Teruyuki Matsumoto

A genetic linkage map of Lentinula edodes (shiitake) based on AFLP markers

A medium-dense genetic linkage map of Lentinula edodes (shiitake) was constructed based on 203 amplified fragment length polymorphism (AFLP) markers and two mating type factors. The segregation of these markers was generated from 95 progeny of a single cross of two distantly related strains. The map consists of 11 linkage groups comprised of eight large (over 100 centimorgans (cM)) and three small (less than 100 cM) groups, and the total genetic distance of the map was 1956.7 cM. The average rate of physical size to genetic distance could be roughly estimated to be less than 18.4 kb cM −1 , which is low compared to the values obtained for other filamentous fungi. Seventeen of the AFLP markers showed highly distorted segregation ratios (X 2 values > 6.63; P

Genetic diversity and strain-typing in cultivated strains of Lentinula edodes (the shii-take mushroom) in Japan by AFLP analysis*

Amplified fragment length polymorphism (AFLP) analysis is based on selective PCR (polymerase chain reaction) amplification of genomic DNA restriction fragments. The present study was performed to: (1) assess the extent of AFLP variation among major cultivated strains of Lentinula edodes (the shii-take mushroom) in Japan; (2) evaluate the usefulness of AFLP as genetic marker for typing the cultivated strains; and (3) infer the genetic relatedness among them. Six AFLP primer pairs detected a total of 304 DNA fragments in a sample of 13 cultivated strains for wood log cultivation and 2 strains for sawdust cultivation, of which 179 DNA fragments (58.9% of detected fragments) were polymorphic between two or more strains. These polymorphic DNA fragments could differentiated all of the cultivated strains. Cluster analysis and principle coordinate analysis based on AFLP data showed two distinct groups; one group was composed of the strains in which fruiting occurred during the middle to low temperature period from autumn to next spring under outdoor wood log cultivation, and the other strains in which fruiting could be induced in the summer under outdoor cultivation and strains for sawdust cultivation under indoor air-controlled condition.

Strain typing of shiitake (Lentinula edodes) cultivars by AFLP analysis, focusing on a heat-dried fruiting body

To validate strain typing by amplified fragment length polymorphism (AFLP) analysis in shiitake (Lentinula edodes) cultivars, the reproducibility of AFLP markers with DNA extracted from the heat-dried fruiting body was evaluated. DNAs were extracted from three different portions of the heat-dried fruiting body – the stipe, pileus, and gill – and AFLP analysis of all parts was carried out using two combinations of selected amplification primer pairs. AFLP profiles of DNA from the gill tissue of heat-dried fruiting body were almost identical to those of cultured mycelia in the same strains, although it was difficult to detect reproducible AFLP profiles from stipe and pileus DNA. These results indicated that AFLP analysis would be applicable for strain typing with heat-dried fruiting bodies of L. edodes by using the DNA extracted from gills.

Mitochondrial DNA restriction fragment length polymorphisms and phenetic relationships in natural populations of the oyster mushroom, Pleurotus ostreatus

Restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) in 34 isolates of the oyster mushroom, Pleurotus ostreatus, from throughout the northern hemisphere, were employed to evaluate genetic relatedness among natural populations. Eighteen isolates were from Japan, ten from Europe, five from U.S.A., and one from Korea. BamHI, EcoRI and EcoRV digests of mtDNAs produced 18, 19 and 19 distinct RFLP patterns, respectively. By combining all RFLP patterns obtained with the three endonucleases, mtDNAs could be assigned to 22 different classes that were clustered phenetically into three major similarity groups, each of which represents a geographically distinct population. The results suggest that geographical distance between natural populations of P. ostreatus is correlated with genetic divergence.

Construction of a Genetic Linkage Map Based on Amplified Fragment Length Polymorphism Markers and Development of Sequence-Tagged Site Markers for Marker-Assisted Selection of the Sporeless Trait in the Oyster Mushroom (Pleurotus eryngii)

ABSTRACT A large number of spores from fruiting bodies can lead to allergic reactions and other problems during the cultivation of edible mushrooms, including Pleurotus eryngii (DC.) Quél. A cultivar harboring a sporulation-deficient (sporeless) mutation would be useful for preventing these problems, but traditional breeding requires extensive time and labor. In this study, using a sporeless P. eryngii strain, we constructed a genetic linkage map to introduce a molecular breeding program like marker-assisted selection. Based on the segregation of 294 amplified fragment length polymorphism markers, two mating type factors, and the sporeless trait, the linkage map consisted of 11 linkage groups with a total length of 837.2 centimorgans (cM). The gene region responsible for the sporeless trait was located in linkage group IX with 32 amplified fragment length polymorphism markers and the B mating type factor. We also identified eight markers closely linked (within 1.2 cM) to the sporeless locus using bulked-segregant analysis-based amplified fragment length polymorphism. One such amplified fragment length polymorphism marker was converted into two sequence-tagged site markers, SD488-I and SD488-II. Using 14 wild isolates, sequence-tagged site analysis indicated the potential usefulness of the combination of two sequence-tagged site markers in cross-breeding of the sporeless strain. It also suggested that a map constructed for P. eryngii has adequate accuracy for marker-assisted selection.

Genetic relationships in the natural population of Pholiota nameko from Japan based on DNA polymorphisms

Abstract This study characterized the genetic relation-ships in the natural population of Pholiota nameko (Strophariaceae) from Japan based on the RFLPs of two regions of nuclear rDNA (ITS and IGS) and mitochondrial DNA and the RAPD profile of nuclear DNA. No intraspecific polymorphism in rDNA was found among 36 isolates of P. nameko used. By contrast, digests of mtDNAs by endonucleases HindIII and BglII produced RFLP patterns that distinguished all isolates except for 2, and clustered 36 wild isolates phenetically into three major similarity groups. However, these groups as obtained by analysis of mtDNA RFLPs did not reflect the geographic origin of the isolates. In RAPD analysis of nuclear DNA using three kinds of primers, every isolate showed its own distinct RAPD profile, but all isolates were clustered only into a large similarity group by phylogenetic analysis based on the RAPD profile. From these results, it is suggested that wild isolates of P. nameko distributed in Japan form a continuous genetic population that has conserved the genetic diversity.

Variation of ITS sequences in a natural Japanese population of Pleurocybella porrigens

Genetic relationships in a natural Japanese population of Pleurocybella porrigens were determined based on ITS sequence data. In a UPGMA similarity tree, all sequences of 23 specimens from 13 different geographic origins were grouped into two distinct clusters (groups A and B). Sequence variation of the ITS region between groups A and B consisted of 33–40 nucleotides, corresponding to 5%–6% of their total length, and specific nucleotide variations characterizing groups A and B were found. Although these results did not show correlation with differences of substrates for fruiting and geographic origins of the specimens, it was suggested that P. porrigens distributed in Japan include at least two genetically different populations.

Fruiting body productivity of protoplast-derived clones in Lentinula edodes*

More than 100 dikaryotic clones (protoclones) derived from mycelial protoplasts of aLentinula edodes dikaryon were examined for their mycelial growth and fruiting body productivity. These protoclones exhibited a variety of vegetative mycelial growth rates, but no apparent difference in colonial morphology compared with the original (parental) dikaryon. Protoclones were cultivated on wood logs under natural conditions, and they exhibited a very wide range of fruiting body yields. Of the 134 protoclones, four were selected that produced a 30–40% increase in dry weight of fruiting body yield over that of the original dikaryon. This high productivity of fruiting bodies was maintained for at least several years. The present results suggest thatL. edodes protoclones can be practically used in strain improvement to increase the capability of fruiting body formation.

An MSH4 homolog, stpp1, from Pleurotus pulmonarius is a "silver bullet" for resolving problems caused by spores in cultivated mushrooms.

ABSTRACT The enormous number of spores produced by fruiting bodies during cultivation of mushrooms can lead to allergic reactions of workers, reduction of commercial value, spread of mushroom disease, pollution of facilities, and depletion of genetic diversity in natural populations. A cultivar harboring a sporulation-deficient (sporeless) mutation would be very useful for preventing these problems, but sporeless commercial cultivars are very limited in usefulness because sporeless traits are often linked with traits that are unfavorable for commercial cultivation. Thus, identifying a causal gene of a sporeless phenotype not linked to the adverse traits in breeding and cultivation is crucial for the establishment of sporeless breeding using a strategy employing targeting induced local lesions in genomes (TILLING) in cultivated mushrooms. We used a Pleurotus pulmonarius (Fr.) Quél. sporeless strain to identify and characterize the single recessive gene controlling the mutation. The 3,853-bp stpp1 gene encodes a protein of 854 amino acids and belongs to the MutS homolog (MSH) family associated with mismatch repair in DNA synthesis or recombination in meiosis. Gene expression analysis of the fruiting body showed that this gene is strongly expressed in the gills. Phenotypic analysis of disruptants formed by gene targeting suggested a reproducible sporeless phenotype. Mutants deficient in a functional copy of this gene have no unfavorable traits for sporeless cultivar breeding, so this gene will be an extremely useful target for efficient and versatile sporeless breeding in P. pulmonarius and various other cultivated mushrooms.

Phylogenetic position of Pholiota nameko in the genus Pholiota inferred from restriction analysis of ribosomal DNA

Abstract To estimate the phylogenetic position of Pholiota nameko in the genus Pholiota, restriction fragment length polymorphisms (RFLPs) for PCR products of 26S ribosomal RNA gene (rDNA), internal transcribed spacers (ITS), and intergenic spacer (IGS) of the rDNA repeat from P. nameko and eight of its closely related species were investigated, and a phylogenetic tree was constructed based on data that resulted from RFLP analysis. P. nameko was clustered together with P. adiposa, P. limonella, and P. aurivella of the subgenus Pholiota (section Adiposae). However, P. nameko and P. albocrenulata, both of which belong to the subgenus Hemipholiota, were phylogenetically separated from each other. Our results suggested that P. nameko is closely related to the members of the section Adiposae. Furthermore, the phylogenetic distance between this section Adiposae group including P. nameko and Kuehneromyces mutabillis was smaller than that between P. malicola var. macropoda of the subgenus Flammula and the members of section Adiposae. Our data indicate that molecular information on rDNA will be useful to reconstruct taxa within the genus Pholiota in the family Strophariacea that have been classified mostly on the basis of morphological characters.