JP4114150B2 - Variety identification method of pre-lotus eringi and its variety identification marker - Google Patents

Description

The present invention relates to a method for discriminating varieties of pre-lotus eringi and a marker comprising a gene amplification primer for amplifying a specific microsatellite region for the discrimination.

  Conventionally, many methods for discriminating plant varieties based on differences in DNA base sequences have been developed (for example, Patent Documents 1 and 2). These methods have been developed by identifying primers for identifying, for example, fingerprint regions of gene sequences of necessary varieties in advance, and by developing primers that amplify the regions of these regions in order to identify the varieties required. The genes of many target species are amplified using the primers, and whether or not the target variety is the target is determined depending on whether or not a predetermined gene has been amplified.

  On the other hand, regarding the cultivar identification of the prelotus genus, few methods using conventional genes have been developed, and it has been done by examining the discrimination by test cultivation and the reaction by counterculture.

  In recent years, the production of eringi has increased rapidly, and the unit price has been higher than that of other mushrooms. However, since the cultivation of eringi has been shallow since it was introduced in Japan and it has not grown in Japan, it is difficult to obtain inoculum, so it is often used separately from commercial products.

When using bacteria from such commercial products, it is necessary to determine whether or not it is a new variety protected by the seedling method.
JP-A-8-89298 Japanese Patent No. 2,697,783

  However, the conventional identification methods using test cultivation and anti-cultivation culture are easily affected by external factors during cultivation or cultivation and cannot be accurately identified. In addition, these methods have a problem that it takes a long time for cultivation or cultivation of bacteria.

  The present invention has been made in view of such conventional problems, and has been made for the purpose of providing a gene amplification primer that enables rapid and accurate discrimination of prelotus genus varieties and a method for distinguishing mushroom varieties using the same. .

The feature of the invention described in claim 1 for solving the conventional problems as described above and achieving the intended purpose is to use the nucleotide sequences on both sides of the CT microsatellite region in the gene of pre-Lotus eringi. The first marker consisting of the primers of SEQ ID NOS: 1 and 2 in the attached sequence listing consisting of the prepared primers, the second marker consisting of the primers 3 and 4, the third marker consisting of the primers 5 and 6, and 7 4th marker consisting of primers 8 and 8, 5th marker consisting of 9 and 10 primers, 6th marker consisting of 11 and 12 primers, and 7th marker consisting of 13 and 14 perform amplification by PCR of the mushroom gene of interest, varieties identification pre Lotus eryngii performing identification of varieties by checking the polymorphism of the amplification product There is the law.

  The feature of the invention described in claim 2 is that, from the first marker consisting of the primers of SEQ ID NOS: 1 and 2, the second marker consisting of the primers 3 and 4, and the primers 5 and 6 The third marker, the fourth marker composed of the primers 7 and 8, the fifth marker composed of the primers 9 and 10, the sixth marker composed of the primers 11 and 12, and the seventh marker composed of the primers 13 and 14. It is a marker for identifying varieties of pre-lotus eringi and related species comprising markers selected from

  In the present invention, it is possible to identify almost all existing eringi varieties by performing amplification of identification target varieties by PCR using a plurality or all of the first to seventh markers described above, Compared with conventional test cultivation and counter cultivation, it is not affected by external factors, and thus it is possible to accurately identify the variety, and it is possible to obtain an identification determination result in a shorter time than these conventional methods.

  In addition, when cultivating eringi, identification data of excellent varieties can be stored and compared with this to contribute to the cultivation of only excellent varieties.

  Next, embodiments of the present invention will be described with reference to the drawings.

  The gene amplification primer of the genus Prerotor according to the present invention comprises a first marker consisting of SEQ ID NOs: 1 and 2, a second marker consisting of 3 and 4, a third marker consisting of 5 and 6, There are seven types: a fourth marker consisting of 7 and 8; a fifth marker consisting of 9 and 10; a sixth marker consisting of 11 and 12; and a seventh marker consisting of 13 and 14; Use and identify varieties by performing PCR analysis on the target Eringi fungus.

  The gene amplification primer is prepared by first cutting out a gene from the total DNA of a specific eringi using a restriction enzyme, and by using the streptavidin magnetic bead method from the entire fragment, the microsatellite region A as shown in FIG. A fragment C containing various gene sequences B on the outside in the microsatellite region is isolated, and an adapter D is attached outside the gene sequence B on both sides of the fragment C (FIG. 1 (1)). . This is inserted into a vector and plasmid amplified using E. coli. Colony hybridization is then performed and only those containing the desired vector are collected.

  Next, the primer E is attached to the outside of the sequence C + D containing the microsatellite region collected in a large amount (FIG. 1 (2)). Among the amplified sequences C + D, the sequences A and B are sequenced to determine the sequence, and the microsatellite region is specifically amplified using the sequence of the region B on both sides of the repeated CT microsatellite region. Design possible primers.

  Amplification of specific microsatellite region genes for various types of eringi using the various types of primers thus obtained as markers, and selecting only those that can be used to identify the varieties of these eringi. The first to seventh markers were determined.

Next, specific examples of the above steps will be described.
Creating a marker DNA Extraction from Materials “Eringi strain” manufactured by Hokuto Co., Ltd. (Institute of Biotechnology, Institute of Biotechnology, No. 7 Seibun No. 1776 (FERM P-15292)) was used.
1 mL of TE solution (10 mM Tris / hydrochloric acid, pH 8.0, 0.1 mM EDTA) and 10% SDS (sodium dodecyl sulfate) are added to and suspended in 5 mg of the lyophilized sample of this eringi and allowed to acclimate for 30 minutes at 65 ° C. Proteinase K 1 mg / mL was added and reacted at 37 ° C. for 4 hours.

  After completion of the reaction, the mixture was centrifuged (10000 × G, 4 ° C., 20 minutes) with a centrifuge to collect the supernatant, and 1/5 amount of 5M NaCl and 10% CTAB (cetyltrimethylammonium bromide) were added at 65 ° C. For 10 minutes, and an equal volume of chloroform / isoamyl alcohol (chloroform: isoamyl alcohol = 24: 1) was added and mixed by inversion.

  Subsequently, the supernatant was collected by centrifugation (10000 × G, 4 ° C., 10 minutes). An equal volume of chloroform / isoamyl alcohol was added to the supernatant and the mixture obtained by repeated inversion 3 times was added to the supernatant, 1/10 volume of 7.5M ammonium acetate was added, and 2.5 times the volume of cooled ethanol was further added. . The precipitate obtained by centrifugation was washed with 70% ethanol, and the final precipitate was dissolved in 500 μL of TE solution.

  After dissolution, 1 mg of RNase was added and reacted at 37 ° C. for 2 hours. After the reaction, the supernatant was collected by centrifugation (10000 × G, 4 ° C., 10 minutes). Add an equal volume of chloroform / isoamyl alcohol to the supernatant and repeat the inversion and mixing 3 times. Add 1/10 volume of 7.5M ammonium acetate to the supernatant, and then add 2.5 volumes of cooled ethanol. It was. The precipitate obtained by centrifugation was washed with 70% ethanol, and the final precipitate was dissolved in 250 μL of TE solution to obtain a DNA sample.

2. Modification of Adapter to DNA Fragment Using a commercially available adapter modification kit (manufactured by Invitrogen), 1 μL of restriction enzyme Eco RI / Mse I was added to a DNA sample (1 μg / 10 μL) and reacted at 37 ° C. for 2 hours. After the reaction, an equal amount of adapter and 1 U / μL of T4 ligase were added and reacted overnight at 4 ° C. to fragment the DNA. After the reaction, 2.5 μL of the fragmented DNA solution was added to Eco RI primer (50 pmol / 1 μL) 1 μ, Mse I primer (10 pmol / 1 μL) 1 μ, 2.5 units of EX Tag polymerase (Takara Shuzo Co., Ltd.) 100 造 dNTPs. (Deoxyribonucleotide triphosphate mixed solution) 2 μL of a solution (manufactured by Takara Shuzo Co., Ltd.), 2.5 μL of 10-fold PCR buffer solution (manufactured by Takara Shuzo Co., Ltd.), and 15.5 μL of sterilized water were prepared.

Amplification of the prepared reaction solution was performed for 20 cycles under the conditions of 94 ° C. for 30 seconds, 56 ° C. for 1 minute, and 72 ° C. for 1 minute to obtain a DNA fragment modified with an adapter.
3. Concentration of DNA fragments having motif sequences using magnetic beads The method for concentrating DNA fragments modified with adapters obtained is based on the method of Kijas et al. (1994. Circle Reader Service No.193.P657-662). It was. A solution in which 3 μL (1 μg / μL) of a label with a motif sequence modified with biotin is bound to a magnetic bead and a solution in which a DNA fragment modified with an adapter is heat-denatured are mixed to modify the adapter with the motif sequence. The obtained DNA fragment was bound to a label bound to magnetic beads. After washing the magnetic beads, the bound DNA fragments were recovered and used as concentrated DNA fragments.

4). Insertion of DNA fragment into plasmid vector Concentrated DNA fragment 5 μL, Eco RI primer (50 pmol / 1 μL) 0.5 μL, Mse I primer (10 pmol / 1 μL) 0.5 μL, 2.5 units of EX Tag polymerase (Takara Shuzo Co., Ltd.) ) A reaction in which 100 μl of dNTP (deoxyribonucleotide 3-phosphate mixed solution) solution (Takara Shuzo Co., Ltd.) 1.2 μL, PCR 10-fold diluted buffer solution (Takara Shuzo Co., Ltd.) 1.5 μL, and sterilized water 5.8 μL were added. A liquid was prepared.

  Amplification of the prepared reaction solution was performed 20 cycles under the conditions of 94 ° C. for 30 seconds, 56 ° C. for 1 minute, and 72 ° C. for 1 minute to obtain an amplified concentrated DNA fragment. A reaction solution was prepared by adding 5 μL of amplified DNA fragment, 10 μL of ligation buffer (Promega), 2 μL of plasmid vector (pGEM-T Easy Vector), 2 μL of T4 ligase (1 U / μL), and 1 μL of sterilized water. The concentrated DNA fragment was inserted into a plasmid vector (pGEM-T Easy Vector) by reacting overnight.

5. Transformation and Genomic Library A plasmid vector (pGEM-T Easy Vector) into which a concentrated DNA fragment has been inserted and a competent cell (E. coli DH5α (manufactured by Toyobo Co., Ltd.)) were reacted in ice for 40 minutes. The reaction was carried out at 45 ° C. for 45 seconds to transform the plasmid vector into competent cells.
The transformed cells were added with 900 μL of SOC medium (manufactured by Toyobo Co., Ltd.), cultured at 37 ° C. for 1 hour, and LB (1% Bacto Tripton, 0.5% Yeast) soaked with 40 mL (40 mg / ml) of X-gal. Extract, 1% NaCl, 1.5% agar, ampicillin (50 μg / ml) were spread on a flat medium and cultured overnight at 37 ° C., and a white colony containing the insert fragment was isolated on an LB agar medium.

6). Hybridization and detection A colony containing a plasmid vector (pGEM-T Easy Vector) having a motif sequence is detected from the isolated colony. The isolated colony was attached to a membrane (Hybond-N +), reacted with an alkali-denaturing solution (0.5N NaOH, 1.5M NaCl), and further neutralized solution (1.5M NaCl, 0.5M Tris / hydrochloric acid, After the treatment at pH 7.4), the membrane is fixed to the membrane surface in an oven at 80 ° C. for 2 hours.

  3 μL (1 μg / μL) of a DIG (digoxigenin) -labeled product having a motif sequence was hybridized with the plasmid on the fixed membrane. A DIG luminescence detection kit (Roche) was used to detect the DIG-labeled product. Unbound DIG-labeled product was washed, and a specific anti-DIG antibody conjugated with alkaline phosphatase was bound to the bound DIG-labeled product, and then reacted with a luminescence detection reagent. The reacted membrane was exposed to an X-ray film to identify colonies having motif sequences.

7). Sequence and primer design and confirmation Decode the base sequence of the DNA fragment inserted in the identified colony. First, the identified colony was cultured overnight in LB (1% Bacto Tripton, 0.5% Vest Extract, 1% NaCl) liquid medium, and the inserted DNA fragment was amplified by PCR .

  For amplification, 3 μL of the culture, 1 μL of T7 primer (20 pmol), 1 μL of SP6 primer (20 pmol), 2.5 units of Hot Star Tag polymerase (manufactured by Qiagen), 100 Μ dNTP (deoxyribonucleotide triphosphate mixed solution) solution (Takara Shuzo Co., Ltd.) 8 μL, PCR 10-fold diluted buffer solution (Takara Shuzo Co., Ltd.) 10 μL, sterile water 76 μL of reaction solution at 94 ° C. for 1 minute, 55 ° C. for 2 minutes, 72 ° C. for 3 minutes For 25 cycles. A part of the amplified product was confirmed by amplification by 1.0% agarose gel electrophoresis, and the remainder was purified.

  For the purified product, a sequence sample was prepared using a Dye-terminator kit (Applied Biosystems). For the sequence, the base sequence of the fragment was decoded using ABI Prism Model 310 (Applied Biosystems).

  A microsatellite region of the motif sequence was found from the decoded base sequence, and a set of primers (F side / R side) capable of reliably amplifying the microsatellite region of the motif sequence was designed according to a standard method.

  The designed primers were amplified by PCR using DNA extracted from several varieties of eringi to confirm polymorphism. The reaction solution was 2 μL of DNA (10 ng / μL) extracted from several types of eringi, 1 μL of F-side primer (10 pmol / μL), 1 μL of R-side primer (10 pmol / μL), 2.5 units of Hot Star Tag polymerase (Qiagen) ), 100 μl of dNTP (deoxyribonucleotide triphosphate mixed solution) solution (Takara Shuzo) 1 μL, PCR 10-fold diluted buffer solution (Takara Shuzo) 2 μL, and sterilized water 14.5 μL The sample was prepared for 33 cycles under the conditions of 92 ° C. for 1 minute, 46 to 60 ° C. for 1 minute, and 72 ° C. for 1 minute depending on the annealing temperature of the primer used.

  Amplified products were separated by 13% non-denaturing polyacrylamide gel electrophoresis. DNA staining and photography were performed according to conventional methods. As a result of the separation, primers capable of obtaining a polymorphism among varieties were selected, and the amplified region was designated as a first marker to a seventh marker using the identification region as an identification marker.

  Thus, the 1st-7th marker which made the F side and R side primer of Table 1 1 set was created.

Identification of varieties of eringi using a marker Amplification by PCR using DNA extracted from several varieties of eringi confirms polymorphism. The reaction solution was 2 μL of DNA (10 ng / μL) extracted from several types of eringi, 1 μL of F side primer (10 pmol / μL), 1 μL of R side primer (10 pmol / μL), 2.5 units of Hot Star Tag polymerase (Qiagen) ), 100 μl of dNTP (deoxyribonucleotide triphosphate mixed solution) solution (Takara Shuzo) 1 μL, PCR 10-fold diluted buffer solution (Takara Shuzo) 2 μL, and sterilized water 14.5 μL The sample was prepared for 33 cycles under the conditions of 92 ° C. for 1 minute, 46 to 60 ° C. for 1 minute, and 72 ° C. for 1 minute depending on the annealing temperature of the primer used. Amplification is performed using two or more combinations of primers. Amplified products were separated by 13% non-denaturing polyacrylamide gel electrophoresis. Staining and photographing of DNA are performed according to a conventional method, and varieties of eringi are identified by confirming DNA polymorphism.

  Table 2 shows the results of identification of 8 types of eringi samples using the first to seventh markers.

第２表は、第１〜第７の各マーカー１〜７を、各識別対象エリンギサンプルに使用して増幅を行った結果が同じであった場合には同じアルファベットを記入し、異なっている場合には異なったアルファベットを記入している。

Table 2 shows that the same alphabet is used when the results of amplification using the first to seventh markers 1 to 7 for each identification target eringi sample are the same, and are different. Is filled with a different alphabet.

That is, in the product type identification using the marker 1, the identification target eringi sample No. 1 and No. No. 5 to 7 cannot be identified, that is, cannot be distinguished from each other. 2-4 and no. No. 8 cannot be identified. 1 and no. 2 means that the identification was successful.
In the product identification using the marker 2, No. 1, no. 3 and no. 5 cannot be identified. 2, no. 6 and no. 7 cannot be identified. 4 and no. 8 cannot be identified. 1, no. 2 and no. 4 could be identified.
Thus, for example, eringi sample no. 2 and No. 3 cannot be identified by marker 1, but can be identified by marker 2, and it can be seen that both eringi samples are of different varieties.

  According to the experiment, it was possible to discriminate all the 15 types of eringi having different varieties by using all the first to seventh markers described above.

  In the above-described embodiment, the identification of the pre-lotus eringi variety is described as the identification target. However, one or more of the first to seventh markers described above are used, and the pre-lotus other than the above 15 types Identification of eringi and related species is also possible.

It is explanatory drawing which shows the state of the gene fragment in the manufacturing process of the marker in this invention.

Explanation of symbols

A Microsatellite region B Base sequence on both sides of microsatellite region C Fragment D Adapter E Primer

Claims (2)

It consists of the first marker consisting of the primers of SEQ ID NOs: 1 and 2 and the primers of 3 and 4 in the attached sequence listing consisting of primers created using the base sequences on both sides of the CT microsatellite region in the gene of pre-lotus eringi Second marker, third marker consisting of primers 5 and 6, fourth marker consisting of primers 7 and 8, fifth marker consisting of primers 9 and 10, and sixth marker consisting of primers 11 and 12 A variety of pre-lotus eringi that uses a marker and a seventh marker consisting of 13 and 14 optionally, amplifies the mushroom gene to be identified by PCR, and identifies the variety by confirming the polymorphism of the amplified product Identification method . From the first marker consisting of the primers of SEQ ID Nos. 1 and 2, the second marker consisting of the primers 3 and 4, the third marker consisting of the primers 5 and 6, and the primers 7 and 8 Varieties of pre-lotus eringi comprising a fourth marker comprising: a fifth marker comprising 9 and 10 primers; a sixth marker comprising 11 and 12 primers; and a seventh marker comprising 13 and 14; Identification marker .

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