Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.

The evolution of mitochondria through variations in mitochondrial DNA (mtDNA) is one of the intriguing questions in eukaryotic cells. In order to assess the causes of the variations in mitochondria, the mtDNAs of the 21 strains of Lentinula edodes were assembled for this study, and analyzed together with four published mtDNA sequences. The mtDNAs were within the sizes of 117 kb ~ 122 kb. The gene number was observed consistent except for two mtDNAs, which carry a duplicated trnG1-trnG2 unit or a putative gene deletion. The size variation was largely attributed to the number of introns, repeated sequences, transposable elements (TEs), and plasmid-related sequences. Intron loss and gain were found from cox1, rnl, and rns of three mtDNAs. Loss of two introns in cox1 of KY217797.1 reduced its size by 2.7 kb, making it the smallest cox1 gene (8.4 kb) among the cox1s of the 25 mtDNAs, whereas gain of a Group II intron (2.65 kb) and loss of a Group I intron (1.7 kb) in cox1 of MF774813.1 resulted in the longest cox1 (12 kb). In rnl of L. edodes, we discovered four intron insertion consensus sequences which were unique to basidiomycetes but not ascomycetes. Differential incorporation of introns was the primary cause of the rnl size polymorphism. Homing endonucleases (HEGs) were suggestively involved in the mobilization of the introns because all of the introns have HEG genes of the LAGRIDADG or GIY-YIG families with the conserved HEG cleavage sites. TEs contributed to 11.04% of the mtDNA size in average, of which 7.08% was LTR-retrotransposon and 3.96% was DNA transposon, whereas the repeated sequences covered 4.6% of the mtDNA. The repeat numbers were variable in a strain-dependent manner. Both the TEs and repeated sequences were mostly found in the intronic and intergenic regions. Lastly, two major deletions were found in the plasmid-related sequence regions (pol2-pol3 and pol1-atp8) in the five mtDNAs. Particularly, the 6.8 kb-long deletion at pol2-pol3 region made MF774813.1 the shortest mtDNA of all. Our results demonstrate that mtDNA is a dynamic molecule that persistently evolves over a short period of time by insertion/deletion and repetition of DNA segments at the strain level.

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Karyotyping in Agaricus bisporus is crucial for both the isolation of homokaryotic strains and the confirmation of dikaryon establishment. For the verification of the karyotype, the A mating type loci of two homokaryotic strains, H39 and H97, were analyzed through comparative sequence analysis. The two loci showed major differences in two sequence regions designated as Region 1 and Region 2. H97 had a putative DNA transposon in Region 1 that had target site duplications (TSDs), terminal inverted repeats (TIRs), and a loop sequence, in contrast to H39, which only had the insertional target sequence. Homologous sequences of the transposon were discovered in the two different chromosomes of H97 and in one of H39, all of which have different TSDs but share high sequence homology in TIR. Region 2 shared three consensus sequences between H97 and H39. However, it was only from H97 that a large insertional sequence of unknown origin was discovered between the first and second consensus sequences. The difference in length in Region 1, employed for the verification of the A mating type, resulted in the successful verification of mating types in the heterokaryotic and homokaryotic strains. This length difference enables the discrimination between homo- and heterokaryotic spores by PCR. The present study suggests that the A mating type locus in A. bisporus H97 has evolved through transposon insertion, allowing the discrimination of the mating type, and thus the nuclear type, between A. bisporus H97 and H39.