Mushroom Science and Biotechnology, Vol. 27(4) 128-133, 2019 Copyright © 2019, Japanese Society of Mushroom Science and Biotechnology

Regular Paper Mating in Mycoleptodonoides aitchisonii is not genetically associated with the monokaryotic clamp cell formation phenotype

Rini RIFFIANI1, 5), Takayuki WADA2), Fu-Chia CHEN3), Norihiro SHIMOMURA4) Takeshi YAMAGUCHI4) and Tadanori AIMI4)*

1) The United Graduate School of Agricultural Sciences, Tottori University, 4-101 Koyama-cho Minami, Tottori 680-8553, Japan 2) Laboratory, Biological Business Department, Ichimasa Kamaboko Co., Ltd., 77-1 Junishin, Agano-shi, Niigata 959-1936, Japan 3) Graduate School of Sustainability Science, Tottori University, 4-101 Koyama-cho Minami, Tottori 680-8553, Japan 4) Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori 680-8553, Japan 5) Research Center of Biology, Indonesia Institute of Science (LIPI), Bogor Cibinong 16911, Indonesia

（Received 10 October 2019 / Accepted 30 October 2019）

Abstract Mycoleptodonoides aitchisonii is a wood-rooting edible mushroom. In response to previous reports that some basidiospore isolates of this mushroom can form complete fruiting bodies and true clamp cells, the frequency of true clamp cell formation was compared between the dikaryotic and the monokaryotic strains. Compared to monokaryotic strains, true clamp cells were observed with greater frequency in dikaryotic strains. Mating incompatibility groups were examined among basidiospore isolates from dikaryotic strain TUFC50005 (P) and TUFC50005-7 × TUFC50005-18 (F1), which were derived from strain TUFC50005. Mating compatibility could be divided into two groups indicating that M. aitchisonii is a bipolar mushroom. Moreover, recombinant mating type strain might not be generated after meiosis, indicating that there may only be a single mating-type locus in M. aitchisonii. No genetic linkage was observed between the phenotype capable of forming monokaryotic clamp cells and mating type, indicating that monokaryotic clamp formation was not linked to the mating-type locus. Key words: Bipolar mating system, Linkage analysis, Monokaryotic clamp cell formation, Mycoleptodonoides aitchisonii

(Bunaharitake) belongs to the family Climacodontaceae Introduction and is commonly found on dead broad-leaved trees In basidiomycete mushrooms, mating compatibility from summer to fall in Asia. The fruiting bodies are is controlled by one or two sets of multiallelic genes effused-reflexed with fan- or spatula-shaped caps. The in what are referred to as bipolar or tetrapolar mating exhibits pharmaceutical properties, including systems, respectively1). These so-called mating-type immunomodulation, lipid-lowering effects and genes regulate nuclear pairing and clamp formation2, 3), antibacterial effects8). Recently, the ability to produce and investigations of mating compatibility increase our monokaryotic fruiting bodies and clamp cells was understanding of the life cycle of individual species. reported in strains isolated from basidiospores9). In Bipolar mating systems are controlled by a single mating- that report, a single dikaryotic M. aitchisonii strain, type locus and only two mating types are produced by TUFC50005, and 20 monokaryons derived from a meiosis. The tetrapolar mating system is based on two basidiospore of strain TUFC50005, exhibited a wide unlinked mating types, commonly referred to as A and spectrum of fruiting body developmental stages. While B loci. In tetrapolar systems, when the two mating type most strains formed primordia, or young fruiting loci are unlinked, four mating types can be generated body-like structures, one of the monokaryons, strain after meiosis among the haploid progeny4, 5). In several TUFC50005-4, formed a complete fruiting body, even higher basidiomycetes, bipolarity has been traced to the though it had only one nucleus and produced only two loss of mating specificity of the B locus. However, when spores after meiosis. We previously demonstrated that B is complete and present, only the A locus segregates in dikaryotization was not required for clamp cell and a mating-type specific manner6, 7). fruiting body formation9). The edible mushroom, Mycoleptodonoides aitchisonii In the present study, we examined the polarity of mating types in M. aitchisonii as well as the genetic *Corresponding author. E-mail: [email protected] linkage between the mating-type locus and the 日本きのこ学会誌 MUSHROOM SCIENCE AND BIOTECHNOLOGY 129 ability to form monokayotic clamp cells by classical observed under a microscope to check for the formation of genetic analysis. In addition, we sought to clarify the clamp connections as evidence of dikaryotization. A slide mechanisms underlying monokaryotic clamp cell culture method was performed to clearly observe clamp formation in M. aitchisonii. connections using TM7 medium. The TM7 medium com- prised 0.2% (w/v) malt agar with 0.2% (w/v) Tween 80 on a thin-layer slide and 2% (w/v) agar, with pH adjusted to Materials and Methods 7.0 with 1 M NaOH (see Shimomura et al.)10). Inoculated 1. Strains and culture conditions mycelia were incubated at 25℃ for 10 days and clamp The strain used in these experiments, M. aitchisonii cells were observed under a microscope (Nikon Eclipse 80i, TUFC 50005, was obtained from the / Nikon Corp., Tokyo, Japan) equipped with a camera (Nikon Mushroom Resource and Research Center at the DS-L2). Faculty of Agriculture, Tottori University, Japan 5. Frequency analysis of clamp cell formation (TUFC). Basidiospore isolates 50005-1 to 50005-20 were Frequency analysis of true clamp-cell formation isolated from fruiting bodies of TUFC 50005. All of the in 10 day-old mycelia on TM7 medium was performed M. aitchisonii strains were maintained on 2% malt agar under a microscope. The frequency of clamp formation slants at room temperature. at the septa that formed between the subterminal cell 2. Cultivation of fruit bodies and the third cell of a mycelium was determined by the The sawdust medium for the cultivation of fruiting following formula10): Clamp formation frequency (%) = bodies consisted of sawdust and rice bran at a ratio of (Number of septa with clamps/number of septa observed) 9:1 (v/v). Water was added to the sawdust-rice bran × 100%. medium so that the moisture content of the substrate 6. Statistical analysis was 65%. Then, 600 g of substrate was bagged into high- The chi-square goodness of fit test is useful for density polyethylene bags (18 × 8 × 28 cm, SE-25ES, comparing a theoretical model to observed data11). At Sakato Sangyo Co. Ltd., Japan), sealed with a heat sealer, first, we attempted to confirm how many progeny in first sterilized by autoclaving (121℃, 60 min), cooled, and then and second generation strains were carrying the A1 and inoculated with an agar plug containing mycelia. The A2 at equal frequencies. To determine the relationship inoculated substrate was incubated at 25℃ in the dark between the mating type and monokaryotic clamp cell for 60 days to facilitate colonization of the substrate by formation we used the chi-square test of independence. the fungus (hereafter referred to as “spawn running”). The chi-square goodness of fit test was estimated Upon completion of spawn running, the fully colonized using the following equation: substrate was irradiated with visible light at 200 lux for a week at 25℃ (Stage 1). Samples were then incubated at 15℃ and a relative humidity of 85% under visible light at 200 lux for 1 month until the initiation of fruiting. To induce fruiting, each culture bag was cut in a cross- shaped pattern using a sterile knife, and incubation was where x2= Chi-square goodness of fit, O = Observed continued until the fruiting bodies matured. value, E = expected value, k = number of categories or 3. Isolation of basidiospore isolates groupings. To prepare a spore suspensions, 1 mL of sterilized The chi-square test of independence was estimated water was pipetted onto spore prints in petri dishes using the following equation: which were then vigorously shaken. The concentration of spores, which was determined using a hemocytometer under a microscope, was adjusted to approximately 1 × 104 to 1 × 106 cells/mL. Then, 0.1 mL of suspension was mixed with 2 mL of soft agar (0.7%) melted at 50℃ 2 in a test tube and then poured onto a 2% malt extract where x = chi-square test of independence, Oi.j = ob- agar plate to prepare a double-layer agar culture. After served value of two nominal variables, incubating the culture at 25℃ for 5 － 7 days, the single Ei.j = expected value of two nominal variables colonies that appeared on the plate were transferred to 2% malt agar slants. These slants were kept at 25℃ before Results being used for crossing experiments. 4. Crossing 1. Frequency of true clamp cell formation The two monokaryotic stocks were placed 1 cm apart In a previous study9), we described true clamp at the center of a 2% malt agar plate. After incubation for 3 cell formation in monokaryons that had been isolated weeks at 25℃, the mycelia on the 2% malt agar plate at the from a basidiospore. In this study, to check the mating contact zone between the two parental monokaryons were compatibility after crossing, we compared the frequency 130 Vol.27 No. 4

Table 1. Frequency of clamp cell formation

Number Total of septa Frequency Strain Nuclear phase number of with (%) septa clamp cells 50005 Dikaryon 68 40 58.8 50005-4 Monokaryon 18 1 5.5 50005-7 Monokaryon 26 1 3.8 50005-18 Monokaryon 17 1 5.9 Fig 1. Visualisation of (A) dikaryotic clamp cell connections (arrows) 50005-7 × Dikaryon 18 12 66.7 and (B) monokaryotic clamp cell formation (arrow) under a light 50005-4 50005-7× microscope. Dikaryon 48 26 54.2 50005-18 TUFC was abbreviated in each strain number

Table 2. Incompatibility groups among basidiospore isolates derived from strain TUFC50005 A1 A2 2* 3* 4* 5 6 9 10 12 15* 16 18* 19* 1* 7* 8* 11 13* 14* 17* 20* 1* + + + + + + + + + + + + − − − − − − − − 7* + + + + + + + + + + + + − − − − − − − − 8* + + + + + + + + + + + + − − − − − − − − 11 + + + + + + + + + + + + A2 − − − − − − − − 13* + + + + + + + + + + + + − − − − − − − − 14* + + + + + + + + + + + + − − − − − − − − 17* + + + + + + + + + + + + − − − − − − − − 20* + + + + + + + + + + + + − − − − − − − − 2* − − − − − − − − − − − − + + + + + + + + 3* − − − − − − − − − − − − + + + + + + + + 4* − − − − − − − − − − − − + + + + + + + + 5 − − − − − − − − − − − − + + + + + + + + 6 − − − − − − − − − − − − + + + + + + + + 9 + + + + + + + + A1 − − − − − − − − − − − − 10 − − − − − − − − − − − − + + + + + + + + 12 − − − − − − − − − − − − + + + + + + + + 15* − − − − − − − − − − − − + + + + + + + + 16 − − − − − − − − − − − − + + + + + + + + 18* − − − − − − − − − − − − + + + + + + + + 19* − − − − − − − − − − − − + + + + + + + + Strain number 50005– is abbreviated +: clamp cells observed in large numbers −: clamp cells were not observed *monokaryotic true clamp cell formation was observed of clamp cell formation between dikaryons produced by Group 2 comprised strains 50005-2, 3, 4, 5, 6, 9, 10, 12, 15, crossing compatible monokaryons and monokaryons 16, 18 and 19. Based on this grouping, we considered that alone (Fig. 1A and B, respectively). The frequency of clamp M. aitchisonii is a bipolar mushroom. The mating types formation in dikaryons was greater than 50%, i.e., more of Group I and II were A2 and A1, respectively. than 50% of septa had clamp cells. On the other hand, the 3. Incompatibility among second generation (F2) maximum frequency of clamp formation in monokaryons basidiospore isolates from dikaryotic strain 50005-7 × was 5.9%, and almost strains were less than 10% (Table 1). 50005-18 The difference in frequencies was thus very clear, and we To identify subloci of the mating locus, a fruiting considered the frequency of clamp cell formation can be body was cultivated using the crossed strain 50005-7 × used to distinguish between dikaryotic cells (dikaryons) 50005-18, and then 86 strains (BRW-2 - BRW-177) were and clamp cells producing monokaryons. isolated from the basidiospore of 50005-7 × 50005-18 and 2. Incompatibility groups among first-generation (F1) crossed with each other. These strains were designated as basidiospore isolates produced from strain TUFC50005. F2 progeny. Microscopic examination of clamp cell forma- Basidiospore isolates from strain TUFC50005 (50005- tion revealed that the 86 strains were clearly divided into 1 to 50005-20) were crossed with each other and clamp two incompatibility groups based on the production of cell formation was investigated under a microscope. true clamp connections at high frequencies after crossing These strains were designated F1 progeny of strain (Table 3). This phenomenon corresponded with the incom- TUFC50005 and the results are shown in Table 2. The 20 patibility group of basidiospore isolates of TUFC50005, in- strains could be clearly divided into two incompatibility dicating that this mushroom is bipolar. Moreover, among groups depending on the production of true clamp cells the 86 isolates, none of the strains were compatible or in- at high frequency after crossing each other. Group 1 compatible with strains belonging to either of the compat- comprised strains 50005-1, 7, 8, 11, 13, 14, 17 and 20, and ibility groups. Since the recombinant mating type strain 日本きのこ学会誌 MUSHROOM SCIENCE AND BIOTECHNOLOGY 131

Table 3. Incompatibility groups among basidiospore isolates Table 4. Monokaryotic clamp cell formation among monospore iso- derived from strains 50005-7 × 50005-18 lates derived from dikaryotic strains (50005-7 × 50005-18)

Clamp Cell A1 A2 A1 A2 formation

2, 3, 4, 6, 7, 8, 9, 12, 13, 14, 18, 10, 11, 15, 16, 17, 23, 26, 31, 36, True clamp 58, 60, 25, 41, 52, 88, 143, 31, 63, 65, 51, 59, 64, 177, 19, 20, 21, 22, 24, 25, 29, 30, 33, 43, 45, 46, 50, 51, 55, 59, 62, 63, 154, 165 175 37, 38, 39, 40, 41, 42, 47, 48, 49, 64, 65, 67, 69, 81, 107, 108, 109, No clamp 2, 3, 4, 6, 7, 8, 9, 12, 13, 14, 10, 11, 15, 16, 17, 23, 26, 52, 53, 57, 58, 60, 70, 71, 74, 75, 110, 125, 129, 145, 175, 177 18, 19, 20, 21, 22, 24, 29, 36, 43, 45, 46, 50, 55, 62, 77, 79, 88, 106, 113, 122, 126, 30, 33, 37, 38, 39, 40, 42, 67, 69, 81, 107, 108, 109, 128, 130, 131, 132, 134, 137, 47, 48, 49, 53, 57, 70, 71, 110, 125, 129, 145 143, 154, 165 74, 75, 77, 79, 106, 113, 122, 126, 128, 130, 131, Strain number BRW － is abbreviated 132, 134, 137

Strain numbers BRW– is abbreviated

Table 5. Chi-squared analysis of the first generation (F1) Table 6. Chi-squared analysis of the second generation (F2)

Chi-squared Chi-squared Test of goodness-of-fitx Test of independencew Test of goodnessx Test of independencew 2 1.8 2 2.967 2 5.069 2 0.880 p 0.180 p 0.085 p 0.024 p 0.348 Χ Χ Χ Χ X Variables are the number of observed (13 : 7) and expected (1:1) mating X Variables are the number of observed (54 : 32) and expected (1:1) types mating types w Variables are mating type and monokaryotic clamp cell phenotype w Variables are mating type and monokaryotic clamp cell phenotype might not be generated after meiosis, the mating type loci A2 in relatively equal frequencies. The progeny of the of M. aitchisonii might not carry the subloci. cross between F1 strains not in accurate compatible 4. Genetic linkage mating type and phenotype of monokary- 50% of its. The relative distribution of A1 and A2 in otic clamp cell formation F1 was approximately 60% and 40%, respectively. In Basidiospore isolates 50005-7 and strains 50005- bipolar mating systems, the progeny of a cross between 18 from TUFC50005 produced true and pseudo-clamps a strain carrying, for example, A1 and A2 will carry in this and previous9) studies. To examine whether the mating types of the two parental types in equal the bipolar A incompatibility factor of M. aitchisonii frequencies. Consequently, the spores from any strain was related to monokaryotic clamp cell formation, we in the F1 generatio will be compatible with 50% of its examined basidiospore isolates from strain TUFC50005 siblings. However, in the second generation, the p- (50005-1 － 50005-20) and 50005-7 × 50005-18 (BRW- value was less than the significance level (p = 0.024 < 2 － BRW-177). The relationships between the true- 0.05), indicating that data did not follow a distribution clamp formation phenotype and mating type among of certain proportions i.e., the progeny did not carry basidiospore isolates are summarized in Table 2 and A1 and A2 in equal frequencies. If the A factor was 4. Strains 50005-7 and 50005-18 were the F1 progeny comprised of two loci, then the inbreeding potential of strain TUFC50005; thus, strain TUFC50005 was would increase to a level determined by the frequency parental (P) . Moreover, both 50005-7 and 50005-18 of recombination between the loci. Thus, the chi-square strains had the ability to produce clamp cells when test of independence showed that there is no relationship they were monokaryons. Segregation analysis of between the A mating type locus and the phenotype of mating type A in F2 progeny showed that segregation monokaryotic clamp cell formation. Moreover, in this was significantly different from expected values. We study some of the progeny of strain 50005-7 × 50005- therefore conducted the chi-square goodness of fit test for 18, in which monokaryotic clamp formation was not the first and second generations to compare the observed observed, basidiospore isolates were able to produce sample distribution with the expected probability monokaryotic clamp cells. Therefore, monokaryotic distribution. To determine the relationship between clamp cell formation is not only affected by the mating the mating type and phenotype of monokaryotic clamp type locus, but also by several other loci. cell formation, the chi-square test of independence Discussion was conducted (Table 5 and 6). Based on the chi-square goodness of fit analysis, the first generation showed a Based on the results of polarity tests, we propose p-value greater than the significance level (p = 0.180 > that M. aitchisonii is a bipolar mating system because 0.05), indicating that the data follow a distribution of only two mating types segregate in meiosis and mating certain proportions and that the progeny carry A1 and incompatibility is controlled by a single mating type 132 Vol.27 No. 4 locus. In bipolar species, this locus can have two or clamp formation is controlled by the expression level of multiple alleles; for example, approximately 25% of homeodomain protein genes and that altered expression basidiomycete species have a bipolar (unifunctional) of A mating type is sufficient to drive true clamp mating system12). In the bipolar incompatibility system, formation. It is therefore possible that dikaryotization only 50% of the F1 produced by random crossing are is not essential for clamp cell or fruiting body formation fertile. As the number of recombinant in a population and that monokaryotic is controlled by increases, so does the probability that non-sister mating the expression level of the mating-type gene. Although will be compatible. Based on the chi-square goodness of both of the monokaryotic parental 50005-7 and 50005- fit test in the F1 generation with 20 monokaryons, the 18 strains can produce monokaryotic clamp connections, p-value was greater than the significance level (p = 0.180 more than 50% of the resulting progeny cannot make > 0.05), indicating that the data follow a distribution of clamp connections. However, it is possible that some of certain proportions, i.e., that the progeny carry A1 and the monokaryotic clamp connections cannot be observed A2 in relatively equal frequencies (1:1). However, in the because the frequency clamp connection is very low. F2 generation with approximately 86 monokaryons, the Alternately, another gene related to clamp connection progeny did not carry A1 and A2 in equal frequencies. formation may control HD2 expression, which location In a hypothetical cross between strains carrying A1 α1- of the gene in different alleles. Thus, A mating-type loci β1 and A2 α2-β2, recombinant spores carrying A3 α1-β2 may have only one HD1/HD2 gene pair with several dif- and A4 α2-β1 would be formed and these recombinants ferent alleles, as is the case in Pholiota nameko15) Pleurotus would be compatible with all of their siblings except djamor16), and Laccaria bicolor17), or two HD1/HD2 subloci, those carrying the identical specify. If the two loci were as in Flammulina velutipes18), or even more than three sub- unlinked the inbreeding potential would be 75%, but if loci, as in Schizophyllum commune19). the loci were linked, the inbreeding potential would be between 50 and 75% depending on the distance between 和 文 摘 要 13) the loci . However, we did not observe the recombinant ブナハリタケのモノカリオンがクランプ細胞を in the progeny tests using M. aitchisonii. It is possible that we need more monospores to detect the recombinant, as 形成する表現型は，交配型と遺伝的に in our previous work on Pholiota nameko14). In that study リンクしていない on the incompatibility factors and mating characteristics of spore isolates in the bipolar mushroom, Pholiota リニー・リファニー1, 5)・和田崇志2)・陳 富嘉3) nameko, only one of 300 monospore isolates carried 霜村典宏4)・山口武視4)・會見忠則4) Ar1 (in addition to the two parental mating types of isolates i.e., 121 of A2 and 152 of A4). Further, it was also 1) 鳥取大学連合農学研究科 found that 26 modified isolates were unable to produce 〒680-8553 鳥取市湖山町南 4-101 a dikaryon with both parental monokaryons. These 2) modified isolates were the product of recombination 一正蒲鉾（株）バイオ事業部バイオ研究室 during sexual reproduction. Isolates carrying the new A 〒950-1936 新潟県阿賀野市純神 77-1 incompatibility factor resulted from successful meiotic 3) 鳥取大学大学院持続性社会創生科学研究科 14) recombination in sexual reproduction . 〒680-8553 鳥取市湖山町南 4-101 The results of the chi-square test for independence 4) 鳥取大学農学部 between mating type and monokaryotic clamp 〒680-8553 鳥取市湖山町南 4-101 connection formation showed that the two groups are 5) weakly related (p > 0.05). In other words, these two インドネシア科学研究所（LIPI） groups are high capability independent. No relationship インドネシア生物科学研究センター , was observed between the A-mating type gene and 16911 インドネシア ボゴール シビノン monokaryotic clamp connection in the F1 and F2 generations. The frequency of true clamp formation ブ ナ ハ リ タ ケ Mycoleptodonoides aitchisonii は 食 用 木 was thus not controlled by genotype of mating type 材腐朽性のきのこである．このきのこの一部の担子胞子分 gene. In the bipolar basidiomycete Pholiota microspora, 離株は，完全な子実体と真のクランプ細胞を形成できるこ a pair of homeodomain protein genes located at the A mating-type locus regulates mating compatibility15). We とが以前に報告された．真のクランプ細胞が観察された頻 previously induced monokaryotic clamp cell formation 度を，ダイカリオンとモノカリオンの間で比較すると，ダ by introducing a compatible homeodomain protein gene イカリオンでの頻度は，モノカリオンでの頻度よりもはる 15) into P. microspora using a recombinant DNA technique . かに高かった． TUFC50004 株からの担子胞子分離株で In that study, the expression levels of the homeodomain ある二核株 TUFC50004（F1）および TUFC50004-7 × protein were affected by the frequency of clamp cell TUFC50004-18（F2）からの担子胞子分離株間で交配不和 formation, gene dosage and activity promoter. True 日本きのこ学会誌 MUSHROOM SCIENCE AND BIOTECHNOLOGY 133

合性グループを調べた．両方の交配不和合性グループは２ T: Monokariotic fruiting body and clamp cell formation in グループに分割されたため，ブナハリタケは二極性きのこ Mycoleptodonoides aitchisonii (Bunaharitake). Mycoscience, 60, 151-155 (2019) https://doi.org/10.1016/j.myc.2019.01.004. であった．さらに，減数分裂後には，交配型に関する組換え 10) Shimomura, N, Egi, T, Sawada, K and Aimi, T: Development 体が分離できなかっため，ブナハリタケの交配型遺伝子座 of detecting medium the clamp formation of Rhizopogon はサブユニット構造を持たない単一遺伝子座である可能性 roseolus mycelium. Mushroom Science and biotechnology, 20, が示唆された．クランプ細胞形成可能なモノカリオンの表 85-88 (2012) 11) McHugh, M L: Thr Chi-square test of independence. 現型とその交配型との間に遺伝的連鎖は観察されなかった． Biochemia Medica, 23(2), 143-149 (2013) したがって，モノカリオンがクランプ細胞を形成能力は，交 12) Bakkeren, G and Kronstad, J W: Linkage of mating-type loci 配型遺伝子座に連鎖していないと考えられる． distinguishes bipolar from tetrapolar mating in basidiomycetous smut fungi. Proc Natl Acad Sci USA, 91, 7085-7089 (1994) 13) Koltin, Y, Stamberg, J and Lemke, P A: Genetic structure and References evolution of incompatibility factors in higher fungi. Bacterio- 1) Whitehouse, H L K: Multiple allelomorph heterothallism in logical Review, 36 (2), 156-171 (1972) the fungi. New Phytol, 48, 212-244 (1949) 14) Ratanatragooldacha, S, Hui, C, Kumata, A and Kitamoto, Y: 2) Fowler, T J, Mitton, M F, Rees, E I and Raper, C: Crossing the The constitution of incompatibility factor and mating char- boundary between the B alpha and B beta mating-type loci in acteristics of spore isolates in a bipolar mushroom, Pholiota Schizophyllum commune. Fungal Genet Biol, 41, 89-101 (2004) nameko. Mycoscience, 43, 113-117 (2002) 3) Iwasa, M, Tanabe, S and Kamada, T: The two nuclei in the 15) Yi, R, Mukaiyama, H, Tachikawa, T, Shimomura, N and dikaryon of the homobasidiomycete Coprinus cinereus change Aimi, T: A-mating-type gene expression can drive clamp for- position after each conjugate division. Fungal Genet Biol, 23, mation in the bipolar mushroom Pholiota microspora (Pholiota 110-116 (1998) nameko). Eukaryotic Cell, 9, 1109-1119 (2010) https://doi. 4) Au, C H, Wong, M C, Bao, D, Zhang, M, Song, C, Song, W, org/10.1128/EC.00374-09. Law, P T, Kües, U and Kwan, H S: The genetic structure of the 16) James, T Y, Kües, U, Rehner, S A and Vilgalys, R: Evolution A mating-type locus of Lentinula edodes. Gene, 535, 184-190 of the gene encoding mitochondrial intermediate peptidase (2013). doi: 10.1016/j.gene.2013.11.036. and its cosegregation with the A mating-type locus of mush- 5) Coelho, M, Bakkeren, G, Sun, S, Hood, M and Giraud, T: room fungi. Fungal Genet Biol, 41, 381-390 (2004a). Fungal Sex: The , pp. 147-175. In Heitman J, 17) Niculita-Hirzel H, Labbé J, Kohler A, le Tacon F, Martin F, Howlett B, Crous P, Stukenbrock E, James T, Gow N (ed), The Sanders I R and Kües U: Gene organization of the mating-type Fungal Kingdom. ASM Press, Washington, DC. (2017) doi: regions in the ectomycorrhizal fungus Laccaria bicolor reveals 10.1128/microbiolspec.FUNK-0046-2016 distinct evolution between the two mating-type loci. New Phy- 6) Aimi, T, Yohida, R, Ishikawa, M, Bao, D and Kitamoto, tol, 180, 329-342 (2008). Y: Identification and linkage mapping of the genes for the 18) van Peer A F, Park S Y, Shin P G, Jang K Y, Yoo Y B, Park Y putative homeodomain protein (hox1) and the putative J, Lee B M, Sung G H, James T Y and Kong W S: Comparative pheromone receptor protein homologue (rcb1) in a bipolar genomics of the mating-type loci of the mushroom Flammuli- basidiomycete, Pholiota nameko. Curr Genet, 48, 184-194 (2005) na velutipes reveals widespread synteny and recent inver- 7) James, T Y, Srivilai, P, Kües, U and Vilgalys, R: Evolution sions. PLoS One 6, e22249 (2011). https://doi: 10.1371/journal. of the bipolar mating system of the mushroom Coprinellus pone.0022249. disseminatus from its tetrapolar ancestors involves loss of 19) Ohm, R A, de, Jong, J F, Lugones, L G, Aerts, A, Kothe, E, mating-type-specific pheromone receptor function. Genetics, Stajich, J E, de, Vries, R P, Record, E, Levasseur, A, Baker, S 172, 1877-1891 (2006) E, Bartholomew, K A, Coutinho, P M, Erdmann, S, Fowler, T 8) Chandrasekaran, G, Oh, D S, and Shin, H J: Versatile applications J, Gathman, A C, Lombard, V, Henrissat, B, Knabe, N, Kües, of the culinary-medicinal mushroom Mycoleptodonoides U, Lilly, W W, Lindquist, E, Lucas, S, Magnuson, J K, Piumi aitchisonii (Berk.) Maas G. (Higher Basidiomycetes): a review. F, Raudaskoski, M, Salamov, A, Schmutz, J, Schwarze, F W, Int J Med Mushrooms, 14, 395-401 (2012) http://dx.doi. vanKuyk, P A, Horton, J S, Grigoriev, I V and Wösten, H A: org/10.1615/IntJMedMushr.v14.i4.70. Genome sequence of the model mushroom Schizophyllum 9) Riffiani, R, Wada, T, Shimomura, N, Yamaguchi, T and Aimi, commune. Nat Biotechnol, 28(9), 957-963 (2010).