JPH11103895A - Discrimination of hop strain - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the method for exactly discriminating the main variety, over 30 kinds, of hops cultivated in the world, the method for detecting the inclusion of different varieties. SOLUTION: The genome DNA extracted from hop and purified is subjected to polymerase chain reaction using a primer as the random primer, STS primer and the like to amplify the DNA that can discriminate the variety of hop. In this process, a high-concentration of salt solution that is insoluble in alcohol such as 5M lithium chloride solution is used for extraction and purification of the genome DNA from hop.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for extracting and purifying genomic DNA from hops and performing PCR (po
The present invention relates to a method for identifying a variety of hops (Humulus lupuls) used in beer production by amplifying DNA that can identify a hop variety by performing a lymerase chain reaction.

[0002]

2. Description of the Related Art Hops are plants belonging to the genus Humulus of the mulberry family, and their cones greatly contribute to the flavor and foam of beer, and are indispensable as one of the raw materials for brewing beer. This hop is cultivated all over the world, including Europe, such as Germany, the Czech Republic, and France, as well as the United States, Australia, New Zealand, and Japan. Hop varieties are largely characterized by the characteristics of their constituents, and have large "aromas" (mild bitterness and delicate flavor), "fine aromas" (having the best flavor among aromas), and "bitter"
(Having many bitter components). In beer brewing, "fine aroma", "aroma",
It is useful in the order of "bitter", and the price is roughly higher accordingly.

[0003] Beer makers that use hops need to confirm that the ordered raw material hops have been correctly delivered. For a long time, differences in the appearance of cones and alpha acid, which causes bitterness, It has been determined based on the difference in component analysis values of homologues and the like. However, most of the hops currently used are processed into pellets (crushed and then compressed), making it impossible to identify them by their appearance, and changing the chemical composition depending on the harvest area and the year of harvest. In the analysis, there were problems such as inaccurate identification or similar values depending on the cultivar, which could not be discriminated, and low detection sensitivity, which made it difficult to confirm when mixed with other varieties.

Recently, Araki et al. Extracted genomic DNA in hops and extracted them with RAPD (random amplified pol).
DNA regions differing between cultivars are found by the ymorphic DNA) method, then the RAPD markers of each cultivar are isolated and cloned, their nucleotide sequences are determined, the nucleotide sequences between each cultivar are compared, and DNA polymorphisms are determined. A method for identifying hop varieties by preparing 10 types of primers and performing PCR so as to clarify (JOURN) was reported.
AL OF FERMENTATION AND BI
OINGINEERING Vol. 84, no. 2,
103-107, 1997). However, only 12 varieties and some 30 of the world's main cultivated hop varieties can be recognized by this method.

[0005] Brady et al. Also identified 34 varieties of hops by extracting genomic DNA from hop leaves and performing PCR using RAPD-PCR and four types of primer sets derived from microsatellite DNA sequences. We reported that we can do it (Euphytica, 9
1, pp. 277-288, 1996), most of which are test varieties or breeding lines with very little planting area mainly in Australia, and do not include most of the world's representative varieties currently in use.

[0006]

SUMMARY OF THE INVENTION The above method for identifying hop varieties by performing PCR on genomic DNA extracted and purified from hops using primers and amplifying DNA capable of identifying hop varieties is disclosed in US Pat. Hop varieties can be said to be effective means for identifying hop varieties regardless of the growth environment, harvest time, etc., but as described above, hop variety identification techniques based on genomic DNA information reported to date can be applied. Because the variety range was narrow and it was not possible to identify other major varieties in the same country of origin where varieties were likely to be mixed or mixed, it was not possible to confirm the raw material hop variety at a practical level.

[0007] An object of the present invention is to perform PCR on genomic DNA extracted and purified from hops using primers.
To amplify DNA that can identify hop varieties to accurately identify more than 30 cultivated hop varieties worldwide, including Germany, the Czech Republic, France, Poland, Slovenia, the United States, New Zealand, Australia, and Japan. And a method for detecting the mixture of different varieties.

[0008]

Means for Solving the Problems The present inventors perform PCR on genomic DNA extracted and purified from hops using primers to identify hop varieties.
Research on the method of identifying hop varieties by amplifying hop varieties revealed that 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. After reaching the knowledge and studying and conducting repeated experiments on methods for extracting and purifying genomic DNA from hops suitable for PCR processing, as a result of using high-concentration lithium chloride, it was possible to efficiently extract genomic DNA suitable for PCR. Finding that it can be purified,
The present invention has been completed.

That is, the present invention provides a method for preparing a genomic DNA extracted and purified from hops, using random primers,
PC using primers such as STS primers
R, and amplifying DNA capable of identifying hop varieties, in a method for identifying hop varieties, wherein extraction and purification of genomic DNA from hops is carried out using a high-concentration salt soluble in alcohols such as 5M lithium chloride. And a method for identifying a hop variety.

[0010] The present invention also relates to genomic DNA from hops.
After using cetyltrimethylammonium chloride for the extraction and purification of the compound, use a high-concentration salt soluble in alcohols such as 5M lithium chloride or a high-concentration salt soluble in alcohols such as 5M lithium chloride. And then using phenol to identify the hop variety.

[0011] The present invention also relates to a primer 1A represented by SEQ ID NO: 1 and a primer 1B represented by SEQ ID NO: 2 for genomic DNA extracted and purified from hops.
A primer set 1 consisting of: primer 2A represented by SEQ ID NO: 3 and primer 2B represented by SEQ ID NO: 4
A primer set 2 consisting of: primer 3A represented by SEQ ID NO: 5 and primer 3B represented by SEQ ID NO: 6
A primer set 3 consisting of: primer 4A represented by SEQ ID NO: 7 and primer 3B represented by SEQ ID NO: 6
A primer set 4 consisting of: primer 5A represented by SEQ ID NO: 8 and primer 3B represented by SEQ ID NO: 6
And a primer set 6-1 comprising a primer set 5 consisting of SEQ ID NO: 9 and a primer set 6A consisting of primer 6A represented by SEQ ID NO: 9 and a primer 3B represented by SEQ ID NO: 6 as STS-modified primers. And DNA which contains the primer DNAs set forth in SEQ ID NOS: 1 to 9 or a part or all of such DNAs and which can amplify DNAs that can identify hop varieties when subjected to PCR using as primers .

Furthermore, the present invention provides the above primer set 6
-1 was further semi-nested (semin) using primer set 4 and primer set 5.
ested) The present invention relates to a method for identifying a hop variety, which comprises performing PCR.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the main cultivated hop varieties in the world to be identified include Hersbrucker, belonging to German fine aroma and aroma.
Hallertauer Trading, Hal
lertauer, Sparter Select, P
erle, Tettnanger, No belonging to bitter
rthern Brewer, Brewers Gol
d, Magnum, S belonging to the Czech fine aroma
aazer, Bor belonging to bitter, Strissel Spalt belonging to aroma in France, Lublin belonging to aroma in Poland, Savinjski belonging to aroma in Slovenia. Golding, Super Styrian belonging to Bitter, Fuggle, American Tettnan belonging to Aroma of the United States
nger, Willamette, Crystal, L
iberty, Nugget, Crus belonging to bitter
ter, chinook, Galena, Pacific. Hallert
au, New Zealland Hallertau, Green. Bullet, Stickl
eBract, Pride of Australia
Ringwood, Kirin 2 belonging to the Japanese bitter,
Toyori Midori, Kita Midori and Eastern Gold.

[0014] From these hop leaves, cones, or pellets or veil of cones, genomic DNA as a template when PCR is applied is extracted and purified, which is currently used. Since most of the hops are processed into pellets (crushed and then compressed), a method that can efficiently extract and purify genomic DNA from hop cone pellets and veils is desirable in consideration of cultivar identification at a practical level. .

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

According to the experiments of the present inventors, a conventional method for preparing DNA which does not use a high concentration of a 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 chloride), the method using Iso-Plant (manufactured by Nippon Gene Co., Ltd.) which is a commercially available DNA extraction kit, and the improved method using 2% CTAB There is a problem that the amount of DNA is small and there are many impurities, and genomic DNA that can be used for PCR cannot be obtained.

For example, the 2% CTAB method is generally well known as a method for extracting genomic DNA from plants, and the improved 2% CTAB method is well known as a method for extracting DNA and RNA from woody plants. These methods allow inexpensive preparation of reagents. However, PCR did not occur at all in the genomic DNA obtained by this extraction method, and it was found that it was necessary to examine other extraction methods and to examine means for further purification. The adoption did not work. In the course of examining the purification means, if a high concentration of a salt soluble in alcohols is used, hop-binding proteins and polysaccharides can be separated, and hop genomic DNA can be efficiently separated from alcohols. It was found that they can be separated. Therefore, by adopting a process of removing protein with phenol after heat treatment at 70 ° C. with 5M lithium chloride,
With a relatively high rate of ~ 5μg / 1g pellet veil, P
It was found that DNA that could be subjected to CR could be obtained. In this way, P from hop cone pellets and veil
For extraction and purification of genomic DNA suitable for CR, it is considered essential to use high-concentration salts soluble in alcohols, and use high-concentration salts soluble in alcohols. If 2% CTAB is used before the step or phenol is used after the step, hop genomic DNA can be efficiently extracted and purified as described above.

Genomic DNA extracted and purified from hops
In contrast, a method of identifying hop varieties by performing PCR using primers and amplifying DNA that can identify hop varieties is a technique utilizing DNA polymorphism between hop varieties. For identification of varieties based on the DNA polymorphism, a large number of random primers are used to amplify a plurality of PCR products by non-specific annealing. PCR that amplifies a DNA polymorphism using the random primer is called RAPD-PCR.

Genomic DNA extracted and purified from hops
Using a large number of these random primers
By performing PD-PCR, the amplified RAPD (random amplified polymorphic DNA) is detected by agarose gel electrophoresis. Then, the hop variety can be identified by utilizing the relationship that the specific random primer among the numerous random primers used amplifies the genomic DNA of the specific variety. However, according to this identification method, there is a possibility that other types of DNA may be amplified in addition to the DNA that can be used for identification.
STS (sequence tagged sit) for amplification by R
It is preferable to determine e), that is, to convert to STS.

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

Further, by comprehensively judging the analysis result of the PCR using the STS-primed primer and the analysis result of the RAPD-PCR using the above-mentioned random primer, it is possible to discriminate from one of the analysis results. Many varieties can be distinguished more advantageously, for example, discrimination between missing varieties becomes possible.

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

The STS-modified primer used in the present invention may be any combination in which 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 of 95 ° C. As the reaction conditions of the PCR method, the denaturation temperature is preferably 90 to 95 ° C., the annealing temperature is 35 to 60 ° C., the extension temperature is 70 to 75 ° C., and the number of cycles is preferably 10 or more.
Any condition can be used as long as it can be used for the R method. 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 order to increase the reproducibility of the PCR program and suppress non-specific reactions, STS
The annealing temperature between the primer and the hop-derived genomic DNA can be set as high as 40 ° C. or higher.

[0024]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. Example 1: Extraction and purification of DNA (Extraction of DNA) Pulverized sample 5 of pellets of various varieties
10 ml of sterilized ultrapure water was added to 0 to 100 mg, and the mixture was incubated at 56 ° C for 10 minutes, and centrifuged (3,00
0 rpm, 30 minutes, room temperature), and the supernatant was discarded. 2% cetyltrimethylammonium chloride (CTA
B) Solution (2% CTAB, 0.1 M Tris (pH 9.
5), 20 mM EDTA, 1.4 M NaCl, 1%
(PVP) 5 ml and put in a water bath at 56 ° C. for 15
Incubated for minutes. Next, 10 ml of a 24: 1 mixture of chloroform and isoamyl alcohol [hereinafter referred to as “CI solution”] was added, and the mixture was shaken at room temperature for 20 minutes.
After centrifugation (3,000 rpm, 30 minutes, room temperature), the collected supernatant was mixed with 5 ml of isopropanol,
Centrifuge again (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)
After discarding the supernatant and removing ethanol by vacuum drying,
5M lithium chloride-TE buffer (10 mM Tris-H
Cl pH 8.0, 1 mM EDTA),
After incubation at 70 ° C. for 1 hour, the precipitate containing the DNA was completely dissolved.

(Purification of DNA for PCR)
In a 5 M lithium chloride-TE buffer containing A, 25 of phenol, chloroform and isoamyl alcohol:
After adding 2 ml of a 24: 1 mixed solution [hereinafter referred to as “PCI solution”], stirring vigorously, centrifugation (3,000 rpm,
10 minutes at room temperature), collect only the upper layer, and add 4 ml of CI solution
After adding vigorously, the mixture was centrifuged again (3,000 rpm).
pm, 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), discarded the supernatant, added 1 ml of 70% ethanol to the precipitate, and centrifuged again (3,000 rpm, 10 minutes, Room temperature), the supernatant was discarded, and ethanol was removed by vacuum drying. After adding 1 ml of 5M lithium chloride-TE buffer, the mixture was incubated at 70 ° C for 1 hour to completely dissolve the DNA-containing precipitate.

Next, 1 ml of PCI solution was added and stirred vigorously, followed by centrifugation (3,000 rpm, 10 minutes, room temperature), collecting only the upper layer, and adding 700 μl of CI solution.
After vigorous stirring, centrifugation again (3,000 rpm,
(10 minutes, room temperature), and only the upper layer was collected. After adding 700 μl of isopropanol and mixing, centrifugation (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 minute) 4 ° C.), the supernatant was discarded, and ethanol was removed by vacuum drying. After adding 900 μl of 5M lithium chloride-TE buffer, the mixture was incubated at 70 ° C. for 30 minutes to completely dissolve the DNA-containing precipitate.

Next, 500 μl of the PCI solution was added and the mixture was vigorously stirred, followed by 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 rpm).
pm, 1 minute, 4 ° C), and only the upper layer was collected. to this,
After adding 700 μl of isopropanol and mixing, the mixture was centrifuged (16,000 rpm, 80 seconds, 4 ° C.), the supernatant was discarded, 1 ml of 70% ethanol was added to the precipitate, and the mixture was centrifuged again (16,000 rpm, 1 minute, 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 Technique
Purification (Part 1) 2% CTAB (cetyltrimethylammonium chloride) method 2% to 50-100 mg of ground sample of hop pellet
CTAB solution (2% CTAB, 0.1 M Tris (pH
9.5), 20 mM EDTA, 1.4 M NaCl,
1% PVP) in a water bath.
Incubated at 10 ° C for 10 minutes. Then, a 24: 1 mixed solution of chloroform and isoamyl alcohol was added to 500 μl.
After shaking for 30 minutes, centrifugation (10,000
rpm, 5 minutes, room temperature), 500 μl of isopropanol was added to the collected supernatant, mixed, and then centrifuged again (10,0
00 rpm, 5 minutes, room temperature), discard the supernatant, and set
After rinsing and flushing with the addition of% ethanol, the supernatant was discarded. After removing ethanol by vacuum drying, TE buffer (1
0 mM Tris-HCl pH 8.0, 1 mM EDT
A) was added by 50 to 100 μl to completely dissolve the precipitate,
1 μl of NaseA (10 mg / ml) was added, and 55
Incubation was performed at 30 ° C. for 30 minutes to obtain DNA.

(Part 2) Iso-Plant method (DNA extraction kit manufactured by Nippon Gene Co., Ltd.) A DNA extraction kit “Solution” manufactured by Nippon Gene Co., Ltd.
I ”300 μl was added and stirred, then“ Solution
nII ”(150 μl) was added thereto, further stirred, and incubated in a water bath at 50 ° C. for 15 minutes.
solution III ”(150 μl), stirred, left on ice for 15 minutes, and centrifuged (15,000 rpm,
15 minutes, 4 ° C.)
After adding 2.5 volumes of ethanol cooled at −20 ° C. and stirring, immediately centrifuge again (15,000 rpm, 15,000 rpm).
(0 min, 4 ° C.), the supernatant was discarded, 70% ethanol was added to the precipitate, and the supernatant was discarded after rinsing and flushing. After removing ethanol by vacuum drying, TE buffer (10 mM Tri)
s-HCl pH 8.0, 1 mM EDTA)
100 μl was added to completely dissolve the precipitate, and RNase A
(10 mg / ml) was added thereto, and the mixture was incubated at 55 ° C. for 30 minutes to obtain DNA.

(Part 3) Improved 2% CTAB method IB pellet was added to 50 to 100 mg of crushed hop pellet sample.
(0.1 M Tris (pH 8.0), 10% Poly-
glyco 16000, 0.35M sorbitol, 0.
After adding 500 μl of 5% spermidine, 0.5% spermine, 0.5% mercaptoethanol) and stirring on ice, centrifugation (15,000 rpm, 10 minutes, 4 ° C.)
After discarding the supernatant, LB (0.1 M Tris (pH
8.0), 250 μl of 0.35 M sorbitol, 0.5% spermidine, 0.5% spermine, 0.5% mercaptoethanol) and stirring, then 25 μl of 10% sacrosine (Sacrosine) and 10 minutes at room temperature.
After standing for 2 minutes, 275 μl of 2% CTAB was added, and the mixture was incubated at 60 ° C. for 10 minutes in a water bath. Next, 24: 1 of chloroform and isoamyl alcohol was used.
After adding 500 μl of the mixture and shaking for 30 minutes, the mixture was centrifuged (10,000 rpm, 5 minutes, room temperature), 500 μl of isopropanol was added to the collected supernatant, shaken for 10 minutes, and then centrifuged again (10,000 rpm, 5 minutes). The supernatant was discarded, 70% ethanol was added to the precipitate, and the supernatant was discarded after rinsing and flushing. After removing ethanol by vacuum drying, TE buffer (10 mM Tris-HC
1 pH 8.0, 1 mM EDTA)
l was added to completely dissolve the precipitate, and RNase A (10 mg
/ Ml), and the mixture was incubated at 55 ° C for 30 minutes to obtain DNA.

The above 2% CTAB method, Iso-Plan
DNA obtained by the t method and the improved 2% CTAB method
There were many contaminants, and no reaction product was observed when PCR was performed. Therefore, the DNA was purified as follows.

(Purification of DNA for PCR) 2% CT
After treating each of the DNA solutions obtained by the AB method, Iso-Plant method and the improved 2% CTAB method with proteinase K (100 μg / ml) at 37 ° C. for 16 hours, an equal amount of PCI solution was added and stirred. Was centrifuged (3,000 rpm, 10 minutes, room temperature), and only the supernatant was recovered. After adding an equal volume of isopropanol and mixing, centrifugation (3,000 rpm, 10 minutes, room temperature)
The supernatant was discarded, 70% ethanol was added to the precipitate, and the supernatant was discarded after rinsing and flushing. After removing ethanol by vacuum drying, a TE buffer was added to dissolve the precipitate to obtain each purified DNA.

PCR was carried out using the 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
In the purification treatment A, the purification treatment in which the operation excluding the proteinase K treatment was repeated 8 to 10 times or more was performed. When the respective purification treatments were carried out, PCR reaction products were observed. Only a DNA amount of 1/10 or less of the method described in No. 1 was obtained, and it was not practically usable.

Example 2: Identification of Varieties by RAPD-PCR and Detection of Contamination (Hop Varieties Used) Hop pellets of the following 12 varieties 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, Yomitori (FA: fire aroma, A: aroma , B: represents bitter (high α acid).)

The template D obtained from 12 varieties of hops
RAPD-P was used for NA using 140 types 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 denatured at 94 ° C. for 5 minutes, and thereafter denatured at 94 ° C. for 1 minute. Annealing at 2 ° C. for 2 minutes and extension at 72 ° C. for 2 minutes as one cycle.
The cycle was repeated. As a DNA amplification device (DNA thermal cycler), Gene Amp PC
R System 9600 (Perkin-Elme
r company).

Next, the reacted solution was subjected to electrophoresis. 1 × Agarose X (Nippon Gene)
TAE buffer (40 mM Tris acetic acid, 1 mM EDT
A) was dissolved to 1.5% in A) to prepare a gel.
Using 1 × TAE buffer as an electrophoresis buffer,
Electrophoresis was performed at 0 V constant for about 30 minutes. After electrophoresis,
The gel was stained by immersion in a 0.5 μg / ml ethidium bromide solution, and irradiated with ultraviolet rays to generate fluorescence due to the DNA fragments. When electrophoresis is performed, DNA
The molecular weight marker (φX174 HincII digest) required to estimate 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 identification of 12 varieties requires at least seven types of random primers from Operon Technology. That is, OPA represented by SEQ ID NO: 10
9, OPA18 represented by SEQ ID NO: 11, SEQ ID NO: 12
OPB12 represented by, OPD represented by SEQ ID NO: 13
03, OPD11 represented by SEQ ID NO: 14, SEQ ID NO: 1
OPE01 shown by No. 5, OP shown by SEQ ID NO: 16
At E15, different bands were observed among the cultivars.

Table 1 shows the names of the random primers, their base sequences, and the number of amplification bands used for identification. Table 2 shows the polymorphism between varieties of the amplified band. In Table 2, the presence or absence of amplified DNA for each random primer was represented by +-.
The band with poor reproducibility represented by ± was not used for discrimination. As can be seen from Table 2, RAPD-PCR using seven random primers
Two varieties: Hersbrucker (HE), Pe
rl (PE), Tettnanger (TE), Nort
hern Brewer (NB), Saazer (SA),
Savinjski. Golding (SG), Suppe
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]

Also, American Tettnan
nger mixed with 5% by weight of Willamette by using random primer OPE01.
When APD-PCR was performed, American T
A faint band appeared in band b of OPE01 which should not appear in ettnnanger, and the contamination of the latter variety could be detected.

Example 3 Identification of Varieties by PCR Using STS-Formed Primers and Detection of Contamination (Hop Varieties Used) Hop pellets of the following 33 varieties 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. , 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 No.2, Toyodori, Kitamidori, Eastern Gold

(RAPD-PCR) The above template DNAs obtained from 33 varieties of hops were screened by RAPD-PCR using 140 random primers (10 bases) of Operon Technology as in Example 2. I went to. Analysis results of electrophoresis, operon
Technology random primers OPA9, OPA
18, OPB12, OPD03, OPD11, OPE0
1. In OPE15 and the like, different bands were observed among the varieties.

(Design of STS-Primed Primers) Of these, target bands 410 (OPA9), 850, and 87 that were observed between varieties on agarose gel when random primers OPA9 and OPA18 were used
0 (OPA18) was cut out using a Prep-A-Gene kit (manufactured by Biorad) and pMOS
Blue-T vector kit (Amersha
m), transformed into Escherichia coli and cloned, and Dye primer
DNA sequence kit (Applied
After PCR by Biosystems, DNA
Sequencer 373A (Perkin-El)
(manufactured by Mer Co., Ltd.).

Based on the determined base sequence, primers of 25 bases were designed (STS conversion) using commercially available primer determination software, and primer sets 1, 2 and 6-1 were prepared. When PCR was performed on genomic DNA extracted and purified from hops using these primer sets, primer set 6-1 showed an additional 6
Since 50 bp, 600 bp and 530 bp bands were detected, the base sequence was determined for each band,
Primers of 5 to 27 bases were designed (STS-ized), and primer sets 3, 4 and 5 were prepared.

The primer set 1 is composed of the primer 1A represented by SEQ ID NO: 1 and the primer 1B represented by SEQ ID NO: 2, and the primer set 2 is composed of the primer 2A represented by SEQ ID NO: 3 and the primer 2B represented by SEQ ID NO: 4. From the above, 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 From the above, the primer set 5 is represented by 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.

Using these primer sets 1, 2, 3, 4 and 5, genomic DN extracted and purified from hops
When PCR is performed on A, primer set 1 has 1300 bp and 750 bp, primer set 2 has 800 bp, and primer set 3 has 600 bp.
And 850 bp, 550 bp and 750 bp for primer set 4, 450 bp for primer set 5,
Each specific band was observed.

When the primer set 6-1 was used, a band was observed at an adjacent position and the discrimination ability was insufficient. Therefore, the primer set 6-1 was used as a template with the PCR product of the primer set 6-1 as a template. Use the primer inside only one of the primer positions used at the time of s
When the PCR is performed with the primer set 4 using the emulated PCR, that is, the PCR product amplified with the primer set 6-1 (hereinafter, referred to as “primer set 6-4” for convenience), a specific band of 500 bp is observed. And P amplified by the primer set 6-1.
When PCR is performed on the CR product using 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-modified primers. Also, se
FIG. 1 shows the difference between the amplified bands by the mined PCR.

[0049]

[Table 3]

(Identification of Varieties and Detection of Contamination) With respect to the template DNAs extracted and purified from the hops of 33 varieties, the primer sets 1, 2, 3, 4, 5, 6-1 and 6-
PCR was performed using 4 and 6-5 (6-4 and 6-6).
5 is a seminate PCR). PCR is TA
Using KARA Taq kit R-001A (manufactured by Takara Shuzo Co., Ltd.), the reaction was performed according to the reaction program shown in Table 4 using a PCR reaction amount of 20 μl per sample and 10 pM of primer. In addition, as a DNA amplifier (DNA thermal cycler), Gene Amp PCR Sys
tem 9600 (Perkin-Elmer) was used.

[0051]

[Table 4]

Then, the reacted solution was added to Agaros
e X (manufactured by Nippon Gene) with 1 × TAE buffer (4
The sample was subjected to electrophoresis using an agarose gel dissolved in 1.5 mM of 0 mM Tris acetic acid and 1 mM EDTA. Using 1 × TAE buffer as an electrophoresis buffer, electrophoresis was performed at a constant voltage of 100 V for about 30 minutes. After the electrophoresis, the gel was stained by immersion in a 0.5 μg / ml ethidium bromide solution, and irradiated with ultraviolet rays to generate fluorescence due to the 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 patterns of the STS-primed primers. As can be seen from Table 5, the created STS
Multiple amplified bands were observed with the modified primers, indicating that the primers are not amplifying one region in the genome, but are amplifying multiple copy regions. Also, according to the present invention, there are 33 major cultivated hop varieties in the world, namely Hersbrucker, Hallerta belonging to German fine aroma, aroma.
user Trading, Hallertaue
r, Sparter Select, Perle, Te
ttnanger, Northern belonging to Bitter
Brewer, Brewers Gold, Magnu
m, Saazer belonging to Czech fine aroma, Bor belonging to bitter, Stt belonging to French aroma
rissel Spalt, Lublin belonging to Polish aroma, Savi belonging to Slovenian aroma
njski. Golding, Bitter's Suppe
r Styrian, Fuggl belonging to US aroma
e, American Tettnnanger, Wi
llamette, Crystal, Liberty,
Nugget, Cluster, chi belonging to bitter
book, Galena, Pacific. Hallertau, NewZ
Ealand Hallertau, G belonging to Bitter
reen. Bullet, StickleBrac,
Australian Pride of Ringwood
d. Kirin No. 2, which belongs to the Japanese bitter, can be identified accurately.

[0054]

[Table 5]

Further, regarding the detection sensitivity for contamination, primer set 2 was used to detect H, a fine aroma from Germany.
Inspection of 5% by weight of Brewers gold, which is also a German bitter, to ersbrucker, a faint band appeared at a band size of 800 bp, and contamination of the latter variety 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 list]

 SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGAACCTTTG CCTAATCCTA AGATT 25

SEQ ID NO: 2 Sequence length: 25 Sequence type: number of nucleic acid chains: single stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GTGTTTTCCG TATCTACGCG CTGCG 2
5

SEQ ID NO: 3 Sequence length: 25 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence ACCATATAATA TCACCCCGAG ACTCA
twenty five

SEQ ID NO: 4 Sequence length: 25 Sequence type: number of nucleic acid chains: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CCATAACAGT TATGGTGAAC CAGAA 25

SEQ ID NO: 5 Sequence length: 27 Sequence type: number of nucleic acid chains: single stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GAAAACTACA CCTAGTATGT GATGCAT 27

Sequence number: 6 Sequence length: 25 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCATCGTCTC GTGTTTTCCG TATCT 25

SEQ ID NO: 7 Sequence length: 27 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CTTGATGCAT GTTGGATGTT TAAGTGT 27

SEQ ID NO: 8 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CCCTGTATAT ACGTGACATG CATGTGT 27

SEQ ID NO: 9 Sequence length: 25 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AGGATCGATC GTTCTCAAGG GTCGT 25

Sequence number: 10 Sequence length: 10 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGGTAACGCC 10

SEQ ID NO: 11 Sequence length: 10 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGGTGACCGT 10

SEQ ID NO: 12 Sequence length: 10 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence CCTTGACGCA 10

SEQ ID NO: 13 Sequence length: 10 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence GTCGCCGTCA 10

SEQ ID NO: 14 Sequence length: 10 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AGCGCCATTG 10

SEQ ID NO: 15 Sequence length: 10 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CCCAAGGTCC 10

SEQ ID NO: 16 Sequence length: 10 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ACGCACAACC 10

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing a difference between amplified bands by a segmented PCR.

Claims (9)

[Claims] 1. Genomic DN extracted and purified from hops
In the method for identifying hop varieties by performing PCR on primer A using primers to amplify DNA capable of identifying hop varieties, genomic DNA from hops
A method for identifying hop varieties, characterized by using a high-concentration salt soluble in alcohols for extraction and purification of hops. 2. The hop variety according to claim 1, wherein a high-concentration salt soluble in alcohols is used after cetyltrimethylammonium chloride is used for extraction and purification of genomic DNA from hops. Identification method. 3. A method for extracting and purifying genomic DNA from hops, using a high-concentration salt soluble in alcohols.
3. The method according to claim 1, wherein phenol is used.
How to identify the hop variety described. 4. A high-concentration salt soluble in alcohols,
4. The composition according to claim 1, wherein said lithium chloride is from 6 to 6 M lithium chloride.
3. The method for identifying a hop variety according to any one of 3. 5. The method for identifying a hop variety according to claim 1, wherein a random primer is used as the primer. 6. The method for identifying a hop variety according to claim 1, wherein an STS-modified primer is used as the primer. 7. STS-modified primers include a primer set 1 comprising a primer 1A represented by SEQ ID NO: 1 and a primer 1B represented by SEQ ID NO: 2, primers 2A represented by SEQ ID NO: 3 and SEQ ID NO: 4. Primer set 2 consisting of primer 2B, primer set 3 consisting of 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 3B, a primer set 5 consisting of a primer 5A represented by SEQ ID NO: 8 and a primer 3B represented by SEQ ID NO: 6, and a primer 6A represented by SEQ ID NO: 9 and a primer 3B represented by SEQ ID NO: 6 Primer set 6 consisting of
7. The hop variety identification method according to claim 6, wherein 1 is used. 8. The PCR product of primer set 6-1 is further combined with primer set 4 and primer set 5
Using semi-nested PC
The method for identifying a hop variety according to claim 7, wherein R is performed. 9. The DNA set forth in any one of SEQ ID NOs: 1 to 9, or the D set forth in any of SEQ ID NOs: 1 to 9
DNA containing a part or all of NA and capable of amplifying DNA that can identify hop varieties when subjected to PCR using as a primer.