表型与亲本不易区分的茯苓同核体菌株新类型

茯苓Wolfiporia hoelen是我国传统中药材之一,也是一种食药兼用的大型真菌,目前已规模化栽培,但由于其交配系统一直不明确,影响了种质改良。前期我们发现了茯苓的同核体,明确了茯苓的交配系统和生活史,并建立了以培养特性和分子标记区分同核体的方法,但未明确是否适用于茯苓种群的不同个体。在对多个菌株的研究中,发现了同核体表型与亲本不易区分的茯苓菌株。本研究主要以来自日本的菌株775 (NBRC 30628)为亲本,对其同核体菌株进行收集鉴定,并对同核体菌株的培养特性、交配现象和杂交等进行了研究。此类菌株的同核体菌株可通过与亲本对峙培养进行鉴定,但菌丝生长、菌落形态和吃料速度等与亲本没有显著差异,不同交配型的同核体之间交配时没有明显的交配现象。rpb2杂合位点标记可以用于鉴定该类型同核体菌株,且能验证是否交配。该类型同核体与之前发现的同核体类型之间可以进行杂交,杂交菌株可与两亲本都产生拮抗现象。该发现补充了之前建立的茯苓同核体鉴定方法,加深了对茯苓物种群体的了解,同时丰富了茯苓的育种资源。     

Formation of interspecies fusants of <Emphasis Type="Italic">Agaricus bisporus</Emphasis> and <Emphasis Type="Italic">Agaricus bitorquis</Emphasis> mushroom by protoplast fusion

Interspecies fusants are formed between Agaricus bisporus and Agaricus bitorquis by protoplast fusion technique. Protoplasts were isolated and regenerated by using Novozyme 234 lytic enzyme. Twenty slow growing isolates were separated from the protoplast regenerated colonies, which were assumed as homokaryons (putative homokaryons). These twenty isolates were subjected to growth rate, colony morphology and spawn run studies for screening of true homokaryons. Antifungal markers were developed for selection of fusants.     

Homokaryons are more combative than heterokaryons of Hericium coralloides

The homokaryotic stage of the basidiomycete lifecycle is generally considered to be short lived, although there is little experimental evidence relating to their longevity in the field. The vast majority of studies on basidiomycete ecology have used only heterokaryons. The few investigations comparing related homokaryons and heterokaryons have revealed no overall trend in differences of extension rate, wood decay or competitive ability. For a rare species the homokaryotic phase may be of greater importance than in common species as it is likely to last longer. Hericium coralloides, a rare wood decay basidiomycete, was used to investigate differences between homokaryons and heterokaryons in terms of extension rate and combative ability. Fifteen homokaryons from three fruit bodies and five heterokaryons (obtained by fruit body tissue isolation) were compared at 5–35 °C on malt agar for extension rate, and paired against heterokaryons of 13 wood decay species to assess combative ability. Homokaryons were paired to create ten artificial heterokaryons whose extension rate at 10 and 20 °C was compared to parental rates. There were some significant differences in extension rates between homokaryons and natural heterokaryons, between homokaryons and heterokaryons created artificially from homokaryons, and between homokaryons from different fruit bodies, but no consistent trends. Homokaryons proved more combative than heterokaryons, which was assessed quantitatively as well as qualitatively using a scoring system for outcome of each pairing. Results are discussed in relation to previous findings and in an ecological context.     

Crosses among Homokaryons from Commercial and Wild-Collected Strains of the Mushroom Agaricus brunnescens (= A. bisporus)

A new technique for the production of hybrid strains of the cultivated mushroom Agaricus brunnescens is described. Homokaryons were recovered from regenerated protoplasts obtained from several heterokaryotic strains. A total of 16 novel hybrids were produced in 63 attempted crosses between paired homokaryons. Recovery of both homokaryons and hybrids was verified by analysis of restriction fragment length polymorphisms. Three of four hybrids fruited in small-scale tests, further confirming that the isolates were true hybrids. Colony morphology alone was found to be a poor indicator of hybrid status. In two instances, three homokaryons crossed successfully in all combinations, suggesting that there are at least three alleles at the putative mating-type locus. Crosses between homokaryons from commercial and wild-collected isolates indicated that these strains belong to the same biological species.     

Nuclear lamins during prophasing and telophasing in heterophasic HeLa and Chinese hamster homokaryons

We have perturbed the dynamics of the nuclear lamins by means of cell fusion between mitotic and interphase cells and have studied redistribution of lamins in fused cells as a function of extracellular pH levels. We show here that in heterophasic M-1 HeLa homokaryons disassembly of interphase lamins predominates at low pH levels between 7.0 to 7.3, whereas deposition of cytoplasmic lamins around condensed metaphase chromosomes was observed at pH 8.0. In HeLa homokaryons lamina disassembly and lamina deposition around chromosomes are mutually exclusive. Using heterophasic M-1 homokaryons of the Chinese hamster cell line DON we observed that disassembly of interphase lamins and deposition of lamins around condensed chromosomes coexisted in the same homokaryon kept at pH 7.0. Disassembly of lamins developed synchronously with premature chromosome condensation (PCC) whereas lamina deposition around the condensed M-chromosomes was followed by telophasing. In fusions kept at pH 8.0 cytoplasmic lamins were exclusively deposited around mitotic chromosomes. The results are interpreted as showing that pH regulates the lamina dynamics in homokaryons of mitotic and interphase cells.     

Extranuclear chloramphenicol resistance mutations in the basidiomyceteSistotrema brinkmannii

Four chloramphenicol resistance mutations were induced in homokaryons ofSistotrema brinkmannii and a heterokaryon test was used to distinguish whether the mutations were nuclear or extranuclear. Dikaryons were recovered from sexually compatible pairings of chloramphenicol-resistant and sensitive strains which also carried chromosomal markers (mating-type and auxotrophic). The homokaryotic components of the dikaryons were recovered from vegetative cells by the production and regeneration of protoplasts. The homokaryons derived from protoplasts were tested for chloramphenicol resistance/sensitivity as well as for the chromosomal markers. In heterokaryon tests, the chloramphenicol resistance/sensitivity determinants reassociated with the chromosomal markers. Only a single recombinant with respect to chromosomal markers was observed among a total of 220 homokaryons analyzed. All pairs of the same chromosomal markers produced numerous recombinants in sexual crosses. These results indicated that the chloramphenicol resistance mutations were extranuclear.     

Analysis of Inbreeding Depression in Agaricus Bisporus

Inbreeding depression was observed in the commercial button mushroom, Agaricus bisporus, by examining two laboratory populations. The outbred population consisted of 20 compatible pairings, 10 homokaryons with each of the homokaryons Ag1-1 and Ag89-65. The inbred population consisted of 104 backcrosses (among which 52 were expected to be sexually compatible) obtained from the pairings of two progenitor homokaryons, Ag1-1 and Ag89-65, with 52 progeny homokaryons derived from the mating between Ag1-1 and Ag89-65. The eight fitness components examined for these two populations were successful matings as identified by the analysis of restriction fragment length polymorphisms, positive mycelial interaction in these successful matings, heterokaryon growth rate, primordium formation by the successful matings, fertile fruiting body formation, time to first break, average number of fruiting bodies per square foot, and average weight per fruiting body. The outcrossed population showed a significant advantage over the inbred population in three of eight fitness components. Two pairs of traits were significantly correlated. The multiplicative fitness ratio of the inbred to the outcrossed population was 0.18. The relevance of inbreeding depression to the evolution of fungal mating systems and to mushroom breeding is discussed.     

Loss of pericellular fibronectin matrix from heterokaryons of normal human fibroblasts and epithelial cells

The expression of fibronectin in heterokaryons of normal human fibroblasts and normal or malignant epithelial cells was studied by indirect immunofluorescence microscopy. Fibroblasts and their homokaryons showed a characteristic pericellular fibronectin matrix, whereas both normal (MDCK) and malignant (HeLa) epithelial cells, and their homokaryons, lacked such a matrix. The fibroblast homokaryons also showed a typical strong, perinuclear cytoplasmic, fibronectin-specific fluorescence. This was much weaker or absent in the MDCK and HeLa cells and their homokaryons. When human fibroblasts were fused with either normal or malignant epithelial cells, no pericellular matrix-like, fibronectin-specific fluorescence could be seen in the heterokaryons. Interestingly, however, a distinct intracellular fluorescence was seen in the heterokaryons, indicating continued production of fibronectin. The results of the present study indicate that both malignant and normal epithelial cells, which do not deposit fibronectin matrix, can cause its loss in heterokaryons with fibroblasts. Thus, discontinued fibronectin matrix formation does not point exclusively to malignancy, but may also reflect the state of differentiation of the parental cells.     

Fibronectin expression is determined by the genotype of the transformed parental cells in heterokaryons between normal and transformed fibroblasts

The expression of fibronectin, a cell surface-associated transformation-sensitive glycoprotein, was studied in hetero- and homokaryons of normal and SV40-transformed human fibroblasts. In immunofluorescence, fibroblast homokaryons had an intense surface-associated and intracelluar fibronectin fluorescence similar to that of normal fibroblasts. Transformed cells and their homokaryons had a minimal surface-associated and a weak intracellular fibronectin fluorescence. In heterokaryons formed between transformed and normal fibroblasts, the expression of fibronectin fell within 24 h to the level of the transformed cell homokaryons. The change was detectable already at 3 h after fusion and was gene-dose dependent. These results show that the transformed genotype determines fibronectin expression in the heterokaryons.     

ISSR as New Markers for Identification of Homokaryotic Protoclones of Agaricus bisporus

To accelerate the breeding of Agaricus bisporus, quick and reliable methods to identify the infrequent homokaryons are necessary. A new marker, inter simple sequence repeat (ISSR) fingerprinting, is described for differentiation of homo- and hetero-karyotic protoclones. Nine slow growing protoclones, two strandy and seven appressed, were analyzed for the first time with ISSR amplifications. The patterns were highly polymorphic and very reproducible. Among 40 primers tested, 7 ISSR primers were selected for the analysis of genomic DNA and generated a total of 68 ISSR fragments. ISSR fingerprinting detected 44.12% polymorphic loci. All appressed homokaryons carried a subset of ISSR markers found in the heterokaryons, and clustered separately in dendrogram. These were not able to produce a fruiting body. A test of cross-fertility and the following fruiting trial proved that 7 of the 9 protoclones with different ISSR fingerprints were homokaryons. These results demonstrated that ISSR markers provide an efficient alternate for identification of homokaryons and suggest these markers be considered as new tools for the survey of Agaricus species.     

Variability in Mating Behaviour and Culture Appearance of Lenzites trabea

Twenty-three homokaryons of Lenzites trabea (Pers.) Fr. wereproduced, mainly by chemical monokaryotization, from dikaryonsisolated from widely separated regions throughout the world.These homokaryons possessed 18 different mating factor alleles,so an estimate was made of the total number of alleles in theworld population of 51, 45, or 36, depending on the method ofestimation. Some variability in the production of clamp connectionsduring mating was noted. It was also found that the appearanceof the cultures varied considerably, especially that of thehomokaryons. A brief study was made of this variability on severalculture media, and the appearance of dikaryons and their constituenthomokaryons was compared on malt agar.     

Control of arabitol formation in Schizophyllum commune development

Summary Dikaryotic cells of S. commune synthesized polyols throughout the life cycle when grown on glucose, cellobiose, or cellulose. Basidiospores contained arabitol and mannitol which were depleted during germination. The mannitol content of the young germlings rose to normal levels within a day; arabitol accumulation remained depressed for 5 to 7 days and then returned to normal levels characteristic of vegetative cells. Individual homokaryons differed in their production of intracellular polyols, which, unlike germlings, remained constant with cultural age. Homokaryon (str. 699) produced low levels of arabitol but high levels of glycerol while another homokaryon (str. 845) was the reverse. Mixtures of these homokaryons as well as the dikaryon (699×845) produced arabitol and glycerol levels intermediate between the parent homokaryons. High concentrations of glucose did not change the nature of the polyols produced. Arabitol formation could be induced prematurely in germlings or elevated in the dikaryon by growth on acetate or ethanol. Both homokaryons responded to growth on acetate with elevated arabitol production; acetate induction of arabitol formation was repressed in all types of cells if glucose were added simultaneously with acetate. Maltose, cellobiose, and trehalose also stimulated arabitol formation in young germlings, suggesting that glucose repression was the cause of decreased arabitol formation in basidiospore germlings. There was no correlation between the formation of arabitol and the derepression of isocitrate lyase or change in specific activities of alkaline and acid phosphatase in germlings grown on various carbon sources.     

An efficient protoplasting/regeneration system forAgaricus bisporus andAgaricus bitorquis

Conditions for efficient protoplasting and regeneration ofAgaricus bisporus andA. bitorquis are described. Especially forA. bisporus protoplasts, high regeneration frequencies were obtained (up to 30%). The protoplasting/regeneration system can be used for routine isolation of homokaryons ofA. bisporus. Such homokaryons, derived from protoplasts containing one type of nucleus only, can easily be identified by analyzing isoenzyme banding patterns.     

Heterokaryon formation with homokaryons derived from protoplasts ofRhizoctonia solani anastomosis group eight

Homokaryons were successfully recovered by regenerating protoplasts prepared from vegetative hyphae of field isolates ofRhizoctonia solani anastomosis group (AG) 8, the causal pathogen of bare-patch disease of cereals. A mating type incompatibility system, which is similar to that reported in AG 1 and AG 4, was demonstrated in AG 8. All homokaryons obtained in AG 8 were able to form tufts with their parent isolates and other heterokaryotic field isolates of AG 8 tested. Heterokaryons were readily recovered from tufts of pairing of certain homokaryon combinations. The synthesized heterokaryons formed tufts with both of the contributing homokaryons. The majority of hyphal tip cultures isolated from tufts resembled one of the contributing homokaryons. These nonheterokaryotic hyphae in tufts are attributed to transient heterokaryon effects.     

Mitotic activity of different variants of heterophase homokaryons

Double labelling with 3H- and 14C-thymidine was used to determine heterophase nuclei (G1-, S-, G2-) in homokaryons of the Chinese hamster cell culture. It was observed that the sequence of the mitosis beginning in homokaryons depends both on the number of nuclei in them and on the combinations of different phase nuclei.     

Genetic evidence for somatic haploidization in developing fruit bodies of Armillaria tabescens

Armillaria spp. have vegetative hyphae with diploid uninucleate cells, but the fruit bodies of many species contain clamped dikaryotic hyphae. Earlier observations suggest that somatic haploidization takes place in developing fruit bodies. To verify this, a uninucleate diploid cell was isolated from each of the 49 mating combinations between single-spore isolates of Armillaria tabescens and they were fruited. Twenty-four isolates produced fruit bodies with at least a partially dikaryotic subhymenium. Dikaryotic hyphae were isolated from fruit-body primordia and homokaryons were obtained by micromanipulation or by protoplasting. Approximately half of the isolates proved to represent recombinant mating types in respect to parent homokaryons, and most of them contained recombinant haploid DNA, based on random-amplified microsatellite markers. The results show that the nuclei in dikaryotic hyphae found in fruit bodies result from somatic haploidization. The mechanism of haploidization remains unclear.     

Biochemistry of Sexual Morphogenesis in Schizophyllum commune: Effect of Mutations Affecting the Incompatibility System on Cell-Wall Metabolism

A number of homokaryons of Schizophyllum commune, which carry various mutations affecting the incompatibility system, and a wild-type homokaryon were grown and examined for differences in the net synthesis of the cell-wall polysaccharides S-glucan, R-glucan, and chitin and in the activity of an enzyme hydrolyzing R-glucan (R-glucanase) in mycelial extracts and culture media. Only slight differences were observed for the accumulation of S-glucan and chitin. In comparison with the wild-type homokaryon, a very high S-glucan/R-glucan ratio was found in a primary B-factor mutant strain. Essentially, wild-type S-glucan/R-glucan ratios were restored in two strains in which additional mutations restored normal morphology: a strain carrying a secondary B-factor mutation and a strain carrying a modifier mutation in addition to the primary B-factor mutation. The S-glucan/R-glucan ratios in three A-factor mutants were intermediate between those of the wild-type homokaryon and the primary B-factor mutant. In young, growing cultures of the various homokaryons, except for the A-factor mutants, a correlation was found between the S-glucan/R-glucan ratios in the cell wall and the activities of R-glucanase in mycelial extracts. A certain specificity of the effect of the studied mutations on enzyme activities was indicated by the fact that, in young cultures, changes in R-glucanase activities were not paralleled by similar changes in the activities of laminarinase and maltase. The results can be correlated with particular morphological features of the homokaryons and, together with earlier results obtained with heterokaryons, indicate the activity of R-glucanase as an integral component of sexual morphogenesis regulated by the incompatibility factors.     

Globin synthesis in fibroblasts fused with erythroblasts. II. Human fibroblasts.

Fusion of human (diploid) fibroblast monolayers with erythroblasts from 3-day chick embryos resulted in cultures containing on the average 14% heterokaryons and 8% fibroblast homokaryons. When these heterokaryon-containing cultures were labeled with radioactive amino acids during the first 24 h after fusion, the proportion of labeled proteins found in the globin region of analytical polyacrylamide gels showed a 40-fold increase compared with fibroblast homokaryons (0.08% vs. 4% of protein synthesized). Incorporation of radioactivity into globin decreased sharply during the second 24 h. Purified 35S-methionine-labeled globin from heterokaryon cultures gave rise to a tryptic fingerprint containing peptides characteristic of chick embryonic globins as late as 4 days after fusion. While fibroblasts in the fusion culture continue to go through the cell cycle normally, heterokaryons stop cycling almost completely soon after fusion.     

Genetic Variability in Pectic Enzymes of Rhizoctonia solani Isolates Causing Bare-patch Disease of Cereals

Field isolates of Rhizoctonia solani obtained from three discrete bare patches in a wheat field in Western Australia were characterized by pectic zymogram grouping. The genetic background of pectic enzymes was analysed by comparing the zymograms of asexual homokaryons and sexual progenies derived from field isolates. The 170 field isolates obtained from the field site produced indistinguishable pectic zymograms. However, variations among field isolates of the same zymogram group were detected, on the basis of zymograms of their resultant protoplast-regenerated cultures. Asexual sibling homokaryons derived from each of the field isolates were heterogeneous for their pectic enzymes. Homokaryons with a common heterokary on incompatibility factor, obtained from a field isolates were homogeneous for pectic enzymes. Basidiospore progenies of a field isolate segregated widely in pectic zymograms. It appeared that the expression of pectic enzymes by field isolates involved multiple genetic factors. The variation of zymograms among homokaryotic strains suggests that each field isolate of R. solani contains two types of nuclei, although cells of vegetative hyphae are multinucleate.     

Expression of Proteins and Glycoproteins Encoded by the Haploid Nuclei in the Dikaryotic State in the Basidiomycete Agrocybe aegerita

The total proteins and concanavalin A-binding glycoproteins of the cultivated mushroom Agrocybe aegerita were studied in homokaryotic siblings and in dikaryotic strains. The glycoproteins exhibited considerable variability compared with the proteins; the genetic diversity detected in homokaryons in the glycoprotein analysis was 30-fold higher than the genetic diversity revealed by protein analysis, and the glycoprotein patterns could be used to characterize individual genotypes. We found that the expression of glycoproteins in haploid nuclei was significantly asymmetric when the nuclei were paired in dikaryons. The expression levels of the two component nuclei depended on their genotypes, and each haploid nucleus was characterized by its level of expression. Furthermore, some specific glycoproteins that were not detected in all of the homokaryons were newly synthesized in the dikaryotic strains. Among these was a glycoprotein designated gpAa-65, which was identified in all of the dikaryotic strains and appeared to be a good molecular marker of the dikaryotic state.