertility. Although MAT1 deletion did not affect microconidia (spermatia) production, microconidia derived from the mutants could not induce perithecia formation. These results indicated that MAT1 is required for microconidia-mediated male fertility in addition to female fertility in P. oryzae .

Proteome dataset of Candida albicans (ATCC10231) opaque cell.

OBJECTIVES: Candida albicans, a polymorphic yeast, is one of the most common, opportunistic fungal pathogens of humans. Among the different morphological forms, opaque form is one of the least-studied ones. This opaque phenotype is essential for mating and is also reported to be involved in colonizing the gastrointestinal tract. Considering the significance of the clinical and sexual reproduction of C. albicans, we have investigated the morphophysiological modulations in opaque form using a proteomic approach. DATA DESCRIPTION: In the current investigation, we have used Micro-Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis to create a protein profile for opaque-specific proteins. Whole-cell proteins from C. albicans (ATCC10231) cells that had been cultured for seven days on synthetic complete dextrose (SCD) medium in both as an opaque (test) and as a white (control) form cells were extracted, digested, and identified using LC-MS/MS. This information is meant to serve the scientific community and represents the proteome profile (SWATH Spectral Libraries) of C. albicans opaque form.

A rapid PCR-based method to determine the Neurospora crassa mating type.

So far mating type determination in Neurospora crassa requires test crosses with strains of known mating type. We present a simple, quick, and reliable polymerase chain reaction-based method for mating type determination in N. crassa.

Unidirectional mating-type switching is underpinned by a conserved MAT1 locus architecture.

Unidirectional mating-type switching is a form of homothallic reproduction known only in a small number of filamentous ascomycetes. Their ascospores can give rise to either self-sterile isolates that require compatible partners for subsequent sexual reproduction, or self-fertile individuals capable of completing this process in isolation. The limited studies previously conducted in these fungi suggest that the differences in mating specificity are determined by the architecture of the MAT1 locus. In self-fertile isolates that have not undergone unidirectional mating-type switching, the locus contains both MAT1-1 and MAT1-2 mating-type genes, typical of primary homothallism. In the self-sterile isolates produced after a switching event, the MAT1-2 genes are lacking from the locus, likely due to a recombination-mediated deletion of the MAT1-2 gene information. To determine whether these arrangements of the MAT1 locus support unidirectional mating-type switching in the Ceratocystidaceae, the largest known fungal assemblage capable of this reproduction strategy, a combination of genetic and genomic approaches were used. The MAT1 locus was annotated in representative species of Ceratocystis, Endoconidiophora, and Davidsoniella. In all cases, MAT1-2 genes interrupted the MAT1-1-1 gene in self-fertile isolates. The MAT1-2 genes were flanked by two copies of a direct repeat that accurately predicted the boundaries of the deletion event that would yield the MAT1 locus of self-sterile isolates. Although the relative position of the MAT1-2 gene region differed among species, it always disrupted the MAT1-1-1 gene and/or its expression in the self-fertile MAT1 locus. Following switching, this gene and/or its expression was restored in the self-sterile arrangement of the locus. This mirrors what has been reported in other species capable of unidirectional mating-type switching, providing the strongest support for a conserved MAT1 locus structure that is associated with this process. This study contributes to our understanding of the evolution of unidirectional mating-type switching.

Engineering heterothallic strains in fission yeast.

In poor nitrogen conditions, fission yeast cells mate, undergo meiosis and form spores that are resistant to deleterious environments. Natural isolates of Schizosaccharomyces pombe are homothallic. This allows them to naturally switch between the two h- and h+ mating types with a high frequency, thereby ensuring the presence of both mating partners in a population of cells. However, alteration of the mating type locus can abolish mating type switching or reduce it to a very low frequency. Such heterothallic strains have been isolated and are common in research laboratories due to the simplicity of their use for Mendelian genetics. In addition to the standard laboratory strains, a large collection of natural S. pombe isolates is now available, representing a powerful resource for investigating the genetic diversity and biology of fission yeast. However, most of these strains are homothallic, and only tedious or mutagenic strategies have been described to obtain heterothallic cells from a homothallic parent. Here, we describe a simple approach to generate heterothallic strains. It takes advantage of an alteration of the mating type locus that was previously identified in a mating type switching-deficient strain and the CRISPR-Cas9 editing tool, allowing for a one-step engineering of heterothallic cells with high efficiency.

Characterisation of the mating-type loci in species of Elsinoe causing scab diseases.

The genus Elsinoe includes many aggressive plant pathogens that infect various economically important agricultural, horticultural and forestry plants. Significant diseases include citrus scab caused by E. fawcettii and E. australis, grapevine spot anthracnose by E. ampelina, and the emerging Eucalyptus scab and shoot malformation disease caused by the recently described E. necatrix. Despite their importance as plant pathogens, little is known regarding the biology of many Elsinoe spp. To gain insights into the reproductive biology of these fungi, we characterized the mating-type loci of seven species using whole genome sequence data. Results showed that the MAT1 locus organization and its flanking genes is relatively conserved in most cases. All seven species manifested a typical heterothallic mating system characterized by having either the MAT1-1 or MAT1-2 idiomorph present in an isolate. These idiomorphs were defined by the MAT1-1-1 or the MAT1-2-1 gene, respectively. A unique MAT1-1 idiomorph containing a truncated MAT1-2-1 gene, and a MAT1-1-1 gene, was identified in E. necatrix and E. fawcettii genomes. Additionally, two idiomorph-specific proteins were found in the MAT1-1 and MAT1-2 idiomorphs of E. australis. Universal mating-type markers confirmed heterothallism across 21 Elsinoe spp., are poised to advance future studies regarding the biology of these fungi.

Invasive Californian death caps develop mushrooms unisexually and bisexually.

Canonical sexual reproduction among basidiomycete fungi involves the fusion of two haploid individuals of different mating types, resulting in a heterokaryotic mycelial body made up of genetically different nuclei. Using population genomics data and experiments, we discover mushrooms of the invasive and deadly Amanita phalloides can also be homokaryotic; evidence of sexual reproduction by single, unmated individuals. In California, genotypes of homokaryotic mushrooms are also found in heterokaryotic mushrooms, implying nuclei of homokaryotic mycelia are also involved in outcrossing. We find death cap mating is controlled by a single mating type locus, but the development of homokaryotic mushrooms appears to bypass mating type gene control. Ultimately, sporulation is enabled by nuclei able to reproduce alone as well as with others, and nuclei competent for both unisexuality and bisexuality have persisted in invaded habitats for at least 17 but potentially as long as 30 years. The diverse reproductive strategies of invasive death caps are likely facilitating its rapid spread, suggesting a profound similarity between plant, animal and fungal invasions.

Variation in transcription regulator expression underlies differences in white-opaque switching between the SC5314 reference strain and the majority of Candida albicans clinical isolates.

Candida albicans, a normal member of the human microbiome and an opportunistic fungal pathogen, undergoes several morphological transitions. One of these transitions is white-opaque switching, where C. albicans alternates between 2 stable cell types with distinct cellular and colony morphologies, metabolic preferences, mating abilities, and interactions with the innate immune system. White-to-opaque switching is regulated by mating type; it is repressed by the a1/α2 heterodimer in a/α cells, but this repression is lifted in a/a and α/α mating type cells (each of which are missing half of the repressor). The widely used C. albicans reference strain, SC5314, is unusual in that white-opaque switching is completely blocked when the cells are a/α; in contrast, most other C. albicans a/α strains can undergo white-opaque switching at an observable level. In this paper, we uncover the reason for this difference. We show that, in addition to repression by the a1/α2 heterodimer, SC5314 contains a second block to white-opaque switching: 4 transcription regulators of filamentous growth are upregulated in this strain and collectively suppress white-opaque switching. This second block is missing in the majority of clinical strains, and, although they still contain the a1/α2 heterodimer repressor, they exhibit a/α white-opaque switching at an observable level. When both blocks are absent, white-opaque switching occurs at very high levels. This work shows that white-opaque switching remains intact across a broad group of clinical strains, but the precise way it is regulated and therefore the frequency at which it occurs varies from strain to strain.

Needles in fungal haystacks: Discovery of a putative a-factor pheromone and a unique mating strategy in the Leotiomycetes.

The Leotiomycetes is a hugely diverse group of fungi, accommodating a wide variety of important plant and animal pathogens, ericoid mycorrhizal fungi, as well as producers of antibiotics. Despite their importance, the genetics of these fungi remain relatively understudied, particularly as they don't include model taxa. For example, sexual reproduction and the genetic mechanisms that underly this process are poorly understood in the Leotiomycetes. We exploited publicly available genomic and transcriptomic resources to identify genes of the mating-type locus and pheromone response pathway in an effort to characterize the mating strategies and behaviors of 124 Leotiomycete species. Our analyses identified a putative a-factor mating pheromone in these species. This significant finding represents the first identification of this gene in Pezizomycotina species outside of the Sordariomycetes. A unique mating strategy was also discovered in Lachnellula species that appear to have lost the need for the primary MAT1-1-1 protein. Ancestral state reconstruction enabled the identification of numerous transitions between homothallism and heterothallism in the Leotiomycetes and suggests a heterothallic ancestor for this group. This comprehensive catalog of mating-related genes from such a large group of fungi provides a rich resource from which in-depth, functional studies can be conducted in these economically and ecologically important species.

Frequent transitions in mating-type locus chromosomal organization in Malassezia and early steps in sexual reproduction.

Fungi in the basidiomycete genus Malassezia are the most prevalent eukaryotic microbes resident on the skin of human and other warm-blooded animals and have been implicated in skin diseases and systemic disorders. Analysis of Malassezia genomes revealed that key adaptations to the skin microenvironment have a direct genomic basis, and the identification of mating/meiotic genes suggests a capacity to reproduce sexually, even though no sexual cycle has yet been observed. In contrast to other bipolar or tetrapolar basidiomycetes that have either two linked mating-type-determining (MAT) loci or two MAT loci on separate chromosomes, in Malassezia species studied thus far the two MAT loci are arranged in a pseudobipolar configuration (linked on the same chromosome but capable of recombining). By generating additional chromosome-level genome assemblies, and an improved Malassezia phylogeny, we infer that the pseudobipolar arrangement was the ancestral state of this group and revealed six independent transitions to tetrapolarity, seemingly driven by centromere fission or translocations in centromere-flanking regions. Additionally, in an approach to uncover a sexual cycle, Malassezia furfur strains were engineered to express different MAT alleles in the same cell. The resulting strains produce hyphae reminiscent of early steps in sexual development and display upregulation of genes associated with sexual development as well as others encoding lipases and a protease potentially relevant for pathogenesis of the fungus. Our study reveals a previously unseen genomic relocation of mating-type loci in fungi and provides insight toward the identification of a sexual cycle in Malassezia, with possible implications for pathogenicity.

Seeing double on Cannabis: Haploids and heteroploids of Bipolaris gigantea on hemp and other dicots.

Bipolaris gigantea (= Drechslera gigantea) causes Bipolaris leaf spot (BLS), a devastating and widespread disease on industrial hemp (Cannabis sativa). An investigation of relationships of isolates from hemp and other plants indicated variation in ploidy that has not previously been reported for Bipolaris. Isolates were obtained from BLS lesions on hemp and nearby weeds in 11 Kentucky counties and were similar to each other in morphology and growth characteristics. In total, 23 isolates were analyzed by multilocus phylogenetics, of which seven were also chosen for whole genome shotgun sequencing. Genes for RNA polymerase II subunit 2 (RPB2), translation elongation factor 1-α (TEF1), and mating type (MAT1) indicated that 13 of the isolates were haploid with only a single allele each of RPB2 and TEF1 and either the MAT1-1 or MAT1-2 idiomorph, whereas 10 were apparently "heteroploid" with two alleles each of RPB2 and TEF1 and both MAT1 idiomorphs. Haploids all had identical RPB2 alleles except for a 1-bp difference in two isolates, identical TEF1 alleles, and (if present) identical MAT1-2 alleles. Those alleles were also present in each heteroploid along with either of two related but distinct alleles for each gene. In contrast, haploids and heteroploids shared allelic variation of MAT1-1. In total, four haploid and two heteroploid genotypes were identified. Genome sequence data assembled to 30-32 Mb for each of four haploid isolates, but 10-31 Mb larger sizes for each of three heteroploids depending on sequencing platform and assembly program. The haploids and heteroploids caused similar disease on hemp.

[Differential transcription of mating-type genes during sexual reproduction of natural Cordyceps sinensis].

Natural Cordyceps sinensis as an insect-fungal complex, which is developed after Ophiocordyceps sinensis infects a larva of Hepialidae family. Seventeen genotypes of O. sinensis have been identified in natural C. sinensis. This paper summarized the literature reports and GenBank database regarding occurrence and transcription of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in natural C. sinensis, in Hirsutella sinensis(GC-biased Genotype #1 of O. sinensis), to infer the mating pattern of O. sinensis in the lifecycle of natural C. sinensis. The mating-type genes and transcripts of MAT1-1 and MAT1-2 idiomorphs were identified in the metagenomes and metatranscriptomes of natural C. sinensis. However, their fungal sources are unclear because of co-colonization of several genotypes of O. sinensis and multiple fungal species in natural C. sinensis. The mating-type genes of MAT1-1 and MAT1-2 idiomorphs were differentially present in 237 H. sinensis strains, constituting the genetic control of the O. sinensis reproduction. Transcriptional control of the O. sinensis reproduction includes: differential transcription or silencing of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs, and the MAT1-2-1 transcript with unspliced intron I that contains 3 stop codons. Research on the H. sinensis transcriptome demonstrated differential and complementary transcriptions of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in Strains L0106 and 1229, which may become mating partners to accomplish physiological heterothallism. The differential occurrence and transcription of the mating-type genes in H. sinensis are inconsistent with the self-fertilization hypothesis under homothallism or pseudohomothallism, but instead indicate the need of mating partners of the same H. sinensis species, either monoecious or dioecious, for physiological heterothallism, or heterospecific species for hybridization. Multiple GC-and AT-biased genotypes of O. sinensis were identified in the stroma, stromal fertile portion(densely covered with numerous ascocarps) and ascospores of natural C. sinensis. It needs to be further explored if the genome-independent O. sinensis genotypes could become mating partners to accomplish sexual reproduction. S. hepiali Strain FENG experienced differential transcription of the mating-type genes with a pattern complementary to that of H. sinensis Strain L0106. Additional evidence is needed to explore a hybridization possibility between S. hepiali and H. sinensis, whether they are able to break the interspecific reproductive isolation. Genotypes #13～14 of O. sinensis feature large DNA segment reciprocal substitutions and genetic material recombination between 2 heterospecific parental fungi, H. sinensis and an AB067719-type fungus, indicating a possibility of hybridization or parasexuality. Our analysis provides important information at the genetic and transcriptional levels regarding the mating-type gene expression and reproduction physiology of O. sinensis in the sexual life of natural C. sinensis and offers crucial reproductive physiology evidence, to assist in the design of the artificial cultivation of C. sinensis to supplement the increasing scarcity of natural resource.