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

Abstract

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.

1. Introduction

Tamogitake mushroom (Pleurotus cornucopiae var. citrinopileatus) is a type of edible mushrooms known to have a unique flavor. Moreover, there is growing evidence that suggests that tamogitake mushroom is beneficial to one’s health, thus increasing its demand for human consumption (Tomiyama et al., 2008; Tanaka, Nishimura, Sato, & Nishihira, 2016). This mushroom contains ergothioneine several times higher than that of other mushrooms, which is a known functional health component.

L-ergothioneine (EGT) is a type of amino acid that has various benefit effects for health. which are antioxidant effects (Franzoni et al., 2006; Bao, Osako, & Ohshima, 2010; Chen, Ho, Hsieh, Wang, & Mau, 2012), anti-inflammatory effects (Ito et al., 2011), protection against rheumatoid arthritis, Crohn’s disease (Cheah & Halliwell, 2012). Furthermore, EGT is thought to act as an antidepressant (Nakamichi et al., 2016), protect against Alzheimer’s disease, and improve memory and learning abilities (Yang et al., 2012). The biosynthetic pathway of EGT has been identified in Mycobacterium tuberculosis (Genghof & Olga, 1964; Seebeck, 2010) and Neurospora crassa (Bello, Barrera-Perez, Morin, & Epstein, 2012) since its discovery in the ergot fungus, Claviceps purpurea (Tanret, 1909). However, the biosynthetic pathway of EGT in mushrooms has not yet been elucidated.

Biosynthesizing EGT in humans is not possible, and it is only synthesized in microorganisms, such as bacteria and fungi. Therefore, EGT must be consumed in food in order for humans to benefit from its effects. The acquirement of EGT-rich strains that contain in the fruiting body is essential in order to maximize its benefits and is one of the important breeding targets in tamogitake mushroom (Chen et al., 2012; Lin et al., 2013).

Recently, the breeding efficiency of various types of crops has improved using the marker-assisted selection (MAS). MAS has been shown to be more effective in the breeding of sporeless mutants obtained due to mutations in Pleurotus pulmonarius (Okuda, Murakami, & Matsumoto, 2009a, 2009b; Okuda, Murakami, Honda, & Matsumoto, 2013) and P. eryngii (Okuda et al., 2012; Im et al., 2016). While EGT is considered a secondary metabolite, which means it is a more complex agricultural characteristic and its levels of content are controlled by several traits. This requires the identification of quantitative trait locus (QTL). DNA polymorphisms will serve as a marker and can be utilized to an identification of QTL due to advances in molecular biological technology (Baars, Sonnenberg, Micosch, & Griensven, 2000). These advances make it possible to construct highly accurate linkage maps. Previous studies have used QTL analysis to link the yield (Ashikari et al., 2005), quality (Ando et al., 2008), and resistance to stress (Sugiura et al., 2004; Fukuoka et al., 2015) with DNA polymorphisms in many types of crops and used their information to the breeding programs. QTL analysis has also been used in edible mushrooms in the same manner as crops. For example, it was used to assess mycelial growth rate (Miyazaki et al., 2008), yield, and the formation of the fruiting body in Lentinula edodes (Going et al., 2016), the yield and cap color in P. ostreatus (Larraya, Alfonso, Arana, Psabarro, & Ramírez, 2003), the brushing sensitivity and cap color in Agaricus bisporus (Gao et al., 2015), the vegetative growth rate in P. ostreatus (Larraya et al., 2002), the fruiting body yield in P. eryngii (Im et al., 2016). In addition, the development and utilization of molecular markers based on the genome information in mushroom have been improved to similar levels as those used in crops.

Next-generation sequencing (NGS) platforms and restriction-site associated DNA sequencing (Rad-seq) technologies have also improved, and they can now be used as reliable methods to detect genome-wide polymorphisms and for diverse genetic investigation, including QTL analysis (Wu et al., 2014). In this study, as a preparation for use new technology above, NGS analysis of the monokaryotic strain, Y1, which is a neohaplont of the cultivar HfpriPc 05-1 of tamogitake mushroom (Yoneyama et al., 2015, 2017), was performed and its genome information was registered in DDBJ (http://www.ddbj.nig.ac.jp, BHFW01000001–BHFW01000088) (Yoneyama et al., 2020).­

In this study, we constructed a first genetic linkage map for tamogitake mushroom using amplified fragment length polymorphisms (AFLP), genes, mating type factors, and Rad markers. We used this map to detect the QTL that is closely associated with a trait of high EGT content in the fruiting body. Subsequently, we identified the single nucleotide polymorphism (SNP) well corresponding with phenotypes of EGT content in wild population.

2. Materials and methods

2.1. Fungal strains and culture conditions

Tamogitake mushroom used in this study was acquired from the wild strains stocked in the Forest products research institute, Hokkaido research organization, which were shown in Table 1. Monokaryotic strain Y12 is one of the neohaplonts of HfpriPc76-4 that is high in EGT content in the fruiting body [8.4 mg/g dry weight (g-dw)]. Meanwhile, Q13 is a monokaryon from the spore isolates of HfpriPc08-3 that contains low EGT content (0.25 mg/g-dw). A total of 105 F1 progenies (single-spore isolates) from the dikaryotic strain Y12Q13 were obtained from mating between Y12 and Q13. Subsequently crossing was performed between 105 progenies and Y11, which was one of the other neohaplonts of HfpriPc76-4 using the same method (Yoneyama et al., 2017). The mating types of 105 progenies were determined by tester strains of each mating type.

Table 1 List of wild tamogitake mushroom strains, and their EGT content of fruiting bodies and DNA type of QTL locus (QTL-EGT).

Strainsa	Origines	EGT content (mg/g-dw)	Rank of EGT contentb	DNA type
Pc07-7	Biei-cho, Kamikawa-gun, Hokkaido, Japan	9.85	Hc	A, T
Pc94-1	Kamui, Asahikawa-shi, Hokkaido, Japan	9.51	Hc	A, A
Pc82-4	Unkown, Hokkaido, Japan	9.15	Hc	A, A
Pc98-9	Kuriyama, Nikko-shi, Tochigi, Japan	8.51	Hc	A, T
Pc76-4	Okuda et al. (2015)	8.40	Hc	A, A
Pc81-1	Saroma-cho, Tokoro-gun, Hokkaido, Japan	8.37	Hc	A, A
Pc08-2	Engaru-cho, Engaru-gun, Hokkaido, Japan	7.85	Hc	A, T
Pc82-3	Kamikawa-cho, Kamikawa-gun, Hokkaido, Japan	7.42	Hc	T, T
Pc93-1	Higashiasahikawa-cho, Kamikawa-gun, Hokkaido, Japan	6.46	Md	A, A
Pc89-3	Unknown, Hokkaido, Japan	6.32	Md	T, T
Pc07-10	Higashiasahikawacho, Asahikawa-shi, Hokkaido, Japan	6.12	Md	A, T
Pc07-9	Kaguraoka, Asahikawa-shi, Hokkaido, Japan	5.48	Md	T, T
Pc99-3	Kaguraoka, Asahikawa-shi, Hokkaido, Japan	5.12	Md	A, T
Pc14-1	Honbestu-cho, Nakagawa-gun, Hokkaido, Japan	4.91	Md	T, T
Pc87-2	Midorimachi, Asahikawa-shi, Hokkaido, Japan	4.69	Md	T, T
Pc98-8	Higashiasahikawacho, Asahikawa-shi, Hokkaido, Japan	4.29	Md	T, T
Pc07-8	Bifuka-cho, Kamikawa-gun, Hokkaido, Japan	3.80	Md	T, T
Pc89-2	Takikawa-shi, Hokkaido, Japan	2.10	Le	T, T
Pc10-1	Okuda et al. (2015)	2.05	Le	A, T
Pc08-1	Engaru-cho, Engaru-gun, Hokkaido, Japan	1.97	Le	T, T
Pc02-1	Kamikawa-cho, Kamikawa-gun, Hokkaido, Japan	1.42	Le	T, T
Pc93-3	Wassamu-cho, Kamikawa-gun, Hokkaido, Japan	1.41	Le	A, A
Pc07-3	Touma-cho, Kamikawa-gun, Hokkaido, Japan	1.31	Le	A, T
Pc97-3	Engaru-cho, Engaru-gun, Hokkaido, Japan	0.96	Le	T, T
Pc07-11	Higashiasahikawacho, Asahikawa-shi, Hokkaido, Japan	0.88	Le	T, T
Pc94-2	Kaguraoka, Asahikawa-shi, Hokkaido, Japan	0.59	Le	A, T
Pc08-6	Okuda et al. (2015)	0.57	Le	T, T
Pc08-3	Okuda et al. (2015)	0.25	Le	T, T
Pc07-6	Okuda et al. (2015)	0.19	Le	T, T
Pc99-2	Okuda et al. (2015)	0.08	Le	T, T

^aAll strains are maintained at Hokkaido Research Organization, Forest Products Research Institute. ^bHigh (H) group was defined as the strain contains higher than 6.62 mg/g-dw. Low (L) group was defined as the strain contains less than 3.41 mg/g-dw. Medium (M) group defined as the strain contains EGT of range from 3.41 to 6.62 mg/g-dw. The different letters denote significant difference between the groups (H, M, L) by Tukey-Krammer test (p < 0.001).

The stock cultures of these strains were maintained on SMYP (soluble starch 20 g, malt extract 10 g, yeast extract 1 g, peptone 1 g and agar 20 g in 1 L distilled water) medium at 1.5 °C. Sawdust spawn was prepared from these stock cultures. The sawdust medium was prepared as a mixture of larch (Larix leptolepis) sawdust, wheat bran, and water (1:1:3.75 ratio by dry weight) for 850 mL plastic bottle cultivation, as described previously (Yoneyama et al., 2015). Subsequently, after autoclaving sawdust medium, each spawn was inoculated on the sawdust medium and cultivation was conducted, as described previously (Yoneyama et al., 2015). The spawn running process was carried out at 22 ± 1 °C, and 70 ± 5% relative humidity (RH), under dark conditions until primordium formation. The primordium was grown at 18 ± 1 °C and 85 ± 5% RH under 350 lux fluorescent lamp for 12 h and under dark condition for 12 h per day. The fruiting body was harvested when the 50% of the cap of the fruiting body grew to 20­­­–25 mm diam. Around 20 g of the harvested fruiting body was freeze-dried and powdered for EGT content analysis. For the analysis, three to five bottles were randomly selected from the four to eight bottles.

2.2. Quantification of EGT content

EGT was extracted from the freeze-dried powder of 0.5 g, which was mixed in 20 mL of deionized water, and the mixture was then suspended by using an ultrasonic vibrator UT-4 (SHARP Corp., Osaka, Japan) for 10 min. The suspension was subsequently centrifuged at 22,000 g at 20 °C, and the resulting supernatant was collected and diluted to a final volume of 25 mL, following the methods previously reported by Bao et al. (2009), with some modifications. Quantification of EGT was performed using high performance liquid chromatography (HPLC) (SHIMADZU Corp., Kyoto, Japan), as described previously by Tepwong et al. (2012). The HPLC system was equipped with a photodiode array detector and the Develosil C30-UG-5 column (5 μm in particle size, 4.6 mm inner diam and 250 mm long; NOMURA CHEMICAL Co., LTD., Seto, Japan). The mobile phase was 0.1% acetic acid in 10% methanol at a flow rate of 0.2 mL/min. EGT was detected at 254 nm, using the absolute calibration curve method by standard reagent (L-(+)-Ergothioneine, Cayman Chemical Co., LTD., Ann Arbor, USA). In addition, differences in EGT content were detected by Tukey–Kramer test and χ²-test using BellCurve for Excel ver. 3.00 software (Social Survey Research Information Co., Ltd., Tokyo, Japan).

2.3. Preparation of DNA, polymorphism analysis and sequencing

Cultures for DNA extraction were grown at 25 °C for 2 wk on SMYP medium overlaid with cellophane. The parental strains, Y12 and Q13, 105 F1 progenies from Y12Q13, and wild strains were cultured. Subsequently, the mycelia were harvested and lyophilized (Okuda et al., 2015). The genomic DNA from each these isolates of the lyophilized mycelium was prepared using a Maxwell 16 Tissue DNA Purification kit (Promega Corp., Madison, USA), as per the manufacturer’s instructions. The genomic DNAs of Y12 and Q13 were prepared for NGS using the CTAB method (Kuhad, Kapoor, & Lal, 2004) with modification

AFLP analysis was performed as described previously by Terashima et al. (2002). The nine primer combinations used for selective amplification were as follows: E+AA/M+TC, E+AC/M+AA, E+AG/M+AC, E+AT/M+CC, E+AC/M+CG, E+AT/M+CG, E+AC/M+CT, E+AT/M+CT, and E+GA/M+CG. All PCR reactions were performed using a Takara Ex Taq (Takara Biomedical. Kusatsu, Japan) DNA polymerase in an iCycler thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA).

The Rad-seq library was prepared as described previously by Sakaguchi et al. (2015), with some modifications. In brief, 20 ng/µL of genomic DNA from the F1 isolates was digested with EcoRI and BglII. The sequence of this library was analyzed in a single read of 51 bp using the Illumina HiSeq 2500 next-generation sequencer (Illumina, CA, USA). The Rad-seq reads were mapped to the genomic DNA of the above reference sequences of tamogitake mushroom (Li & Durbin, 2009), and SNP was identified using Stacks v.1.19 software (Catchen, Hohenlohe, Bassham, Amores, & Cresko, 2013).

The genomic DNAs of the parental strains (Y12 and Q13) were sequenced using paired-end sequencing, and the reads were mapped to the reference sequence of the Y1 genome using SAMtools (Li et al., 2009) and BCFtools (Danecek & McCarthy, 2017). Selection of the polymorphism was confirmed and visualized using the Integrative Genomics Viewer (http://broadinstitute.org/software/igv/). In order to increase the mapping markers in the predicted QTL regions [linkage group 3 (LG3) and LG10] which had been estimated by preliminary analysis of the specific polymorphic sites, candidate genes were predicted using AUGUSTUS software (http://bioinf.uni-greifswald.de/augustus/), and their gene function were determined using the EuKaryotic Orthologous Groups (KOG) and evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) databases. The above information analysis was performed by Takara Bio Inc. (Kusatsu, Shiga, Japan). If the gene function of the polymorphic site regions had been estimated, it was used as a gene marker (Supplementary Table S1).

In order to determine the SNP genotype, DNA sequencing was performed using BigDye Terminator v3.1 (Applied Biosystems, Foster City, USA), as per the manufacturer’s protocol.

2.4. Construction of linkage maps and QTL analysis

Chi-squared (χ²) tests were performed to determine if the segregation ratio of the trait was significantly different from the expected 1:1 ratio among the isolates of the progeny. Linkage maps were constructed using MAPMAKER/EXP version 3.0b (Lander et al., 1987). Logarithm of the odds (LOD) score for the distance of maps were assessed using 3.0–5.0 and Kosambi, with the maximum distance set at 40 centimorgans (cM). QTL analysis was performed using the composite interval mapping (CIM) program of Windows QTL Cartographer, version 2.5 (Wang, Basten, & Zeng, 2007). Threshold value was used by a default value, 11.5 (Lander & Botstein, 1989).

3. Results

3.1. The range of EGT content

The EGT contents of 30 wild strains of tamogitake mushroom were determined to be ranging from 0.08 to 9.85 mg/g-dw, with an average of 4.33 ± 3.28 mg/g-dw (n = 30) suggesting quantitative differences between strains (Table 1).

Dikaryotic population obtained from crossing between the F1 progenies from Y12Q13 and Y11 were determined to contain EGT ranging from 3.41 to 9.69 mg/g-dw with an average of 6.62 ± 1.47 mg/g-dw (n = 105) (Fig. 1). There was a 22.2% coefficient of variation. The normal distribution of EGT content in the F1 dikaryotic population was confirmed using the Kolmogorov-Smirnov test and the Shapiro-Wilk test. Therefore, the EGT content was presumed to distribute continuously. These data suggest that EGT content of fruiting body is controlled by multiple loci and that the obtained F1 monokaryotic progenies are suitable group to perform QTL analysis on.

Fig. 1 Distribution of ergothioneine content in the 105 F1 progenies and proportion of the gene-type of QTL locus (QTL-EGT) in each content class. 105 F1 population shown by asterisk (*) was divided between Y12 type and Q13 type by the hyps gene marker.

To characterize the level of EGT contents of wild strains and F1 crossed strains, they were divided into low content or high content groups by standardized at 3.41 mg/g-dw, which was the lowest content of all F1, as shown in Fig. 1. Furthermore, in order to classify the F1 crossed strains with higher EGT content than 3.41 mg/g-dw, these strains were divided into two groups of M (intermediate content) and H (high content) based on the average content of EGT, 6.62 mg/g-dw.

3.2. Marker development and genetic map construction

The frequency in the appearance of each mating type was approximately equal to 1:1:1:1 (χ² = 0.64, 0.75 < p < 0.90) in the F1 dikaryotic population (Supplementary Table S2). EGT contents of each four mating type were approximately same level at 6.77 ± 1.57，6.17 ± 1.44, 6.63 ± 1.65, and 6.88 ± 1.26, respectively. From these results, it is considered that mating factors were not linked to the quantity of EGT as supporting by the result of Fig. 2. In addition to the two mating factors, the polymorphisms of both of Y12 and Q13 were assessed using AFLP, Rad-Seq, and gene markers. As a result, we could try to make the map after preparing a candidate for 427 markers composed of 40 AFLP, 315 AFLP markers, and 72 gene markers. A linkage map was constructed using the MAPMAKER software version 3.0b (Lander et al., 1987; Lander & Boststein, 1989). Although several markers were discarded as they were not linearly linked to neighboring markers, the remaining 245 markers, including 195 Rad markers, 10 AFLP markers, 2 mating type factors, and 38 gene markers (Table 2), were located on the linkage map.

Fig. 2 Genetic linkage maps and QTL affecting the ergothioneine content in tamogitake mushroom. The genetic distance (cM Kosambi) of the markers is indicated on the left side of each LG; the marker names are on the right. AFLP markers are designated by the primer combination and their size. Rad markers indicate the position of the polymorphic site in their respective contigs. Gene makers are shown in Supplementary Table S1. Superscripts of alphabet represent skewed markers (0.01 < P < 0.05) (Supplementary Table S3). The round mark represents the estimated location of QTL-EGT in the LG10.

Table 2. Characteristics of the tamogitake mushroom genetic linkage map.

Linkage group	No. of markersa					Observed length (cM)	Average marker interval (cM)b	Largest interval (cM)
	AFLP	Rad	Gene	Mating type factor	Total
LG1	3	20	4	1	28	83.5	2.9	23.4
LG2	0	25	1	0	26	86.4	3.3	20.7
LG3	4	21	9	0	34	177.8	5.2	23.7
LG4	1	17	2	0	20	48.8	2.4	11.4
LG5	1	32	2	1	36	89.4	2.4	37.1
LG6	0	16	1	0	17	53.6	3.1	21.1
LG7	0	7	2	0	9	16.1	1.7	9.1
LG8	0	25	0	0	25	85.2	3.4	22.5
LG9	0	5	0	0	5	50.5	10.1	26.5
LG10	1	8	10	0	19	91.4	4.8	15.5
LG11	0	5	0	0	5	18	3.6	8.2
LG12	0	14	7	0	21	106.1	5.1	20.2
Total	10	195	38	2	245	906.8
Average					20.4	75.5	4.0
aAFLP, Amplified fragment length polymorphism; Rad, Restriction-site associated DNA; Gene, shown in Supplemental Table 1; bThe average marker interval did not contain the same location markers.

The constructed genetic linkage map consisted of 12 linkage groups and a total length of 906.8 cM, with an average marker interval of 4.0 cM. The minimum length of the linkage group was estimated to be at 16.1 cM of LG7, and the maximum length was 177.8 cM of LG3 (Table 2; Fig. 2).

Genome sequences were used to confirm that the homolog PcoEgt1 (LC317066) of the EGT biosynthesis locus was located on LG8 and that mating factors A and B were located on LG1 and LG5, respectively. The locus and the primer sequences for detection by PCR are shown in Supplementary Table S1. Among the 195 of Rad markers, 112 markers formed 2–8 clusters composed of 37 loci (Fig. 2; Supplementary Table S3).

3.3. QTL Analysis and estimation of QTL controlling the EGT content on the LG10.

The QTL analysis for EGT content of the fruiting body was conducted using CIM method with the software application Windows QTL Cartographer 2.5. As a result, the QTL was presumed to be located on LG10 with a LOD score of 2.53 and 10.1% explanation of variance (Fig. 2; Supplementary Fig. S1). The region of the presumed QTL was located in 24.6 cM of the sum of 6 markers from sam2 to the znf gene marker (Fig. 2).

From the sequence information of the markers, we extracted the location of the reference sequence on the estimated region of the QTL. Consequently, it was predicted that it was in an 87 kb region (Fig. 3). We also performed sequence analysis of parental strains Y12 and Q13 using Illumina HiSeq. De novo assembly based on the sequences obtained was performed in order to assess whether there are differences in the genome sequence between parental strains Y12 and Q13. Then, we extracted the region corresponding to the markers on the linkage map. The extracted regions of parental genomes of Y12 and Q13 were much the same in the length to that of the reference genome Y1. They also, including the reference genome, were composed of same 27 genes although the order of some genes was different (Fig. 3; Table 3). Therefore, it was considered that information of this 87 kb reference sequence, which was estimated as a location of QTL, could be used for the identification of marker locus responsible for target QTL.

Fig. 3 QTL locus (QTL-EGT) on the LG10 and its estimated corresponding region in contig 21 of the reference genome (Y1) and parental genomes (Y12 and Q13). The horizontal line indicates threshold value of 2.50. The LOD confidence interval is represented by the black bar around the circle. Marker names and their genetic positions (cM) were described in Fig. 2. Abbreviations for the genes used in this figure are shown in Supplementary Tables S1 and S3. The gene composition and order in this region of the Y12 and Q13 genomes were identical.

Table 3. Gene order from 808,570 bp to 899,628 bp in contig 21 of reference genome (Y1) of tamogitake mushroom.

Abbreviation in Fig. 2 and 3	No. of databasea	Estimated gene function	5' position of gene
tfIId	KOG3219	Transcription initiation factor TFIID, subunit TAF11	808570
znf	KOG4124	Zinc finger protein	811052
	KOG4510	Permease of the drug/metabolite transporter (DMT) superfamily	821352
	KOG0225	Pyruvate dehydrogenase E1, alpha subunit	823775
hyp3	XP_003022574	Hypothetical protein	826890
cad	KOG0370	Multifunctional pyrimidine synthesis protein CAD	833667
	KOG0247	domain protein	842388
nc2	KOG1659	Class 2 transcription repressor NC2, alpha subunit (DRAP1)	844612
hyps	KDQ28398	Hypothetical protein	846458
	KOG3176	Predicted alpha-helical protein, potentially involved in replication/repair	848109
dap	KOG2100	Dipeptidyl aminopeptidase	849303
c4	KOG0873	C-4 sterol methyl oxidase	855423
priA	KOG0243	Kinesin-like protein	857642
	NOG177524	Fungal hydrophobin	862061
fr	KOG1502	Flavonol reductase/cinnamoyl-CoA reductase	867324
dap	KOG2100	Dipeptidyl aminopeptidase	868403
fr	KOG1502	Flavonol reductase/cinnamoyl-CoA reductase	873914
	NOG21759	HypA-like protein	875146
	KOG0854	Alkyl hydroperoxide reductase, thiol specific antioxidant and related enzyme	877925
	KOG3656	FOG: 7 transmembrane receptor	880788
gt	KOG4696	Glycosyl transferase, family 2	883466
gs	KOG0399	Glutamate synthase	885770
	NOG11304	Fungalysin	887872
	KOG0607	MAP kinase-interacting kinase and related serine/threonine protein kinases	891397
pgd	NOG09647	6-phosphogluconate dehydrogenase	893197
	XP454996	Uncharacterized protein	895387
	NOG63708	Unnamed protein	897373
sam2	KOG4300	Predicted methyltransferase	898341
	KOG0274	Cdc4 and related F-box and WD-40 proteins	899628
aKOG: EuKaryotic Orthologous Groups, NOG: Non-supervised Orthologous Groups Groups

3.4. SNP in the QTL region corresponding to EGT content of wild strains.

To estimate the responsible gene for the target QTL, the genome sequences between Y12 and Q13 were compared and polymorphism were detected. Around 225 non-synonymous substitutions [insertions and deletions (INDELs) and SNPs] polymorphisms were predicted in the coding region of the 27 genomes. In order to presume the relationship between these polymorphisms and the QTL peak, we sequentially assessed between the polymorphisms in the neighborhood of the peak and the EGT content in 105 F1 progenies and 30 wild strains. As a result, the SNP, A/T, located in the 747th base in the coding region of “hyps” gene (Length: 825 bp, DDBJ, LC553556), which is next to the “nc2” gene, was remarked (Fig. 3; Table 3). This SNP led to the conversion of the 249th amino acid, Q (CAA), to H (CAT) as a non-synonymous substitution. The genotype, represented by T and A, coincided with the parental Y12 and Q13 genotypes, respectively. Furthermore, as shown Table 1, with a few exceptions, we observed that high EGT (more than 6.62 mg/g-dw) wild strains (H type group) had the A, T or A, A genotypes occupied 87.5% of H type group. On the other hand, low EGT (less than 3.41 mg/g-dw) wild strains (L type group) had the T, T genotype and occupied 69.2% of L type group. No other polymorphisms with a relationship to EGT content were identified.

4. Discussion

EGT content in the fruiting body of tamogitake mushroom has been shown to be higher than that of other mushrooms (Bao et al., 2010; Chen et al., 2012). In this study, we aimed to detect the region of the QTL that is controlling the EGT content in the fruiting body of tamogitake mushroom.

4.1. Linkage Maps

The genetic linkage map was constructed using AFLP markers, Rad markers, mating factors and gene markers, which were composed of 12 linkage groups with a total length of 906.8 cM and an average marker interval of 4.0 cM (Table 2; Supplementary Table S3). This is the first genetic linkage map of tamogitake mushroom.

Estimation of the genomic region, using the genetic linkage maps, such as the locus of the mutation causing genes and the QTLs for agricultural traits has been previously reported in P. eryngii [6.17 cM/marker (Okuda et al., 2012), 4.09 cM/marker (Im et al., 2016)], P. ostreatus [5.3 cM/marker (Larraya, Pérez, Ritter, Pisabarro, & Ramírez, 2000)] and P. pulmonarius [5.2 cM/marker (Okuda et al., 2009b)]. We suggest that these findings offer valuable genome data for basic and applied genetic research of tamogitake mushroom, as the marker density in this linkage map is homogeneously distributed, is approximately the same level as the other Pleurotus spp. described above.

4.2. EGT content

In this study, to validate the EGT content trait of the F1 progenies were crossed. These dikaryotic progenies displayed a wide distribution in EGT content, with a minimum of 3.41 mg/g-dw to a maximum of 9.69 mg/g-dw. The progenies did not have the same low level of EGT (under 3.0 mg/g-dw), and the low EGT characteristic from the other parent had also improved (Fig. 1). These results suggest that the characteristic of having high EGT content has incomplete dominant heritability against low content. Therefore, retaining the loci which produce more EGT would be useful for breeding strains with high EGT content in the fruiting body.

4.3. QTL analysis

One QTL related to EGT content in the fruiting body was detected, with over 10% of the contribution rate explained in analysis of dikaryotic population from crossing and cultivation environment. Although several reports have used this approach to assess mycelial growth (Larraya et al., 2002; Miyazaki et al., 2008), color of the fruiting body (Larraya et al., 2003; Gao et al., 2015), productivity, and disease resistance (Gao et al., 2015), as far as we know, this is the first report that uses QTL analysis to assess the content of functional substances in mushrooms.

The EGT biosynthesis pathway, which includes histidine as a starter, was investigated in N. crassa (Bello et al., 2012). The PcoEgt1 is a homolog of main gene in the EGT biosynthesis pathway of tamogitake mushroom’s genome. However, this homolog positioned on LG8, which is different from LG10 includes QTL region (Fig. 2). In addition, from investigation of the EGT biosynthesis pathway as described above, although it was presumed that the existence of multiple QTLs related to EGT content, this study only identified one QTL (Fig. 2; Supplementary Fig. S1). The detecting number of QTL generally affected the marker density on the constructed linkage map (Hori et al., 2003). On the other hand, the marker density in this study was the same level on the linkage map as previous reports (Larraya et al., 2000; Okuda et al., 2009b; Okuda et al., 2012; Im et al., 2016). Hereafter, for increasing the estimation number of QTLs, it is necessary to prepare the strains and retain the different backgrounds based on this result.

4.4. The SNP related to QTL for EGT content

The QTL region (QTL-EGT) was estimated to be about 87 kb in length on the physical map, and this region is expected to include 27 genes, based on the comparison of Y12 and Q13 genomes (Fig. 3). The nearest genome marker to the QTL peak is nc2. The phenotypes of 105 progeny isolates were clearly divided in a 1:1 segregation, which coincided mainly with Y12 and Q13, as shown in Fig. 1. The QTL peak in the high EGT group and low EGT group tended to coincide with Y12 and Q13, respectively. However, a position of the QTL peak had a LOD value of 2.52, which was obtained by the CIM method, and did not perfectly correspond to this marker. Accordingly, we investigated the estimated SNP to generate the peak in the 87 kb region of the QTL-EGT. Based on the investigation of the relationship between the genome type and EGT content in 30 wild strains, we identified the SNP as either an A or a T, which coincided with Y12 or Q13, respectively. This SNP was 500 bp from the nc2 marker, which indicates that the gene type coincides with EGT content. The other polymorphism did not coincide with the EGT content of wild strains. Therefore, it was suggested that this SNP was possibly related to the generation of the peak of the QTL-EGT. The distribution frequency of the gene type of 105 progenies was also found to resemble the nc2 marker. The gene type of the cross dikaryon between the F1 progenies and Y11 (A) could reveal either the homo-type (A/A) or the hetero-type (A/T). EGT contents of these strains ranged higher than 3.41 mg/g-dw and were not found to be in the lower range (0.25 mg/g-dw) of the parental strain. This suggests that the QTL trait from parental Y12 exhibited incomplete dominant inheritance.

As described above, the homo-type (A/A) occupied 73.3% of the isolates that contained over 8.0 mg/g-dw high levels of EGT. In contrast, 15 strains with the homo-type (T/T) occupied 69.2% of the isolates and had lower than the average levels of EGT (4.33 mg/g-dw). Therefore, we speculated the homo-type (A/A) and hetero-type (A/T) were linked to high EGT levels. Thus, the SNP, A/T, could provide the possibility to the development an efficient MAS program in breeding for high EGT content of tamogitake mushroom.

The SNP was located at a region of unknown functional significance, with a gene encoding 275 amino acids. The SNP resulted in a polymorphism leading to the substitution of an amino acid from Q to H by using AUGUSTUS software. Although we could not identify the protein function of the predicted gene hyps in this study, it is expected as a candidate gene responsible for the regulation of EGT levels in the fruiting body of tamogitake mushroom. Therefore, it will be very interesting to elucidate of its protein function in the future for the progress of the medicinal application of this mushroom.

In conclusion, we constructed a genetic linkage map based on the segregation analysis of 245 markers in the monokaryon through meiosis in tamogitake mushroom. With the CIM method, the QTL for EGT content was found which explained 22.1% of the variance in EGT contents. Furthermore, we identified the SNP, A/T, in the gene site at the EGT region, which is in good agreement with the EGT content in 30 strains from wild population. Our results might provide fundamental information for biosynthesis of the EGT in tamogitake mushroom and breeding its higher content strain.

Disclosure

The authors declare no conflicts of interest.

Acknowledgments

This research was supported by Innovation Creation Research Promotion Project (No. 27036C) from Ministry of Agriculture, Forestry and Fisheries of Japan.

References

• Ando, T. , Yamamoto, T. , Shimizu, T. , Ma, X. F. , Shomura, A. , Takeuchi, Y. , Lin, S. Y. ,& Yano, M. (2008). Genetic dissection and pyramiding of quantitative traits for panicle architecture by using chromosomal segment substitution lines in rice. Theoretical and Applied Genetics, 116, 881–890. https://doi.org/10.1007/s00122-008-0722-6.
• Ashikari, M. , Sakakibara, H. , Lin, S. , Yamamoto, T. , Takashi, T. , Nishimura, A. , Angeles, E. R., Qian, Q. , Kitano, H. ,& Matsuoka, M. (2005). Cytokinin oxidase regulates rice grain production. Science, 309,741–745. https://doi.org/10.1126/science.1113373.
• Baars, J. J. P. , Sonnenberg, A. S. M. , Micosch, T. S. P. ,& Griensven, L. J. L. D. V. (2000). Science and cultivation of edible fungi. In: L. J. L. D. V. Griensven (Ed.), Development of sporeless strain of oyster mushroom Pleurotus ostreatus (pp. 317–323). Rotterdam, Netherland: Balkema.
• Bao, H. N. D. , Osako K. ,& Ohshima, T. (2010). Value-added use of mushroom ergothioneine as a color stabilizer in processed fish meats. Journal of the Science of Food and Agriculture, 90, 1634–1641. https://doi.org/10.1002/jsfa.3992.
• Bao, H. N. D. , Shinomiya, Y. , Ikeda, H. ,& Ohshima, T. (2009). Preventing discoloration and lipid oxidation in dark muscle of yellowtail by feeding an extract prepared from mushroom (Flammulina velutipes) cultured medium. Aquaculture, 295, 243–249. https://doi.org/10.1016/j.aquaculture.2009.06.042.
• Bello, M. H. , Barrera-Perez, V. , Morin, D. ,& Epstein, L. (2012). The Neurospora crassa Nc∆Egt-1 identifies an ergothioneine biosynthetic gene and demonstrates that ergothioneine enhances conidial survival and protects against peroxide toxicity during conidial germination. Fungal Genetics and Biology, 49, 160–172. https://doi.org/10.1016/j.fgb.2011.12.007.
• Catchen, J. , Hohenlohe, P. A. , Bassham, S. , Amores, A. ,& Cresko, W. A. (2013). Stacks: an analysis tool set for population genomics. Molecular Ecology, 22, 3124–3140. https://doi.org/10.1111/mec.12354.
• Cheah I. K. ,& Halliwell, B. (2012). Ergothioneine; antioxidant potential, physiological function and role in disease. Biochemica et Biophysica Acta, 1822, 784–793. https://doi.org/10.1016/j.bbadis.2011.09.017.
• Chen, S. Y. , Ho, K. J. , Hsieh, Y. J. , Wang, L. T. ,& Mau, J. L. (2012). Contents of lovastatin, γ-aminobutyric acid and ergothioneine in mushroom fruiting bodies and mycelia. LWT-Food Science and Technology, 47, 274–278. https://doi.org/10.1016/j.lwt.2012.01.019.
• Danecek, P. ,& McCarthy, S. A. (2017). BCFtools/csq: haplotype-aware variant consequences. Bioinformatics, 33, 2037–2039. https://doi.org/10.1093/bioinformatics/btx100.
• Franzoni, F. , Colognato, R. , Galetta, F. , Laurenza, I. , Barsotti, M. , Stefano, R. D. , Bocchetti, R. , Regoli, F. , Carpi, A. , Balbarini, A. Migliore, L. ,& Santoro, G. (2006). Scavenging capacity of ergothioneine: comparison with reduced glutathione, uric acid and trolox. Biomedicine&Pharmacotherapy, 60, 453–457. https://doi.org/10.1016/j.biopha.2006.07.015.
• Fukuoka, S. , Saka, N. , Mizukami, Y. , Koga, H. , Yamanouchi, U. , Yoshioka, Y. , Hayashi, N. , Ebana, K. , Mizobuchi, R. ,& Yano, M. (2015). Gene pyramiding enhances durable blast disease resistance in rice. Scientific Reports, 5, 7773. https://www.nature.com/articles/srep07773/
• Gao, W. , Weijn, A. , Baars, J. J. P. , Mes, J. J. , Visser, R. G. F. ,& Sonnenberg, A. S. M. (2015). Quantitative trait locus mapping for brushing sensitivity and cap color of Agaricus bisporus (button mushrooms). Fungal Genetics and Biology, 77, 69–81. https://doi.org/10.1016/j.fgb.2015.04.003.
• Genghof, D. S. ,& Olga, V. D. (1964). Biosynthesis of ergothioneine and hercynine by mycobacteria. Journal of Bacteriology, 87, 852–862.
• Going, W. , Li, L. , Zhou, Y. , Bian, Y. , Kwan, H. , Cheung, M. ,& Xiao, Y. (2016). Genetic dissection of fruiting body-related traits using quantitative trait loci mapping in Lentinula edodes. Applied Microbiology and Biotechnology, 100, 5437–5452. https://doi.org/10.1007/s00253-016-7347-5.
• Hori, K. , Kobayashi, T. , Shimizu, A. , Sato, K. , Takeda, K. ,& Kawasaki, S. (2003). Efficient construction of high-density linkage map and it’s application to QTL analysis in barley. Theoretical and Applied Genetics, 107, 806–813. https://doi.org/ 10.1007/s00122-003-1342-9.
• Im, C.H. , Park, Y.H. , Hammel, K. E. , Park, B. , Kwon, S.W. , Ryu, H. ,& Ryu, J. S. (2016). Construction of a genetic linkage map and analysis of quantitative trait loci associated with the agronomically important traits of Pleurotus eryngii. Fungal Genetics and Biology, 92, 50–64. https://doi.org/10.1016/j.fgb.2016.05.002.
• Ito, T. , Kato, M. , Tsuchida, H. , Harada, E. , Niwa, T. ,& Osawa, T. (2011). Ergothioneine as an anti-oxidative/anti-inflammatory component in several edible mushrooms. Food Science and Technology Research, 17, 103–110. https://doi.org/10.3136/fstr.17.103.
• Kuhad, R. C. , Kapoor, R. K. ,& Lal, R.    (2004).   Improving the yield and quality of DNA isolated from white-rot fungi.  Folia Microbiology, 49, 112–116. https://doi.org/10.1007/BF02931383.
• Lander, E. S. ,& Botstein, D. (1989). Mapping mendelian factors underlying quantitative traits using linkage maps. Genetics, 121, 185–199.
• Lander, E. S. , Green, P. , Abrahamson, J. , Barlow, A. , Daly, M. J. , Lincoln, S. E. ,& Newburg, L. (1987). MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics, 1, 174–181.
• Larraya, L. M. , Alfonso, M. , Arana, D. , Psabarro, A. G. ,& Ramírez, L. (2003). Mapping of genomic region (quantitative trait loci) controlling production and quality in industrial culture of the edible basidiomycete Pleurotus ostreatus. Applied and Environmental Microbiology, 69, 3617–3625.
• Larraya, L. M. , Idereta, E. , Arana, D. , Ritter, E. , Pisabarro, A. G. ,& Ramírez, L. (2002). Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus. Applied and Environmental Microbiology, 68, 1109–1114. https://doi.org/10.1128/AEM.68.3.1109-1114.2002.
• Larraya, L. M. , Pérez, G. , Ritter, E. , Pisabarro, A. G. ,& Ramírez, L. (2000). Genetic linkage map of the edible basidiomycete Pleurotus ostreatus. Applied and Environmental Microbiology, 66, 5290–5300. https://doi.org/10.1128/AEM.66.12.5290-5300.2000.
• Li, H. ,& Durbin, R. (2009). Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics, 25, 1754–1760. https://doi.org/10.1093/bioinformatics/btp324.
• Li, H. , Handsaker, B. , Wysoker, A. , Fennell, T. , Ruan, J. , Homer, N. , Marth, G. , Abecasis, G. ,& Richard, D. (2009). 1000 Genome Project Data Processing Subgroup. The sequence alignment/map format and SAMtools. Bioinformatics, 25, 2078–2079. https://doi.org/10.1093/bioinformatics/btp352.
• Lin, S. Y. , Chen, Y. K. , Yu, H. T. , Barseghyan, G. S. , Asatiani, M. D. , Wasser, S. P. ,& Mau, J. L. (2013). Comparative study of contents of several bioactive components in fruiting bodies and mycelia of culinary-medicinal mushrooms. International Journal of Medicinal Mushrooms, 15, 315–323. https://doi.org/10.1615/IntJMedMushr.v15.i3.80.
• Miyazaki, K. , Huang, F. , Zhang, B. , Shiraishi, S. , Sakai, M. Shimaya, C. ,& Shishido, K. (2008). Genetic map of a basidiomycete fungus, Lentinula edodes (shiitake mushroom), constructed by tetrad analysis. Breeding Science, 58, 23–30. https://doi.org/10.1270/jsbbs.58.23.
• Nakamichi, N. , Nakayama, K. , Ishimoto, T. , Masuo, Y. , Wakayama, T. , Sekiguchi, H. , Sutoh, K. , Usumi, K. , Iseki, S. ,& Kato, Y. (2016) Food-derived hydrophilic antioxidant ergothioneine is distributed to the brain and exerts antidepressant effect in mice. Brain and Behavior, 6, e00477. https://doi.org/10.1002/brb3.477.
• Okuda, Y. , Ueda, J. , Obatake, Y. , Murakami, S. , Fukumasa, Y. ,& Matsumoto, T. (2012). Construction of a genetic linkage map based on AFLP markers and development of STS markers for marker-assisted selection of the sporeless trait in oyster mushroom (Pleurotus eryngii). Applied and Environmental Microbiology, 78, 1496–1504. https://doi.org/10.1128/AEM.07052-11.
• Okuda, Y. , Murakami, S. ,& Matsumoto, T. (2009a) Development of STS markers suitable for marker-assisted selection of sporeless trait in oyster mushroom, Pleurotus pulmonarius. Breeding Science, 59, 315–319. https://doi.org/10.1270/jsbbs.59.315.
• Okuda, Y. , MurakamI, S. ,& Matsumoto, T. (2009b). A genetic linkage map of Pleurotus pulmonarius based on AFLP markers, and localization of the gene region for the sporeless mutation. Genome, 52, 438–446. https://doi.org/10.1139/G09-021.
• Okuda, Y. , Murakami, S. , Honda, Y. ,& Matsumoto, T. (2013). An MSH4 homolog, stpp1, from Pleurotus pulmonarius is a “silver bullet” for resolving problems caused by spores in cultivated mushrooms. Applied and Environmental Microbiology, 79, 4520–4527. https://doi.org/ 10.1128/AEM.00561-13.
• Okuda, Y. , Yoneyama, S. , Gisusi, S. , Harada, A. , Sato, M. ,& Matsumoto, T. (2015). Development of STS markers suitable for marker-assisted selection of sporeless trait in golden oyster mushroom, Pleurotus cornucopiae var. citrinopileatus. Mushroom Science and Biotechnology, 23, 85–88. https://doi.org/10.24465/msb.23.2_85.
• Sakaguchi, S. , Sugino, T. , Tsumura, Y. , Ito, M. , Crisp, M. D. , Bowman, D. M. J. S. , Nagano, A. J. , Honjo, M. N. , Yasugi, M. , Kudoh, H. , Matsuki, Y. , Suyama, Y. ,& Isagi, Y. (2015). High-throughput linkage mapping of Australian white cypress pine (Callitris glaucophylla) and map transferability to related species. Tree Genetics and Genomes, 11, 121. https://doi.org/10.1007/s11295-015-0944-0.
• Seebeck, F. P. (2010). In vitro reconstitution of mycobacterial ergothioneine biosynthesis. Journal of the American Chemical. Society, 132, 6632–6633. https://10.1021/ja101721e.
• Sugiura, N. , Tsuji, T. , Fujii, K. , Kato, T. , Saka, N. , Touyama, T. , Hayano-Saito, Y. ,& Izawa, T. (2004). Molecular marker-assisted selection in a recurrent cross breeding for the incorporation of resistance to rice stripe virus and panicle blast in rice (Oryza sativa L.) (in Japanese). Breeding Research, 6, 143–148. https://doi.org/10.1270/jsbbr.6.143
• Tanaka, A. , Nishimura, M. , Sato, Y. ,& Nishihira, J. (2016). Enhancement of the Th1-phenotype immune system by the intake of Oyster mushroom （Tamogitake） extract in a double-blind, placebo-controlled study. Journal of Traditional and Complementary Medicine, 6, 424–430. https://doi.org/10.1016/j.jtcme.2015.11.004.
• Tanret, M. C. (1909). Sur une nouvelle retiree du seigle ergot, I’ergothioneine (in French). Comptes Rendus de I’Académiedes Sciences, 149, 222–224.
• Tepwong, P. , Giri, A. ,& Ohshima, T. (2012). Effect of mycelial morphology on ergothioneine production during liquid fermentation of Lentinula edodes. Mycoscience, 53, 102–112. https://doi.org/10.1007%2Fs10267-011-0145-0.
• Terashima, K. , Matsumoto, T. , Hayashi, E. ,& Fukumasa-Nakai, Y. (2002) A genetic linkage map of Lentinula edodes （shiitake） based on AFLP markers. Mycological Research, 106, 911–917. https://doi.org/10.1017/S0953756202006275.
• Tomiyama, T. , Kaihou, S. , Ishida, M. , Nishikawa, H. , Yamazaki, N. , Tsuji, K. , Mitsutake, S. ,& Igarashi, Y. (2008). The water retention effects and action for atopic dermatitis-like symptoms of ethyl alcohol extract (from Tamogi-take Mushroom) on animal model of atopic dermatitis (in Japanese). Journal of Japan Society of Nutrition and Food Science, 61, 21–26. https://doi.org/10.4327/jsnfs.61.21
• Wang, S. , Basten, C. J.,& Zeng, Z. B. (2007). Windows QTL cartographer 2.0. Raleigh, NC: Department of Statistics, North Carolina State University.
• Wu, K. , Liu, H. , Yang, M. , Tao, Y. , Ma, H. , Wu, W. , Zuo, Y. ,& Zhao, Y. (2014). High-density genetic map construction and QTLs analysis of grain yield-related traits in sesame (Sesamum indicum L.) based on RAD-seq technology. BMC Plant Biology, 274, 1–14. https://doi.org/10.1186/s12870-014-0274-7.
• Yang, N. C. , Lin, H. C. , Wu, J. H. , Ou, H. C. , Chai, Y. C. , Tseng, C. Y. , Liao, J. W. ,& Song, T. Y. (2012). Ergothioneine protects against neuronal injury induced by β-amyloid in mice. Food and Chemical Toxicology, 50, 3902–3911. https://doi.org/10.1016/j.fct.2012.08.021.
• Yoneyama, S. , Ando, N. , Azuma, T. , Sato, M. , Ushijima, S. ,& Matsumoto, T. (2017). Genetic and cytological analysis of a sporulation-deficient (sporeless) mutant of Pleurotus cornucopiae var. citrinopileatus induced by UV irradiation (in Japanese). Japanese Journal of Mycology, 58, 41–50. https://doi.org/10.18962/jjom.jjom.H29-04.
• Yoneyama, S. , Gisusi, S. , Sato, M. , Harada, A. , Muraguchi.,& Matsumoto, T. (2015). Induction of sporulation-deficient mutations in Pleurotus cornucopiae var. citrinopileatus induced by UV irradiation (in Japanese). Mushroom Science and Biotechnology, 23, 20–25. https://doi.org/10.24465/msb.23.1_20.
• Yoneyama, S. , Shirai, N. , Ando, N. , Azuma, T. , Tsuda, M. ,& Matsumoto, T. (2020). 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. Breeding Science. https//doi.org/10.1270/jsbbs 20039.