Monokaryotization by protoplasting heterothallic species of edible mushrooms

After regeneration of protoplasts of seven heterothallic of edible mushrooms, two types of monokaryons were recovered, being identified by the absence of clamp connections. In regenerated colonies, monokaryons grew more slowly than dikaryons and so could be distinguished from them. In all species, the yield of monokaryons in the regenerated colonies was over 45%. The mating types and auxotrophic markers were the same in the protoplasted monokaryons and parental monokaryons. In comparison with other monokaryotization methods, protoplasting is rapid, simple and effective; its applications in breeding of edible mushrooms are discussed.     

黑木耳交配型的研究*

从黑木耳Auricularia auricula沪3菌株的子实体收集、分离、鉴定得到157株单孢子萌发的单核菌丝体,以其中73株孢子单核体为材料进行交配实验,经过三轮交配反应,结果表明,木耳的交配型符合四极性交配系统的分布规律。根据四种交配反应情况把73株孢子单核体分为四组,并从四组中挑选出B17、B177、B101、B65作为四种交配型的标准菌株;与此同时,从沪3菌株的双核菌丝体制备的原生质体中获得53株原生质体单核体,对这些单核体进行交配,确定出两种亲本交配型,以Y150(A1B1)和Y121(A2B2)作为标准菌株,将两个亲本交配型标准菌株与四个孢子单核体标准菌株进行交配反应,初步确定出四种孢子单核体的交配型分别为: B17 (A2B2)、B177(A1B1)、B101(A2B1)和B65(A1B2)。     

香菇135菌株特异SCAR标记在其原生质体单核中的分布

以香菇保藏菌种庆元135以及庆元出菇的135新鲜子实体组织分离得到的菌丝为材料制备原生质体,分别获得58和83个原生质体单核体;经交配型鉴定后,用已获得的135特异SCAR引物对单核进行PCR扩增,统计SCAR条带的分布。结果显示：原生质体单核体分为A1B1和A2B2两种交配型,而特异条带仅存在于后者中,证明135特异SCAR标记是稳定遗传的,也为鉴定以带标记核为亲本之一的杂交后代提供了依据,这就进一步拓宽了分子标记应用于菌株鉴定的适用范围。     

基于F2群体的香菇遗传图谱构建及其在QTL定位中的应用

以171个F2双核体菌株为作图群体,通过相互配对的2个单核体的基因型推断双核体基因型,构建了第一张基于双核体群体的香菇遗传图谱。该图谱包含分布于15个连锁群的459个标记,覆盖长度为989.7cM,平均标记间隔为2.2cM。此外,以此双核体群体作为表型分离群体,定位了6个与香菇双核体菌丝生长速度相关的QTLs,位于5个连锁群上。采用全同胞单核体随机交配策略,易于构建相对大的双核体群体,用于连锁图构建和QTL定位。研究表明,在食用菌连锁图谱构建及QTL定位研究中,利用F2群体,可能为提高遗传作图效率,解决作图群体与表型分离群体间不一致问题提供新的途径。     

香菇135菌株特异SCAR标记在其原生质体单核中的分布

以香菇保藏菌种庆元135以及庆元出菇的135新鲜子实体组织分离得到的菌丝为材料制备原生质体,分别获得58和83个原生质体单核体;经交配型鉴定后,用已获得的135特异SCAR引物对单核进行PCR扩增,统计SCAR条带的分布。结果显示:原生质体单核体分为A1B1和A2B2两种交配型,而特异条带仅存在于后者中,证明135特异SCAR标记是稳定遗传的,也为鉴定以带标记核为亲本之一的杂交后代提供了依据,这就进一步拓宽了分子标记应用于菌株鉴定的适用范围。     

Genetical variability of glutamate dehydrogenase activity in monokaryotic and dikaryotic mycelia of the ectomycorrhizal fungus Hebeloma cylindrosporum

Summary Glutamate dehydrogenase (GDH) is the key enzyme of ammonium assimilation by ectomycorrhizal fungi. Its activity might be use as a criterion to select mycelia capable of enhancing the nitrogen nutrition of the host plants. Genetical variability of the GDH activity of the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnési was studied in an attempt to determine if this enzyme activity could be improved by way of chromosomal genetics. The activity of 11 wild strains was compared with that of 70 mycelia obtained as the progeny of a laboratory fruiting strain HC1. These 70 mycelia were 20 monokaryons (5 for each mating type) and the 50 synthesized dikaryons obtained from all the compatible fusions between these monokaryons. The specific GDH activity of the 11 wild strains ranged from 1.5 to 11.6 nkat mg^-1 fungal protein. The activity of the monokaryotic progeny of the HC1 strain was, on average, three times lower (2.85 n kat mg^-1 fungal protein) than that of the parental dikaryon. In contrast, synthesized dikaryons originating from these monokaryons were very variable and had an average values similar to that of the parental dikaryon (9.1 nkat mg^-1 fungal protein). Eighteen of these synthesized dikaryons contained an activity higher than that of the original HC1 strain. The variation of the GDH activity of these dikaryons involves additive and non additive (interactive) components, each of them accounting for ca. 50% of the genetical variation. The non additive variation could not be explained by a model involving only dominance. These results are discussed with reference to the genetical improvement of mycorrhizal fungi in order to enhance nitrogen nutrition of the host plants.Abbreviations GDH glutamate dehydrogenase - IAA indole-3-acetic acid - NADP nicotimamide adenine dinucleotide phosphate     

Control of extracellular slime accumulation in monokaryons and resultant dikaryons of Schizophyllum commune.

Extracellular slime accumulation, as alcohol-precipitable material was measured after eight days of growth in glucose-asparagine-salts broth in twenty-two different monokaryons and six resultant dikaryons of Schizophyllum commune. The nutritional control of slime accumulation was also examined in monokaryotic mycelium. Slime occurred after growth in sucrose, glucose, fructose and xylose, with glycerol best. Low inorganic phosphates limited both slime and mycelial growth while limiting MgSO4 decreased growth and enhanced slime. In glucose-asparagine broth, various monokaryons differed widely in slime accumulation, ranging from none (e.g., strain 19) to nearly 800 mg per 100 ml filtrate (strain 1) after eight days growth, followed by a marked decline in slime (eleven days to twenty-one days). Resultant dikaryons all showed less slime accumulation, even when established from two high slime-accumulating monokaryons. In contrast, conditions which arrested dikaryotic fruit-body morphogenesis led to increased slime accumulation.     

Genetische Untersuchungen zur Fruchtkörper- und Artbildung bei Basidiomyceten: Genetische Kontrolle der Fruchtkörperbildung beiPolyporus ciliatus

Using mainly the basidiomycetePolyporus ciliatus, we were able to produce fully developed fruitbodies not only in the normal meiotic cycle (teleomorphic = perfect) but in some stocks also in the mitotic cycle (anamorphic = imperfect) of exclusively haploid mycelia. These haploid and mitotic fruitbodies can be distinguished from dikaryotic ones only by microscopic examination, i.e. by their lack of clamp connections, karyogamy and meiosis, the production of two-spored basidia with few, but larger basidiospores. Using three other stocks of isogenic mycelial strains that did not form any fruiting structures from either haploid or dikaryotic mycelia with clamp connections, we could not find any evidence for a “fruiting initiation gene” as was postulated byEsser & al. (1977). No fruitbodies occurred in compatible combinations within one isogenic stock, but normal fruitbodies are produced abundantly in crossings of different isogenic stocks. We were able to confirm these results with the help of the non-fruiting strains originally used byStahl & Esser (1976).—Our experimental results are compatible with the hypothesis suggested byKniep (1930): The genetic control of the sexual cycle by the mating type factors A and B on the one hand, and of the fruitbody formation on the other, are essentially independent. For the latter, a multitude of different genes has been proposed. Fruitbody formation thus is a typical polygenic character. The results of experiments on two other morphological characters i.e. primordia and coralloid structures, also correspond with this genetic concept.     

大球盖菇原生质体再生及单核化特性的研究*

大球盖菇原生质体再生条件及单核化特性结果。原生质体再生速度极快,涂布平板３ｄ后肉眼即可见明显的再生菌落形成,在ＰＧＰＭ再生培养基上再生率为０．９７～２．０％,渗稳剂种类对再生率无明显影响,但可影响再生菌落形态,液体预培养１～２ｄ,再生率明显下降;大球盖菇原生质体单核化率高达７７．６％,且再生双核体和再生单核体在形成再生菌落时无时间差,其生长速度亦无快慢之分,液体预培养可显著减少单核化率,再生单核体中存在亲本两种交配型,但二者的比率不为１。     

Relationship between monokaryotic growth rate and mating type in the edible basidiomycete Pleurotus ostreatus

The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the matB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matB locus that allowed us to dissect it into two genetically linked subunits (matBalpha and matBbeta) and to correlate the presence of specific matBalpha and matA alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.     

The A mating-type genes of the mushroom Coprinus cinereus are not differentially transcribed in monokaryons and dikaryons

Summary The A mating type factor of Coprinus cinereus regulates part of a developmental sequence that leads to the conversion of the asexual monokaryon into the fertile dikaryon. The A42 factor is a complex of seven genes, at least four of which are involved in determining the specificity of mating interactions. In this report we show that the A42 genes are constitutively expressed in both monokaryons and dikaryons. This has important implications with respect to intracellular recognition of a compatible mating, which requires an interaction between proteins already present within the cells of the mating partners, and for the subsequent maintenance of dikaryotic growth.     

The constitution of incompatibility factor and mating characteristics of spore isolates in a bipolar mushroom, <Emphasis Type="Italic">Pholiota nameko</Emphasis>

Pholiota nameko is a wood-rotting edible mushroom that carries a bipolar A incompatibility factor gene. The linkage analysis of the multiple allelomorphic A factor gene demonstrated that sexual reproduction produced a monospore isolate carrying a new A factor gene in addition to two parental mating types of isolates. However, 10%–30% of the modified monospore isolates could not produce a dikaryon with both of the parental monokaryons by crossing. It is concluded that the bipolar A incompatibility factor gene of P. nameko is constituted of two functional subunits, Aα and Aβ, which might be successively located beside each other with an apparent genetic distance of 0.3 centi-Morgan between them on the same chromosome. Further, some monospore isolates that did not conjugate with both parental monokaryons could produce dikaryons with different monokaryotic stocks with either one of the parental mating types. This result suggests that the crossing capability of these isolates were essentially those for one of the mating types of the parental monokaryons, but that their function for mating activity was made partially by unequal crossing-over in the process of sexual recombination. Received: May 1, 2001 / Accepted: December 5, 2001     

Nuclear and Non-nuclear Factors influencing Clamp Connection Formation in Coprinus disseminatus

Matings between sister single spore lines of Coprinus disseminatusshowed a cryptic tetrapolar pattern. The two groups of matingsthat resulted in formation of mycelia with clamp connections(apparent dikaryons) differed in rate of nuclear penetrationduring mating. In one group penetration occurred at rates comparablewith nuclear migration in other species and in the other groupit was often extensive but at a rate similar to, or less than,the dikaryon growth-rate. No differences were detected betweenthese two groups in stability, colony extension rate, frequencyof clamp connections, proportions of true clamp connectionsand pseudoclamps, or number of nuclei per hyphal tip cell. Cytological studies and the isolation of hyphal tips showedthat both groups of apparent dikaryons were heterokaryotic di-or trikaryons. The di- and trikaryotic conditions co-existedin the same mycelium, but adjacent cells of individual branchingsystems usually contained equal numbers of nuclei. Within apparentdikaryons the number and kinds of nuclei per cell were similarin hyphae with clamp connections and those with simple septa.Treatments that prevented clamp connection formation did notalter the nuclear status of most of the hyphae. Irregularities in nuclear distribution were infrequent and mostwere associated with pseudoclamps. Forty per cent of nodes withprobable pseudoclamps yielded homokaryotic branches, which wereof either constituent mating type. There was some indicationthat irregularities in nuclear distribution could also occurduring divisions associated with simple septa.     

基于等位基因InDel快速检测黑木耳原生质体单核体

以黑木耳 Auricularia auricula-judae栽培菌株Au916三磷酸甘油醛脱氢酶（GAPDH）两个等位基因中的InDel为基础,以原生质体再生菌株为研究材料,建立了基于InDel的黑木耳原生质体单核体检测技术,并采用荧光显微镜检和配对试验对此方法进行了验证。应用InDel能清晰地区分黑木耳双核体和两种原生质体单核体,且不同的单核体之间可以进行交配,其杂交双核体能正常出耳,结果表明基于等位基因InDel标记可以快速准确地检测黑木耳原生质体单核体。在大规模测序获得真菌基因组信息的基础上,InDel标记在原生质体单核体鉴定中的应用将更加普遍。     

ISSR标记在黑木耳单核体遗传分析中的应用

采用原生质体单核化和单孢分离法分别获得黑木耳Auricularia auricula栽培菌株新科5号两个亲本单核体(T1、T2)和33个F1代孢子单核体。从73条ISSR引物中筛选出13条可区分两个亲本单核体(T1、T2)的引物,对新科5号及F1代孢子单核体进行扩增,共扩增出70条带,其中63条在供试菌株间表现出多态性,多态位点百分率为90.0%。根据扩增结果采用软件NTsys2.10e计算菌株间的遗传相似系数并进行聚类分析,36个菌株间的遗传相似系数(GS值)的变化范围为0.2500～0.8382,其中T1和T2之间的GS值最小,遗传相似程度最低;F1代33个孢子单核体中24个与T1聚为一类,其余9个与T2聚为另一类,但并非严格按照交配型进行归类。试验表明黑木耳子实体上各个担子在减数分裂中染色体交换极具多样性,F1代孢子单核体表现出偏向其中一个亲本的现象,ISSR标记在黑木耳杂交育种中具有潜在的应用价值。     

Fungal fidelity: Nuclear divorce from a dikaryon by mating or monokaryon regeneration

Basidiomycete fungi perform fertilizations by incorporation of nuclei into a monokaryotic mycelium to establish a dikaryon. The dikaryon cannot incorporate another type of nucleus, but can still act as a nucleus donor in a dikaryon–monokaryon (di–mon) mating, known as the Buller phenomenon. Previously, it has been observed that: (1) in a particular di–mon mating, one of the nuclear types of the dikaryon generally performs better as a donor than the other, and (2) when nuclei from a dikaryon are separated to form monokaryons again (dedikaryotisation), recovery of monokaryons of the two nuclear types is usually unequal. In this study, we investigated if these two observations of asymmetry are functionally related. We tested this hypothesis by performing both di–mon matings and dedikaryotisation of dikaryons derived from five different monokaryons. When a single mechanism controls both processes, the nucleus better at fertilizing a monokaryon in a Buller pairing should also be recovered upon dedikaryotisation with a higher frequency. The results showed a hierarchical structure for recovery among nuclei in dedikaryotisation, but this hierarchy did not correspond to the fertilization success during di–mon mating. These findings thus show that the mechanism causing asymmetric regeneration of nuclei, is most likely not the same as the mechanism responsible for increased chance of fertilization in di–mon matings. We discuss the complexity of the interactions that occur during di–mon matings with regards to the mating type loci.     

Life history and developmental processes in the basidiomycete Coprinus cinereus.

Coprinus cinereus has two main types of mycelia, the asexual monokaryon and the sexual dikaryon, formed by fusion of compatible monokaryons. Syngamy (plasmogamy) and karyogamy are spatially and temporally separated, which is typical for basidiomycetous fungi. This property of the dikaryon enables an easy exchange of nuclear partners in further dikaryotic-monokaryotic and dikaryotic-dikaryotic mycelial fusions. Fruiting bodies normally develop on the dikaryon, and the cytological process of fruiting-body development has been described in its principles. Within the specialized basidia, present within the gills of the fruiting bodies, karyogamy occurs in a synchronized manner. It is directly followed by meiosis and by the production of the meiotic basidiospores. The synchrony of karyogamy and meiosis has made the fungus a classical object to study meiotic cytology and recombination. Several genes involved in these processes have been identified. Both monokaryons and dikaryons can form multicellular resting bodies (sclerotia) and different types of mitotic spores, the small uninucleate aerial oidia, and, within submerged mycelium, the large thick-walled chlamydospores. The decision about whether a structure will be formed is made on the basis of environmental signals (light, temperature, humidity, and nutrients). Of the intrinsic factors that control development, the products of the two mating type loci are most important. Mutant complementation and PCR approaches identified further genes which possibly link the two mating-type pathways with each other and with nutritional regulation, for example with the cAMP signaling pathway. Among genes specifically expressed within the fruiting body are those for two galectins, beta-galactoside binding lectins that probably act in hyphal aggregation. These genes serve as molecular markers to study development in wild-type and mutant strains. The isolation of genes for potential non-DNA methyltransferases, needed for tissue formation within the fruiting body, promises the discovery of new signaling pathways, possibly involving secondary fungal metabolites.     

Relationship between Monokaryotic Growth Rate and Mating Type in the Edible Basidiomycete Pleurotus ostreatus

The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the matB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matB locus that allowed us to dissect it into two genetically linked subunits (matBα and matBβ) and to correlate the presence of specific matBα and matA alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.     

Monokariotic fruiting body and clamp cell formation in Mycoleptodonoides aitchisonii (Bunaharitake)

The ability to produce monokaryotic fruiting bodies and clamp cells in culture was examined in monokaryotic strain isolated from several dikaryotic parental strains of the edible mushroom, Mycoleptodonoides aitchisonii (Bunaharitake). We describe a single dikaryotic M. aitchisonii strain, TUFC50005, and 20 monokaryons derived from it, which exhibited a wide spectrum of monokaryotic fruiting types. Most strains formed primordia, or young fruiting body-like structures, but only one of the monokaryons, strain TUFC50005-4, formed a fruiting body, even though it had only one nucleus and produced only two spores after meiosis. We demonstrated that dikariotization was not required for clamp cell formation, fruiting body formation, or meiosis, in this mushroom.