Identification of a SNP and development of a PCR-based allele-specific marker of the sporulation-deficient (sporeless) trait of the Tamogitake 108Y2D mutant using next-generation sequencing.

The mass scattering of basidiospores during the cultivation of edible mushrooms causes serious problems, such as allergic reactions in workers. Sporulation-deficient (sporeless) cultivars would be very useful for preventing these issues. We aimed to identify the single-nucleotide polymorphism (SNP) that is responsible for the single dominant sporeless mutation of the Tamogitake 108Y2D mutant using next-generation sequencing (NGS) and TILLING technology and to develop an allele-specific PCR marker for sporeless breeding. By comparing the sequences of the wild-type and its mutant genomes, we identified 685 mutation loci in gene regions and pinpointed one SNP only consistent with sporeless phenotype for 105 segregants, i.e., a C to T located at position 1,950 of the exonic region of a putative fungal transcription factor that generated a stop codon. We developed an allele-specific marker based on the identified SNP, and its high practicality was validated using tests against progenies from several hybrids and wild isolates from different geographical origins. Thus, the allele-specific PCR marker developed here will be useful for marker-assisted selection in the breeding of the sporeless trait of this mushroom. Furthermore, the technical success of SNP identification and marker development based on NGS genome data can help achieve efficient mutation breeding in mushrooms.

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

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.

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).

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.

Karyological characterization of meiosis, post-meiotic mitosis and nuclear migration in the ectomycorrhizal fungus Rhizopogon roseolus (= R. rubescens).

Karyological characteristics during basidiosporogenesis of Rhizopogon roseolus, a member of the hypogeous Agaricomycetes, were studied by light and transmission electron microscopy. More than 1000 tissue fragments of young basidiomata were stained with HCl-Giemsa and observed by a light microscopy to evaluate nuclear behavior. Basidium morphology in the hymenium was observed by transmission electron microscopy. Meiosis and post-meiotic mitosis took place in the center of the basidium. Sterigmata appeared when the first meiotic division occurred, and the center of the basidium became constricted when the second meiotic division occurred. Asynchronous nuclear migration from the basidium into the basidiospores occurred after post-meiotic mitosis, producing eight uninucleate basidiospores. The nucleus migrated patchily into basidiospores. The pattern of post-meiotic mitosis of R. roseolus, in which post-meiotic mitosis took place in the center of the basidium, is reported for the first time.

PCR-based gene marker with a high efficiency genome scanning (HEGS) system: application for distinguishing among cultivars in Lentinula edodes.

To establish a high throughput and cost-efficiency procedure for distinguishing among cultivars in Lentinula edodes, the polymorphism in the genes reported previously in this fungus was examined using PCR or PCR-RFLP techniques with a high-efficiency genome scanning (HEGS) system. As a result, PCR-based markers derived from eight genes (tyr, cap, ppa, IGS-RFLP, pri B-RFLP, mfbC-RFLP, gla-RFLP, xy-RFLP) showed polymorphisms among cultivars in this fungus and consequently, enabled to distinguish seventy-nine cultivars used in this study.

Disease resistance and growth promotion activities of chitin/cellulose nanofiber from spent mushroom substrate to plant.

Some studies have reported the method for treating the spent mushroom substrate (SMS). However, the effective use as a functional raw material based on properties of SMS remains a formidable challenge. In this study, we investigated the usefulness of SMS in agriculture to develop a new method for treating and utilizing it. First, we attempted to isolate chitin/cellulose nanofiber complex (CCNFC) from SMS using chemical pretreatment and mechanical fibrillation. The characterization results like SEM, FT-IR, and XRD showed that we successfully isolated the CCNFC from SMS. Second, we explored the biological activities of the CCNFC for its potential application as a functional agricultural nanomaterial. CCNFC water dispersion with low concentration (0.1 and 1 mg/mL) exhibited significant plant disease resistance and plant growth promotion activities. Our results suggested that SMS may provide a useful source of functional agricultural nanomaterial, which may contribute to treating and applying it in agriculture.

Construction of a genetic linkage map and detection of quantitative trait locus for the ergothioneine content in tamogitake mushroom (Pleurotus cornucopiae var. citrinopileatus).

Developing high-content strains of L-ergothioneine (EGT), an antioxidant amino acid, is an important breeding target for tamogitake mushroom, Pleurotus cornucopiae var. citrinopileatus. We constructed a genetic linkage map based on segregation analysis of markers in 105 F1 progenies. The loci of 245 markers, including 10 AFLP markers, 195 Rad markers, 2 mating type factors, and 38 gene markers, were mapped. The map contained 12 linkage groups with a total genetic distance of 906.8 cM, and an average marker interval of 4.0 cM. The population from crossing between tester monokaryon and F1 progenies was used to characterize quantitative trait loci (QTL) for EGT content. With composite interval mapping (CIM) method, QTL of EGT content were found to be located in linkage group 10, having a Logarithm of the odds (LOD) score of 2.53 with a 10.1% contribution rate. Moreover, a single nucleotide polymorphism (SNP), A/T, was identified in a gene region of the genome in the neighborhood where the QTL peak existed. This SNP genotype was in good agreement with the EGT phenotypes of each strain in the both QTL population and wild population. Thus, this SNP would have great potential value to use the marker-assisted selection (MAS) for this mushroom with high EGT content.

Morphological, cytological and genetic analyzes of the 'sango' mutant with the defects in basidiocarp development in edible mushroom Pleurotus pulmonarius.

A spontaneous, morphological variation 'sango' was observed in the progeny of a Pleurotus pulmonarius (Fr.) Quél. wild-type basidiocarp (also known as fruiting body) collected from the field. This variant developed wart- and coral-like structures instead of normal basidiocarps. Microscopic analysis showed that the sango phenotype had defects in the differentiation of the pileus and hymenium. Basidiocarp phenotypic data analysis in the progenies revealed that the sango trait is a heritable mutation character controlled by a single recessive gene. This mutation locus was mapped on linkage group III of a previously constructed genetic linkage map by amplified fragment length polymorphism (AFLP) technique in P. pulmonarius. Four AFLP markers identified by bulked segregant analysis showed linkage to the sango mutation locus, with the genetic distance ranging from 0 to 2.1 cM. Of these markers, one marker was co-segregated with the sango mutation locus. This knowledge will be a useful foundation for practical breeding as well as for elucidating molecular mechanisms in basidiocarp development of main edible mushrooms.

Communities of arbuscular mycorrhizal fungi in Pyrus pyrifolia var. culta (Japanese pear) and an understory herbaceous plant Plantago asiatica.

We investigated communities of arbuscular mycorrhizal fungi (AMF) in the fine roots of Pyrus pyrifolia var. culta, and Plantago asiatica to consider the relationship between orchard trees and herbaceous plants in AMF symbioses. The AMF communities were analyzed on the basis of the partial fungal DNA sequences of the nuclear small subunit ribosomal RNA gene (SSU rDNA), which were amplified using the AMF-specific primers AML1 and AML2. Phylogenetic analysis showed that the obtained AMF sequences were divided into 23 phylotypes. Among them, 12 phylotypes included AMF from both host plants, and most of the obtained sequences (689/811) were affiliated to them. Canonical correspondence analysis showed that the host plant species did not have a significant effect on the distribution of AMF phylotypes, whereas the effects of sampling site, soil total C, soil total N and soil-available P were significant. It was also found that the mean observed overlaps of AMF phylotypes between the paired host plants in the same soil cores (27.1% of phylotypes shared) were significantly higher than the mean 1,000 simulated overlaps (14.2%). Furthermore, the same AMF sequences (100% sequence identity) were detected from both host plants in 8/12 soil cores having both roots. Accordingly, we concluded that Py. pyrifolia and Pl. asiatica examined shared some AMF communities, which suggested that understory herbaceous plants may function as AMF inoculum sources for orchard trees.

Communities of arbuscular mycorrhizal fungi in the roots of Pyrus pyrifolia var. culta (Japanese pear) in orchards with variable amounts of soil-available phosphorus.

We examined the colonization rate and communities of arbuscular mycorrhizal fungi (AMF) in the roots of Pyrus pyrifolia var. culta (Japanese pear) in orchards to investigate the effect of phosphorus (P) fertilization on AMF. Soil cores containing the roots of Japanese pear were collected from 13 orchards in Tottori Prefecture, Japan. Soil-available P in the examined orchards was 75.7 to 1,200 mg kg(-1), showing the extreme accumulation of soil P in many orchards. The AMF colonization rate was negatively correlated with soil-available P (P <0.01). AMF communities were examined on the basis of the partial fungal DNA sequences of the nuclear small-subunit ribosomal RNA gene (SSU rDNA) amplified by AMF-specific primers AML1 and AML2. The obtained AMF sequences were divided into 14 phylotypes, and the number of phylotypes (species richness) was also negatively correlated with soil-available P (P <0.05). It was also suggested that some AM fungi may be adapted to high soil-available P conditions. Redundancy analysis showed the significant effects of soil pH, available P in soil, and P content in leaves of P. pyrifolia var. culta trees on AMF distribution. These results suggested that the accumulation of soil-available P affected AMF communities in the roots of Japanese pear in the orchard environment.

A genetic linkage map of Pleurotus pulmonarius based on AFLP markers, and localization of the gene region for the sporeless mutation.

In the cultivation of edible mushrooms, including Pleurotus pulmonarius (Fr.) Quel., the enormous number of spores produced by fruiting bodies can adversely affect mushroom growers' health, mushroom cultivation facilities, and the genetic diversity of natural populations. In this study, we constructed a primary genetic linkage map and identified the locus associated with the sporulation-deficient (sporeless) mutation of P. pulmonarius using 150 progeny isolates derived from a cross between sporeless and wild-type isolates. Based on the segregation of 300 AFLP markers, two mating-type factors, and the sporeless trait, a linkage map was generated consisting of 12 linkage groups. The map covered a total genetic distance of 971 cM, with an average marker interval of 5.2 cM. The gene region responsible for the sporeless mutation was located in linkage group II including 40 AFLP markers and the A mating-type factor locus. Of these markers, the nearest marker to the sporeless locus was located 1.4 cM away. Construction of this P. pulmonarius genetic linkage map and identification of markers that are closely linked to the sporeless locus will facilitate marker-assisted selective breeding of a sporeless strain with economically important traits.