Diversity of A mating type in Lentinula edodes has been assessed by analysis of A mating loci in 127 strains collected from East Asia. It was discovered that hypervariable sequence region with an approximate length of 1 kb in the A mating locus, spanning 5' region of HD2-intergenic region-5' region of HD1, could represent individual A mating type as evidenced by comprehensive mating analysis. The sequence analysis revealed 27 A mating type alleles from 96 cultivated strains and 48 alleles from 31 wild strains. Twelve of them commonly appeared, leaving 63 unique A mating type alleles. It was also revealed that only A few A mating type alleles such as A1, A4, A5, and A7 were prevalent in the cultivated strains, accounting for 62.5% of all A mating types. This implies preferred selection of certain A mating types in the process of strain development and suggests potential role of A mating genes in the expression of genes governing mushroom quality. Dominant expression of an A mating gene HD1 was observed from A1 mating locus, the most prevalent A allele, in A1-containing dikaryons. However, connections between HD1 expression and A1 preference in the cultivated strains remain to be verified. The A mating type was highly diverse in the wild strains. Thirty-six unique A alleles were discovered from relatively small and confined area of mountainous region in Korean peninsula. The number will further increase because no A allele has been recurrently observed in the wild strains and thus newly discovered strain will have good chances to contain new A allele. The high diversity in small area also suggests that the A mating locus has evolved rapidly and thus its diversity will further increase.
Variable number tandem repeats (VNTRs) in mitochondrial DNA (mtDNA) of Lentinula edodes are of interest for their role in mtDNA variation and their application as genetic marker. Sequence analysis of three L. edodes mtDNAs revealed the presence of VNTRs of two categories. Type I VNTRs consist of two types of repeat units in a symmetric distribution, whereas Type II VNTRs contain tandemly arrayed repeats of 7- or 17-bp DNA sequences. The number of repeat units was variable depending on the mtDNA of different strains. Using the variations in VNTRs as a mitochondrial marker and the A mating type as a nuclear type marker, we demonstrated that one of the two nuclei in the donor dikaryon preferentially enters into the monokaryotic cytoplasm to establish a new dikaryon which still retains the mitochondria of the monokaryon in the individual mating. Interestingly, we found 6 VNTRs with newly added repeat units from the 22 mates, indicating that elongation of VNTRs occurs during replication of mtDNA. This, together with comparative analysis of the repeating pattern, enables us to propose a mechanistic model that explains the elongation of Type I VNTRs through reciprocal incorporation of basic repeat units, 5’-TCCCTTTAGGG-3’ and its complementary sequence (5’-CCCTAAAGGGA-3’).
In mating of Lentinula edodes, dikaryotic strains generated from certain monokaryotic strains such as the B2 used in this study tend to show better quality of fruiting bodies regardless of the mated monokaryotic strains. Unlike B2, dikaryotic strains generated from B16 generally show low yields, with deformed or underdeveloped fruiting bodies. This indicates that the two nuclei in the cytoplasm do not contribute equally to the physiology of dikaryotic L. edodes, suggesting an expression bias in the allelic genes of the two nuclei. To understand the role of each nucleus in dikaryotic strains, we investigated single nucleotide polymorphisms (SNPs) in laccase genes of monokaryotic strains to reveal nuclear origin of the expressed mRNAs in dikaryotic strain. We performed reverse transcription PCR (RT-PCR) analysis using total RNAs extracted from dikaryotic strains (A5B2, A18B2, and A2B16) as well as from compatible monokaryotic strains (A5, A18, and B2 for A5B2 and A18B2; A2 and B16 for A2B16). RT-PCR results revealed that Lcc1, Lcc2, Lcc4, Lcc7, and Lcc10 were the mainly expressed laccase genes in the L. edodes genome. To determine the nuclear origin of these laccase genes, the genomic DNA sequences in monokaryotic strains were analyzed, thereby revealing five SNPs in Lcc4 and two in Lcc7. Subsequent sequence analysis of laccase mRNAs expressed in dikaryotic strains revealed that these were almost exclusively expressed from B2-originated nuclei in A5B2 and A18B2 whereas B16 nucleus did not contribute to laccase expression in A2B16 strain. This suggests that B2 nucleus dominates the expression of allelic genes, thereby governing the physiology of dikaryons.
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