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Article

Identification of Critical Candidate Genes Controlling Monokaryon Fruiting in Flammulina filiformis Using Genetic Population Construction and Bulked Segregant Analysis Sequencing

by
Peng Wang
1,†,
Ya Yu
1,†,
Lei Xia
1,
Qi Yan
2,
Xiao Tan
1,
Dongyin Wang
3,
Xue Wang
3,
Zhibin Zhang
4,
Jiawei Wen
1,* and
Xiao Huang
1,*
1
Jilin Academy of Agricultural Sciences (Northeast Agricultural Research Center of China), Changchun 130033, China
2
College of Horticulture, Jilin Agricultural University, Changchun 130118, China
3
Institute of Science and Technology Information of Jilin Province, Changchun 130033, China
4
School of Life Science, Northeast Normal University, Changchun 130024, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Fungi 2025, 11(7), 512; https://doi.org/10.3390/jof11070512
Submission received: 1 June 2025 / Revised: 29 June 2025 / Accepted: 5 July 2025 / Published: 8 July 2025
(This article belongs to the Section Fungal Genomics, Genetics and Molecular Biology)
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Abstract

Fruiting body formation in edible fungi is a critical development process for both scientific understanding and industrial cultivation, yet the underlying genetic mechanisms remain poorly elucidated. This study aimed to identify key genes regulating monokaryotic fruiting in Flammulina filiformis. A genetic segregation population was constructed through selfing purification and hybrid segregation of the FF002 strain, followed by mapping candidate genes with bulked segregant analysis sequencing (BSA-seq). A 10 kb genomic region on scaffold19 was identified, pinpointing the gene FV-L110034160, which encodes a U2 snRNP complex component involved in pre-mRNA splicing. A T→G SNP located 121 bp downstream of the ATG codon caused a serine-to-alanine substitution, disrupting a conserved domain and altering fruiting phenotypes. Phylogenetic analysis further revealed conservation of this gene in fungal genera. These findings elucidate a key regulatory gene controlling monokaryotic fruiting in F. filiformis, providing novel insights into fruiting body formation mechanisms and establishing a foundation for genetic studies in other edible fungi.

1. Introduction

The mechanism of fruiting body formation is both a fundamental scientific question for understanding the growth and development of edible fungi and a critical practical issue for their efficient domestication and cultivation. Extensive investigations have explored this from multiple perspectives, such as environmental factors, nutritional physiology, and mutant analyses, leading to the identification of numerous associated genes [1,2,3,4]. Notably, the rapid advancement and application of genome and transcriptome sequencing technologies in life sciences have significantly enhanced the efficiency of gene discovery. Notably, recent studies have employed multi-omics approaches to investigate the regulatory genes involved in fruiting body formation in various edible and medicinal fungi, including Schizophyllum commune [5], Agaricus bisporus [6], Coprinus cinereus [7], F. filiformis [8], Ganoderma lucidum [9], Lentinula edodes [10], Hypsizygus marmoreus [11], and Cyclocybe aegerita [12]. These studies revealed that upregulated genes are predominantly associated with signal transduction, RNA transcription, protein synthesis, carbohydrate metabolism, and cell wall biogenesis pathways, while downregulated genes were primarily involved in sugar transport and glycolysis pathways. However, the omics-based approaches often yield an overwhelming number of regulatory genes, making it significantly challenging to identify the key control genes that directly control fruiting body formation.
Bulked segregant analysis (BSA) is a method that utilizes phenotypic differences to construct segregating populations for rapid screening and mapping of genes associated with target traits [13]. This approach has been extensively applied in functional gene mapping studies in plants and animals [14,15], and is increasingly utilized in edible fungi research [16]. However, challenges such as difficulties in marker development and labor-intensive screening remain persist. BSA-seq, which integrates BSA with high-throughput sequencing, employs SNPs and InDels directly as markers. This method offers advantages including shorter experimental timelines, precise mapping, uniform marker distribution, and high marker density, establishing it as one of the most effective strategies for functional gene discovery [17,18,19]. For fungal gene mapping, Song employed BSA-seq to dissect the gene network regulating pseudohyphal growth in Saccharomyces cerevisiae and identified multiple loci associated with invasive growth, including known pseudohyphal growth genes FLO8 and FLO11, as well as the negative regulator SFL1 [20]. Using BSA-seq, Jared identified a putative xylitol dehydrogenase gene, XDH1, demonstrating that wild S. cerevisiae can use xylose as its sole carbon source for biomass accumulation [21]. Moreover, Swart performed BSA-seq to map mutation sites linked to metabolic or growth traits, demonstrating its applicability for asexual fungi [22]. Despite challenges such as dependency on reference genomes and complexities in population construction, BSA-seq remains a critical tool in fungal genetic research due to its precision and efficiency.
F. filiformis, commonly known as enoki or winter mushroom [23], is prized for its culinary qualities and delicate flavor. It exhibits a well-characterized life cycle, rapid growth rate, and readily obtainable sexual and asexual spores [24], enabling efficient purification of genetic backgrounds through genetic manipulation. Additionally, F. filiformis possesses monokaryotic fruiting capability and can form fruiting bodies in culture media [25,26,27]. This characteristic not only effectively mitigates the influence of environmental and nutritional differences on fruiting body development but also minimizes interference from mating-type genes and allelic variations, making F. filiformis an ideal model for studying the mechanisms underlying fruiting body formation in edible fungi.
Here, through genetic population constriction and BSA-seq, we identified key regulatory genes involved in fruiting body formation in F. filiformis and delineated their associated metabolic pathways. These findings not only advance our understanding of the molecular mechanisms governing fruiting body formation in F. filiformis but also provide valuable insights for studying fruiting body development in other edible fungi. Furthermore, this research has significant implications for developing novel domesticated varieties and promoting sustainable practices in the edible mushroom industry.

2. Materials and Methods

2.1. Strain Identification

The wild F. filiformis strain FF002 was isolated from poplar tree roots in Rongting Street, Nanguan District, Changchun City, Jilin Province, China (125°23′26″E, 43°49′29″N). Monokaryotic strains derived from FF002 are categorized into two distinct groups based on their fruiting capabilities: fruiting and non-fruiting (Figure 1).

2.2. Identification of Monokaryotic Fruiting Traits

The ventral surface of the FF002 mature fruiting body was placed face down in a sterile 9 cm Petri dish. After 3–6 h of static incubation at 25 °C, white spore prints were obtained by removing the fruiting bodies. The spore suspension mother liquor was made in a clean bench. The spore concentration was quantified using a hemocytometer and diluted to 100–200 spores/mL. A 100 μL aliquot of the suspension was evenly spread onto PDA medium using a sterile bent glass rod, and the plates were incubated at 25 °C. Spores germinated within 3–5 days. A single colony was picked out and inoculated into a test tube containing PDA medium and labeled. Monokaryotic strains were identified using double-fluorescence staining [28].
These strains were cultured at a constant temperature of 25 °C in the dark, and their monokaryotic fruiting status was recorded after 30 days. As the objective of this study was to identify genes controlling the transition from vegetative to reproductive growth in fruiting body formation in F. filiformis, rather than genes regulating fruiting body growth and development, monokaryotic strains that formed only primordia without further differentiation were also classified as exhibiting fruiting traits.

2.3. Genetic Population Construction

2.3.1. Preparation of Homozygous Strains with Monokaryotic Fruiting and Non-Fruiting Traits

Monokaryotic strains of FF002, exhibiting either fruiting or non-fruiting traits, were self-crossed within each trait group to produce dikaryotic strains. After fruiting body formation, monokaryotic strains were reisolated from these dikaryons, and their fruiting capacities were assessed. Monokaryotic strains maintaining consistent trait types were selected for repeated cycles of (i) self-crossing, (ii) fruiting, (iii) monokaryon preparation, and (iv) fruiting trait assessment, until the monokaryotic progeny of self-crossed strains exhibited no trait segregation. This iterative process ultimately yielded near-isogenic self-crossed lines that differed exclusively in monokaryotic fruiting trait, resulting in homozygous dikaryotic strains with stable monokaryotic fruiting or non-fruiting traits (Figure 2).

2.3.2. Construction of a Genetic Segregating Population for Monokaryotic Fruiting Traits

The two homozygous parental strains were crossed, and basidiospores were collected from the fruiting bodies of the hybrid strain. Monokaryotic strains derived from these spores were phenotypically evaluated for fruiting capability. This monokaryotic strain population represents a genetic segregation population for monokaryotic fruiting traits and serves as the mapping population for associated regulatory genes (Figure 2).

2.4. Genomic DNA Extraction

Inoculum blocks (5 mm × 5 mm) from strains of the genetic segregation population were inoculated into PD medium and incubated at 25 °C with shaking at 120 rpm under dark conditions for 10 days. The resulting mycelia were harvested, pressed with sterile filter paper to remove excess moisture, flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent use. Genomic DNA was extracted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The quality of purified genomic DNA was examined using a NanoDrop 2000 UV spectrophotometer (Thermo Scientific, Waltham, MA, USA) and a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA).

2.5. BSA-Sequencing

A total of 24 DNAs were selected from the two types of monokaryotic strains (fruiting and non-fruiting) and pooled separately. Whole-genome resequencing was performed on DNA extracted from the two bulked pools, including the monokaryotic fruiting FF002-F and the monokaryotic non-fruiting FF002-N, as well as two parental strains (FF002-PL-30F and FF002-PL-8N). Genomic DNA was sonicated, and the adapters were ligated to the fragment ends. After purification, the library was amplified for 150 bp paired-end sequencing on the Illumina X-Ten platform (Genedenovo Biotechnology Co., Ltd., Guangzhou, China). After removing the adapters and low-quality reads, the clean reads from each sample were aligned against the FV-L11 genome [29] using the Burrows–Wheeler Aligner (0.7.17) [30]. Then, alignment files were converted to BAM format using SAMtools (1.22) [31]. Variant calling was identified using the Genome Analysis Toolkit3 (3.8-1) [32], followed by single-nucleotide polymorphisms (SNPs) or insertion/deletion polymorphisms (InDels) annotation using ANNOVAR (http://annovar.openbioinformatics.org, accessed on 12 January 2025) [33]. Association analysis was performed using the SNP-index [34], Δ(SNP-index) [17], calculation of G statistic [35], Euclidean distance (ED) [36], and two-tailed Fisher exact test [37] based on the SNPs. Finally, the overlapping interval of the four methods was considered the final QTL interval.

2.6. Functional Annotation of Candidate Genes

The nucleotide and protein sequences of candidate genes within the mapped interval were retrieved according to genomic data and annotation information. Protein homology analysis and conserved domain annotations were performed using NCBI Blastp (https://blast.ncbi.nlm.nih.gov, accessed on 18 January 2025) and the Conserved Domains Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, accessed on 18 January 2025), respectively. SNP mutation within the coding sequences of the candidate genes was identified based on the BSA-seq results. Genes harboring missense mutations that altered the encoded protein within conserved domains, and showing significant association between mutation status (in both parents and pools), were selected as target genes.

2.7. Functional Validation and Phylogenetic Analysis of Target Genes

Primers were designed based on the target gene sequences, and PCR amplification was performed using DNA from individual strains of the segregated population. The amplified products were sequenced to further validate the association between SNP mutation sites and monokaryotic fruiting traits.
Protein sequences from representative fungi species were retrieved from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 18 January 2025) using target gene sequences as a query for phylogenetic analysis. Multiple sequence alignment was performed using the ClustalX software (http://www.clustal.org, accessed on 18 January 2025)) with default parameters. A phylogenetic tree was constructed using MEGA 7, using the maximum likelihood (ML) method with the JTT substitution model, supported by 500 bootstrap replications.

3. Results

3.1. Construction of a Genetic Segregation Population for Monokaryotic Fruiting Control Genes

A total of 80 FF002 monokaryotic strains were obtained through basidiospore collection and monokaryotic strain preparation. Among these, 60 strains (75%) exhibited monokaryotic fruiting trait, while the remaining 20 strains (25%) were non-fruiting, with no other phenotypic variations observed (Supplementary Table S1).
Monokaryotic fruiting and non-fruiting strains of FF002 were selected for selfing. All 47 monokaryotic strains derived from the Sf1 (43 × 56) successfully produced fruiting bodies, and monokaryotic strains from three Sf2 (1 × 4, 3 × 12, and 19 × 40) generations also consistently formed fruiting bodies. These results indicate that the Sf1 (43 × 56) and Sf2 (1 × 4, 3 × 12, and 19 × 40) generation strains were homozygous for the monokaryotic fruiting trait. Among the 53 monokaryotic strains derived from the Sn1 (36 × 62), 15 formed fruiting bodies, whereas 38 did not. Three Sn2 (5 × 14, 10 × 11, and 13 × 25) generations were established from the non-fruiting monokaryotic strains. Notably, all 33 monokaryotic strains from the Sn2 (5 × 14) failed to produce fruiting bodies. Furthermore, monokaryotic strains from three Sn3 (2 × 9, 3 × 4, and 8 × 32) generations derived from 5 × 14 also consistently failed to form fruiting bodies. These findings indicate that the Sn2 (5 × 14) and Sn3 (2 × 9, 3 × 4, and 8 × 32) generation strains were homozygous for the monokaryotic non-fruiting trait (Figure 2, Table A1, Supplementary Table S1).
A dikaryotic strain Sf*n 30 × 8 was constructed by crossing the fruiting monokaryotic strain (FF002-PL-30F) from Sf2 19 × 40 with the non-fruiting monokaryotic strain (FF002-PL-8N) from Sn3 8 × 32. In addition, 156 monokaryotic strains were collected and established as a genetic segregation population for studying monokaryotic fruiting control genes. Of these, 74 strains (FF002-F) produced monokaryotic fruiting bodies, while 82 did not (FF002-N) (Figure 2, Supplementary Table S1). The observed 1:1 segregation ratio (χ2 test, χ2 = 0.41, p > 0.05) suggests monogenic control of this trait.
To identify candidate genes associated with fruiting, we constructed two bulked segregant populations (BSA pools)—each comprising 24 monokaryotic strains (FF002-F and FF002-N)—for high-throughput sequencing library and performed BSA-seq analysis. Strains of two parental lines (FF002-PL-30F and FF002-PL-8N) were collected for further library construction and BSA-seq.

3.2. Identification of Monokaryotic Fruiting Control Genes Through BSA-Seq Analysis

After trimming the adapter and removing low-quality data, 20.4–30.7 Gb of high-quality clean data were maintained per library. Alignment to the reference genome yielded uniquely mapped reads ranging from 30.5% to 38.8%, with an average sequencing depth of 68.80 X (Table A3).
A total of 7021 SNPs and 292 InDels with high-quality between two parent lines (fruiting and non-fruiting lines) were selected for association analyses. These variants were evaluated using four complementary methods: Δ(SNP-index), Euclidean distance (ED), G-value, and Fisher’s exact test. With a 99% confidence threshold, all four analytical methods consistently pinpointed a significant association between scaffold19 (45,000–55,000) and the monokaryotic fruiting trait, establishing this region as the candidate (Figure 3E).
Five candidate genes were identified within the target genomic interval, based on gene annotation analysis (Figure 3F, Table 1). Among them, a total of 12 non-synonymous mutations were detected within the coding sequence (CDS) regions by integrating SNP data from resequencing data of segregated pools (Table A2). Intriguingly, the FV-L110034160 gene contained a mutation 121 bp downstream of the ATG start codon in its third exon, with the two parental lines displaying homozygous reference (0|0) and homozygous variant (1|1) genotypes. This mutation was consistently reflected in the dominant alleles of the two gene pools, corresponding to the parental genotypes. This T→G transversion contributed to a substitution from a hydrophilic serine (Ser) to a hydrophobic alanine (Ala) (Figure 3G). The FV-L110034160 gene spans 2193 bp, containing seven exons and six introns, with a 1710 bp coding sequence (CDS) that encodes a 569-amino acid protein. This protein functions as a component of the U2 small nuclear ribonucleoprotein (snRNP) complex, which mediates precursor mRNA (pre-mRNA) splicing. The protein contains a conserved SF3B2 domain (also designated SAP145 or SF3b145), a key element of the SF3b complex that facilitates intron excision during U2 snRNP-dependent pre-mRNA splicing. This finding aligns with previous studies reporting significant upregulation of RNA splicing-related genes during fruiting body formation in F. velutipes [38] and Cyclocybe aegerita [12]. Collectively, these results indicate a significant association between FV-L110034160 and monokaryotic fruiting, supporting it as a strong candidate gene governing this developmental trait.

3.3. Validation of the Target Gene and Phylogenetics Analysis

Using primers specific to FV-L110034160, we performed PCR amplification and sequencing of individual strains from both gene pools. The results revealed complete genotype–phenotype correlation: all non-fruiting monokaryotic strains were consistent with the FF002-PL-8N parental genotype (T) at the SNP locus scaffold19-47,775-T-G, while fruiting monokaryotic strains matched the FF002-PL-30F parental genotype (G) (Figure 4). This alteration likely contributes to the loss of function in the conserved domain of the encoded protein and is associated with the phenotypic change in the fruiting trait of monokaryotic strains, suggesting that FV-L110034160 may be a candidate gene associated with the monokaryotic fruiting trait.
Phylogenetic analysis of the FV-L110034160 gene revealed distinct evolutionary patterns. The protein showed high conservation among species within the same genus, such as Armillaria borealis and Armillaria mellea within the genus Armillaria, as well as Amanita muscaria and Amanita rubescens within the genus Amanita. However, significant divergence was observed at taxonomic levels above the genus, particularly between F. filiformis and Lentinula edodes (Figure 5). The conservation of such genes among edible fungi species suggested their important roles in the development of the fruiting body.

4. Discussion

The mechanism of fruiting body formation has long been a focal point in edible fungi research, with investigators employing multiple perspectives, including physical environment, nutritional physiology and mutant analyses, to identify key regulatory genes. Among these approaches, the use of mutant strains defective in fruiting body formation, among model species, has proven to be one of the most critical and effective strategies. Mutant-based approaches have successfully identified several key genes involved in fruiting body formation, including the blue light receptor genes wc-1 and wc-2 [39]; the development switch gene Cc.rmt1 [40], which encodes arginine methyltransferase, and, when regulated, leads to sclerotia formation instead of fruiting bodies following hyphal knotting; the inhibit fruiting body formation and dikaryotic hyphal growth gene Cc.snf5 [41]; the regulatory stipe elongation gene eln2 [4]; the influence clamp connection and fruiting body formation genes clp1 [42], Poclp1 [43], Fvclp1 [44], and ich1 [45]; and the prevent primordia knotting gene thn [46]. Thus, studying mutants defective in fruiting body formation in model species has proven to be an effective strategy for identifying key control genes. However, this approach is constrained by its reliance on the detection of dominant mutations and the randomness of mutation sites, which significantly hinders the elucidation of fruiting body formation mechanisms. Therefore, increasing the diversity of key control genes for fruiting body formation and transitioning from studies limited to dominant mutations to those capable of identifying both dominant and recessive mutations represent critical directions for advancing our understanding of fruiting body formation in edible fungi.
Monokaryotic fruiting refers to the phenomenon in which monokaryotic strains of basidiomycetes with a heterothallic sexual reproductive system form fruiting bodies without mating. This trait has been observed in over 30 basidiomycete species [47]. In addition to F. filiformis [25,26], common edible fungi species such as Auricularia heimuer [48], Agrocybe aegerita [49], and Pholiota microspora [50] also exhibit monokaryotic fruiting. Since monokaryotic strains possess only one set of chromosomes, both dominant and recessive mutations in key fruiting body control genes can disrupt fruiting body formation, thereby expanding the range of genes amenable to study. Consequently, the phenomenon of monokaryotic fruiting provides a novel approach for investigating key control genes by directly correlating phenotypic traits with genotypes through eliminating interference from allelic variations. This trait offers significant advantages for studying key control genes involved in fruiting body formation.
Building on the identification of control genes for fruiting body formation in F. filiformis through monokaryotic fruiting trait analysis, we further employed multiple rounds of selfing purification to generate homozygous strains with highly uniform genetic backgrounds, differing only in the genetic loci controlling monokaryotic fruiting. This approach minimized interference from genetic variability. Our experiments revealed that homozygous strains for the fruiting trait could typically be obtained after 2–3 generations of selfing, and in some cases, after just one generation, are far fewer than the generations required in plants. This efficiency likely stems from the direct observability of the monokaryotic fruiting trait, which allows targeted selection of monokaryotic strains with identical fruiting phenotypes for selfing crosses, significantly reducing the time required for genetic manipulation. Moreover, since the segregation population was derived from continuous selfing of a single starting strain, the genetic background remained highly consistent. This enabled direct localization of candidate genes to a 10 kb interval using BSA-seq alone, eliminating the need for subsequent development of molecular markers for fine-mapping and achieving a mapping resolution comparable to map-based cloning methods [51,52]. However, challenges arose during selfing, including the accumulation of deleterious mutations and segregation distortion. By the S3 generation, the probability of successful mating among monokaryotic strains significantly decreased, posing substantial difficulties for applying this technique to map quantitative traits controlled by multiple genes (as selfing may become infeasible before major-effect genes are purified). These limitations warrant further investigation.
The mapping rate of reads to the reference genome ranged from 30~38%, which is relatively low for fungal resequencing studies. To investigate potential causes, we aligned our resequencing data to a high-quality reference genome of F. filiformis strain KACC42780 (GenBank: GCA_000633125.1), noted for its superior assembly. However, the mapping rates remained modest, ranging from 35.83~46.55%. This result suggests that reference genome incompleteness is unlikely to be the primary factor contributing to the low mapping rates. Instead, we hypothesize that the low mapping rates may result from substantial genetic divergence between the wild, undomesticated F. filiformis strain used in this study and cultivated strains. This interpretation is consistent with findings by Zhao et al. [53], who reported significant genomic differences between wild and domesticated F. filiformis populations, including variations in genome size and gene content. While the lower mapping rates could potentially lead to the oversight of additional loci closely associated with monokaryotic fruiting traits, we conducted rigorous statistical analyses, including Δ(SNP-index), Euclidean distance (ED), G-value, and Fisher’s exact test, to validate the identified loci. These analyses consistently demonstrated a significant correlation between the mapped loci and monokaryotic fruiting traits, suggesting the accuracy of our results.
Although this study successfully identified the candidate gene FV-L110034160 associated with monokaryotic fruiting in F. filiformis through selfing purification and BSA-seq analysis, and established a strong correlation between SNPs in this gene and the observed phenotype. The protein encoded by FV-L110034160 functions as a component of the U2 small nuclear ribonucleoprotein (snRNP) complex, playing a critical role in pre-mRNA splicing as a core constituent of the spliceosomal U2 snRNP. This component is highly conserved across eukaryotes [54]. During fruiting body formation, precise temporal regulation of gene expression is essential, and the spliceosome is pivotal for post-transcriptional regulation, including alternative splicing [55]. Studies have demonstrated that mutations in U2 snRNP components in Candida albicans lead to defects in hyphal morphogenesis [56]. Similarly, in Saccharomyces cerevisiae, the absence of U2 snRNP-related proteins directly impairs late-stage development [57]. Moreover, significant differential expression of U2 snRNP components has been observed during fruiting body formation in F. filiformis [38] and C. aegerita [12]. However, the direct impact of this gene on monokaryotic fruiting body formation has not yet been validated. In future studies, the gene candidates of this research will be further validated through CRISPR and RNAi techniques. Additionally, building on our efficient target gene mapping pipeline—which combines trait-specific selfing purification, genetic population construction, BSA-seq, and candidate gene validation—we will investigate genes controlling diverse monokaryotic fruiting traits (e.g., primordia presence/absence, differentiation extent, and morphological characteristics) across additional F. filiformis strains. Furthermore, we plan to employ transcriptome sequencing to analyze the mycelial and primordium stages of the F. filiformis strain FF002. This approach aims to identify candidate genes associated with fruiting body formation, thereby providing deeper insights into the mechanisms underlying fruiting body formation in F. filiformis. This systematic approach will help construct a comprehensive regulatory network of fruiting body development in F. filiformis, advancing both mechanistic understanding and practical applications in edible fungi research.

5. Conclusions

This study successfully established F. filiformis as the model organism to identify genes related to fruit body formation. By employing standardized growth conditions, genetic population construction, and BSA-seq analysis, we identified FV-L110034160, a key gene encoding a U2 snRNP component critical for pre-mRNA splicing. A functional SNP (scaffold19-47,775 bp, T→G) causing a Ser→Ala substitution was found to alter gene function, directly influencing monokaryotic fruiting. PCR-based validation confirmed the mutation’s phenotypic association. These findings not only advance our understanding of fruiting body development in F. filiformis but also provide a methodological framework for studying similar mechanisms in other edible fungi. This work further offers potential strategies for improving fruiting body induction in economically important basidiomycetes that remain challenging to cultivate artificially. Future studies should explore the broader regulatory network involving FV-L110034160 and assess its applicability across fungal species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof11070512/s1, Table S1: Statistical analysis of monokaryotic fruiting traits in constructed genetic population strains.

Author Contributions

Conceptualization, J.W. and X.H.; methodology, P.W. and Y.Y.; software, D.W., X.W., and Z.Z.; validation, X.T., L.X., and X.H.; formal analysis, P.W. and Q.Y.; investigation, X.T., Q.Y., Y.Y., and X.H.; resources, P.W.; data curation, Q.Y., P.W., and L.X.; writing original draft preparation, P.W.; writing—review and editing, J.W. and Z.Z.; visualization, X.T.; supervision, X.H. and J.W.; project administration, J.W., X.H., and P.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Jilin Province Agricultural Science and Technology Innovation Project (CXGC2024ZY040), the Science-Technology Development Plan Project of Jilin Province (20250102307JC), and the earmarked fund for JLARS (JLARS-2025-060308).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included within the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

Thanks to Youjin Deng and Xinrui Liu from the College of Life Sciences, Fujian Agricultural and Forestry University, for providing the whole genome sequence and annotation information of FV_L11; thanks to Chengcheng Lu for drawing the genetic population construction diagram.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Table A1. Preparation of the homozygous strains for fruiting and non-fruiting traits of FF002.
Table A1. Preparation of the homozygous strains for fruiting and non-fruiting traits of FF002.
Selfing StrainMonokaryotic Fruiting Selfed Line of FF002 Selfing Strain Monokaryotic Non-Fruiting Selfed Line of FF002
Selfing
Generation		 Selfing
Combination		Segregation of Gamete Fruiting TraitsHomozygosity of Fruiting TraitsSelfing
Generation		 Selfing
Combination		Segregation of Gamete Fruiting TraitsHomozygosity of Non-Fruiting Traits
(Fruiting: Non-Fruiting)(Fruiting: Non-Fruiting)
Sf143 × 5647:0100%Sn136 × 6215:3871.69%
Sf21 × 439:0100%Sn25 × 140:33100%
3 × 1233:0100%10 × 118:1970.37%
19 × 4035:0100%13 × 25Forming primordia but failing to form fruiting bodies-
Sf3---Sn32 × 90:20100%
3 × 40:20100%
8 × 320:20100%
Table A2. Non-synonymous SNP loci in candidate genes.
Table A2. Non-synonymous SNP loci in candidate genes.
SNP_idFF002-F Type
(and Read)		FF002-N_Type
(and Read)		FF002-PL-8N_Type
(and Read)		FF002-PL-30F_Type
(and Read)		Gene StructureGene Function
scaffold19-46186-C-A1|1 (0|27)1|1 (0|45)1|1 (0|54)1|1 (0|35)FV-L110034150mRNA-1:exon4:c.G863T:p.S288I
scaffold19-46310-T-C1|1 (0|25)1|1 (0|45)1|1 (0|40)1|1 (0|37)FV-L110034150mRNA-1:exon4:c.A739G:p.N247D
scaffold19-46687-G-T1|1 (0|19)1|1 (0|41)1|1 (0|49)1|1 (0|35)FV-L110034150mRNA-1:exon2:c.C455A:p.A152D
scaffold19-46698-T-G1|1 (0|23)1|1 (0|38)1|1 (0|49)1|1 (0|31)FV-L110034150mRNA-1:exon2:c.A444C:p.E148D
scaffold19-46841-T-A1|1 (0|11)1|1 (0|20)1|1 (0|19)1|1 (0|7)FV-L110034150mRNA-1:exon2:c.A301T:p.M101L
scaffold19-46863-G-T1|1 (0|5)1|1 (0|14)1|1 (0|14)1|1 (0|7)FV-L110034150mRNA-1:exon2:c.C279A:p.S93R
scaffold19-46864-C-T1|1 (0|5)1|1 (0|14)1|1 (0|13)1|1 (0|7)FV-L110034150mRNA-1:exon2:c.G278A:p.S93N
scaffold19-47775-T-G0|1 (21|13)0|1 (12|25)0|0 (0|63)1|1 (44|0)FV-L110034160mRNA-1:exon3:c.T121G:p.S41A
scaffold19-47855-G-C1|1 (0|31)1|1 (0|32)1|1 (0|43)1|1 (0|37)FV-L110034160mRNA-1:exon3:c.G201C:p.E67D
scaffold19-47922-T-G1|1 (0|19)1|1 (0|38)1|1 (1|44)1|1 (0|45)FV-L110034160mRNA-1:exon4:c.T214G:p.L72V
scaffold19-51884-T-C1|1 (1|48)1|1 (0|75)1|1 (0|72)1|1 (0|72)FV-L110034180mRNA-1:exon7:c.A604G:p.I202V
scaffold19-54000-G-C1|1 (0|19)1|1 (0|49)1|1 (0|43)1|1 (0|46)FV-L110034190mRNA-1:exon6:c.C857G:p.A286G
Note: genotype: 1|1 (homozygous variant); 0|1 (heterozygous variant); 0|0 (homozygous reference). Read: numerator: number of reads supporting the reference allele (0); denominator: number of reads supporting the variant allele (1).
Table A3. Sequencing data evaluation and comparison with reference genome statistics.
Table A3. Sequencing data evaluation and comparison with reference genome statistics.
SampleClean ReadsClean Bases (bp)Q20 (%)Q30 (%)Mapped (%)GC (%)
FF002-F13628972204434580097.1992.3730.5449.10
FF002-N20510422307656330096.4290.9238.7550.06
FF002-PL-30F21785042326775630096.9192.8937.0749.70
FF002-PL-8N17372780260591700096.6091.2936.3950.11
Note: sample, sample number; clean reads, number of clean reads filtered; clean bases, number of bases filtered; Q20 (%), percentage of bases with mass value greater than or equal to 20 in total bases; Q30 (%), percentage of bases with mass value greater than or equal to 30 in total bases; mapped (%), percentage of clean reads to reference genome in total clean reads; GC (%), sample GC content that is percentage of G and C type bases in total bases.

References

  1. Kües, U.; Liu, Y. Fruiting Body Production in Basidiomycetes. Appl. Microbiol. Biotechnol. 2000, 54, 141–152. [Google Scholar] [CrossRef] [PubMed]
  2. Yang, T.; Xiong, W.; Dong, C. Cloning and Analysis of the Oswc-1 Gene Encoding a Putative Blue Light Photoreceptor from Ophiocordyceps sinensis. Mycoscience 2014, 55, 241–245. [Google Scholar] [CrossRef]
  3. Eastwood, D.C.; Herman, B.; Noble, R.; Dobrovin-Pennington, A.; Sreenivasaprasad, S.; Burton, K.S. Environmental Regulation of Reproductive Phase Change in Agaricus bisporus by 1-Octen-3-ol, Temperature and CO2. Fungal Genet. Biol. 2013, 55, 54–66. [Google Scholar] [CrossRef]
  4. Muraguchi, H.; Kamada, T. A Mutation in the eln2 Gene Encoding a Cytochrome P450 of Coprinus cinereus Affects Mushroom Morphogenesis. Fungal Genet. Biol. 2000, 29, 49–59. [Google Scholar] [CrossRef]
  5. Ohm, R.A.; De Jong, J.F.; Lugones, L.G.; Aerts, A.; Kothe, E.; Stajich, J.E.; De Vries, R.P.; Record, E.; Levasseur, A.; Baker, S.E.; et al. Genome Sequence of the Model Mushroom Schizophyllum commune. Nat. Biotechnol. 2010, 28, 957–963. [Google Scholar] [CrossRef]
  6. Morin, E.; Kohler, A.; Baker, A.R.; Foulongne-Oriol, M.; Lombard, V.; Nagy, L.G.; Ohm, R.A.; Patyshakuliyeva, A.; Brun, A.; Aerts, A.L.; et al. Genome Sequence of the Button Mushroom Agaricus bisporus Reveals Mechanisms Governing Adaptation to a Humic-Rich Ecological Niche. Proc. Natl. Acad. Sci. USA 2012, 109, 17501–17506. [Google Scholar] [CrossRef] [PubMed]
  7. Muraguchi, H.; Umezawa, K.; Niikura, M.; Yoshida, M.; Kozaki, T.; Ishii, K.; Sakai, K.; Shimizu, M.; Nakahori, K.; Sakamoto, Y.; et al. Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea. PLoS ONE 2015, 10, e0141586. [Google Scholar] [CrossRef]
  8. Yu, Y.H.; Wu, T.H.; Ye, Z.W.; Chen, B.X.; Guo, L.Q.; Lin, J.F. Transcriptional Profiling Reveals the Regulatory Network of Cold-Induced Primordium Formation in Flammulina velutipes. Acta Mycol. Sin. 2020, 39, 1065–1076. (In Chinese) [Google Scholar] [CrossRef]
  9. Liu, D.; Sun, X.; Diao, W.; Qi, X.; Bai, Y.; Yu, X.; Li, L.; Fang, H.; Chen, Z.; Liu, Q.; et al. Comparative Transcriptome Analysis Revealed Candidate Genes Involved in Fruiting Body Development and Sporulation in Ganoderma lucidum. Arch. Microbiol. 2022, 204, 514. [Google Scholar] [CrossRef]
  10. Zhang, L.; Gong, W.; Li, C.; Shi, N.; Yang, Y.; Zhao, Y.; Yuan, Y.; Qin, L.; Chen, S.; Zhou, Y. RNA-Seq-Based High-Resolution Linkage Map Reveals the Genetic Architecture of Fruiting Body Development in Shiitake Mushroom, Lentinula edodes. Comput. Struct. Biotechnol. J. 2021, 19, 1641–1653. [Google Scholar] [CrossRef]
  11. Quan, X.; Arshad, M.; Ya, L.; Wang, J.; Li, Y.; Song, C.; Zhang, D. Transcriptomic Profiling Revealed Important Roles of Amino Acid Metabolism in Fruiting Body Formation at Different Ripening Times in Hypsizygus marmoreus. Front. Microbiol. 2023, 14, 1169881. [Google Scholar] [CrossRef]
  12. Orban, A.; Weber, A.; Herzog, R.; Hennicke, F.; Rühl, M. Transcriptome of Different Fruiting Stages in the Cultivated Mushroom Cyclocybe aegerita Suggests a Complex Regulation of Fruiting and Reveals Enzymes Putatively Involved in Fungal Oxylipin Biosynthesis. BMC Genom. 2021, 22, 324. [Google Scholar] [CrossRef]
  13. Michelmore, R.W.; Paran, I.; Kesseli, R.V. Identification of Markers Linked to Disease-Resistance Genes by Bulked Segregant Analysis: A Rapid Method to Detect Markers in Specific Genomic Regions by Using Segregating Populations. Proc. Natl. Acad. Sci. USA 1991, 88, 9828–9832. [Google Scholar] [CrossRef]
  14. Zou, C.; Wang, P.; Xu, Y. Bulked Sample Analysis in Genetics, Genomics and Crop Improvement. Plant Biotechnol. J. 2016, 14, 1941–1955. [Google Scholar] [CrossRef]
  15. Pool, J.E. Genetic Mapping by Bulk Segregant Analysis in Drosophila: Experimental Design and Simulation-Based Inference. Genetics 2016, 204, 1295–1306. [Google Scholar] [CrossRef] [PubMed]
  16. Gui, Y.; Wang, C.C.; Bian, Y.B.; Wang, Z.S.; Ma, X.L.; Zhou, Y. Advances in the Application of Bulked Segregant Analysis in Edible Fungi Genetic Research. Acta Edulis Fungi 2020, 27, 179–187. (In Chinese) [Google Scholar] [CrossRef]
  17. Takagi, H.; Abe, A.; Yoshida, K.; Kosugi, S.; Natsume, S.; Mitsuoka, C.; Uemura, A.; Utsushi, H.; Tamiru, M.; Takuno, S.; et al. QTL-seq: Rapid Mapping of Quantitative Trait Loci in Rice by Whole Genome Resequencing of DNA from Two Bulked Populations. Plant J. 2013, 74, 174–183. [Google Scholar] [CrossRef]
  18. Wang, L.; Lu, Z.; Regulski, M.; Jiao, Y.; Chen, J.; Ware, D.; Xin, Z. BSA-seq: An Interactive and Integrated Web-Based Workflow for Identification of Causal Mutations in Bulked F2 Populations. Bioinformatics 2021, 37, 382–387. [Google Scholar] [CrossRef]
  19. Fang, X.; Sun, X.; Yang, X.; Li, Q.; Lin, C.; Xu, J.; Gong, W.; Wang, Y.; Liu, L.; Zhao LLiu, B.; et al. MS1 Is Essential for Male Fertility by Regulating the Microsporocyte Cell Plate Expansion in Soybean. Sci. China Life Sci 2021, 64, 1533–1545. [Google Scholar] [CrossRef]
  20. Song, Q.; Johnson, C.; Wilson, T.E.; Kumar, R.; Hyten, D.; Klepadlo, M.; Cregan, P.; Li, Z. Pooled Segregant Sequencing Reveals Genetic Determinants of Yeast Pseudohyphal Growth. PLoS Genet. 2014, 10, e1004570. [Google Scholar] [CrossRef]
  21. Wenger, J.W.; Schwartz, K.; Sherlock, G. Bulk Segregant Analysis by High-Throughput Sequencing Reveals a Novel Xylose Utilization Gene from Saccharomyces cerevisiae. PLoS Genet. 2010, 6, e1000942. [Google Scholar] [CrossRef] [PubMed]
  22. Swart, K.; Van der Vondervoort, P.J.I.; De Vries, R.P.; Hondel, C.A.M.J.J.; Visser, J. Parasexual Crossings for Bulk Segregant Analysis in Aspergillus niger to Facilitate Mutant Identification via Whole Genome Sequencing. Fungal Genet. Biol. 2017, 103, 1–10. [Google Scholar] [CrossRef]
  23. Dai, Y.C.; Yang, Z.L. New Annotations on the Scientific Names of Five Important Edible Fungi in China. Acta Mycol. Sin 2018, 37, 1572–1577. (In Chinese) [Google Scholar] [CrossRef]
  24. Tan, Y.; Wang, B.; Zhao, R.L. Nuclear Phase Changes in the Life Cycle of Flammulina velutipes. Acta Edulis Fungi 2015, 22, 13–19. (In Chinese) [Google Scholar] [CrossRef]
  25. Yi, R.R.; Cao, H.; Pan, Y.J. Monokaryotic Fruiting and Color of the Fruiting Body of Flammulina velutipes. Acta Edulis Fungi 2007, 14, 33–36. (In Chinese) [Google Scholar] [CrossRef]
  26. Pan, B.H.; Li, C.P. Research and Application of the Monospore Fruiting of Flammulina velutipes. China Edible Fungi 1994, 13, 21–22. (In Chinese) [Google Scholar]
  27. Zhang, P.; Zhang, Z.G. Research on Monokaryotic Fruiting Bodies of Flammulina velutipes. Edible Fungi China 1998, 17, 17. (In Chinese) [Google Scholar]
  28. Kang, Z.S.; Li, Z.Q.; Shang, H.S.; Ma, Q.; Wang, H.W. Technique of Double Fluorescence Staining for Nuclei and Septa of Plant Pathogenic Fungi. Acta Mycol. Sin. 1993, 12, 174–176. (In Chinese) [Google Scholar]
  29. Liu, F. Preliminary Study of Flammulina velutipes Genome and Transcriptome. Ph.D. Dissertation, Fujian Agriculture and Forestry University, Fuzhou, China, 2014. (In Chinese). [Google Scholar]
  30. Li, H.; Durbin, R. Fast and Accurate Short Read Alignment with Burrows-Wheeler Transform. Bioinformatics 2009, 25, 1754–1760. [Google Scholar] [CrossRef]
  31. Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G.; Abecasis, G.; Durbin, R. The Sequence Alignment/Map Format and SAMtools. Bioinformatics 2009, 25, 2078–2079. [Google Scholar] [CrossRef]
  32. McKenna, A.; Hanna, M.; Banks, E.; Sivachenko, A.; Cibulskis, K.; Kernytsky, A.; Garimella, K.; Altshuler, D.; Gabriel, S.; Daly, M.; et al. The Genome Analysis Toolkit: A MapReduce Framework for Analyzing Next-Generation DNA Sequencing Data. Genome Res. 2010, 20, 1297–1303. [Google Scholar] [CrossRef]
  33. Wang, K.; Li, M.; Hakonarson, H. ANNOVAR: Functional Annotation of Genetic Variants from High-Throughput Sequencing Data. Nucleic Acids Res. 2010, 38, e164. [Google Scholar] [CrossRef] [PubMed]
  34. Abe, A.; Kosugi, S.; Yoshida, K.; Natsume, S.; Takagi, H.; Kanzaki, H.; Matsumura, H.; Mitsuoka, C.; Tamiru, M.; Innan, H.; et al. Genome Sequencing Reveals Agronomically Important Loci in Rice Using MutMap. Nat. Biotechnol. 2012, 30, 174–178. [Google Scholar] [CrossRef]
  35. Magwene, P.M.; Willis, J.H.; Kelly, J.K. The Statistics of Bulk Segregant Analysis Using Next Generation Sequencing. PLoS Comput. Biol. 2011, 7, e1002255. [Google Scholar] [CrossRef] [PubMed]
  36. Hill, J.T.; Demarest, B.L.; Bisgrove, B.W.; Gorsi, B.; Su, Y.C.; Yost, H.J. MMAPPR: Mutation Mapping Analysis Pipeline for Pooled RNA-seq. Genome Res. 2013, 23, 687–697. [Google Scholar] [CrossRef]
  37. Fisher, R.A. On the Interpretation of χ2 from Contingency Tables, and the Calculation of P. J. R. Stat. Soc. 1922, 85, 87–94. [Google Scholar] [CrossRef]
  38. Bao, D.; Gong, M.; Zheng, H.; Chen, M.; Zhang, L.; Wang, H.; Jiang, J.; Wu, L.; Zhu, Y.; Zhu, G.; et al. Transcriptomic Analysis of Flammulina velutipes Reveals Differentially Expressed Genes during Development Stages. BMC Genom. 2013, 14, 413. [Google Scholar] [CrossRef]
  39. Ohm, R.A.; Aerts, D.; Han, A.B.; Wösten, H.A.B. The Blue Light Receptor Complex WC-1/2 of Schizophyllum commune Is Involved in Mushroom Formation and Protection against Phototoxicity. Environ. Microbiol. 2013, 15, 943–955. [Google Scholar] [CrossRef]
  40. Nakazawa, T.; Tatsuta, Y.; Fujita, T.; Nakahori, K.; Kamada, T. Mutations in the Cc.rmt1 Gene Encoding a Putative Protein Arginine Methyltransferase Alter Developmental Programs in the Basidiomycete Coprinopsis cinerea. Curr. Genet. 2010, 56, 361–367. [Google Scholar] [CrossRef]
  41. Ando, Y.; Nakazawa, T.; Oka, K.; Nakahori, K.; Kamada, T. Cc.snf5, a Gene Encoding a Putative Component of the SWI/SNF Chromatin Remodeling Complex, Is Essential for Sexual Development in the Agaricomycete Coprinopsis cinerea. Fungal Genet. Biol. 2013, 50, 82–89. [Google Scholar] [CrossRef]
  42. Inada, K.; Morimoto, Y.; Arima, T.; Murata, Y.; Kamada, T. The clp1 Gene of the Mushroom Coprinus cinereus Is Essential for A-Regulated Sexual Development. Genetics 2001, 157, 133–140. [Google Scholar] [CrossRef] [PubMed]
  43. Boontawon, T.; Nakazawa, T.; Horii, M.; Ohnuki, S.; Nakamoto, M.; Sakamoto, M.; Inoue, Y.; Izumitsu, K.; Shigenobu, S.; Kaneko, S.; et al. Functional Analyses of Pleurotus ostreatus pcc1 and clp1 Using CRISPR/Cas9. Fungal Genet. Biol. 2021, 154, 103599. [Google Scholar] [CrossRef] [PubMed]
  44. Lyu, X.; Jiang, S.; Wang, L.; Chou, T.; Wang, Q.; Meng, L.; Mukhtar, I.; Xie, B.; Wang, W. The Fvclp1 Gene Regulates Mycelial Growth and Fruiting Body Development in Edible Mushroom Flammulina velutipes. Arch. Microbiol. 2021, 203, 5373–5380. [Google Scholar] [CrossRef]
  45. Muraguchi, H.; Kamada, T. The ich1 Gene of the Mushroom Coprinus cinereus Is Essential for Pileus Formation in Fruiting. In Proceedings of the Annual Meeting of the Japanese Society for Molecular Biology, Nagoya, Japan, 18 December 1998; p. 3133. [Google Scholar]
  46. Schuren, F.H.J. Atypical Interactions between thn and Wild-Type Mycelia of Schizophyllum commune. Mycol. Res. 1999, 103, 1540–1544. [Google Scholar] [CrossRef]
  47. Stahl, U.; Esser, K. Genetics of Fruit Body Production in Higher Basidiomycetes. I. Monokaryotic Fruiting and Its Correlation with Dikaryotic Fruiting in Polyporus ciliatus. Mol. Gen. Genet. 1976, 148, 183–197. [Google Scholar] [CrossRef]
  48. Huang, Y.D. Research on the Polarity of Wood Ear Fungus. Master’s Thesis, Huazhong Agricultural University, Wuhan, China, 2004. (In Chinese). [Google Scholar]
  49. Bao, D.P.; Wang, N.; Tan, Q.; Pan, Y.J. Research Progress on the Cultivation Physiology and Life Cycle of Pleurotus eryngii. Acta Edulis Fungi 1999, 6, 55–60. (In Chinese) [Google Scholar] [CrossRef]
  50. Zhang, M.; Zhang, J.J.; Liu, J.J.; Liu, N.; Li, H. Research on the Morphology of Monokaryotic Mycelium and Fruiting of Pleurotus ostreatus. Acta Agric. Boreali-Sin. 2011, 26, 219–222. (In Chinese) [Google Scholar] [CrossRef]
  51. Peng, W.; Yao, F.J.; Lu, L.X.; Wang, Z.S.; Li, Y.; Wu, X.L. Map-Based Cloning of Genes Encoding Key Enzymes for Pigment Synthesis in Auricularia cornea. Fungal Biol. 2019, 123, 843–853. [Google Scholar] [CrossRef]
  52. Ma, X.X.; Lu, L.X.; Zhang, Y.M.; Yao, F.J.; Li, Y.; Wu, X.L. Velvet Family Members Regulate Pigment Synthesis of the Fruiting Bodies of Auricularia cornea. J. Fungi 2023, 9, 412. [Google Scholar] [CrossRef]
  53. Liu, F.; Ma, X.B.; Han, B.; Wang, B.; Xu, J.P.; Cao, B.; Ling, Z.L.; He, M.Q.; Zhu, X.Y.; Zhao, R.-L. Pan-genome analysis reveals genomic variations during enoki mushroom domestication, with emphasis on genetic signatures of cap color and stipe length. J. Adv. Res. 2024; in press. [Google Scholar] [CrossRef]
  54. van der Feltz, C.; Anthony, K.; Brilot, A.; Pomeranz Krummel, D.A. Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A. Crit. Rev. Biochem. Mol. Biol. 2019, 54, 327–347. [Google Scholar] [CrossRef]
  55. Grützmann, K.; Sasso, S.; Deshpande, A.M.; Schöler, F.; Lohse, M.B.; Nowrousian, M. The occurrence and function of alternative splicing in fungi. Trends Genet. 2014, 30, 6–13. [Google Scholar] [CrossRef]
  56. Pelloth, C.; Schafer, K.; Sparber, F.; Převorovský, M.; Hogan, D.A.; Schüller, C. The spliceosome impacts morphogenesis in the human fungal pathogen Candida albicans. mBio 2023, 14, e00409-23. [Google Scholar] [CrossRef]
  57. Gottschalk, A.; Bartels, C.; Neubauer, G.; Luhrmann, R.; Fabrizio, P. A novel yeast U2 snRNP protein, Snu17p, is required for the first catalytic step of splicing and for progression of spliceosome assembly. MCB 2001, 21, 3037–3046. [Google Scholar] [CrossRef]
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Figure 1. Fruiting bodies and monokaryotic strains of F. filiformis strain FF002. (A) Wild F. filiformis strain fruiting bodies; (B) non-fruiting monokaryotic strains; (C) fruiting monokaryotic strains.
Figure 1. Fruiting bodies and monokaryotic strains of F. filiformis strain FF002. (A) Wild F. filiformis strain fruiting bodies; (B) non-fruiting monokaryotic strains; (C) fruiting monokaryotic strains.
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Figure 2. Process of constructing a genetic segregation population for monokaryotic fruiting traits.
Figure 2. Process of constructing a genetic segregation population for monokaryotic fruiting traits.
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Figure 3. Identification of the monokaryotic fruiting control genes. (A) Manhattan plot showing the distribution of Δ(SNP-index) on scaffold19; (B) Manhattan plot showing the distribution of Euclidean distance (ED2) on scaffold19; (C) Manhattan plot showing the distribution of G-value on scaffold19; (D) Manhattan plot showing the distribution of log-transformed Fisher’s exact test p-value distribution –log10(p) on scaffold19; (E) physical map of scaffold19; (F) the 9.8 kb region of the monokaryotic fruiting region based on four independent BSA-seq mappings containing 5 predicted genes according to the FV-L11 reference genome; (G) schematic illustration and characterization of FV-L110034160, exons and introns are shown in boxes and lines, respectively. The mutation site was indicated.
Figure 3. Identification of the monokaryotic fruiting control genes. (A) Manhattan plot showing the distribution of Δ(SNP-index) on scaffold19; (B) Manhattan plot showing the distribution of Euclidean distance (ED2) on scaffold19; (C) Manhattan plot showing the distribution of G-value on scaffold19; (D) Manhattan plot showing the distribution of log-transformed Fisher’s exact test p-value distribution –log10(p) on scaffold19; (E) physical map of scaffold19; (F) the 9.8 kb region of the monokaryotic fruiting region based on four independent BSA-seq mappings containing 5 predicted genes according to the FV-L11 reference genome; (G) schematic illustration and characterization of FV-L110034160, exons and introns are shown in boxes and lines, respectively. The mutation site was indicated.
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Figure 4. Validation of the FV-L110034160 gene of fruiting and non-fruiting monokaryotic strains. (A) Morphological characteristics and DNA sequences near the mutation site of non-fruiting monokaryotic strains in the genetic population, alongside sequencing chromatograms of the mutation site in non-fruiting monokaryotic parental strain FF002-PL-8N used for population construction (in the genetic population, the phenotype of monokaryotic non-fruiting strains consistently exhibits a T base at the mutation site, identical to the monokaryotic non-fruiting parental strain); (B) morphological characteristics and DNA sequences near the mutation site of fruiting monokaryotic strains in the genetic population, alongside sequencing chromatograms of the mutation site in fruiting monokaryotic parental strain FF002-PL-30F used for population construction (in the genetic population, the phenotype of monokaryotic fruiting strains consistently exhibits a G base at the mutation site, identical to the monokaryotic fruiting parental strain).
Figure 4. Validation of the FV-L110034160 gene of fruiting and non-fruiting monokaryotic strains. (A) Morphological characteristics and DNA sequences near the mutation site of non-fruiting monokaryotic strains in the genetic population, alongside sequencing chromatograms of the mutation site in non-fruiting monokaryotic parental strain FF002-PL-8N used for population construction (in the genetic population, the phenotype of monokaryotic non-fruiting strains consistently exhibits a T base at the mutation site, identical to the monokaryotic non-fruiting parental strain); (B) morphological characteristics and DNA sequences near the mutation site of fruiting monokaryotic strains in the genetic population, alongside sequencing chromatograms of the mutation site in fruiting monokaryotic parental strain FF002-PL-30F used for population construction (in the genetic population, the phenotype of monokaryotic fruiting strains consistently exhibits a G base at the mutation site, identical to the monokaryotic fruiting parental strain).
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Figure 5. Phylogenetic tree of mushroom DUF382-domain-containing protein. The tree for DUF382-domain-containing protein homologue amino acid sequences was calculated and constructed using the MEGA program (The protein encoded by the target gene was highlighted with a red square frame).
Figure 5. Phylogenetic tree of mushroom DUF382-domain-containing protein. The tree for DUF382-domain-containing protein homologue amino acid sequences was calculated and constructed using the MEGA program (The protein encoded by the target gene was highlighted with a red square frame).
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Table 1. Five candidate genes screened out by annotation information.
Table 1. Five candidate genes screened out by annotation information.
Gene IDLocationPFAMsPathwayFunction Description
FV-L110034150Scaffold19 (45,969–47,338)ASXH-Asx homology domain
FV-L110034160Scaffold19 (47,374–49,566)DUF382 (CUS1)ko03040 (pentose phosphate pathway)DUF382-domain-containing protein
FV-L110034170Scaffold19 (49,778–51,300)BCDHK_Adom3, HATPase_c-Dehydrogenase
FV-L110034180Scaffold19 (51,399–52,780)3Beta_HSD, Epimerase, NAD_binding_4-Dihydrokaempferol 4-reductase activity
FV-L110034190Scaffold19 (53,799–55,299)HAD_2ko00561 (glycerolipid metabolism), ko01100 (metabolic pathways)HAD-hyrolase-like
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Wang, P.; Yu, Y.; Xia, L.; Yan, Q.; Tan, X.; Wang, D.; Wang, X.; Zhang, Z.; Wen, J.; Huang, X. Identification of Critical Candidate Genes Controlling Monokaryon Fruiting in Flammulina filiformis Using Genetic Population Construction and Bulked Segregant Analysis Sequencing. J. Fungi 2025, 11, 512. https://doi.org/10.3390/jof11070512

AMA Style

Wang P, Yu Y, Xia L, Yan Q, Tan X, Wang D, Wang X, Zhang Z, Wen J, Huang X. Identification of Critical Candidate Genes Controlling Monokaryon Fruiting in Flammulina filiformis Using Genetic Population Construction and Bulked Segregant Analysis Sequencing. Journal of Fungi. 2025; 11(7):512. https://doi.org/10.3390/jof11070512

Chicago/Turabian Style

Wang, Peng, Ya Yu, Lei Xia, Qi Yan, Xiao Tan, Dongyin Wang, Xue Wang, Zhibin Zhang, Jiawei Wen, and Xiao Huang. 2025. "Identification of Critical Candidate Genes Controlling Monokaryon Fruiting in Flammulina filiformis Using Genetic Population Construction and Bulked Segregant Analysis Sequencing" Journal of Fungi 11, no. 7: 512. https://doi.org/10.3390/jof11070512

APA Style

Wang, P., Yu, Y., Xia, L., Yan, Q., Tan, X., Wang, D., Wang, X., Zhang, Z., Wen, J., & Huang, X. (2025). Identification of Critical Candidate Genes Controlling Monokaryon Fruiting in Flammulina filiformis Using Genetic Population Construction and Bulked Segregant Analysis Sequencing. Journal of Fungi, 11(7), 512. https://doi.org/10.3390/jof11070512

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