JP2006034142A - Method for judging kind of hop using microsatellite dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a rapid/ready method for identifying main kinds recently circulated in large amounts in a hop market by carrying out a PCR of a genome DNA extracted from the hop using a primer to amplify a microsatellite region by which the kind of the hop can be identified. <P>SOLUTION: The method for judging the kind of the hop comprises carrying out the polymerase chain reaction (PCR) of the DNA of the hop which is a specimen by using the kind-identifying primer designed so that the microsatellite DNA containing polymorphism on base sequences among kinds may be amplified to amplify the microsatellite DNA, and analyzing the amplified DNA fragments. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a hop variety identification method, and further relates to a hop variety identification method using microsatellite DNA.

Hop is a plant belonging to the genus Humulus of the mulberry family. One. However, since the effects of these hops vary greatly depending on the variety, accurate selection and identification of the hop variety is important for brewing stable and good quality beer. In addition, the market price of hops varies greatly depending on the varieties.

Until now, cultivar identification of hops has been performed by quantifying the appearance characteristics of the spikelets and the bitter and essential oil components obtained from spikelets using gas chromatography or liquid chromatography. However, since most of the hops are crushed and compressed in the harvested area and transported in the form of pellets, it is very difficult to distinguish varieties from appearance characteristics, while analyzing the components In the method, the index component is greatly influenced by the climate and soil quality of the area where hops are cultivated, the hop preservation method, etc., and the analysis results differ depending on the harvest year and harvest area even for the same variety. There was a problem that it was difficult to identify varieties and required a lot of labor for analysis work.

On the other hand, in recent years, cultivar identification methods applying some genetic engineering have been developed. In these methods, a variety is identified by detecting a difference in base sequence between varieties, that is, a polymorphism. In other words, since the genetic features that are not affected by the cultivation environment and the storage method are used as indices, it is possible to accurately identify the variety. Variety identification methods using genetic engineering such as RAPD (random amplified polymorphic DNA) method, RFLP (Restriction fragment length polymorphisms) method, AFLP (amplified fragment length polymorphisms) method, and microsatellite method have already been developed in many commercial crops. It is applied.

In hops, Araki et al. Finds different DNA regions among varieties by the RAPD method, creates 10 primers that clarify the polymorphism of DNA, and performs PCR to identify hop varieties. (Patent Document 1). Similarly, Murakami et al. Also reported a hop variety identification method using the RAPD method (Patent Document 2). However, the RAPD method has a small amount of information obtained from a single marker, and it requires a lot of markers for actual variety identification. Therefore, it requires a lot of time and labor.

Brady et al. Also developed a variety identification method combining the RAPD method and the microsatellite region, and reported that 34 types of hops can be identified (Non-Patent Document 1), but many of them are not distributed in the hop market. It is a test variety or breeding line, and currently does not include varieties with a large distribution volume.
Republished WO97 / 05281 JP-A-11-103895 Euphytica, 91, 277-288, 1996

The above method for identifying hop varieties by using polymorphisms on the hop genome is an effective means for identifying hop varieties regardless of the hop cultivation environment, harvest time or storage method. As described above, hop variety identification methods based on polymorphisms in genomic DNA reported to date have little information obtained from a single marker, which requires a lot of time and effort, or actually enters the hop market. Since it is not possible to identify the main varieties in circulation, it is difficult to efficiently identify hop varieties.

An object of the present invention is to perform PCR on genomic DNA extracted from hops using primers and amplify a microsatellite region that can identify hop varieties. The object is to provide a quick and simple method of identification.

The hop variety identification method of the present invention is a method of detecting and identifying this polymorphism by a genetic technique based on the difference in the number of repetitions in the microsatellite region between varieties, that is, polymorphism. A microsatellite region containing polymorphism among varieties among hop DNAs as analytes is amplified by polymerase chain reaction (hereinafter referred to as PCR) using a cultivar identification primer, and the cultivar is analyzed by analyzing the amplified DNA. Is to identify.

In order to achieve the above method, it is necessary to know the polymorphism in the microsatellite region between hop varieties. This polymorphism specifically includes a difference in the number of repeats of a repetitive sequence in the microsatellite region, and the length of the sequence involved in the polymorphism may be 1 bp.

As a method for detecting a microsatellite region containing a polymorphism on the sequence between varieties as described above, a microsatellite region cloning method developed by Kijas et al., Rajiendara et al. Or Isabel et al. In this cloning method, a microsatellite region in genomic DNA of unknown sequence is isolated using a synthetic biotinylated probe. Specifically, hop genomic DNA is prepared by treating hop genomic DNA with a restriction enzyme and incorporating it into a vector. Single-stranded DNA is synthesized based on this library, and the microsatellite region is concentrated using a biotinylated probe. The microsatellite region can be isolated by performing PCR based on this DNA solution.

On the other hand, in addition to the method for isolating the microsatellite in the present invention as described above, the above method can be used as a method for selecting a primer capable of detecting a polymorphism by PCR, that is, as a primer design method. it can. Therefore, any polymorphic region between varieties in the hop microsatellite region detected based on the isolation method described above is included in the subject of the present invention. In addition, any primer capable of amplifying the polymorphic region detected based on the above method can be used as the breed identification primer of the present invention.

When the product identification is performed using the product identification primer, amplified DNA having a repetitive sequence within the product and different sizes depending on the case is generated by PCR. The amplified DNA generated here can be fractionated by direct electrophoresis, and the variety can be identified by the electrophoresis pattern.

  As described above, as a result of intensive studies, the present inventors have found microsatellite DNA having 13 polymorphisms from hop DNA, and designed primers described in SEQ ID NOs: 1 to 26 that can amplify them by PCR. Among them, five types of microsatellite most suitable for variety identification were selected, primer pairs capable of amplifying those microsatellite were selected, and a variety identification method capable of identifying a large number of varieties was developed to complete the present invention.

That is, the first of the present invention is to perform polymerase chain reaction using a variety identification primer designed to amplify microsatellite DNA containing a polymorphism on the base sequence between varieties of hop DNA as a subject. By performing the amplification, the microsatellite DNA is amplified, and the cultivar identification method is characterized by identifying the cultivar by analyzing the difference in length of the amplified DNA fragment.
The second aspect of the present invention provides the hop variety identification method according to the first aspect, wherein the variety identification primer is SEQ ID NO: 1-26.

  The hop variety identification method of the present invention makes it possible to identify hops more clearly and easily than before, and improve raw material management and quality control in the beer production process.

A. Cloning of Microsatellite Region Using Biotinylated Primer The microsatellite cloning method using a biotinylated primer is the method of Kijas et al. (Kijas JM, Fowler JC, Garbett CA, Thomas MR. (1994) Enrichment of microsatellite from the citrus genome using biotinylated oligonucleotide sequences bound to streptavidin-coated magnetic particles. Biotechniques 16,656-60, 662) and Rajiendara (Rajendra P. Kandpal, Geeta Kandpal, Sherman M. Weissman (1994) Construction of libraries enriched for sequence repeats and jumping. clones, hybridization selection for region-specific markers, Proc, Natl, Acad, Sci, USA 91, 88-92) and others, Isabel (Isabel Tenser, Stefania Degli Ivanissevich, Michele Morgante, Cesare Gessler (1999) Identification of Microsatellite markers and their application to population genetics of Venturia inaequalis, see Phytopathology 89: 748-753) Cloning was performed by a method with some modifications.

(1) Extraction of genome from hops DNA was extracted from leaves of hop "Southern early growth" which is an Asahi beer registered cultivar cultivated at Iwate hop test farm. One hop leaf was placed in a 50 ml centrifuge tube, immersed in liquid nitrogen for about 1 minute to freeze, immediately set in a multi-bead shocker (Yasui Kikai), and crushed at 1400 rpm for 20 seconds. Genomic DNA was extracted from the obtained powder using genomic-tip (QIAGEN).

(2) Preparation of hop genomic library After digesting 100 μg of hop genomic DNA with restriction enzymes EcoRI (TaKaRa) and MseI (New England Biolabs), it was separated by agarose gel electrophoresis, and a gel of about 300 bp to 1,500 bp was obtained. Cut out. DNA was recovered from the excised gel using GeneCleanKit (BIOlOl). Next, an oligo DNA having the sequence shown in FIG. 1 is prepared, incubated at 95 ° C. for 5 minutes, and annealed by gradually lowering the temperature so that the ends become protruding ends by EcoRI digestion or MseI digestion. Adapter DNA was prepared. The hop genomic DNA digested with EcoRI and MseI was ligated with the Ligation Kit ver. Ligation was performed using II (TaKaRa).

PCR (polymerase chain reaction) was performed using the DNA in the ligation solution as a template. PCR was performed using AmpliTaq Gold (Applied Biosystems) as the polymerase and 0.01 μM EcoRI primer and MseI primer (FIG. 2) annealing to the adapter part of FIG. For the PCR reaction, a thermal cycler manufactured by MJ Research was used. The reaction temperature was 94 ° C. for 4 minutes, 94 ° C. for 1 minute, 37 ° C. for 1 minute, and 72 ° C. for 1 minute 33 times, followed by extension reaction at 72 ° C. for 4 minutes.

(3) Isolation of microsatellite
To a 1.5 ml microcentrifuge tube containing 0.6 ml of Streptavidin-Coated Magnetic Beads (Promega), 3 μg of a biotinylated primer (either one of FIG. 3) was added and incubated at room temperature for 15 minutes. To remove excess primer, after washing 3 times with 0.5 × SSC, the biotin-apidine conjugate was suspended in 350 μl of 10 × SSC. To 100 μl of the PCR reaction solution of (2), 550 μl of sterilized water was added, heat-denatured at 95 ° C. for 5 minutes and then rapidly cooled, added to the biotin-apidine conjugate and left at room temperature for 20 minutes. Then, it was washed 4 times with 2 × SSC and 4 times with 1 × SSC. To the collected magnetic beads, 200 μl of 1.5 NaOH was added and mixed, and the mixture was left at room temperature for 20 minutes. The supernatant was recovered, neutralized with 1.25 M acetic acid, desalted using SUPREC-02 (TaKaRa), and used as a microsatellite DNA concentrated solution.

(4) PCR
PCR was performed using the desalted microsatellite DNA concentrated solution as a template. PCR was performed using AmpliTaq Gold (Applied Systems) as the polymerase and 0.01 μM EcoRI primer and MseI primer (FIG. 2) as primers. The equipment and reaction conditions used for the reaction were the same as the PCR in (2). After the PCR reaction, the reaction solution was purified using PCR Purification Kit (QIAGEN).

(5) Ligation and sequencing The purified DNA was ligated with pGEM vector using pGEM-T Easy Vector System (Promega) and transformed into E. coli JM109. The grown colonies were subjected to insert check, and the base sequence was determined by an ordinary method for inserts having a size of 300 to 800 bp.

(6) Sequence analysis After analyzing the sequence using a capillary sequencer (BECKMAN COULTER; CEQ8000), primers were designed and synthesized so as to sandwich the microsatellite region only in the microsatellite region having a large number of repetitions. At that time, a fluorescent dye of FAM (blue), HEX (green), or NED (yellow) was added to one primer.

B. Polymorphism analysis In the world, whether the microsatellite region to be amplified has a polymorphism by performing PCR using the synthetic oligonucleotide designed to sandwich the microsatellite obtained as described above as a primer set. The results of examining 37 varieties with a large circulation are shown in (1) to (3) below.
(1) Extraction of hop genomic DNA We obtained 37 hop varieties (Table 1) that are currently in circulation in the world and extracted DNA from the sample. Place one spike of 37 varieties in the table into a 10 ml centrifuge tube (manufactured by Yasui Kikai Co., Ltd.), immerse in liquid nitrogen for about 1 minute and freeze, immediately set the multi-bead shocker to 20 at 1400 rpm. Milled for seconds. DNA was extracted from 50 mg of the obtained powder using DNeasy Plant Mini Kit (QIAGEN) according to the protocol. As a result, 30-50 μg of genomic DNA of various hop varieties was obtained.

(2) PCR
PCR was performed using 5 ng of extracted hop DNA as a template. AmpliTaqGold (Applied Biosystems) was used as the polymerase, and the primer pair designed and synthesized by adding fluorescence was used as the primer. For the PCR reaction, a thermal cycler manufactured by MJ Research was used. The reaction temperature was 94 ° C. for 13 minutes, followed by 27 times of 94 ° C. for 45 seconds, 55 ° C. for 39 seconds, and 72 ° C. for 90 seconds, followed by extension reaction at 72 ° C. for 10 minutes.

(3) Electrophoresis using a DNA sequencer 2.5 μl of formamide (amresco), 0.5 μl of size standard marker (Applied Biosystems) GENESCAN-400HD [ROX], 1 μl of PCR reaction solution, loading buffer (attached to the size standard marker) 0 Each 5 μl was mixed and heat denatured at 95 ° C. for 5 minutes to obtain a loading sample. This was separated and analyzed by an electrophoresis apparatus (ABIPRISM 377 DNA Sequencer). As a result, primer pairs described in SEQ ID NOs: 1-26 capable of amplifying polysatellite having a polymorphism were selected. Among them, 5 primer pairs of SEQ ID NOS: 1 and 2, 5 and 6, 17 and 18, 21 and 22, 23 and 24, which are particularly clear results, were selected and used for breeding. .

Selection of microsatellite markers By performing PCR using the synthetic oligonucleotides obtained as described above designed to sandwich microsatellite as a primer set, it is determined whether the microsatellite region to be amplified has a polymorphism. Examples 1 to 13 show the results of research on 37 varieties with a large circulation volume in the world.

Of the primers obtained as described above, the fluorescent addition primer described in SEQ ID NO: 1 in the sequence listing and the primer described in SEQ ID NO: 2 were each used at a final concentration of 0.1 μM. Furthermore, 0.2 ng of Ampli Taq Gold polymerase (Applied Biosystems), 200 μM of each of 4 types of bases (dATP, dTTP, dCTP, dGTP) and 5 ng of hop genomic DNA prepared from each of 37 hop varieties were added. Polymerase chain reaction was carried out in 10 mM Tris-HCl buffer (pH 8.3) containing 50 mM KCl, 1.5 mM MgCl. The reaction volume was 20 μl. Each step in the above polymerase chain reaction was carried out under the following conditions. First hold at 94 ° C. for 13 minutes, then the denaturation step is heated at 94 ° C. for 45 seconds, the primer annealing step is incubated at 55 ° C. for 39 seconds, and the DNA polymerase extension step is cycled at 72 ° C. for 90 seconds. 27 times and held at 72 ° C. for 10 minutes. The amplified DNA obtained by the polymerase chain reaction was separated by electrophoresis using a 36 cm acrylamide gel for ABI Prism 377 DNA sequencer (Applied Biosystems). At that time, GS400HD (Applied Biosystems) was used as a size marker. For analysis after electrophoresis, Gene Scan (Applied Biosystems) was used, and the band size was calculated. The gel image obtained by electrophoresis is shown in FIG. As a result of analyzing the gel image using Gene Scan, as shown in Table 2, when this primer set was used, 11 types of polymorphisms were observed, and 37 types of hops were combined into 17 types by combining the positions of the bands. I was able to identify. These polymorphisms were easy to distinguish and were observed with good reproducibility.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 3 and 4 as primer sequences. The obtained results are shown in Table 3. As is apparent from the table, when the synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 3 and 4 were used as primers, seven types of polymorphism were observed, and 37 hops were selected depending on the combination of band positions. 7 types could be identified.

The synthesis was carried out under the same conditions as in Example 1, using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 5 and 6 as primer sequences. Table 4 shows the obtained results. As is apparent from the table, when the synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOS: 5 and 6 were used as primers, nine types of polymorphism were observed, and 37 hops were selected depending on the combination of band positions. 14 types could be identified.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 7 and 8 as primer sequences. The results obtained are shown in Table 5. As is apparent from the table, when the synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 7 and 8 were used as primers, nine types of polymorphisms were observed, and 37 hops were selected depending on the combination of band positions. 14 types could be identified.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 9 and 10 as primer sequences. The results obtained are shown in Table 6. As is clear from the table, when the synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 9 and 10 were used as primers, 5 types of polymorphisms were observed, and 37 hops were selected depending on the combination of band positions. Eight types could be identified.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 11 and 12 as primer sequences. The results obtained are shown in Table 7. As is apparent from the table, when the synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 11 and 12 were used as primers, three types of polymorphisms were observed, and 37 hops were selected depending on the combination of band positions. Four types could be identified.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 13 and 14 as primer sequences. Table 8 shows the obtained results. As is apparent from the table, when the synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 13 and 14 were used as primers, 11 types of polymorphisms were observed, and 37 hops were selected depending on the combination of band positions. 17 types could be identified.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 15 and 16 as primer sequences. Table 9 shows the obtained results. As is apparent from the table, when the synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 15 and 16 were used as primers, 5 types of polymorphisms were observed, and 37 hops were selected depending on the combination of band positions. Eight types could be identified.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 17 and 18 as primer sequences. Table 10 shows the obtained results. As is apparent from the table, when synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 17 and 18 were used as primers, 8 types of polymorphisms were observed, and 37 hops were selected depending on the combination of band positions. 12 types could be identified.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 19 and 20 as primer sequences. The obtained results are shown in Table 11. As is apparent from the table, when the synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 19 and 20 were used as primers, 11 types of polymorphism were observed, and 37 hops were selected depending on the combination of band positions. 14 types could be identified.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 21 and 22 as primer sequences. The results obtained are shown in Table 12. As is apparent from the table, when synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 21 and 22 were used as primers, 12 types of polymorphisms were observed, and 37 hops were selected depending on the combination of band positions. 14 types could be identified.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 23 and 24 as primer sequences. The obtained results are shown in Table 13. As is apparent from the table, when the synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 23 and 24 were used as primers, 13 types of polymorphisms were observed, and 37 hops were selected depending on the combination of band positions. 22 types could be identified.

The synthesis was carried out under the same conditions as in Example 1 using synthetic oligonucleotides having the base sequences described in SEQ ID NOs: 25 and 26 as primer sequences. The obtained results are shown in Table 14. As is apparent from the table, when the synthetic oligonucleotides having the nucleotide sequences set forth in SEQ ID NOs: 25 and 26 were used as primers, three types of polymorphisms were observed, and 37 hops were selected depending on the combination of band positions. It was possible to identify 5 types.

Selection of microsatellite to be used for hop variety identification and hop variety identification by PCR As described above, cloning of microsatellite was performed by concentrating the microsatellite region using a biotinylated primer. Then, polymorphism analysis was performed by selecting only those having a relatively large number of repetitions. As a result, as shown in Table 15, 13 types of microsatellite obtained different band patterns depending on the variety (polymorphism).

The number of microsatellite iterations is indicated by a subscript number in the “Microsatellite Type” column. Since the number of microsatellite repeats varies depending on the type, the band size varies depending on the type when the microsatellite region is amplified by PCR and electrophoresed. The number of types of the band size is shown as “Allele”.

Among the 13 types of microsatellite from which the polymorphism was obtained, SEQ ID NOS: 1 and 2, 5 and 6, 17 and 18, 21 and which were primer pairs that amplify microsatellite whose band sizes differ greatly depending on the variety The combination of 22, 23 and 24 was used for cultivar identification, and primers for specifically amplifying these microsatellite were prepared as shown in Table 16. It was devised to be able to distinguish varieties by a single run by adjusting the fluorescent color and size of DNA to be amplified. (Fig. 5)

Among the primer pairs that amplify microsatellite regions having polymorphism among varieties obtained this time, five primer pairs of SEQ ID NOs: 1 and 2, 5 and 6, 17 and 18, 21 and 22, 23 and 24 were used. In combination, as a result of class identification for 37 hop varieties, it was possible to classify 37 varieties into 27 groups (Table 17). Nugget and U. are the main varieties that cannot be classified. S. There were Nugget, Hersbrucker and Hersbrucker Spat, Saazer and Tettnanger (Barth), and these were varieties widely known as very close varieties. In addition, this method, which applied the microsatellite region polymorphism to variety identification, can identify varieties more easily with fewer markers than the variety identification method using the RAPD method reported by Araki et al. It was possible.

A numerical representation of the pattern of bands that appear as a result of electrophoresis. The same number indicates the same band pattern. In the rightmost column, varieties having the same pattern are indicated by the same mark.

Generation of adapter DNA in cloning of hop microsatellite regions using biotinylated primers. EcoRI and MseI primers in cloning of hop microsatellite regions using biotinylated primers. Biotinylated primer in cloning of hop microsatellite region using biotinylated primer. Results obtained by performing PCR and electrophoresis on 37 hops using the primers of SEQ ID NOs: 1 and 2. The red band is a size marker. Results of acrylamide electrophoresis of fragments containing microsatellite regions amplified by the primers of SEQ ID NOs: 1 and 2, 5 and 6, 17 and 18, 21 and 22, 23 and 24 for 37 hop varieties. The numbers from the leftmost lane to 37 lanes correspond to the numbers assigned to the varieties in Table 1.

Claims (2)

The microsatellite DNA is obtained by performing polymerase chain reaction using a variety identification primer designed to amplify microsatellite DNA containing a polymorphism on the base sequence between the varieties of the hop DNA as the analyte. A method for identifying a variety of hops, wherein the variety is identified by amplification and analysis of the amplified DNA fragment.
The hop breed identification method according to claim 1, wherein the breed identification primer is SEQ ID NO: 1-26.