Phenylalanine Ammonia-Lyase: A Key Gene for Color Discrimination of Edible Mushroom Flammulina velutipes

In nature; Flammulina velutipes, also known as winter mushrooms, vary in the color of their fruiting bodies, from black, yellow, pale yellow, or beige to white. The purpose of this study was to compare the genome sequences of different colored strains of F. velutipes and to identify variations in the genes associated with fruiting body color. Comparative genomics of six F. velutipes strains revealed 70 white-strain-specific variations, including single nucleotide polymorphisms (SNPs) and insertions/deletions (indels), in the genome sequences. Among them, 36 variations were located in the open reading frames, and only one variation was identified as a mutation with a disruptive in-frame deletion (ΔGCGCAC) within the annotated gene phenylalanine ammonia-lyase 1 (Fvpal1). This mutation was found to cause a deletion, without a frameshift, of two amino acids at positions 112 and 113 (arginine and threonine, respectively) in the Fvpal1 gene of the white strain. Specific primers to detect this mutation were designed, and amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) was performed to evaluate whether the mutation is color specific for the F. velutipes fruiting body. PCR analysis of a total of 95 F. velutipes strains revealed that this mutation was present only in white strains. In addition, monospores of the heterozygous mutant were isolated, and whether this mutation was related to the color of the fruiting body was evaluated by a mating assay. In the mating analysis of monospores with mutations in Fvpal1, it was found that this mutation plays an important role in determining the color of the fruiting body. Furthermore, the deletion (Δ112RT113) in Fvpal1 is located between motifs that play a key role in the catalytic function of FvPAL1. These results suggest that this mutation can be used as an effective marker for the color-specific breeding of F. velutipes, a representative edible mushroom.


Introduction
The edible mushroom Flammulina velutipes belongs to the family Tricholomataceae within Agaricales and grows on old trees or the stumps of various broadleaf trees from late autumn to the following spring. This mushroom is a cold-resistant fungus that occurs even in winter; therefore, it is also called the winter mushroom [1,2]. Wild-type F. velutipes strains vary in color from yellowish-brown to dark brown, whereas artificial cultivars are predominantly white [3] (Figure 1). The first artificially cultivated variety was a brown F. velutipes strain, but the cultivation of F. velutipes began in earnest after white varieties were bred in Japan, and these are still the main cultivated varieties today [3,4]. The white F. velutipes mainly cultivated in Korea and Japan were discovered by chance, and the genetic cause of the discoloration of the fruiting body is not known. Since the artificial cultivation of F. velutipes began, various white cultivars have been actively developed,

Fungal Strain Culture and Genomic DNA Isolation
F. velutipes strains (Table 1) were obtained from the Mushroom Research Division, National Institute of Horticultural and Herbal Science (Rural Development Administration, Jeonju, Republic of Korea) and were grown on potato dextrose agar (PDA; 4 g potato starch, 20 g dextrose, 15 g agar per liter) at 25 °C for 15 days. Genomic DNA was then extracted using extraction buffer (0.25 M Tris-HCl, 100 mM NaCl, 50 mM ethylenediaminetetraacetic acid, 5% SDS), 2 × CTAB buffer (100 mM Tris-HCl pH 8, 20 mM EDTA pH 8, 2% CTAB, 1.4 M NaCl, and 1% polyvinyl pyrrolidone), and phenol-chloroform-isoamyl alcohol (25:24:1) as previously described [29]. The extracted DNA samples were treated with RNase A (Qiagen, Hilden, Germany).  As the genome sequences of F. velutipes species have been determined and in-depth genetic information has been revealed, comparative studies on the biological characteristics and diversity of these mushrooms have been actively conducted [1,[25][26][27][28][29]. The genetic repertoire of F. velutipes mushrooms revealed through genomic studies has proven that this mushroom can be used for a wide variety of industrial applications and has ample potential for the development of new varieties. In this study, comparative genome analysis of F. velutipes was performed to identify key genes or mutations related to the color of the F. velutipes fruiting body. The color-related genes or mutations of F. velutipes identified in this study can be used to selectively develop the color of this mushroom variety in the future.

Genome Sequencing and Identification of Variations
Genome sequencing of F. velutipes strains was performed using the HiSeq 2000 platform (Illumina, Inc., San Diego, CA, USA). Sequenced reads were processed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 11 August 2022) and Trimmomatic (version 0.39) [30] for quality control. The final reads were used for mapping to the reference genome (F. velutipes KACC42780; accession number) [1] and to identify sequence variations using the genome analysis toolkit (GATK) pipeline [31] with the Burrows-Wheeler Aligner (BWA) [32], SAMtools [33], and PICARD [34]. SnpEff [35] and BEDTools [36] were used to identify the locations of variations within the genes. Gene modeling of the reference genome was performed by the Funannotate pipelines [37], using F. velutipes KACC43778 transcriptome data as "evidence". Gene models of F. velutipes KACC42780 were annotated using KEGG, InterPro, and UniProt databases. Genome sequencing reads were deposited in NABIC (National Agricultural Biotechnology Information Center, RDA, Korea) (see Data Availability Statement).

Primer Design and Amplification Refractory Mutation System PCRs
Primers that amplified specific variation sites within the phenylalanine ammonialyase 1 (Fvpal1) gene of white and non-white F. velutipes strains were designed based on the amplification refractory mutation system (ARMS) [38]. Three primer sets were designed with expected amplicons of 464 bp, 293 bp, and 200 bp from all F. velutipes strains, F. velutipes white strains, and F. velutipes non-white strains, respectively ( Table 2). Genomic DNA was used as a template (100 ng/µL) for ARMS PCR reactions using the Taq PreMix kit (TNT Research, Anyang, Korea) and 0.25 pmol of each primer in a 20 µL reaction mixture. PCR conditions were 10 min of initial denaturation at 94 • C, followed by 30 cycles of denaturation at 94 • C for 30 s, annealing at 56 • C for 30 s, extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min using Bio-Rad thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA). The amplified products were separated on a 2% agarose gel in 0.5 × TAE buffer (Tris-acetic acid-EDTA, TNT Research, Anyang, Korea) buffer and visualized with ethidium bromide on a UV transilluminator.

Mating of F. velutipes Strains
For spore collection of F. velutipes ASI4175 (non-white, heterozygote for ∆GCGCAC Fvpal1) and F. velutipes w1-8 (white, homozygote for ∆GCGCAC Fvpal1) strains, the stipe of the fruiting body was removed, and only part of the pileus was separated and placed in a Petri dish with the fold of the pileus facing down, and spores were collected for 24 h. The collected spores were diluted to a concentration of 1.0 × 10 −3 cfu/mL, and 100 µL of this dilution was spread on PDA medium and cultured for 7 d in a 25 • C incubator, protected from light. The germinated spores were inoculated into PDA medium and further cultured for 7 d in an incubator at 25 • C. Monokaryons without clamp connection were selected among the cultured mycelia. The Fvpal1 genotype of the isolated monokaryons was analyzed by ARMS PCR to select those for mating.
The monokaryons isolated from F. velutipes ASI4175 and F. velutipes w1-8 were inoculated on PDA medium at intervals of 1-2 cm and cultured for 7 d in a 25 • C incubator; hybrid dikaryons with clamp connection were then selected for fruiting. The hybrid dikaryons were inoculated into fruiting medium (80% sawdust and 20% rice bran) and incubated at 20 • C and 65% humidity for 30 days; the temperature was then sequentially changed from 14 • C (95% humidity) to 7 • C (80% humidity) for fruiting.

Identification of Fruiting Body Color-Specific Mutation of F. velutipes
The quality-trimmed reads of the six F. velutipes genome sequences were mapped to the reference genome sequence (F. velutipes KACC42870) at a rate of 73.15-80.13% (Table 3). A total of 70 white-color-specific variations, including 57 single nucleotide polymorphisms (SNPs) and 13 indels, were identified from the genome comparison of non-white and white F. velutipes strains (Table S1). Among the 11 chromosomes of F. velutipes, chromosome 7 had the highest number of variations (60%, 33 SNPs, and 9 indels), suggesting that genetic variations among F. velutipes strains mainly occurred on chromosome 7. Gene modeling of the reference strain (KACC42870) was conducted to identify variations in the genes. Using transcriptome data from Funannotate pipelines, a total of 15,874 gene models were identified from the reference genome (KACC42870) ( Table S2). Gene annotations revealed that 36 predicted genes were associated with the identified variations (Table S1). Among the variations in the genes, only one variation (indel) in Fvpal1 was identified; with a disruptive in-frame deletion in the exon of white F. velutipes strains. This variation was caused by a six-nucleotide (GCGCAC) deletion in the Fvpal1 gene of the white F. velutipes strains ( Figure 2 and Figure S1). The six-nucleotide deletion was located in the third exon of the Fvpal1 gene and resulted in arginine and threonine deletions without frameshift or reading frame interruption. genes. Using transcriptome data from Funannotate pipelines, a total of 15,874 gene models were identified from the reference genome (KACC42870) ( Table S2). Gene annotations revealed that 36 predicted genes were associated with the identified variations (Table S1). Among the variations in the genes, only one variation (indel) in Fvpal1 was identified; with a disruptive in-frame deletion in the exon of white F. velutipes strains. This variation was caused by a six-nucleotide (GCGCAC) deletion in the Fvpal1 gene of the white F. velutipes strains (Figures 2 and S1). The six-nucleotide deletion was located in the third exon of the Fvpal1 gene and resulted in arginine and threonine deletions without frameshift or reading frame interruption.  Figure 3 shows the scheme of ARMS PCR primer design to detect the specific variation ΔGCGCAC in the Fvpal1 gene and to discriminate the fruiting body color of F. velutipes. A four-primer set was designed to amplify all F. velutipes, non-white, or white strains ( Table 2). The FveF and FveR primers were expected to amplify the 464 bp product from all F. velutipes strains. In addition, the FveF/FveW and FveR/FveB primer sets were expected to amplify 293 bp and 200 bp products for F. velutipes white and non-white strains, respectively.

Primers Design and ARMS PCR for Fruiting Body Color Discrimination of F. velutipes
ARMS is a simple and reliable method for identifying single nucleotide variations (SNVs) or deletions [38]. Since ARMS uses PCR primers that allow the amplification of  Figure 3 shows the scheme of ARMS PCR primer design to detect the specific variation ∆GCGCAC in the Fvpal1 gene and to discriminate the fruiting body color of F. velutipes. A four-primer set was designed to amplify all F. velutipes, non-white, or white strains ( Table 2). The FveF and FveR primers were expected to amplify the 464 bp product from all F. velutipes strains. In addition, the FveF/FveW and FveR/FveB primer sets were expected to amplify 293 bp and 200 bp products for F. velutipes white and non-white strains, respectively. strains were detected using specific primers and ARMS PCR analysis, and as a result, the fruiting body color of F. velutipes strains was accurately discriminated.

Primers Design and ARMS PCR for Fruiting Body Color Discrimination of F. velutipes
Although the color of the fruiting body was accurately discriminated by analysis of the 95 strains used in this study, future research should continuously analyze an additional F. velutipes strain to determine whether the variation in Fvpal1 affects the color of the fruiting body.   ARMS is a simple and reliable method for identifying single nucleotide variations (SNVs) or deletions [38]. Since ARMS uses PCR primers that allow the amplification of DNA only in the presence of specific mutations, the amplification of ARMS PCR products determines the presence of mutations. As shown in Figure 4, ARMS PCR analysis revealed the specific detection of the ∆GCGCAC variation in Fvpal1, as well as the specific discrimination of non-white and white F. velutipes strains. Among the 95 F. velutipes strains tested, ARMS PCR amplified 464 bp and 293 bp products in all white strains. The non-white strains amplified either 464 bp and 200 bp or 464 bp, 200 bp, and 293 bp products. The F. velutipes ASI4175 non-white strain amplified all three products, indicating that it was heterozygous for the normal and ∆GCGCAC Fvpal1 gene. These results suggest that the normal Fvpal1 gene has a dominant effect on the fruiting body color of F. velutipes, as F. velutipes ASI4175 is a non-white strain. In this study, Fvpal1 gene mutations in 50 white strains were detected using specific primers and ARMS PCR analysis, and as a result, the fruiting body color of F. velutipes strains was accurately discriminated.
Although the color of the fruiting body was accurately discriminated by analysis of the 95 strains used in this study, future research should continuously analyze an additional F. velutipes strain to determine whether the variation in Fvpal1 affects the color of the fruiting body.

Mutation in the Fvpal1 Gene Affect Fruiting Body Color of F. velutipes
Phenylalanine ammonia-lyase (PAL; EC 4.3.1.24) catalyzes the deamination of Lphenylalanine to trans-cinnamic acid and is commonly found in plants and fungi [39,40]. PAL is involved in the first step of the phenylpropanoid pathway, leading to the synthesis of various phenylpropanoids such as flavonoids, isoflavonoids, anthocyanins, lignins, and other phenolic compounds [40]. Therefore, PAL is considered to be a key initiator of the phenylpropanoid pathway, a transition process from primary to secondary metabolism.
In plants, particularly in lettuce, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, caffeoyltartaric acid, and dicaffeoyltartaric acid have been reported to be associated with browning [41,42]. It has also been reported that the phenylpropanoid pathway is activated by wounds or hormones, such as ethylene, to increase the synthesis of phenolic compounds [41,43]. Therefore, PAL has been extensively studied to increase the understanding of metabolic processes as well as browning. It has also been reported that PAL enzyme and phenolic compounds are essential for the browning of mung bean sprouts [44]. In addition, phenolic compounds, including trans-caffeoyltartronic acid and trans-coumaroyltartronic acid, are substrates of polyphenol oxidase (PPO; EC 1.10.3.1) in mung bean sprouts and act as major factors for browning [44]. It has been suggested that the polyphenols in mung bean sprouts increase gradually during storage and are oxidized by PPO to form a brown pigment [45,46].
It has been reported that the fungal PAL enzyme degrades phenylalanine via a pathway similar to that in plants [47,48]. Further research revealed the phenylalanine metabolic pathway in some basidiomycetes, including Rhodotorula glutinis, Schizophyllum commune, and Sporobolomyces roseus [48][49][50]. In the phytopathogenic fungi Moniliophthora perniciosa, PAL has been found to accumulate during the infection stage, suggesting that it may be associated with pathogenicity [51]. Additionally, Tricholoma matsutake and F. velutipes PAL mRNAs were expressed specifically at the developmental stage, and in F. velutipes, the highest expression was found in the mycelium and when L-tyrosine was added [52,53]. Although there have been reports of various roles for PAL, it has not been reported that it is associated with the color of fungi, including mushrooms.

Structural Characteristics of the PAL of F. velutipes
The catalytic prosthetic 3,5-dihydro-5-methylidine-4H-imidazol-4-one group (MIO) is essential for the catalytic activity of PAL and is produced by the autocatalytic crystallization of amino acids, including alanine, serine, and glycine [40,54,55]. The MIO group is commonly found in ammonia lyases, including PAL, tyrosine ammonia lyase (TAL), and histidine ammonia lyase (HAL), and the ASG motif plays an important role in this enzymatic activity [56][57][58][59]. A highly conserved MIO group (ASG motif) was also found at positions 238-240 in Fvpal1 of F. velutipes in both the white and non-white strains ( Figure 6). Another highly conserved motif was also found in Fvpal1 of F. velutipes, including stabilizing residues for the MIO group (N 303 and Y 401 ) and carboxylic-acid-binding residues for the substrate (R 404 ).
Among the PAL and TAL motifs, specific residues for substrate specificity are phenylalanine-leucine (FL) for phenylalanine and histidine-leucine (HL) for tyrosine. A characteristic histidine-glycine (HQ) motif of the PAL enzyme, which exhibits substrate activity for both tyrosine and phenylalanine, has also been reported [60,61]. For PAL and TAL enzymes with an HQ motif, it has been reported to have dual substrate activity with a Phenylalanine/Tyrosine ratio greater than one [61][62][63][64]. A histidine-glycine ( 160 HQ 161 ) motif was also found in the FvPAL1 of F. velutipes as well as other species, including P. ostreatus and A. bisporus ( Figure 6). Although specific activity assays for phenylalanine and tyrosine are In this study, mating analysis revealed that the Fvpal1 gene plays an important role in the fruiting body color of F. velutipes, suggesting that this gene could be a useful marker for the selective breeding of new varieties of F. velutipes, especially for fruiting body color.

Structural Characteristics of the PAL of F. velutipes
The catalytic prosthetic 3,5-dihydro-5-methylidine-4H-imidazol-4-one group (MIO) is essential for the catalytic activity of PAL and is produced by the autocatalytic crystallization of amino acids, including alanine, serine, and glycine [40,54,55]. The MIO group is commonly found in ammonia lyases, including PAL, tyrosine ammonia lyase (TAL), and histidine ammonia lyase (HAL), and the ASG motif plays an important role in this enzymatic activity [56][57][58][59]. A highly conserved MIO group (ASG motif) was also found at positions 238-240 in Fvpal1 of F. velutipes in both the white and non-white strains ( Figure 6). Another highly conserved motif was also found in Fvpal1 of F. velutipes, including stabilizing residues for the MIO group (N 303 and Y 401 ) and carboxylic-acid-binding residues for the substrate (R 404 ). In this study, two PAL genes, Fvpal1 and Fvpal2, were identified in the genome of F. velutipes. No mutations were found in the sequence of the Fvpal2 gene ( Figure S3). As shown in Figure 7, the fruiting body color-related mutation Δ 112 RT 113 was not found in the Fvpal2 gene of either non-white or white F. velutipes strains. Motifs essential for the catalytic activity of PAL enzymes were found in Fvpal2 genes. However, substrate-specific residues of Fvpal2 ( 138 MQ 139 ) were found to be different from those of the Fvpal1 gene ( 160 HQ 161 ) but identical to those of the PoPAL1 gene ( 160 MQ 161 ) of P. ostreatus (Figures 6 and  7).
In a previous study [53], Fvpal was identified in F. velutipes and was found to have the same sequence as the Fvpal1 gene of the F. velutipes non-white strain identified in this study, which consisted of 2746 bp and 12 exons and showed 100% identity to Fvpal, with 724 amino acids (2175 bp cDNA). However, Δ 112 RT 113 deletions identified in the Fvpal1 gene were not found in the Fvpal gene sequence. These results indicate that the previously reported Fvpal gene [53] was identified in non-white F. velutipes, and this was confirmed with the information that the non-white strain F. velutipes 4164 was used for the identification of the Fvpal gene [53]. The Fvpal2 gene is 2617 bp in size with eight exons and consists of a 2208 bp cDNA that is translated into 735 amino acids ( Figure S3). Furthermore, among the Fvpal2 genes identified from F. velutipes strains, no mutations specific to nonwhite or white strains were found, except for variations for each strain. Although further studies are required, these results suggest that the Fvpal2 gene is essential for the physiological and metabolic functions of F. velutipes but that the Fvpal1 gene has the potential to function selectively in determining the color of the F. velutipes fruiting body. Among the PAL and TAL motifs, specific residues for substrate specificity are phenylalanine-leucine (FL) for phenylalanine and histidine-leucine (HL) for tyrosine. A characteristic histidine-glycine (HQ) motif of the PAL enzyme, which exhibits substrate activity for both tyrosine and phenylalanine, has also been reported [60,61]. For PAL and TAL enzymes with an HQ motif, it has been reported to have dual substrate activity with a K mPhenylalanine/Tyrosine ratio greater than one [61][62][63][64]. A histidine-glycine ( 160 HQ 161 ) motif was also found in the FvPAL1 of F. velutipes as well as other species, including P. ostreatus and A. bisporus ( Figure 6). Although specific activity assays for phenylalanine and tyrosine are required, the HQ motif in the FvPAL1 of F. velutipes suggests that this enzyme could possibly catalyze both substrates.
In this study, two PAL genes, Fvpal1 and Fvpal2, were identified in the genome of F. velutipes. No mutations were found in the sequence of the Fvpal2 gene ( Figure S3). As shown in Figure 7, the fruiting body color-related mutation ∆ 112 RT 113 was not found in the Fvpal2 gene of either non-white or white F. velutipes strains. Motifs essential for the catalytic activity of PAL enzymes were found in Fvpal2 genes. However, substrate-specific residues of Fvpal2 ( 138 MQ 139 ) were found to be different from those of the Fvpal1 gene ( 160 HQ 161 ) but identical to those of the PoPAL1 gene ( 160 MQ 161 ) of P. ostreatus (Figures 6 and 7).

Conclusions
In nature, Flammulina velutipes forms non-white fruiting bodies, but white fruiting body varieties were accidentally developed by artificial breeding. However, until recently the physiological, biochemical, and genetic causes associated with the formation of white fruiting bodies in F. velutipes had not been elucidated. A recent comparative analysis reported that components of non-white F. velutipes strains, such as amino acids, saccharides, and β-glucan, were relatively higher or lower than those of white strains [6,[12][13][14][15][16][17]. These results suggest that the fruiting body color of F. velutipes can be used as a criterion for breeding new varieties. Therefore, the selective breeding of fruiting body color is considered a great advantage for efficient breeding.
In this study, comparative genomics of six F. velutipes strains showed 70 variations unique to white strains, including SNPs and indels. Of these, 36 were found in open reading frames and only one caused a disruptive in-frame deletion (ΔGCGCAC) in the Fvpal1 gene, resulting in the deletion of two amino acids (arginine and threonine) at positions 112 and 113. Specific primers were designed to detect this mutation, and PCR analysis of 95 F. velutipes strains revealed that this mutation was present only in white strains. In addition, monospores of the heterozygous mutant were isolated, and a mating assay was performed to evaluate the mutation's relationship to fruiting body color. As a result, progeny resulting from mating monospores with and without the ΔGCGCAC deletion in the Fvpal1 gene showed non-white fruiting bodies. However, mating monospores with the mutated Fvpal1 gene resulted in progeny with only white-colored fruiting bodies.
Although the effect of mutations in the Fvpal1 gene of F. velutipes on enzyme activity and metabolic function remains to be studied, it is considered that this gene can be effectively used for selective breeding of this mushroom.   In a previous study [53], Fvpal was identified in F. velutipes and was found to have the same sequence as the Fvpal1 gene of the F. velutipes non-white strain identified in this study, which consisted of 2746 bp and 12 exons and showed 100% identity to Fvpal, with 724 amino acids (2175 bp cDNA). However, ∆ 112 RT 113 deletions identified in the Fvpal1 gene were not found in the Fvpal gene sequence. These results indicate that the previously reported Fvpal gene [53] was identified in non-white F. velutipes, and this was confirmed with the information that the non-white strain F. velutipes 4164 was used for the identification of the Fvpal gene [53]. The Fvpal2 gene is 2617 bp in size with eight exons and consists of a 2208 bp cDNA that is translated into 735 amino acids ( Figure S3). Furthermore, among the Fvpal2 genes identified from F. velutipes strains, no mutations specific to non-white or white strains were found, except for variations for each strain. Although further studies are required, these results suggest that the Fvpal2 gene is essential for the physiological and metabolic functions of F. velutipes but that the Fvpal1 gene has the potential to function selectively in determining the color of the F. velutipes fruiting body.

Conclusions
In nature, Flammulina velutipes forms non-white fruiting bodies, but white fruiting body varieties were accidentally developed by artificial breeding. However, until recently the physiological, biochemical, and genetic causes associated with the formation of white fruiting bodies in F. velutipes had not been elucidated. A recent comparative analysis reported that components of non-white F. velutipes strains, such as amino acids, saccharides, and β-glucan, were relatively higher or lower than those of white strains [6,[12][13][14][15][16][17]. These results suggest that the fruiting body color of F. velutipes can be used as a criterion for breeding new varieties. Therefore, the selective breeding of fruiting body color is considered a great advantage for efficient breeding.
In this study, comparative genomics of six F. velutipes strains showed 70 variations unique to white strains, including SNPs and indels. Of these, 36 were found in open reading frames and only one caused a disruptive in-frame deletion (∆GCGCAC) in the Fvpal1 gene, resulting in the deletion of two amino acids (arginine and threonine) at positions 112 and 113. Specific primers were designed to detect this mutation, and PCR analysis of 95 F. velutipes strains revealed that this mutation was present only in white strains. In addition, monospores of the heterozygous mutant were isolated, and a mating assay was performed to evaluate the mutation's relationship to fruiting body color. As a result, progeny resulting from mating monospores with and without the ∆GCGCAC deletion in the Fvpal1 gene showed non-white fruiting bodies. However, mating monospores with the mutated Fvpal1 gene resulted in progeny with only white-colored fruiting bodies.
Although the effect of mutations in the Fvpal1 gene of F. velutipes on enzyme activity and metabolic function remains to be studied, it is considered that this gene can be effectively used for selective breeding of this mushroom.