Origin of the paternal gametes of Japanese whitish truffle (<i>Tuber japonicum</i>) in the second year of truffle production at a konara oak (<i>Quercus serrata</i>) plantation in Kyoto Prefecture, Japan

Tuber japonicum is an ectomycorrhizal ascomycete fungus native to Japan that produces whitish truffles as one of their reproductive organs. In 2019, a truffle plantation was established in Kyoto Prefecture, Japan, by planting 14 seedlings of konara oak inoculated with two distinct inocula. In November 2022, the first ascocarp production was detected. To contribute to our understanding of the origin of the paternal gametes in this species, we determined the genotypes of 91 second-year ascocarps collected from the site in the autumn of 2023. All of the successfully-analyzed ascocarps possessed one of three maternal multilocus genotypes (MLGs), as identified by simple sequence repeat (SSR) marker analysis, two of which were identical to the paternal and maternal genotypes identified in ascocarps harvested the previous year. The number of paternal MLGs identified increased markedly from 2 in 2022 to 53 in 2023, 48 of which were found in only a single ascocarp. All paternal MLGs were either identical to a maternal one or a random recombination between them, indicating that the paternal gametes were derived from ascospores formed the previous year. Thus, this study clearly demonstrates that the majority of paternal mating partners in T. japonicum originate from the previous year’s ascospores.