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.

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.