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

In nature; <i>Flammulina velutipes</i>, 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 <i>F. velutipes</i> and to identify variations in the genes associated with fruiting body color. Comparative genomics of six <i>F. velutipes</i> 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 (<i>Fvpal1</i>). 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 <i>Fvpal1</i> 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 <i>F. velutipes</i> fruiting body. PCR analysis of a total of 95 <i>F. velutipes</i> 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 <i>Fvpal1</i>, it was found that this mutation plays an important role in determining the color of the fruiting body. Furthermore, the deletion (Δ¹¹²RT¹¹³) in <i>Fvpal1</i> 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 <i>F. velutipes</i>, a representative edible mushroom.