Generation of Iron-Independent Siderophore-Producing Agaricus bisporus through the Constitutive Expression of hapX.

Agaricus bisporus secretes siderophore to uptake environmental iron. Siderophore secretion in A. bisporus was enabled only in the iron-free minimal medium due to iron repression of hapX, a transcriptional activator of siderophore biosynthetic genes. Aiming to produce siderophore using conventional iron-containing complex media, we constructed a recombinant strain of A. bisporus that escapes hapX gene repression. For this, the A. bisporushapX gene was inserted next to the glyceraldehyde 3-phosphate dehydrogenase promoter (pGPD) in a binary vector, pBGgHg, for the constitutive expression of hapX. Transformants of A. bisporus were generated using the binary vector through Agrobacterium tumefaciens-mediated transformation. PCR and Northern blot analyses of the chromosomal DNA of the transformants confirmed the successful integration of pGPD-hapX at different locations with different copy numbers. The stable integration of pGPD-hapX was supported by PCR analysis of chromosomal DNA obtained from the 20 passages of the transformant. The transformants constitutively over-expressed hapX by 3- to 5-fold and sidD, a key gene in the siderophore biosynthetic pathway, by 1.5- to 4-fold in mRNA levels compared to the wild-type strain (without Fe3+), regardless of the presence of iron. Lastly, HPLC analysis of the culture supernatants grown in minimal medium with or without Fe3+ ions presented a peak corresponding to iron-chelating siderophore at a retention time of 5.12 min. The siderophore concentrations of the transformant T2 in the culture supernatant were 9.3-fold (-Fe3+) and 8-fold (+Fe3+) higher than that of the wild-type A. bisporus grown without Fe3+ ions, while no siderophore was detected in the wild-type supernatant grown with Fe3+. The results described here demonstrate the iron-independent production of siderophore by a recombinant strain of A. bisporus, suggesting a new application for mushrooms through molecular biological manipulation.

SNP-Based Genetic Linkage Map and Quantitative Trait Locus Mapping Associated with the Agronomically Important Traits of Hypsizygus marmoreus.

White strains of Hypsizygus marmoreus are more difficult to cultivate than are brown strains; therefore, new white strain breeding strategies are required. Accordingly, we constructed the genetic map of H. marmoreus with 1996 SNP markers on 11 linkage groups (LGs) spanning 1380.49 cM. Prior to analysis, 82 backcrossed strains (HM8 lines) were generated by mating between KMCC03106-31 and the progenies of the F1 hybrid (Hami-18 × KMCC03106-93). Using HM8, the first 23 quantitative trait loci (QTLs) of yield-related traits were detected with high limit of detection (LOD) scores (1.98-9.86). The length, thickness, and hardness of the stipe were colocated on LG 1. Especially, length of stipe and thickness of stipe were highly correlated given that the correlation coefficients were negative (-0.39, p value ≤ .01). And a typical biomodal distribution was observed for lightness of the pileus and the lightness of the pileus trait belonged to the LG 8, as did traits of earliness and mycelial growth in potato dextrose agar (PDA) medium. Therefore, results for color traits can be suggested that color is controlled by a multi-gene of one locus. The yield trait was highly negatively correlated with the traits for thickness of the stipe (-0.45, p value ≤ .01). Based on additive effects, the white strain was confirmed as recessive; however, traits of mycelial growth, lightness, and quality were inherited by backcrossed HM8 lines. This new genetic map, finely mapped QTLs, and the strong selection markers could be used in molecular breeding of H. marmoreus.