JP3408955B2 - How to identify hop varieties - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for extracting and purifying genomic DNA from hops, and using primers to carry out PCR (po
lymerase chain reaction) and amplifying DNA capable of identifying hop varieties, thereby identifying hop (Humulus lupulus) varieties used in beer production.

[0002]

BACKGROUND ART Hop is a plant belonging to the genus Humulus of the mulberry family, and its citrus flowers make a great contribution to the flavor and foam of beer and are an essential ingredient for brewing beer. This hop is cultivated not only in Europe such as Germany, Czech Republic and France, but also in the United States, Australia, New Zealand, Japan and all over the world, and its cultivars are diverse. The hop varieties are largely “aroma” (having a mild bitterness and delicate flavor), “fine aroma” (having a particularly excellent flavor among aromas), and “bitter” depending on the characteristics of the components contained in hops.
It is divided into 3 types (having many bitterness components). In beer brewing, "fine aroma", "aroma",
They are said to be useful in the order of "bitter," and the prices are increasing accordingly.

Beer makers who use hops need to confirm whether the ordered raw material hops have been delivered correctly. Since ancient times, the difference in the appearance of the flowers and the alpha acid that causes bitterness We have made distinctions based on differences in component analysis values for homologues. However, most of the hops currently used are processed into pellets (crushed and then compressed), and it is not possible to distinguish them by their appearance. In addition, the chemical composition changes depending on the harvesting area and harvest year, and the chemical composition There are problems that the analysis is inaccurate or that similar values cannot be distinguished depending on the variety, and that the detection sensitivity is low and it is difficult to confirm when a small amount of other variety is mixed.

Recently, Araki et al. Extracted the genomic DNA in the hops to obtain RAPD (random amplified pol).
DNA regions that differ between cultivars are found by the ymorphic DNA) method, and then RAPD markers of each cultivar are isolated and cloned, the nucleotide sequences are determined, and the nucleotide sequences between the cultivars are compared to determine the DNA polymorphism. We have reported a method to identify hop varieties by making 10 kinds of primers and PCR that make clear (JOURN
AL OF FERMENTATION AND BI
OENGINEERING Vol. 84, No. Two
103-107, 1997). However, this method was able to recognize only 12 varieties and some of the main cultivated hop varieties in the world exceeding 30 varieties.

In addition, Brady et al. Also identified hops of 34 varieties by extracting genomic DNA from hop leaves and performing PCR using RAPD-PCR and four kinds of primer sets derived from microsatellite DNA sequences. I reported that I could do it (Euphytica, 9
1, p277-288, 1996), most of which are mainly test varieties or breeding lines with a very small planting area in Australia, and do not include most of the representative cultivars in the world currently used.

[0006]

[Problems to be Solved by the Invention] The above method for identifying hop varieties by performing PCR using primers on the genomic DNA extracted and purified from hops and amplifying DNA that can identify hop varieties is It can be said that it is an effective means for discriminating hop varieties irrespective of the growing environment, harvesting time, etc., but as described above, the hop variety discriminating technology based on the genomic DNA information reported so far can be applied. It is not possible to confirm the raw material hop variety at a practical level because it is not possible to identify other major varieties of the same producing country that have a narrow variety range and are likely to be mixed up or mixed.

An object of the present invention is to perform PCR on the genomic DNA extracted and purified from hops by using a primer.
By amplifying DNA that can identify hop varieties, we can accurately detect over 30 major cultivated hop varieties in the world including Germany, Czech Republic, France, Poland, Slovenia, USA, New Zealand, Australia and Japan. It is to provide a method for identifying and mixing of different kinds.

[0008]

[Means for Solving the Problems] The present inventors carry out PCR using a primer on genomic DNA extracted and purified from hops to identify a hop variety.
The inventors have earnestly studied the method of identifying hop varieties by amplifying hops, and the problem in the method of identifying hop varieties using PCR lies in the process of extracting and purifying genomic DNA suitable for PCR treatment from hops. As a result of reaching the knowledge and examining / experimenting a method of extracting and purifying genomic DNA suitable for PCR treatment from hops, efficient extraction of genomic DNA suitable for PCR by using a high concentration of lithium chloride Find that it can be purified,
The present invention has been completed.

That is, the present invention provides a random primer for genomic DNA extracted and purified from hops,
PC using a primer such as an STS primer
A method for identifying a hop variety by performing R and amplifying a DNA capable of identifying the hop variety, comprising:
Limethylammonium bromide (CTAB) treatment, or
A chloroform / isoamyl alcohol mixed solution (C
I) Treatment and / or phenol / chloroform / isoa
Is it hops using milled alcohol mixed liquid (PCI) treatment together ?
When extracting and purifying genomic DNA from
After that, the precipitate that was precipitated with isopropanol was
Use a high-concentration salt that is soluble in alcohols of at least 2M or more.
Treatment (however, at a temperature such as -20 ° C, RNA
(Excluding treatments to remove
A method for identifying a characteristic hop variety .

The present invention also relates to genomic DNA from hops.
CTAB treatment, CI treatment, or PCI
After the treatment , a high-concentration salt soluble in alcohols such as 5M lithium chloride is used, or a high-concentration salt soluble in alcohols such as 5M lithium chloride is used, and then protein is removed with phenol . The present invention relates to a method for identifying the above-mentioned hop varieties that employs processing .

The present invention is also directed to the primer 1A shown in SEQ ID NO: 1 and the primer 1B shown in SEQ ID NO: 2 with respect to the genomic DNA extracted and purified from hops.
A primer set 1 consisting of, a primer 2A represented by SEQ ID NO: 3 and a primer 2B represented by SEQ ID NO: 4
A primer set 2 consisting of, a primer 3A represented by SEQ ID NO: 5 and a primer 3B represented by SEQ ID NO: 6
A primer set 3 consisting of, a primer 4A represented by SEQ ID NO: 7 and a primer 3B represented by SEQ ID NO: 6
A primer set 4 consisting of, a primer 5A represented by SEQ ID NO: 8 and a primer 3B represented by SEQ ID NO: 6
The hop variety characterized in that the primer set 5 consisting of and the primer set 6-1 consisting of the primer 6A represented by SEQ ID NO: 9 and the primer 3B represented by SEQ ID NO: 6 are used as STS-ized primers. And a DNA which includes the primer DNAs described in SEQ ID NOS: 1 to 9 and can be used as a primer to perform PCR to amplify a DNA that can identify a hop variety.

Furthermore, the present invention provides the above-mentioned primer set 6
The PCR product of -1 was further subjected to seminested (semin) using primer set 4 and primer set 5.
ested) PCR is performed.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the main cultivated hop varieties in the world to be distinguished are German fine aroma, Hersbrucker belonging to aroma,
Hallertauer Tradition, Hal
lertauer, Salter Select, P
erle, Tettnanger, Bitter No
rtern Brewer, Brewers Gol
d, Magnum, S belonging to Czech fine aroma
aazer, Bor which belongs to bitter, Strissel Spalt which belongs to aroma of France, Lubin which belongs to aroma of Poland, Savinjski Golding which belongs to aroma of Slovenia, Super Styrian which belongs to bitter, and Fuganle, Americ which belongs to aroma in the United States.
nger, Willamette, Crystal, L
iberty, Nugget, Clus belonging to Bitter
ter, Chinak, Galena, Pacific Hallerta belonging to aroma of New Zealand
u, NewZealand Hallertau, Green Bullet, Stickle belonging to Bitter
Pride of R from Bract, Australia
ingwood, Kirin No. 2 which belongs to Japanese bitter, Yomidori, Kitamidori and Eastern Gold can be mentioned.

Genomic DNA serving as a template for PCR is extracted and purified from these hop leaves, flowers, or pellets or bales of flowers, which are currently used. Since most hops are processed into pellets (crushed and then compressed), considering the variety identification at a practical level, a method that can efficiently extract and purify genomic DNA from hop peony pellets or bales is desirable. .

As the method for extracting and purifying genomic DNA in the present invention, as long as a genomic DNA applicable to PCR can be obtained, any extraction and purification method can be used as long as a high concentration salt soluble in alcohol is used. It may be the law. Here, the high concentration means a molar concentration of 2 M or more, preferably 4 to 6 M, and lithium chloride is a salt soluble in alcohols,
Sodium chloride, guanidine chloride, ammonium sulfate,
Although sodium phosphate and the like can be exemplified, 5 M lithium chloride is particularly preferable as a high-concentration salt soluble in alcohols.

According to the experiments conducted by the present inventors, a general method for preparing DNA which does not use a high-concentration salt soluble in alcohols, for example, a method using proteinase, phenol, chloroform, guanidine hydrochloride, EDT
The method using A, the method using 2% CTAB (cetyltrimethylammonium bromide ), the method using a commercially available DNA extraction kit Iso-Plant (Nippon Gene Co., Ltd.), the improved method using 2% CTAB, There is a problem that the amount of DNA is small and many impurities are present, and it is not possible to obtain a genomic DNA that can be subjected to PCR.

For example, the 2% CTAB method is generally well known as a method for extracting genomic DNA of plants, and the improved 2% CTAB method is well known as a method for extracting DNA / RNA of woody plants. These methods allow inexpensive reagent preparation. However, it was found that PCR did not occur at all in the genomic DNA obtained by this extraction method, and it was necessary to examine other extraction methods and means for further purification. Even if I adopted it, it didn't work. Then, in the course of studying the purification means, if a high concentration salt soluble in alcohol is used, hop binding protein and polysaccharide can be separated, and hop genomic DNA can be efficiently obtained from alcohol. We have found that they can be separated. Therefore, by adopting a process of removing protein with phenol after heat-treating with 5 M lithium chloride at 70 ° C.,
At a relatively high rate of ~ 5μg / 1g pellet bale, P
It was found that a DNA that can be used for CR can be obtained. In this way, P from hops and flowers
In order to extract and purify genomic DNA suitable for CR, it is considered essential to use a high-concentration salt soluble in alcohols, and a high-concentration salt soluble in alcohols is used. If 2% CTAB is used before the step or phenol is used after the step, the hop genomic DNA can be efficiently extracted and purified as described above.

Genomic DNA extracted and purified from hops
On the other hand, a method of identifying hop varieties by performing PCR using a primer and amplifying DNA that can identify hop varieties is a technique that utilizes DNA polymorphism between hop varieties. For the identification of cultivars based on the DNA polymorphism, first, a large number of random primers are used in order to amplify a plurality of PCR products by non-specific annealing. PCR for amplifying a DNA polymorphism using this random primer is called RAPD-PCR.

Genomic DNA extracted and purified from hops
In contrast, many random primers were used for RA
By performing PD-PCR, the amplified RAPD (random amplified polymorphic DNA) is detected by agarose gel electrophoresis. The hop variety can be identified by utilizing the relationship that the specific random primer among the many random primers used amplifies the genomic DNA of the specific variety. However, according to this identification method, DNA of other species may be amplified in addition to DNA that can be used for identification.
STS (sequence tagged sit) for amplification by R
It is preferable to determine e), that is, to implement STS.

Therefore, R using a random primer is used.
RAPD amplified by APD-PCR was separated by agarose gel electrophoresis, and then DNA isolated and purified from agarose gel was incorporated into an E. coli cloning vector and cloned, and the nucleotide sequence of the DNA was determined by a usual method. It is desirable to prepare STS-ized primers of 15 to 30 bases based on the nucleotide sequence, subject them to PCR, and discriminate the hop variety by the presence or absence of DNA to be amplified.

Further, by comprehensively judging the analysis result of PCR using this STS-ized primer and the analysis result of RAPD-PCR using the above-mentioned random primer, it is possible to discriminate from the analysis result of one of them. It is also possible to more advantageously carry out the identification of many varieties, such as the identification between the varieties that did not exist.

As the random primer, a commercially available product, for example, a random primer (10 bases) manufactured by Operon Technology can be used. In order to select, from these random primers, one that can amplify RAPD that can be used for discrimination of cultivars by RAPD-PCR, it is usually desirable to screen 100 or more random primers.

The STS-form primers used in the present invention may be a combination such that one is a sense primer and the other is an antisense primer.
The origin of the DNA polymerase used for R is not limited as long as it has heat resistance at 95 ° C. As the reaction conditions of the PCR method, a denaturation temperature of 90 to 95 ° C., an annealing temperature of 35 to 60 ° C., an extension temperature of 70 to 75 ° C., and a cycle number of 10 or more are preferable, but not limited thereto, and usually PC
Any condition that can be used in the R method may be used. The obtained PCR reaction product is detected by separation or identification means such as electrophoresis using an agarose gel, and the presence or absence of an amplification product by PCR is confirmed. In addition, regarding the PCR program, to improve reproducibility and suppress non-specific reactions, STS
The annealing temperature between the modified primer and the hop-derived genomic DNA can be set as high as 40 ° C. or higher.

[0024]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Example 1: Extraction and purification of DNA (extraction of DNA) 10 ml of sterilized ultrapure water was added to 50 to 100 mg of crushed samples of pellets of various varieties, and the mixture was incubated at 56 ° C for 10 minutes and centrifuged (3,000 rpm, 30).
2% cetyltrimethylammonium bromide (CTAB) solution (2% C
TAB, 0.1M Tris -HCl (pH 9.5), 2
0 mM EDTA, 1.4 M NaCl, 1% PVP)
5 ml was added and incubated in a water bath at 56 ° C for 15 minutes. Then, 10 ml of a 24: 1 mixed solution of chloroform and isoamyl alcohol [hereinafter referred to as "CI solution"] was added, shaken at room temperature for 20 minutes, and then centrifuged (3,000 rpm, 30 minutes, room temperature) and recovered. After adding 5 ml of isopropanol to the supernatant and mixing, centrifuging again (3,000 rpm, 10 minutes, room temperature), discarding the supernatant, adding 1 ml of 70% ethanol to the precipitate, and centrifuging again (3,000 rpm, 10 minutes, Room temperature), the supernatant is discarded, ethanol is removed by vacuum drying, and then 5M lithium chloride-TE buffer (10 mM Tris-HCl pH is used).
4 ml of 8.0, 1 mM EDTA) was added and
Incubated for a period of time to completely dissolve the precipitate containing the DNA.

(Purification of DNA for PCR) 2 ml of a 25: 24: 1 mixed solution of phenol, chloroform and isoamyl alcohol [hereinafter referred to as "PCI solution"] was added to 5M lithium chloride-TE buffer containing the above DNA. After vigorous stirring, centrifuge (3,000
(rpm, 10 minutes, room temperature), collect only the upper layer, add 4 ml of CI solution, stir vigorously, and then centrifuge again (3, 3).
(000 rpm, 10 minutes, room temperature), and only the upper layer was collected. To this, 4 ml of isopropanol was added and mixed, followed by centrifugation (3,000 rpm, 10 minutes, room temperature),
Discard the supernatant, add 1 ml of 70% ethanol to the precipitate, and centrifuge again (3,000 rpm, 10 minutes, room temperature).
, Discard the supernatant, remove the ethanol by vacuum drying,
1 ml of 5 M lithium chloride-TE buffer was added and incubated at 70 ° C. for 1 hour to completely dissolve the DNA-containing precipitate.

Then, after adding 1 ml of PCI solution and stirring vigorously, it was centrifuged (3,000 rpm, 10 minutes, room temperature), only the upper layer was collected, and 700 μl of CI solution was added,
After vigorous stirring, centrifuge again (3,000 rpm,
After 10 minutes at room temperature), only the upper layer was collected. To this, 700 μl of isopropanol was added and mixed, followed by centrifugation (16,000 rpm, 80 seconds, 4 ° C.), discarding the supernatant, adding 1 ml of 70% ethanol to the precipitate, and centrifuging again (16,000 rpm, 1 minute). 4 ° C.), the supernatant was discarded, ethanol was removed by vacuum drying, 900 μl of 5M lithium chloride-TE buffer was added, and the mixture was incubated at 70 ° C. for 30 minutes to completely dissolve the DNA-containing precipitate.

Then, after adding 500 μl of PCI solution and stirring vigorously, centrifugation (16,000 rpm, 1 minute,
4 ° C.), collect only the upper layer, add 500 μl of CI solution, vigorously stir, and centrifuge again (16,000 r).
pm, 1 minute, 4 ° C.), and only the upper layer was collected. to this,
After adding 700 μl of isopropanol and mixing, centrifuge (16,000 rpm, 80 seconds, 4 ° C.), discard the supernatant, add 1 ml of 70% ethanol to the precipitate, and centrifuge again (16,000 rpm, 1 min, 4 ° C.). ), Discard the supernatant, remove ethanol by vacuum drying, add 200 μl of TE buffer, incubate at 70 ° C. for 1 hour,
DNA for PCR was prepared.

Comparative Example 1: Extraction of DNA by conventional technology
Purification (1) 2% CTAB (cetyltrimethylammonium bromide ) method 2% for 50-100 mg of ground sample of hop pellets
CTAB solution (2% CTAB, 0.1 MTris- HC
1 (pH 9.5), 20 mM EDTA, 1.4M N
aCl, 1% PVP) (500 μl) was added, and the mixture was incubated in a water bath at 60 ° C. for 10 minutes. Next, 500 μl of a 24: 1 mixed solution of chloroform and isoamyl alcohol was added, and the mixture was shaken for 30 minutes and then centrifuged (1
50,000 rpm, 5 minutes, room temperature), 500 μl of isopropanol was added to the collected supernatant, mixed and centrifuged again (10,000 rpm, 5 minutes, room temperature), and the supernatant was discarded.
70% ethanol was added to the precipitate, rinsed and flushed, and the supernatant was discarded. After removing ethanol by vacuum drying, TE
Buffer solution (10 mM Tris-HCl pH 8.0, 1 m
50 to 100 μl of M EDTA) was added to completely dissolve the precipitate, 1 μl of RNaseA (10 mg / ml) was added, and the mixture was incubated at 55 ° C. for 30 minutes to obtain DNA.

(Part 2) Iso-Plant Method (DNA Extraction Kit manufactured by Nippon Gene Co., Ltd.) A 50% to 100 mg crushed sample of hop pellets is added to a DNA extraction kit "Solution manufactured by Nippon Gene Co., Ltd."
I ”300 μl was added and stirred, and then“ Solutio
n II ”(150 μl) was added, and the mixture was further stirred and incubated in a water bath at 50 ° C. for 15 minutes.
After adding 150 μl of "solution III" and stirring, leave on ice for 15 minutes and then centrifuge (15,000 rpm,
15 minutes, 4 ° C.), and the supernatant consisting of the recovered aqueous phase
Add 2.5 times the volume of ethanol cooled at -20 ° C, stir, and immediately centrifuge again (15,000 rpm, 1
The supernatant was discarded, 70% ethanol was added to the precipitate to rinse and flush, and the supernatant was discarded. After removing ethanol by vacuum drying, TE buffer (10 mM Tri
s-HCl pH 8.0, 1 mM EDTA) 50-
100 μl was added to completely dissolve the precipitate, and RNaseA was added.
1 μl of (10 mg / ml) was added and incubated at 55 ° C. for 30 minutes to obtain DNA.

(Part 3) Improved 2% CTAB method Hop pellets ground sample 50 to 100 mg IB
(0.1 M Tris -HCl (pH 8.0), 10% P
oly-glyco16000, 0.35M sorbitol, 0.5% spermidine, 0.5% spermine,
After adding 500 μl of 0.5% mercaptoethanol) and stirring on ice, centrifugation (15,000 rpm, 10
Min (4 ° C), discard the supernatant, and then remove LB (0.1M Tr
is -HCl (pH 8.0), 0.35M sorbitol,
0.5% spermidin, 0.5% spermine, 0.5
% Mercaptoethanol) 250 μl and stirred, and then 10% Sacrosine 25 μl
Was added and left at room temperature for 10 minutes, then 2% CTAB275
μl was added, and the mixture was incubated in a water bath at 60 ° C. for 10 minutes. Next, after adding 500 μl of a 24: 1 mixed solution of chloroform and isoamyl alcohol and shaking for 30 minutes, centrifugation (10,000 rpm, 5 minutes, room temperature)
Then, add 500 μl of isopropanol to the collected supernatant, shake for 10 minutes, and centrifuge again (10,000 r
pm, 5 minutes, room temperature), the supernatant was discarded, 70% ethanol was added to the precipitate to rinse and flush, and the supernatant was discarded.
After removing ethanol by vacuum drying, TE buffer (10 mM
Tris-HCl pH 8.0, 1 mM EDTA) was added in an amount of 50 to 100 μl to completely dissolve the precipitate, and RNas
1 μl of eA (10 mg / ml) was added, and the temperature was 55 ° C., 30
Minutes incubator and base over door, to obtain a DNA.

The above-mentioned 2% CTAB method, Iso-Plan
The DNA obtained by the t method and the modified 2% CTAB method is
There were many impurities and no reaction product was observed when PCR was performed. Therefore, the purification treatment of DNA was carried out as follows.

(Purification of DNA for PCR) 2% CTAB method, Iso-Plant method and improved 2% C
Each of the DNA solutions obtained by the TAB method was treated with proteinase K (100 μg / ml) at 37 ° C. for 16 hours.
After the time treatment, an equal amount of the PCI solution was added and stirred, and the mixture was centrifuged (3,000 rpm, 10 minutes, room temperature) to collect only the supernatant. An equal amount of isopropanol was added to and mixed with this, followed by centrifugation (3,000 rpm, 10 minutes, room temperature), discarding the supernatant, adding 70% ethanol to the precipitate, rinsing and flushing, and then discarding the supernatant. After removing the ethanol by vacuum drying, TE buffer was added to dissolve the precipitate, whereby each purified DNA was obtained.

PCR was carried out using the thus-prepared purified DNA obtained by the 2% CTAB method, the purified DNA obtained by the Iso-Plant method, and the purified DNA obtained by the improved 2% CTAB method. As a result, no reaction product was observed. Therefore, the above DN
Of the A purification treatments, the procedure except the proteinase K treatment was repeated 8 to 10 times or more. When PCR treatment products were observed, the recovery rate was extremely low. Only 1/10 or less of the DNA amount of the method described in 1 was obtained, which was not practically applicable.

Example 2: Identification of varieties by RAPD-PCR and detection of contamination (test hop varieties) The following 12 hop pellets were used. Germany FA, A Hersbrucker, Perle, Tettnanger B Northern Brewer Czech Republic FA Saazer Slovenia A Savinjski Golding B Super Styrian USA A American Tettnannger, Willamette B Nugget Japan B Kirin No. 2, Todoromi (F: Fireroma, A: Aroma , B: represents bitter (high α acid).

The above mold D obtained from 12 varieties of hops
RAPD-P against NA using 140 kinds of random primers (10 bases) from Operon Technology
Screening by CR was performed. PCR is TAK
Using ARA Taq kit R-001A (manufactured by Takara Shuzo Co., Ltd.), a primer was used at 1.0 μM per sample, and the reaction program was first denaturing at 94 ° C. for 5 minutes, and thereafter denaturing at 94 ° C. for 1 minute, 40 Anneal at 2 ℃ for 2 minutes, extension reaction at 72 ℃ for 2 minutes as one cycle 40
The cycle was repeated. As a DNA amplification device (DNA thermal cycler), Gene Amp PC
R System 9600 (Perkin-Elme
r company) was used.

Then, the reacted solution was subjected to electrophoresis. 1 x Agarose X (Nippon Gene)
TAE buffer (40 mM Tris-acetate, 1 mM EDT
A gel was prepared by dissolving it in A) so as to be 1.5%.
Use 1x TAE buffer as the electrophoresis buffer and use 10
Electrophoresis was carried out at a constant voltage of 0 V for about 30 minutes. After electrophoresis,
The gel was dipped in a 0.5 μg / ml ethidium bromide solution for staining and irradiated with ultraviolet rays to generate fluorescence due to DNA fragments. When performing electrophoresis, DNA
A molecular weight marker (φX174 HincII digest) necessary for estimating the molecular weight of the fragment was used.

(Identification of varieties and detection of contamination) As a result of electrophoresis analysis, it was found that at least 7 kinds of random primers of Operon Technology Co., Ltd. were required for identification of 12 varieties. That is, OPA9 represented by SEQ ID NO: 10, OPA18 represented by SEQ ID NO: 11,
OPB12 shown by SEQ ID NO: 12, OPD03 shown by SEQ ID NO: 13, OPD1 shown by SEQ ID NO: 14
1, OPE01 represented by SEQ ID NO: 15, SEQ ID NO: 16
In OPE15 indicated by, different bands were observed among varieties.

Table 1 shows the names of random primers, their nucleotide sequences, and the number of amplification bands used for identification. In addition, Table 2 shows the polymorphism among amplified bands. In Table 2, the presence or absence of amplified DNA for each random primer is represented by +-.
Bands with poor reproducibility represented by ± were not used for identification. As can be seen from Table 2, the above 1 was determined by RAPD-PCR using 7 kinds of random primers.
Two varieties, namely Hersbrucker (HE), Pe
rle (PE), Tettnanger (TE), Nort
hern Brewer (NB), Saazer (SA),
Savinjski Golding (SG), Supe
r Styrian (SS), American Tet
tnanger (AT), Willamette (W
I), Nugget (NU), Kirin No. 2 (K2), and Yomidori (TY) can be identified.

[0039]

[Table 1]

[0040]

[Table 2]

In addition, American Tettnan
Nerger mixed with 5% by weight WillaMette was used to add R using random primer OPE01.
When APD-PCR was performed, American T
A faint band appeared in the band b of OPE01 which should not appear in ettnanger, and the latter cultivar could be detected.

Example 3: Identification of varieties by PCR using STS-modified primers, detection of contamination (test hop varieties) The following 33 hop pellets were used. Germany FA, A Hersbrucker, Hallertauer Tradition, Hallertauer Spalter Select, Perle, Tettnanger B Northern Brewer, Brewers Gold, Magnum Czech Republic FA Saazer B Bor France A Sttrissel Spalt Poland A Lublin Slovenia A Savinjski Golding B Super Styanton America Willamette, Crystal, Liberty B Nugget, Cluster, Chinook, Galena New Zealand A Pacific Hallertau, NewZealand Hallertau B Green Bullet, Stickle Bract Australia Pride of Ringwood Japan B Kirin 2, Tomidori, Kitamimidori, Eastern Gold

(RAPD-PCR) Screening by RAPD-PCR was performed on the above-mentioned template DNA obtained from 33 varieties of hops by using 140 kinds of random primers (10 bases) from Operon Technology Co., as in Example 2. Went to. Electrophoresis analysis results, random primers OPA9, OPA18, OPB12, OPDO from Operon Technology
3, OPD11, OPE01, OPE15, etc.,
Different bands were observed among the varieties.

(Design of STS Primer) Of these, random primers OPA9 and OPA18
Target bands 410 (OPA9), 850, the presence / absence of which was observed between the varieties on the agarose gel when using
870 (OPA18), Prep-A-Gen
Cut out using e-kit (manufactured by Biorad) and pM
OS Blue-T vector kit (Amers
(manufactured by HAM) and transformed into Escherichia coli and cloned to obtain Dye prim.
er DNA sequence kit (Applie
after PCR by d Biosystems),
NA Sequencer 373A (Perkin-
A part of the nucleotide sequence was determined by Elmer).

Based on the determined nucleotide sequence, a commercially available primer determination software was used to design a primer of 25 bases (STS conversion) to prepare primer sets 1, 2 and 6-1. Using these primer sets, PCR was performed on the genomic DNA extracted and purified from hops.
Bands of 50 bp, 600 bp, and 530 bp were detected.
A primer of 5 to 27 bases was designed (STS conversion) to prepare primer sets 3, 4 and 5.

Primer set 1 comprises primer 1A represented by SEQ ID NO: 1 and primer 1B represented by SEQ ID NO: 2, and primer set 2 comprises primer 2A represented by SEQ ID NO: 3 and primer 2B represented by SEQ ID NO: 4. Therefore, the primer set 3 is composed of the primer 3A represented by SEQ ID NO: 5 and the primer 3B represented by SEQ ID NO: 6, and the primer set 4 is composed of the primer 4A represented by SEQ ID NO: 7 and the primer 3B represented by SEQ ID NO: 6. Therefore, the primer set 5 is composed of the primer 5A represented by SEQ ID NO: 8 and the primer 3B represented by SEQ ID NO: 6, and the primer set 6-1 is represented by the primer 6A represented by SEQ ID NO: 9 and SEQ ID NO: 6. And primer 3B.

Genomic DN extracted and purified from hops using these primer sets 1, 2, 3, 4 and 5
When PCR was performed on A, the primer set 1 had 1300 bp and 750 bp, the primer set 2 had 800 bp, and the primer set 3 had 600 bp.
And 850 bp, 550 bp and 750 bp in primer set 4, and 450 bp in primer set 5,
Each specific band was seen.

In addition, when the primer set 6-1 was used, bands were observed at close positions and the discrimination ability was insufficient. Therefore, the PCR product of the primer set 6-1 was used as a template, and the primer set 6-1 was used. Use only the primer on the inside of the primer position used when
A specific band of 500 bp was observed by using eminished PCR, that is, when PCR was performed on the PCR product amplified by the primer set 6-1 with the primer set 4 (hereinafter referred to as "primer set 6-4" for convenience). , P amplified by primer set 6-1
When PCR is performed on the CR product with the primer set 5 (hereinafter, referred to as “primer set 6-5” for convenience) 45
A specific band of 0 bp was seen. Table 3 shows the types and base sequences of the STS-ized primers. Also, se
The difference in the amplified band by the minested PCR is shown in FIG.

[0049]

[Table 3]

(Identification of cultivars and detection of contamination) For the template DNAs extracted and purified from hops of 33 cultivars, the above-mentioned primer sets 1, 2, 3, 4, 5,
PCR was performed using 6-1, 6-4 and 6-5 (6
-4 and 6-5 are semi-stated PCR). P
For the CR, TAKARA Taq kit R-001A (manufactured by Takara Shuzo Co., Ltd.) was used and the PCR reaction amount was 2 per sample.
The reaction program shown in Table 4 was performed using 0 μl and 10 pM of the primer. In addition, a DNA amplification device (DNA
As a thermal cycler, Gene Amp P
CR System 9600 (Perkin-Elm
er) was used.

[0051]

[Table 4]

Then, the reacted solution is treated with Agaros.
eX (manufactured by Nippon Gene Co., Ltd.) was added to 1 × TAE buffer (4
It was subjected to electrophoresis using an agarose gel dissolved in 0 mM Tris-acetate, 1 mM EDTA) to a concentration of 1.5%. Using 1 × TAE buffer as the electrophoresis buffer, electrophoresis was performed at 100 V for about 30 minutes. After the electrophoresis, the gel was immersed in a 0.5 μg / ml ethidium bromide solution for staining, and was irradiated with ultraviolet rays to generate fluorescence due to DNA fragments. When performing electrophoresis, a molecular weight marker (φX174 HincII digest) necessary for estimating the molecular weight of the DNA fragment was used.

Table 5 shows the polymorphism pattern by the STS-ized primer. As you can see from Table 5, the created STS
Multiple amplified bands were observed with the modified primer, indicating that this primer does not amplify one region in the genome, but multiple copy regions. Further, according to the present invention, 33 major cultivated hop varieties of the world, that is, Hersbrucker, Hallerta belonging to German fine aroma and aroma.
uer Tradition, Hallertau
r, Salter Select, Perle, Te
ttnanger, Northern belonging to bitter
Brewer, Brewers Gold, Magnu
m, Sazer, which belongs to Czech fine aroma, Bor, which belongs to Bitter, and Stt, which belongs to French aroma
rissel Spalt, Lublin belonging to Polish aroma, Savi belonging to Slovenian aroma
njski Golding, Super belonging to Bitter
r Styrian, Fuggl belonging to US aroma
e, American Tettnnanger, Wi
llamette, Crystal, Liberty,
Nugget, Cluster, Chi belonging to Bitter
nook, Galena, Pacific Hallertau, NewZ belonging to aroma of New Zealand
ealand Hallertau, G belonging to Bitter
reen Bullet, StickyBract,
Australian Prime of Ringwood
d, Kirin No. 2, which belongs to Japanese bitters, can be accurately identified as green, kitamidori and eastern gold.

[0054]

[Table 5]

Regarding the detection sensitivity of contamination, the primer set 2 was used to detect H which is a fine aroma of Germany.
When the ersbrucker was inspected for a product in which the German bitter, Brewers gold, was mixed in a pellet weight ratio of 5%, a faint band appeared at a band size of 800 bp, and the latter varieties could be detected.

[0056]

According to the present invention, more than 30 major cultivated hop varieties in the world can be accurately identified with good reproducibility, and contamination of other varieties can be detected.

[0057]

[Sequence listing] SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array GGAACCTTTG CCTAATCCTA AGATT 25

SEQ ID NO: 2 Sequence length: 25 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array GTGTTTTCCG TATCTACGCG CTGCG 25

SEQ ID NO: 3 Sequence length: 25 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array ACCATAAATA TCACCCCGAG ACTCA 25

SEQ ID NO: 4 Sequence length: 25 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array CCATAACAGT TATGGTGAAC CAGAA 25

SEQ ID NO: 5 Sequence length: 27 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array GAAAACTACA CCTAGTATGT GATGCAT 27

SEQ ID NO: 6 Sequence length: 25 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array GCATCGTCTC GTGTTTTCCG TATCT 25

SEQ ID NO: 7 Sequence length: 27 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array CTTGATGCAT GTTGGATGTT TAAGTGT 27

SEQ ID NO: 8 Sequence length: 27 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array CCCTGTATAT ACGTGACATG CATGTGT 27

SEQ ID NO: 9 Sequence length: 25 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array AGGATCGATC GTTCTCAAGG GTCGT 25

SEQ ID NO: 10 Sequence length: 10 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array GGGTAACGCC 10

SEQ ID NO: 11 Sequence length: 10 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array AGGTGACCGT 10

SEQ ID NO: 12 Sequence length: 10 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array CCTTGACGCA 10

SEQ ID NO: 13 Sequence length: 10 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array GTCGCCGTCA 10

SEQ ID NO: 14 Sequence length: 10 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array AGCGCCATTG 10

SEQ ID NO: 15 Sequence length: 10 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array CCCAAGGTCC 10

SEQ ID NO: 16 Sequence length: 10 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acids Synthetic DNA Array ACGCACAACC 10

[Brief description of drawings]

FIG. 1 is a diagram showing a difference in amplification band by semi-stated PCR.

Continued Front Page (56) References International Publication 97/005281 (WO, A1) TAXON, 1995, Vol. 44, p. 379-386 BioTechniques, 1993, Vol. 14, No. 5, p. 748-751 Breed Sci, 1994, Vol. 44, separate volume 2, p. 199 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12Q 1/68 C12N 15/09-15/90 REGISTRY (STN) BIOSIS / WPI (DIALOG)

Claims (8)

(57) [Claims] 1. A genomic DN extracted and purified from hops
Against A, PCR was performed using primers, a method of identifying hop varieties by amplifying DNA capable of identifying hop varieties, cetyl trimethylammonium Moni
Umbromide treatment or chloroform / isoaure for the treatment
Mill alcohol mixed liquid treatment and / or phenol / chloro
A combination of form and isoamyl alcohol mixed solution
When extracting and purifying genomic DNA from the
After the treatment, the precipitate precipitated with isopropanol,
High-concentration salt soluble in at least 2M alcohol
(However, at a temperature such as -20 ° C, R
(Excluding the treatment to remove NA)
And a method for identifying hop varieties characterized by. Wherein on the occasion to the extraction and purification <br/> of genomic DNA from hops, after treatment using a high concentration of salt soluble in at least 2M or more alcohols, depending on the phenol
Claims characterized by adopting a process for removing proteins by
Item 1. A method for identifying a hop variety according to Item 1 . 3. A high-concentration salt soluble in alcohols is 4
Claim, characterized in that a ~6M lithium chloride 1 also
Is the hop variety identification method described in 2 . 4. The method for identifying a hop variety according to claim 1 , wherein a random primer is used as the primer. 5. The method for identifying a hop variety according to any one of claims 1 to 3 , wherein an STS-ized primer is used as the primer. 6. The STS-ized primer is a primer set 1 consisting of a primer 1A represented by SEQ ID NO: 1 and a primer 1B represented by SEQ ID NO: 2, a primer 2A represented by SEQ ID NO: 3 and a primer represented by SEQ ID NO: 4. Primer set 2 consisting of primer 2B, primer 3A represented by SEQ ID NO: 5 and primer 3B represented by SEQ ID NO: 6, primer 4A represented by SEQ ID NO: 7 and primer represented by SEQ ID NO: 6 Primer set 4 consisting of 3B, primer set 5 consisting of primer 5A represented by SEQ ID NO: 8 and primer 3B represented by SEQ ID NO: 6, and primer 6A represented by SEQ ID NO: 9 and primer 3B represented by SEQ ID NO: 6 Primer set consisting of 6-
The method for identifying a hop variety according to claim 5, wherein 1 is used. 7. The PCR product of primer set 6-1 is further combined with primer set 4 and primer set 5.
Using a semi-nested PC
The method for identifying a hop variety according to claim 6, wherein R is performed. 8. The DNA described in any one of SEQ ID NOS: 1 to 9 or the D described in any one of SEQ ID NOS: 1 to 9.
A DNA containing NA and used as a primer to amplify a DNA capable of identifying a hop variety D
NA.