Identification of the Pol Gene as a Species-Specific Diagnostic Marker for Qualitative and Quantitative PCR Detection of Tricholoma matsutake

Tricholoma matsutake is a rare, precious, and wild edible fungus that could not be cultivated artificially until now. This situation has given way to the introduction of fake T. matsutake commodities to the mushroom market. Among the methods used to detect food adulteration, amplification of species-specific diagnostic marker is particularly important and accurate. In this study, the Pol gene is reported as a species-specific diagnostic marker to identify three T. matsutake varieties and 10 other types of edible mushrooms through qualitative and quantitative PCR. The PCR results did not reveal variations in the amplified region, and the detection limits of qualitative and quantitative PCR were found to be 8 ng and 32 pg, respectively. Southern blot showed that the Pol gene exists as a single copy in the T. matsutake genome. The method that produced the purest DNA of T. matsutake in this study was also determined, and the high-concentration salt precipitation method was confirmed to be the most suitable among the methods tested. The assay proposed in this work is applicable not only to the detection of raw materials but also to the examination of processed products containing T. matsutake.


Introduction
Tricholoma matsutake is an ectomycorrhizal agaricomycete predominantly associated with pines and oaks. It is a commercially valuable edible mushroom [1] with great significance, not only because of its delicate flavor but also because of its diverse biological properties [2], which include multiple immunostimulatory, hematopoietic, antineoplastic, antimutation, and antioxidation activities [3].
The growing number of T. matsutake consumers has steadily expanded market demands for this mushroom. On account of the continued deterioration of ecological systems and the environment and excessive picking, the natural productivity of this gourmet mushroom has gradually declined. To date, no artificial cultivation method has yet been developed for T. matsutake due to the lack of information concerning precise soil requirements and cues for sporophore formation [4]. Therefore, wild T. matsutake and its products are in a short supply in the market, and, as such, its economic value has risen sharply. To address demands for the mushroom and reap higher profits, merchants frequently pass off counterfeit or adulterated T. matsutake products as genuine items, for example, sliced dried Agaricus blazei [5]. Establishing an effective and convenient method for identifying authentic T. matsutake is an urgent necessity.

Comparison of DNA Extraction Methods
The DNA of T. matsutake fruiting bodies from Yunnan was obtained using the CTAB, SDS-CTAB, high-concentration salt precipitation, and kit methods. The results of 1% agarose gel for genomic DNA ( Figure 1A) indicated that the four DNA extraction methods could successfully extract genomic DNA with good integrity. All of the OD260/280 and DNA concentrations obtained are listed in Table 1, there are significant differences in the DNA concentrations, and the OD260/280 ratio of the High-concentration salt precipitation method compared with other three extraction methods reveals that the purity of genomic DNA extracted by this method is the highest.

Species-Specific Diagnostic Marker of T. Matsutake
Among the genes selected, the Pol gene (Tricholoma matsutake pol gene for polyprotein encoding RNase H and integrase, Genbank No. AB016926) showed no homology with other genes from different varieties of the mushroom.
It has been reported that retrotransposons have been incorporated into the genome of their hosts and inherited to the host progenies since the earliest establishment of their parasitism. In a previous study, this Pol gene was a retroelement from T. matsutake, which related to RNase H and integrase of retrotransposons [44]. The reverse transcriptase domain was found in T. matsutake worldwide, this finding suggested that retroelements associate with ectomycorrhizal basidiomycetes and might be useful as genetic markers for identification, phylogenetic analysis, and mutagenesis of this fungal group [45]. After the BLAST analysis of Pol gene, it just confirmed the statement above. Hence, we chose the Pol gene as the candidate species-specific diagnostic marker of T. matsutake.

Species Specificity of Qualitative PCR aSSAYS
The genomic DNA isolated from 10 non-T. matsutake species (R. virescens, A. deliciosus, B. speciosus, T. albuminosus, A. blazei, L. edodes, P. eryngii, F. velutipes, and P. ostreatus) and three T. matsutake strains (Yunnan, Sichuan, and Jilin) was subjected to 18s rDNA amplification (Figure 2A). The 18s rDNA primer pair was used to confirm all the extracted DNA can be effectively amplified, and the quality of the extracted DNA clearly met the conditions of PCR (Figure 2A).  The restriction enzyme digestion result in Figure 1B showed that the genomic DNA extracted by the four methods could be digested by EcoR I, and the products were in a dispersive state. However, the digestibility of genomic DNA extracted by the CTAB, SDS-CTAB, and kit methods was lower than that of the high-concentration salt precipitation method, and there were a large number of large fragments, which showed brighter digestion products in the upstream part of the lanes, while the high-concentration salt precipitation method was more uniform. It revealed the high-concentration salt precipitation method was superior in quality and has less enzyme inhibitors.
The PCR products of the 18S rRNA region amplified with the fungal universal 18S primers were staining to identify whether the extracted T. matsutake genomic DNA can be used for subsequent PCR amplification. As shown in Figure 1C, the 18S rRNA fragments were efficiently amplified using the DNA extracted by the four methods.
In summary, by integrating the above four indicators shown in Table 1 and Figure 1, all genomic DNA extracted by the four methods could meet with the requirements of the PCR experiment, however, the high-concentration salt precipitation method was confirmed to be the most suitable DNA extraction way among the methods tested.

Species-Specific Diagnostic Marker of T. Matsutake
Among the genes selected, the Pol gene (Tricholoma matsutake pol gene for polyprotein encoding RNase H and integrase, Genbank No. AB016926) showed no homology with other genes from different varieties of the mushroom.
It has been reported that retrotransposons have been incorporated into the genome of their hosts and inherited to the host progenies since the earliest establishment of their parasitism. In a previous study, this Pol gene was a retroelement from T. matsutake, which related to RNase H and integrase of retrotransposons [44]. The reverse transcriptase domain was found in T. matsutake worldwide, this finding suggested that retroelements associate with ectomycorrhizal basidiomycetes and might be useful as genetic markers for identification, phylogenetic analysis, and mutagenesis of this fungal group [45]. After the BLAST analysis of Pol gene, it just confirmed the statement above. Hence, we chose the Pol gene as the candidate species-specific diagnostic marker of T. matsutake.

Species Specificity of Qualitative PCR aSSAYS
The genomic DNA isolated from 10 non-T. matsutake species (R. virescens, A. deliciosus, B. speciosus, T. albuminosus, A. blazei, L. edodes, P. eryngii, F. velutipes, and P. ostreatus) and three T. matsutake strains (Yunnan, Sichuan, and Jilin) was subjected to 18s rDNA amplification ( Figure 2A). The 18s rDNA primer pair was used to confirm all the extracted DNA can be effectively amplified, and the quality of the extracted DNA clearly met the conditions of PCR ( Figure 2A). The primer pair Pol-F/R was applied to the qualitative PCR of the Pol gene; PCRs were also conducted using the genomic DNA of the 13 mushroom samples indicated above. Electrophoretic analysis of all qualitative PCR products ( Figure 2B) revealed no objective product, except in the PCR products of the three T. matsutake samples. The results confirm that the qualitative PCR applied in this work are highly specific for T. matsutake.

Homology Analysis of the Pol Gene among Different T. Matsutake Varieties
An excellent species-specific diagnostic marker should have low heterogeneity and a consistent copy number in the same species. We carried out qualitative PCR using identical amounts of DNA from the three T. matsutake strains to determine whether the Pol gene undergoes any sort of variation. The primer pair Pol-F/R was applied to the qualitative PCR of the Pol gene; PCRs were also conducted using the genomic DNA of the 13 mushroom samples indicated above. Electrophoretic analysis of all qualitative PCR products ( Figure 2B) revealed no objective product, except in the PCR products of the three T. matsutake samples. The results confirm that the qualitative PCR applied in this work are highly specific for T. matsutake.

Homology Analysis of the Pol Gene among Different T. Matsutake Varieties
An excellent species-specific diagnostic marker should have low heterogeneity and a consistent copy number in the same species. We carried out qualitative PCR using identical amounts of DNA from the three T. matsutake strains to determine whether the Pol gene undergoes any sort of variation. Amplification products with identical sizes and relative intensities were obtained for all varieties after qualitative PCR ( Figure 2B, lines 2-4), and the slight differences, which were attributed to the quality of the isolated DNA [13], were considered negligible. As shown in Figure 3, the homologous similarity sequence identity between the PCR products and the reference Pol gene was 94.51%. These results indicate that the Pol gene did not show the sequence variation among the T. matsutake varieties studied.

Homology Analysis of the Pol Gene among Different T. Matsutake Varieties
An excellent species-specific diagnostic marker should have low heterogeneity and a consistent copy number in the same species. We carried out qualitative PCR using identical amounts of DNA from the three T. matsutake strains to determine whether the Pol gene undergoes any sort of variation. Amplification products with identical sizes and relative intensities were obtained for all varieties after qualitative PCR ( Figure 2B, lines 2-4), and the slight differences, which were attributed to the quality of the isolated DNA [13], were considered negligible. As shown in Figure 3, the homologous similarity sequence identity between the PCR products and the reference Pol gene was 94.51%. These results indicate that the Pol gene did not show the sequence variation among the T. matsutake varieties studied.

Confirmation of the Pol Gene Copy Number by Southern Blot
Besides species specificity, low sequence variation, and a consistent copy number, an excellent species-specific diagnostic marker is expected to possess a low copy number. Therefore, using

Confirmation of the Pol Gene Copy Number by Southern Blot
Besides species specificity, low sequence variation, and a consistent copy number, an excellent species-specific diagnostic marker is expected to possess a low copy number. Therefore, using Southern blot, we analyzed the copy number of the Pol gene in two T. matsutake varieties gathered from Yunnan and Jilin Provinces. Whether Hind III or EcoR I was used to digest the genomic DNA of T. matsutake, only one hybridization band was found in the nylon membrane (Figure 4), which means the Pol gene is only present as a single copy in the T. matsutake genome. Southern blot, we analyzed the copy number of the Pol gene in two T. matsutake varieties gathered from Yunnan and Jilin Provinces. Whether Hind III or EcoR I was used to digest the genomic DNA of T. matsutake, only one hybridization band was found in the nylon membrane (Figure 4), which means the Pol gene is only present as a single copy in the T. matsutake genome.

Sensitivity of the Qualitative and Taqman-Based Real-Time Quantitative PCR Assays
Genomic DNA from T. matsutake was diluted five times from 200 ng/µL to 12.8 pg/µL over a gradient, and the results ( Figure 5A) showed a detection limit of 8 ng for qualitative PCR. With the same way, in Figure 5B, the sensitivity of Taqman quantitative PCR was found to be 32 pg. A standard

Sensitivity of the Qualitative and Taqman-Based Real-Time Quantitative PCR Assays
Genomic DNA from T. matsutake was diluted five times from 200 ng/µL to 12.8 pg/µL over a gradient, and the results ( Figure 5A) showed a detection limit of 8 ng for qualitative PCR. With the same way, in Figure 5B, the sensitivity of Taqman quantitative PCR was found to be 32 pg. A standard curve of the Pol gene was then generated by using the proposed quantitative PCR system, and a linear relationship (R 2 = 0.993) with a slope of −3.081 was determined between the DNA quantities and Ct values ( Figure 5C).

Application of the Pol gene to Detect Processed T. Matsutake Products
We used the established Taqman-based PCR system to detect the source of T. matsutake in processed products, including mushroom biscuit, oil, and sauce. The target products could be amplified by employing Taqman-based quantitative PCR with the primer Pol-F/R and the probe Pol-P. The PCR results ( Figure 6A) were consistent with the ingredients listed on the packaging of each products. The results of real-time quantitative PCR ( Figure 6B) were in keeping with those of qualitative PCR. So, the results of both assays were prove that T. matsutake biscuits tested contained T. matsutake and the Pol gene was a practical and precise species-specific diagnostic marker for T. matsutake in highly processed foods, and affirmed its absence, as necessary.

Application of the Pol gene to Detect Processed T. Matsutake Products
We used the established Taqman-based PCR system to detect the source of T. matsutake in processed products, including mushroom biscuit, oil, and sauce. The target products could be amplified by employing Taqman-based quantitative PCR with the primer Pol-F/R and the probe Pol-P. The PCR results ( Figure 6A) were consistent with the ingredients listed on the packaging of each products. The results of real-time quantitative PCR ( Figure 6B) were in keeping with those of qualitative PCR. So, the results of both assays were prove that T. matsutake biscuits tested contained T. matsutake and the Pol gene was a practical and precise species-specific diagnostic marker for T. matsutake in highly processed foods, and affirmed its absence, as necessary.

Materials
The following mushroom species were purchased from local farmers' markets: Russula virescens, Agaricus deliciosus, Boletus speciosus, Termitornyces albuminosus, Agaricus blazei, Agrocybe cylindracea, Lentinula edodes, Pleurotus eryngii, Flammulina velutipes, and Pleurotus ostreatus. Three T. matsutake varieties strains collected from the provinces of Yunnan, Sichuan, and Jilin, China, were kindly supplied by all China Federation of Supply and Marketing Cooperative's Kunming Institute of Edible Fungi. All of the samples were collected in the quantity of 200g.
Three processed products labeled containing T. matsutake component, including mushroom biscuit, oil, and sauce, were gathered from Internet and local markets in Kunming, Yunnan province. They were used to verify the application of this selected species-specific diagnostic marker.

Genomic DNA Extraction
Four methods, including an improved CTAB method [46], the SDS-CTAB method [47], highconcentration salt precipitation method [48], and the kit method (Rapid Fungi Genomic DNA Isolation Kit, B518229, Sangon, Shanghai, China), were compared to determine the most suitable protocol for extracting the genomic DNA of T. matsutake. The T. matsutake fruiting bodies from Yunnan was used as the sample, each method was performed in triplicate. First, added the equal silica into 5 g mushroom sample, and then grounded them with liquid nitrogen. The target genomic DNA was isolated from the 0.2 g sample powder. All DNA extraction steps were performed in accordance with the references and manufacturer protocol. At last, the DNA was eluted in 50 µL EB buffer.
The subsequent DNA extraction was followed the selected optimal method. All of the OD260/280 and DNA concentrations were measured with a NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), the quality of genomic DNA was further analyzed on 1% agarose gel with ethidium bromide (0.1 µg /mL), and run with 1 × TAE buffer. Finally, the DNA solutions were stored at −20°C.

Materials
The following mushroom species were purchased from local farmers' markets: Russula virescens, Agaricus deliciosus, Boletus speciosus, Termitornyces albuminosus, Agaricus blazei, Agrocybe cylindracea, Lentinula edodes, Pleurotus eryngii, Flammulina velutipes, and Pleurotus ostreatus. Three T. matsutake varieties strains collected from the provinces of Yunnan, Sichuan, and Jilin, China, were kindly supplied by all China Federation of Supply and Marketing Cooperative's Kunming Institute of Edible Fungi. All of the samples were collected in the quantity of 200 g.
Three processed products labeled containing T. matsutake component, including mushroom biscuit, oil, and sauce, were gathered from Internet and local markets in Kunming, Yunnan province. They were used to verify the application of this selected species-specific diagnostic marker.

Genomic DNA Extraction
Four methods, including an improved CTAB method [46], the SDS-CTAB method [47], high-concentration salt precipitation method [48], and the kit method (Rapid Fungi Genomic DNA Isolation Kit, B518229, Sangon, Shanghai, China), were compared to determine the most suitable protocol for extracting the genomic DNA of T. matsutake. The T. matsutake fruiting bodies from Yunnan was used as the sample, each method was performed in triplicate. First, added the equal silica into 5 g mushroom sample, and then grounded them with liquid nitrogen. The target genomic DNA was isolated from the 0.2 g sample powder. All DNA extraction steps were performed in accordance with the references and manufacturer protocol. At last, the DNA was eluted in 50 µL EB buffer.
The subsequent DNA extraction was followed the selected optimal method. All of the OD260/280 and DNA concentrations were measured with a NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), the quality of genomic DNA was further analyzed on 1% agarose gel with ethidium bromide (0.1 µg /mL), and run with 1 × TAE buffer. Finally, the DNA solutions were stored at −20 • C.

Enzyme Digestion of T. Matsutake Genomic DNA
The DNA digestion was performed in a 20 µL reaction system containing 2 µL of 10× H Buffer, 1 µL EcoR I (15 U/µL) (TaKaRa Biotechnology Co. Ltd., Dalian, China), 3 µg genomic DNA, and added ultrapure water to 20 µL, and put the tube in a water bath (37 • C) for 2 h, after that added 2 µL 10× loading Buffer (Tiangen, Beijing, China) to stop the reaction. The digestion products was further analyzed on 1% agarose gel with ethidium bromide (0.1 µg /mL), and run with 1× TAE buffer.

Southern Blot
Complete enzyme cleavage of DNA from T. matsutake fruiting bodies obtained from Yunnan and Jilin was performed using Hind III and EcoR I, respectively, according to the manufacturer's instructions (TaKaRa) to ascertain the copy number of the Pol gene in the same species. The cleaved DNA was separated by 0.8% garose gel in 1×TAE at a constant voltage of 20 V overnight. Thereafter, the DNA fragments were transferred to a nylon membrane (Amersham Biosciences Shanghai Ltd., Darmstadt, Germany) from the 0.8% agarose gel. A 775 bp DNA fragment of the Pol gene was amplified with Southern blot-F/R and used as the hybridized probe, which was labeled with DIG-dUTP (DIG Hybridization Detection Kit, Mylab Co., Beijing, China). Pre-hybridization was carried out at 42 • C for 2 h, after which the pre-hybridization solution was poured off. The probe was denatured into single-strand DNA for 10 min at 100 • C and then cooled for 5 min. Exactly 4 µL of the denatured probe and 8 mL of the hybridization solution were added to a hybridization bag, and hybridization was performed at 42 • C overnight. Finally, the nylon membrane was washed twice with 3× SSC/0.5% SDS, and autoradiography was performed for 2-3 days.

Species-Specific Diagnostic Marker Selection of T. Matsutake
Genes belonging to T. matsutake were sought in Genbank, and several genes were preliminarily selected as detection targets according to their detailed gene information. Afterword, BLAST analysis was performed to determine the homology of the targets. The gene that with the lowest homology compared with all the DNA sequences in Genbank was selected as the candidate.

Primers and Probe
All primers and TaqMan fluorescent dye-labeled probes were designed using ABI Prism Primer Express version 3.0 software (Applied Biosystems, Foster City, CA, USA) and synthesized by Sangon Co. Ltd. (Shanghai, China).
The universal primer 18S rDNA-F/R was used to evaluate DNA quality. The Pol gene was assessed through qualitative PCR with the primer pol-F/R and through quantitative PCR with the primer pol-F/R, as well as the probe pol-P. Southern blot-F/R was used for Southern blot assay. The detailed sequences of these primers and probe are listed in Table 2. During Pol gene amplification, the procedures applied for 18S rDNA amplification were followed, but annealing was optimized at 56 • C, 58 • C, 60 • C, 62 • C, and 64 • C separately. After comparison, the annealing temperature 60 • C was chosen.
The PCR amplified products were analyzed on 2% agarose gel with ethidium bromide (0.1 µg/mL), and run with 1× TAE buffer. The PCR products, that conducted the homology analysis, were sequenced using pol-F/R separately by Sangon (Shanghai, China) company.
Real-time quantitative PCR of the Pol gene was also conducted in an ABI StepOne Plus Real-Time System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) using the TaqMan probe methods. TaqMan probe-based qPCR was performed using a 20 µL reaction system containing 1×TaqMan Gene Expression Master Mix (TaKaRa, Dalian, China), 200 nM primers, 200 nM probe, and 50 ng of DNA. Each sample was quantified twice for each biological replicate.

Sensitivity of the Qualitative and Taqman-Based Real-Time Quantitative PCR Assays
Genomic DNA from T. matsutake was diluted seven times from 200 ng/µL to 12.8 pg/µL (five-fold serial dilutions) over a gradient using the non-T. matsutake DNA, and a series of PCRs with 1 µL DNA sample were conducted to determine the detection limit of qualitative PCR, and the products were analyzed using 2% agarose.
To evaluate the sensitivity of Taqman-based quantitative PCR, genomic DNA was serially diluted six times to final concentrations ranging from 100 ng/µL to 32 pg/µL (five-fold serial dilutions) using the non-T. matsutake DNA, and with 1 µL DNA sample, the detection limit of Taqman quantitative PCR was determined.

Conclusions
In conclusion, this work demonstrated that High-quality DNA from T. matsutake fruiting bodies was obtained following the high-concentration salt precipitation method, and the Pol gene was selected and validated as an ideal species-specific diagnostic marker for the PCR-based detection of T. matsutake sources after assay of its species specificity, copy number, high homology in different varieties, and sensitivity. The detection limit of Taqman-based quantitative PCR analysis was 32 pg, which means this method could be used to detect processed T. matsutake products containing low amounts of the target genomic DNA.