JP4719917B2 - Azuki raw material varieties judgment method of azuki bean processed food - Google Patents

Description

  The present invention relates to a method for accurately determining a variety of azuki bean varieties of processed food, particularly azuki bean processed food containing azuki bean as a raw material.

  The rights of new plant breeders are protected internationally by a plant variety protection system. However, in recent years, fraudulent acts such as cultivating new varieties of crops developed by domestic agricultural research institutes overseas without permission and importing them as processed foods are increasing, and there is a problem of squeezing domestic farmers. . In addition, from the viewpoint of consumer protection, it is also an important issue to prevent unauthorized labeling of processed food ingredients. However, a technique for determining the varieties of plants used as raw materials for processed foods has not yet been developed, and development of a practical application technique for identifying the raw material varieties of processed foods is awaited.

  On the other hand, a DNA polymorphism detection method has attracted attention as a technique for identifying plant varieties. Specifically, RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism), SSR (Simple Sequence Repeat), CAPS (Cleaved Amplified Polymorphic Sequence), ISSR (Inter-Simple Sequence) Repeat), SNP (Single Nucleotide Polymorphism) and the like have been studied (see Non-Patent Document 1), and rice, rush, strawberries, green beans, etc. have been put to practical use a variety identification technique using the above method.

  There are transposable elements in the genome of plants. A transposable element, also called a “metastatic genetic factor” or “mobile genetic factor”, has the ability to metastasize from one site on another chromosome to another site, or is transferred on the chromosome during the evolution process. This is a DNA sequence that has been shown to be. With the transfer of transposable elements, DNA duplication, deletion, or chromosomal rearrangement occurs. About 70-80% of mutations that occur in nature are attributed to the activity of such transposable elements.

  Retrotransposons are a type of transposable element. Retrotransposon transfer involves a process in which a sequence in the genome is transcribed, and then a DNA that is a copy of the original sequence is inserted into the genome by a reverse transcription reaction. The inserted sequence is stably inherited. To do. There are many sequences in the plant genome that have been replicated in this way during evolution, most of which have lost their translocation activity. In addition, many replica sequences of retrotransposon are scattered in the genome and are known to be excellent gene markers.

  As a technique for identifying varieties using such a retrotransposon as a genetic marker, for example, Patent Documents 1 and 2 identify varieties of sweet potato raw materials by detecting whether or not a transposon is inserted into the sweet potato genome. A method of determining is disclosed.

Non-Patent Documents 2 and 3 describe a method for identifying azuki bean varieties using the RAPD method and the SSR method.
JP 2005-229921 A (published on September 2, 2005) JP 2006-42808 A (published February 16, 2006) Hiroshi Yano, Possibility of variety identification by DNA polymorphism analysis-DNA polymorphism detection technology in plants and its application-, Agriculture and Horticulture, 79: 131-136 (2004) Hokkaido Central Agricultural Experiment Station, (Reference Material 5) “Identification of red bean varieties by DNA analysis”, [online], [Search March 26, 2007], Internet <URL: http: //www.hinsyu.maff. go.jp/DNAgaido/san5.pdf> Hokkaido Central Agricultural Experiment Station (Reference 6) “Identification of red bean varieties by DNA analysis”, [online], [March 26, 2007 search], Internet <URL: http: //www.hinsyu. maff.go.jp/DNAgaido/san6.pdf> Waugh, R., K. McLean, AJ Flavell, SR Pearce, A. Kumar, BBT Thomas and W. Powell. Genetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S- SAP). Mol Gen Genet 253: 687-694 (1997)

  As described above, it is very important to determine the varieties of raw material plants from processed foods from the viewpoint of protecting the rights of new breeders and protecting consumers. However, a technique for determining azuki bean varieties used as a raw material for processed azuki bean has not yet been developed, and development of a practical application technique for determining a raw cultivar for azuki bean processed food is awaited.

  The reason why the raw material variety determination technology for azuki bean processed food has not yet been developed is that it is difficult to apply the above-described plant variety determination technology as it is to the raw material variety determination for processed food. Like azuki bean cultivar determination method using DNA polymorphism for the determination of raw material varieties of azuki bean processed food and sequence-specific amplification polymorphism method (non-patent document 4) to display (display) polymorphisms of retrotransposon insertion sites at once The reason why it is difficult to apply the method for determining azuki bean varieties is that the DNA derived from the azuki bean raw material varieties contained in the azuki bean processed food is deteriorated and fragmented by processing in the manufacturing process. Therefore, a specific example will be described below.

  Of the plant variety determination methods using the above-described DNA polymorphism, the variety determination method using SSR (Simple Sequence Repeat) is now becoming mainstream. In this method, a portion on the genome in which a short sequence repeatedly appears, such as a so-called microsatellite, is amplified and detected by PCR. Such repeat numbers are likely to be mutated, and polymorphisms appear frequently. In addition, since PCR is performed by setting a primer in a unique region adjacent to a repetitive sequence, each SSR marker can be treated as a specific locus, and the difference in the number of repeats can be treated as an allele. Since a large number of repetitive sequences exist in various places on the genome of a plant, a variety is identified by a combination of a plurality of alleles at a plurality of loci.

  When this method is applied to various types of crops, and when the cultivar used as a raw material is identified from those processed products, first, the DNA fragment to be subjected to nucleic acid amplification such as PCR is a repetitive sequence and its both sides. Therefore, there is a problem that the present invention is larger than a DNA fragment to be subjected to nucleic acid amplification for variety identification.

  Since the DNA derived from the raw material varieties contained in the processed food is deteriorated and fragmented by processing in the manufacturing process, the SSR method with a large DNA fragment targeted for nucleic acid amplification for cultivar identification is the method according to the present invention. It is unsuitable compared.

  Of the plant variety determination methods using the above-described DNA polymorphism, a variety determination method using RAPD (Random Amplified Polymorphic DNA) or CAPS (Cleaved Amplified Polymorphic Sequence) has been put into practical use. These methods are premised on amplification of a specific fragment, but it is difficult to find a large number of sites on the genome so that determination can be made using only amplified fragments shorter than a certain length. In particular, since the method using RAPD uses one kind of primer, it is not possible to find a site where a short fragment is amplified so that there is no problem in breed comparison in DNA that has deteriorated and fragmented by processing. It seems almost impossible.

  Moreover, the conventional plant variety determination method using the polymorphism of the insertion site of the retrotransposon performs variety identification by comprehensively showing the differences between the varieties of the insertion site of the retrotransposon (for example, S described above) -Perform display like SAP method, Southern analysis, etc.). In order to perform a comprehensive analysis in this way, the quality of the DNA to be used is good, that is, that it is not fragmented by an operation such as DNA extraction is an essential condition for the reproducibility and reliability of the results. Therefore, these methods are not suitable for determining the raw material varieties of processed foods.

  On the other hand, in processed products manufactured through the process of making raw materials into kneaded products or powders, such as rice cakes, flour, noodles, and bread, the contents of processed products when multiple varieties are used as raw materials It is technically impossible to sort the goods according to the raw material varieties used. Therefore, in identifying the raw material varieties, the processed product itself is analyzed. In such a case, the conventional technology for discriminating varieties using DNA uses polymorphisms of genetic loci indicating varietal polymorphisms, and combines the types of alleles possessed by individual varieties at the respective loci. Therefore, the type of allele of the mixed varieties is detected at the same time. For this reason, even if a combination of allele types of a specific variety is detected in the processed products that have been investigated, are those types detected because the specific variety is actually included in the product? Or, it is difficult to identify whether the combination was detected by chance using alleles of multiple varieties used in the product. A simple example of this difficulty is that for products that use both parental varieties of the cultivar being investigated, all of the alleles of the cultivar being investigated are available, even if the cultivar being investigated is not actually used. Detected from the product.

  The present invention has been made in view of the above-mentioned problems, and its object can be applied to degraded and fragmented DNA contained in azuki bean processed food, and the azuki varieties of the raw plant of the azuki bean processed food can be improved. An object of the present invention is to provide a method capable of accurately determining. Another object of the present invention is to provide a method capable of accurately determining a variety of azuki bean varieties of processed food even in a processed food containing a plurality of varieties of azuki bean raw materials.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the azuki bean variety “Kita no Otome” is present in the insertion site of Phare-1 retrotransposon, which is one of the transposable elements present in the azuki bean genome. We found a part that is present in cultivars that are currently cultivated other than “Kita no Otome”. And even if there are multiple varieties of Azuki raw food varieties due to the presence of the insertion site unique to “Kitaotome”, it is possible to accurately and quickly determine whether the processed food contains the varieties. After confirming that this can be done, the present invention has been completed.

  Furthermore, the present inventors are present in the adzuki cultivar “Shumari” among the insertion sites of the RLVA retrotransposon, one of the transposable elements present in the Azuki genome, but are currently cultivated other than “Shumari”. I found a part that does not exist in the varieties. And even if there are multiple varieties of azuki bean raw material varieties based on the presence or absence of the insertion site unique to "Sumari", it is possible to accurately and quickly determine whether the varieties are included in the processed food. It was confirmed that this was possible, and the present invention was completed.

  That is, the method for determining the variety of azuki bean raw material according to the present invention is a method for determining the variety of azuki bean raw material contained in azuki bean processed food, and detects whether a retrotransposon has been inserted at a specific site in the azuki bean genome. It is characterized by including a detection step of determining the variety of azuki bean raw material by examining the presence or absence of a retrotransposon insertion site unique to the adzuki bean variety.

  In addition, the method for determining the variety of azuki bean raw material according to the present invention is a method for determining the variety of azuki bean raw material contained in azuki bean processed food, and detects whether or not a Phare-1 retrotransposon is inserted at a specific site in the azuki bean genome. Thus, the method includes a detection step of determining a variety of azuki bean raw material. Therefore, it becomes possible to make a simple and accurate determination of azuki bean cultivar using the inter-variety polymorphism of the Phare-1 retrotransposon insertion site.

  Further, the method for determining the varieties of azuki bean raw material according to the present invention is a method for determining the varieties of azuki bean raw material contained in azuki bean processed food, and whether or not RLVA retrotransposon is inserted as a retrotransposon at a specific site in the azuki bean genome. It is characterized by including a detection step of determining the type of azuki bean raw material by detecting the above. Therefore, it is possible to make a simple and accurate determination of azuki bean cultivar using the RLVA retrotransposon insertion site polymorphism.

  In the method for determining azuki bean cultivar according to the present invention, in the detection step, a primer designed based on the base sequence of the genomic region adjacent to the retrotransposon of the azuki bean cultivar in which the retrotransposon is inserted at a specific site of the azuki bean genome. And a primer set that combines a primer designed based on the base sequence of a retrotransposon and a method for confirming the presence or absence of an amplification product by performing a nucleic acid amplification reaction using DNA prepared from azuki bean processed food as a template It is preferable. Since the length of the fragment amplified by the above primer set can be designed arbitrarily, if the primer set is designed so that short fragments are amplified, this can be achieved even when the DNA in azuki bean processed food is highly fragmented. The presence or absence of an amplified fragment can be confirmed as a template. Further, since the presence or absence of the amplification product is confirmed, it can be confirmed by simple electrophoresis, and skill is not required for determination of the result.

  In the method for determining azuki bean cultivar according to the present invention, the fragment length of the amplification product is preferably 40 bp to 600 bp. By designing the primer so that the length of the amplification product is shortened, even if the DNA extracted from azuki bean processed food is highly fragmented, it can be used as a template for nucleic acid amplification reaction Become.

  In the method for determining azuki bean cultivar according to the present invention, when detecting whether Phare-1 retrotransposon is inserted as the retrotransposon in the detection step, the base sequence of the genomic region adjacent to the Phare-1 retrotransposon The primer designed based on the base sequence shown in SEQ ID NO: 4 is a primer consisting of the base sequence shown in SEQ ID NO: 4, and the primer designed based on the Phare-1 retrotransposon base sequence is based on the base sequence shown in SEQ ID NO: 3. It is preferable that it is a primer. In addition, the primer which consists of a base sequence shown by sequence number 3 or 4 is also contained in this invention.

  In the method for determining azuki bean cultivar according to the present invention, when detecting whether or not RLVA retrotransposon is inserted as the retrotransposon in the detection step, the design is based on the base sequence of the genomic region adjacent to the RLVA retrotransposon. The primer to be designed is a primer consisting of the base sequence shown in SEQ ID NO: 7, and the primer designed based on the base sequence of the RLVA retrotransposon is a primer consisting of the base sequence shown in SEQ ID NO: 6. preferable. In addition, the primer which consists of a base sequence shown by sequence number 6 or 7 is also contained in this invention.

  Azuki raw material variety discriminating kit of the present invention is an Azuki raw material type discriminating kit for determining the type of azuki bean raw material contained in azuki bean processed food, and the retrotransposon is inserted into the retrotransposon insertion site unique to a specific azuki bean variety. It comprises a primer designed based on the base sequence of the adjacent genomic region and a primer designed based on the base sequence of the retrotransposon. As a result, it is possible to carry out the azuki raw material variety determination method of the present invention and perform a simple and accurate determination of the azuki raw material variety.

  The kit for discriminating varieties of azuki bean according to the present invention is designed based on the primer designed based on the base sequence of the genomic region adjacent to Phare-1 retrotransposon as the retrotransposon and the base sequence of Phare-1 retrotransposon. And a primer. As a result, it is possible to carry out the azuki raw material variety determination method of the present invention and perform a simple and accurate determination of the azuki raw material variety.

  Examples of the primer contained in the kit include a primer consisting of the base sequence shown in SEQ ID NO: 3 and a primer consisting of the base sequence shown in SEQ ID NO: 4.

  In the kit for discriminating varieties of raw material of the present invention, a primer designed based on the base sequence of the genomic region adjacent to the RLVA retrotransposon as the retrotransposon and a primer designed based on the base sequence of the RLVA retrotransposon It is preferable to include.

  Examples of the primer included in the kit include a primer consisting of the base sequence shown in SEQ ID NO: 6 and a primer consisting of the base sequence shown in SEQ ID NO: 7.

  As described above, the method for determining azuki bean cultivar according to the present invention detects whether or not a retrotransposon is inserted at a specific site of the azuki bean genome, and examines the presence or absence of a retrotransposon insertion site specific to a specific azuki bean cultivar, It is the structure including the detection process which determines the kind of azuki bean raw material.

  In addition, as described above, the method for determining azuki bean cultivar according to the present invention is a detection step of determining a variety of azuki bean by detecting whether or not a Phare-1 retrotransposon is inserted at a specific site in the azuki bean genome. It is the structure containing.

  Further, the method for determining azuki bean raw material varieties of the present invention includes a detection step of determining the variety of azuki bean raw material by detecting whether or not the RLVA retrotransposon is inserted at a specific site of the azuki bean genome as described above. It is a configuration.

  Therefore, there is an effect that it is possible to easily and accurately determine azuki bean raw material type.

  In addition, when a nucleic acid amplification reaction is used in the detection step, it can be detected even if the amount of DNA used as a template is extremely small. Therefore, there is an effect that the DNA extracted from azuki bean processed food is deteriorated and the variety of azuki bean raw material can be determined even if the amount of extractable DNA is very small. In addition, by designing primers so that the fragments amplified by the nucleic acid amplification reaction are extremely short, even if the DNA extracted from azuki bean processed food is highly fragmented, the variety of azuki bean raw material is determined. There is an effect that can be.

  Moreover, in processed foods containing azuki bean of multiple varieties by detecting whether or not a retrotransposon has been inserted for the insertion site that is unique to the Phare-1 retrotransposon or RLVA retrotransposon varieties In addition, it is possible to determine with high accuracy whether a variety having such a unique insertion site is included.

  Moreover, since the azuki raw material kind determination kit of this invention is a kit for implementing the azuki raw material kind determination method of this invention, there exists an effect that this method can be implemented more rapidly and simply.

  As described above, according to the present invention, the raw material varieties of azuki bean processed food can be determined quickly, simply, and accurately. The right of the person can be protected, and the illegal display of the raw material varieties of the processed food can be prevented.

  An embodiment of the present invention will be described as follows. Note that the present invention is not limited to this.

  The method for determining azuki bean cultivar according to the present invention is a method for determining the varieties of azuki bean raw material contained in azuki bean processed foods, detecting whether or not a retrotransposon is inserted at a specific site of the azuki bean genome, and a specific azuki bean cultivar It is characterized by including a detection step of determining the type of azuki bean raw material by examining the presence or absence of a unique retrotransposon insertion site. Thereby, even when the azuki raw material varieties of the azuki processed food are a plurality of varieties, it is possible to accurately and quickly determine whether the processed food contains the varieties.

  The present inventors have found an insertion site unique to "Kitaotome" (Embodiment 1) and an insertion site unique to "Sumari" (Embodiment 2) as the retrotransposon insertion site unique to the Azuki bean breed. It was. The unique insertion site of “Kitaotome” is the insertion site of Phare-1 retrotransposon, one of the transposable elements present in the azuki bean genome. Moreover, the insertion site unique to “Shumari” is the insertion site of RLVA retrotransposon, one of the transposable elements present in the azuki bean genome. By examining the presence or absence of a retrotransposon insertion site unique to these azuki bean varieties, it becomes possible to determine the variety of azuki bean raw materials.

[Embodiment 1]
The method for determining a variety of azuki bean according to the present invention is a method for determining a variety of azuki bean raw material contained in azuki bean processed food, and detects whether or not a Phare-1 retrotransposon is inserted at a specific site in the azuki bean genome. By this, it is a method including the detection process which determines the kind of azuki bean raw material.

  The target azuki bean processed food is not particularly limited as long as it contains azuki bean in the raw material and can extract azuki bean raw material DNA from the azuki bean processed food. In addition, azuki bean processed food is a product obtained by subjecting all or a part of azuki bean raw material plant or a harvested product (fruit, seed, etc.) obtained from the raw material plant to heating and drying. Therefore, the processed food of azuki bean includes not only the final product but also an intermediate processed product.

  Moreover, the azuki bean processed food is not limited to a processed food containing a single variety of raw material plants, and may be a processed food containing a plurality of types of raw material plants. As will be described later, by detecting whether or not the Phare-1 retrotransposon has been inserted at the insertion site unique to the Phare-1 retrotransposon azuki bean variety, raw material plants of a plurality of Azuki bean varieties are included. It is also possible to determine whether azuki bean varieties having such unique insertion sites are included in azuki bean processed food.

  Retrotransposons are a type of transposable element that has been transferred in the form of replicating itself in the process of evolution and inserting it into the genome. In many plants, many types of retrotransposons exist in a replicated state. In the current plant genome, most retrotransposon sequences have lost their translocation activity and are stably inherited when inserted into the genome. The genomic sequence adjacent to the retrotransposon is unique for each insertion site. Furthermore, there is a polymorphism between varieties where insertion of retrotransposon is observed in one cultivar and insertion is not seen in another cultivar.

  LTR-type retrotransposons, which have a repetitive sequence called LTR (Long Terminal Repeat) at both ends of the sequence, are present in all higher plants to the extent investigated so far. Numerous sequences have been isolated and named (Johns et al (1985) A low copy number, copia-like transposon in maize.EMBO J 8: 1093-1102., Manninen and Schulman (1993) BARE-1, a copia-like retroelement in barley (Hordeum vulgare L.). Plant Mol Biol 22: 829-846., Laten et al. (1998) SIRE-1, a copia / Ty1-like retroelement from soybean, encodes a retroviral envelope-like Proc Natl Acad Sci USA 95: 6897-6902., Kumekawa et al. (1999) Identification and characterization of novel retrotransposons of the gypsy type in rice. Mol Gen Genet 260: 593-602.).

  Retrotransposons that do not have LTR are called non-LTR types. Non-LTR type retrotransposons lack the frame of enzyme sequence required for transfer called LINE (Long Interspersed Nucleotide Element) and most of such frame called SINE (Short Interspersed Nucleotide Element). There is a missing short sequence. LINE is present in all higher plants to the extent investigated so far, and individual sequences have also been isolated and named (Leeton, PRJ and Smyth, DR (1993) An abundant LINE-like Element amplified in the genome of Lilium speciosum.Mol. Gen. Genet., 237, 97-104.Noma, K., Ohtsubo, E. and Ohtsubo, H. (1999) Non-LTR retrotransposons (LINEs) as ubiquitous components of The plant genomes. Mol. Gen. Genet., 261, 71-79. Schwarz-Sommer, Z., Leclercq, L., Gobel, E. and Saedler, H. (1987) Cin4, an insert altering the structure of the A1 gene in Zea mays, exhibits properties of non-viral retrotransposons. EMBO J, 6, 3873-3880.).

  Among LTR-type retrotransposons, those classified as Ty1-Copia type have been isolated from many plants by PCR targeting amino acid conserved sequences present in the reverse transcriptase region. (Voytas et al. (1992) copia-like retrotransposons are ubiquitous among plants.Proc Natl Acad Sci USA 89: 7124-7128., Hirochika et al. (1992) Retrotransposon families in rice. Mol Gen Genet 233: 209- 216.). Therefore, even if the full-length retrotransposon sequence has not been examined, TAIL (Thermal Asymmetric) can be used to isolate the extension sequence if a known reverse transcriptase region sequence is identified in the DNA database. InterLaced) PCR (Liu et al. (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant Journal 8: 457-463.), Suppression PCR (Siebert et al. (1995) An Improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 23: 1087-1088. and other methods (Pearse et al. (1999) Rapid isolation of plant Ty1-copia group retrotransposon LTR sequences for molecular marker studies. Plant Journal 19: 711-717.), The terminal LTR sequence can be identified.

  The Phare-1 retrotransposon in the present invention is a transposon classified into the above Ty1-Copia type, and a large number of Phare-1 retrotransposon replication sequences exist in the azuki bean genome. Among the Phare-1 retrotransposon replication sequences, the base sequence of one sequence is determined from the reverse transcriptase region to the terminal LTR sequence of the full-length sequence, and the base sequence shown in SEQ ID NO: 1 corresponds to this. . As will be described later, the present inventor has an insertion site of Phare-1 retrotransposon, which is one of the transposable elements present in the azuki bean genome, unique to the azuki bean variety “Kita no Otome” and is currently cultivated. It has been found that there are no varieties other than Kitaotome.

  In the above detection step, “detecting whether or not a Phare-1 retrotransposon is inserted at a specific site of the azuki bean genome” means detecting the presence or absence of the Phare-1 retrotransposon at a specific site of the azuki bean genome. means. As described above, there is a variety between varietal polymorphisms at the retrotransposon insertion site, that is, there is a phenomenon in which insertion is not observed in another cultivar at the place of the genome where Phare-1 retrotransposon insertion is observed in one cultivar. Therefore, the variety is determined by clarifying the presence or absence of the Phare-1 retrotransposon at a specific site.

  A method for detecting whether or not a retrotransposon is inserted at a specific site in the azuki bean genome is not particularly limited, and a known method may be appropriately selected and used, but a nucleic acid amplification reaction is preferably used. As a method using the nucleic acid amplification reaction, for example, based on the nucleotide sequence of the genomic region adjacent to the Phare-1 retrotransposon of the breed “Kitaotome” in which Phare-1 retrotransposon is inserted at a specific site of the azuki bean genome. Using a primer set that combines the designed primer and the primer designed based on the base sequence of Phare-1 retrotransposon, a nucleic acid amplification reaction is performed using DNA prepared from processed food as a template, and the presence or absence of the amplified product The method of confirming can be mentioned.

  The genomic region adjacent to the Phare-1 retrotransposon may be a region adjacent to the 5 'side or a region adjacent to the 3' side of the retrotransposon sequence. Primers designed based on the nucleotide sequence of the genomic region adjacent to Phare-1 retrotransposon (hereinafter abbreviated as “adjacent primer”) and primers designed based on the nucleotide sequence of Phare-1 retrotransposon (hereinafter referred to as “adjacent primer”) The abbreviation “RTP primer” is only required to be designed in such a direction as to allow nucleic acid amplification by a primer set combining these.

  Adjacent primers may be designed based on the base sequence of the genomic region adjacent to the Phare-1 retrotransposon, but should be designed based on the base sequence of a highly specific portion of the base sequence of the genomic region. Is preferred. This is because if a primer with high sequence specificity can be designed, the appearance of non-specific amplification products can be suppressed, and the determination of the result becomes accurate and easy.

  The RTP primer is preferably designed based on a highly conserved sequence portion of the base sequence of Phare-1 retrotransposon. If RTP primers are designed based on highly conserved sequence parts, there is no need to separately design RTP primers corresponding to each adjacent primer, and a combination of a predesigned RTP primer and each adjacent primer can be used. It becomes possible to constitute a primer set.

  The template DNA for the nucleic acid amplification reaction is DNA prepared from azuki bean processed food. The method for preparing DNA from azuki bean processed food is not particularly limited, and a known method may be used. For example, the method described in “Manual for Testing and Analysis of Genetically Modified Foods” (JAS Analytical Test Handbook 2002) can be mentioned.

  The nucleic acid amplification reaction may be appropriately selected from known nucleic acid amplification means. Specific examples include PCR method, ICAN method, UCAN method, LAMP method, primer extension method and the like, but are not limited thereto. Of these, the PCR method is suitable as an amplification method used in the present raw material type determination method.

  In addition, when adopting the PCR method as an amplification method used in this raw material variety determination method, by using real-time PCR, the proportion of the raw material of the adzuki bean variety “Kita no Otome” to be determined in the azuki bean processed food It is possible to estimate whether it is included. Specifically, a primer set designed based on the retrotransposon insertion site common to all Azuki bean varieties is prepared, and real-time PCR is performed using this primer set using DNA extracted from azuki bean processed food as a template. As a result, the total amount of DNA corresponding to the DNA of all azuki bean varieties used in azuki bean processed foods is determined. Next, real-time PCR is performed using DNA extracted from azuki bean processed food as a template using a primer set designed based on the insertion site unique to Phare-1 retrotransposon “Kita no Otome”. As a result, the amount of “Kita no Otome” DNA corresponding to the DNA of the Azuki cultivar “Kita no Otome” used in azuki bean processed foods is determined. By calculating the ratio of the “Kita no Otome” DNA amount to the total DNA amount, it is possible to quantify how much the raw material of the Azuki cultivar “Kita no Otome” is contained in the azuki processed food.

  Examples of a method for confirming the presence or absence of an amplification product include electrophoresis, but are not limited thereto. The major feature of the method for determining the variety of azuki bean varieties of the present invention is that it detects the presence or absence of an amplification product of an approximate size of interest rather than detecting the difference in length of amplification products. is there. Therefore, it is not necessary to perform strict electrophoresis such as detecting a difference of several bases using a device such as a sequencer, and accurate determination can be performed by simple electrophoresis such as agarose gel electrophoresis. Therefore, there is an advantage that it can be carried out at a low cost and does not require skill in determination and operation of the device.

  As described above, azuki bean variety determination can also be performed based on the presence or absence of an amplification product only at the insertion site if it is an insertion site present only in a specific variety having Phare-1 retrotransposon insertion.

  As a feature point that the present method is particularly suitable for determining azuki bean varieties of azuki bean processed food, the length of the amplified product can be changed. That is, if the adjacent primer is designed at a position far from the RTP primer, the length of the amplified fragment becomes long, and if the adjacent primer is designed at a position near the RTP primer, the length of the amplified fragment becomes short. For example, if the RTP primer is designed at the 5 ′ end or 3 ′ end of the Phare-1 retrotransposon sequence and the adjacent primer is designed to contact the RTP primer, theoretically, the length of both primers combined is about It can also be an amplified fragment. Since DNA obtained from processed foods of azuki bean is usually fragmented, if the length of the amplified fragment is designed to be as short as possible, it can be cultivated even when a highly-fragmented processed food-derived DNA is used as a template. Judgment is possible. Therefore, it can be said that the method for determining the varieties of azuki bean according to the present invention is very suitable for determining the varieties of processed foods in which DNA has deteriorated or fragmented. However, it is not necessary to design the primer so that the length of the amplified fragment is always the shortest. Check how much the DNA contained in the processed azuki bean food is fragmented, and the DNA fragmented to that extent. Primers may be designed so that amplification can be performed using as a template.

  Specifically, the adjacent primer is preferably designed based on the base sequence of the adjacent genomic region within 500 bp from either end of the Phare-1 retrotransposon sequence. More preferably, it is 400 bp or less, More preferably, it is 300 bp or less. Moreover, it is preferable that the fragment length of the amplification product by the combination of the adjacent primer and the RTP primer is 40 bp to 600 bp. More preferably, it is 40 bp-400 bp, More preferably, it is 40 bp-300 bp. If the adjacent primer is designed in such a range and the length of the amplification product is controlled, it becomes possible to apply the method for determining azuki bean cultivar according to the present invention to more azuki bean processed foods.

  The breeding of new varieties of plants is carried out by crossing between different varieties, and in the case of vegetatively breeding plants, by selecting a desired individual from the hybrid first generation individuals obtained by the crossing, In the case of seed-propagating crops, it is promoted by selecting a desired individual or a combination of its parents, etc. from hybrids obtained by the mating or individuals thereof. The insertion site of Phare-1 retrotransposon according to the present invention is the insertion position of Phare-1 retrotransposon between the cultivar thus grown and other varieties currently used for red beans production. And was found as an insertion site that exists only in "Kita no Otome".

  The inventors of the present invention are studying variety determination for “red bean cake”, which is a processed food using azuki bean (Vigna angularis) as a raw material, using the method for determining a variety of azuki bean raw material according to the present invention. The “red bean rice cake” targeted by the inventors is an example of an azuki bean processed food made from azuki bean. The processed food made from azuki bean is particularly limited as long as it contains azuki bean. It is not something.

  In addition, processed foods made from azuki bean such as red bean koji are crushed into a paste in the process of processing. For this reason, it is technically impossible to sort the contents of a product for each used raw material variety in the case where multiple azuki bean varieties are used for processing. It is necessary to determine whether or not a specific adzuki cultivar is used as a raw material from the product itself that is mixed. The Phare-1 retrotransposon according to the present invention determines whether the azuki bean variety “Kita no Otome” is used as a raw material from the product itself in which a plurality of azuki bean varieties including the azuki bean variety “Kita no Otome” is mixed. Nevertheless, it is a preferred retrotransposon. Hereinafter, the unique insertion site of Phare-1 retrotransposon “Kitaotome” will be described.

  First, according to the method of Hirochika et al. (Hirochika et al. (1992) Retrotransposon families in rice. Mol Gen Genet 233: 209-216.), The present inventor applied the reverse transcriptase region of the Ty1-Copia type retrotransposon. The sequence of the enzyme region was isolated from the genome of azuki bean variety “Shumari” by PCR targeting the existing amino acid conserved sequence. Suppression PCR (Siebert et al. (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 23: 1087-1088.) And the terminal LTR sequence was identified and named Phare-1 retrotransposon. The base sequence shown in SEQ ID NO: 1 corresponds to this. Next, in order to find an insertion position that seems to be unique to Phare-1, an adapter addition method to the restriction enzyme-treated end (Sequence-Specific Amplification Polymorphism method, see Non-Patent Document 4, hereinafter abbreviated as “S-SAP method”) ) Was used to compare the insertion site of Phare-1 retrotransposon for the varieties in Table 1.

  As a result, an insertion site that was present in “Kitaotome” but was not present in other cultivated azuki bean varieties was found, and the nucleotide sequence was determined by cloning the insertion site. This base sequence is the base sequence shown in SEQ ID NO: 2. Regarding SEQ ID NO: 2, the base sequence portion of the genomic region adjacent to the Phare-1 retrotransposon is the base sequence of positions 1 to 63, and positions 64 to 203 are present in the Phare- It is the sequence of the terminal part of 1. In order to confirm that this insertion site does not exist in the currently cultivated varieties other than those in Table 1, a sequence based on the nucleotide sequence of the genomic region adjacent to the Phare-1 retrotransposon among these insertion sites cloned. The adjacent primer of No. 4 and the RTP primer of SEQ ID No. 3 were designed based on the base sequence of Phare-1 retrotransposon. PCR was performed using the primers designed in this way, and the Genetic Resources Department of Hokkaido Central Agricultural Experiment Station collected from Japan, China, Korea, etc. -1 The retrotransposon insertion site was examined. As a result, it was found that the insertion site of Phare-1 retrotransposon is unique to the azuki bean cultivar “Kita no Otome” and does not exist in the currently cultivated varieties other than “Kita no Otome” (Table 2, Figure). 1).

  As described above, when the method for determining azuki bean cultivar according to the present invention is performed using the primer consisting of the base sequence shown in SEQ ID NO: 4 and the RTP primer consisting of the base sequence shown in SEQ ID NO: 3, the cultivar “Kitaotome” Can be identified.

  The sequence of SEQ ID NO: 2 is unique to the azuki bean cultivar “Kita no Otome” used in the experiment, and is inserted in the Phare-1 retrotransposon that is not present in other azuki cultivars currently cultivated other than “Kita no Otome”. (Phare-1 retrotransposon sequence and adjacent genomic sequence).

  Azuki bean cultivar identification kit according to the present invention is a kit for carrying out the azuki bean cultivar identification method according to the present invention, and is a primer designed based on the base sequence of the genomic region adjacent to Phare-1 retrotransposon And a primer designed based on the base sequence of Phare-1 retrotransposon. In addition to the primers, the kit preferably contains reagents / instruments used for extracting and purifying DNA from the target processed food, reagents / instruments used for nucleic acid amplification reactions, reagents / instruments used for electrophoresis, and the like.

  Moreover, the azuki bean variety identification kit according to the present invention is a kit for carrying out the azuki raw material variety determining method according to the present invention for azuki bean processed foods containing azuki bean, from the base sequence shown in SEQ ID NO: 3 above. And a primer consisting of the base sequence shown in SEQ ID NO: 4 may be used. In addition to the primers, the kit preferably contains reagents and instruments used for extracting and purifying DNA from the processed food of interest, reagents and instruments used for nucleic acid amplification reactions, reagents and instruments used for electrophoresis, and the like. .

[Embodiment 2]
The method for determining azuki bean varieties according to the present invention is a method for determining the varieties of azuki bean raw materials contained in azuki bean processed food, and whether or not RLVA (Retrotransposon like Vigna angularis) retrotransposon is inserted at a specific site of the azuki bean genome. It is a method including the detection process which determines the kind of azuki bean raw material by detecting this.

  In the above detection step, “detecting whether or not an RLVA retrotransposon is inserted into a specific site of the azuki genome” means detecting the presence or absence of the RLVA retrotransposon at a specific site of the azuki genome. As mentioned above, because there is a polymorphism between varieties at the insertion site of retrotransposon, that is, there is a phenomenon in which insertion is not seen in another cultivar at the place of the genome where RLVA retrotransposon insertion is seen in one cultivar, By determining the presence or absence of RLVA retrotransposon at a specific site, breed determination is performed.

  The RLVA retrotransposon according to the present invention is suitable for determining whether the azuki bean variety “Shumari” is used as a raw material from the product itself in which a plurality of azuki bean varieties including the azuki bean variety “Shumari” are mixed. A retrotransposon. Hereinafter, the insertion site unique to “Sumari” of RLVA retrotransposon will be described.

  The RLVA retrotransposon in the present invention is presumed to be a transposon classified into the LTR (LTR: Long Terminal Repeat) type. The LTR type retrotransposon has repetitive sequences in the same direction at both ends of the full-length sequence. On the other hand, the full-length sequence of RLVA retrotransposon has not been cloned, and only a clone that appears to be a single LTR (Solo LTR) has been identified.

  The RLVA sequence was found as an insert into another azuki bean retrotransposon sequence (SGr-7 family). RLVA discovered as an insertion sequence had the characteristics of an LTR sequence in which the base sequence starts with TG and ends with CA. However, no repetitive sequence (LTR) was found at both ends of the sequence, and no homology with the enzyme region of retrotransposon was found in the sequence itself. As a result of the transposon display (S-SAP: Sequence-Specific Amplification Polymorphism: see Non-patent Document 4) based on the sequence presumed to be LTR, it was found that a large number of copies exist in the genome of azuki bean. It was. From this, it was estimated that the RLVA sequence is a single LTR sequence (Solo LTR).

  Next, in order to find the insertion position considered to be unique to RLVA, the insertion sites of RLVA retrotransposon were compared for the varieties shown in Table 3 using the S-SAP method based on the Solo LTR sequence in RLVA.

  As a result, an insertion site of RLVA retrotransposon and an insertion site specifically found in "Sumari" was found among cultivars currently cultivated. Thus, since an insertion site that was present in "Shumari" but was not present in other cultivated azuki bean cultivars was found, the insertion site was cloned and the nucleotide sequence was determined. This base sequence is the base sequence shown in SEQ ID NO: 5. Regarding SEQ ID NO: 5, the base sequence portion of the genomic region adjacent to the RLVA retrotransposon is the base sequence of positions 99 to 600, and positions 1 to 98 are the end portions of RLVA present at this insertion site Is an array of In order to confirm the specificity of this insertion site, among the cloned insertion sites, the RLVA squeeze primer (RTP primer) of SEQ ID NO: 6 based on the sequence of the terminal portion of RLVA present at this insertion site, An adjacent primer of SEQ ID NO: 7 was designed based on the base sequence of the genomic region adjacent to the RLVA retrotransposon. PCR was carried out using the primers designed in this manner, and the presence of this insertion site of RLVA retrotransposon was examined for 26 types of azuki cultivar shown in Table 3. As a result, it was found that this insertion site of the RLVA retrotransposon is unique to the adzuki cultivar “Shumari” among the currently cultivated varieties (see Table 3 and FIG. 3). Furthermore, in a study conducted at the Hokkaido Central Agricultural Experiment Station targeting 32 domestic azuki varieties and 136 genetic resources introduced from overseas, this insertion site of RLVA retrotransposon It was found to be unique to “mari” and not present in cultivars other than “syumari” (see Tables 4 and 5).

  As described above, when the method for determining azuki bean cultivar according to the present invention is carried out using the RTP primer consisting of the base sequence shown in SEQ ID NO: 6 and the adjacent primer consisting of the base sequence shown in SEQ ID NO: 7, It is possible to identify “mari”.

  The sequence of SEQ ID NO: 5 is unique to the Azuki cultivar “Shumari” used in the experiment, and is an RLVA retrotransposon insertion site (RLVA retrotransposon that does not exist in other cultivated Azuki cultivars other than “Shumari”. Sequence and adjacent genomic sequence).

  Azuki raw material variety identification kit according to the present invention is a kit for carrying out the azuki raw material variety determination method according to the present invention, a primer designed based on the base sequence of the genomic region adjacent to the RLVA retrotransposon, and Any primer including a primer designed based on the base sequence of RLVA retrotransposon may be used. In addition to the primers, the kit preferably contains reagents / instruments used for extracting and purifying DNA from the target processed food, reagents / instruments used for nucleic acid amplification reactions, reagents / instruments used for electrophoresis, and the like.

  Moreover, the azuki bean variety identification kit according to the present invention is a kit for carrying out the azuki raw material variety determining method according to the present invention for azuki bean processed foods containing azuki bean, from the base sequence represented by SEQ ID NO: 6 above. And a primer having the base sequence shown in SEQ ID NO: 7 may be used. In addition to the primers, the kit preferably contains reagents and instruments used for extracting and purifying DNA from the processed food of interest, reagents and instruments used for nucleic acid amplification reactions, reagents and instruments used for electrophoresis, and the like. .

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the embodiments can be obtained by appropriately combining technical means disclosed in different embodiments. The form is also included in the technical scope of the present invention.

[Example 1. Detection of Phare-1 retrotransposon insertion site among azuki bean varieties]
The present inventor examined the insertion site of Phare-1 retrotransposon for 26 kinds of azuki bean varieties and strains possessed by the Hokkaido Central Agricultural Experiment Station Genetic Resources Department. The insertion site of Phare-1 retrotransposon was also investigated in 89 kinds of Azuki bean cultivars and lines collected from Japan, China, Korea, etc.

(1) Material Table 1 shows 26 azuki cultivars and 26 types of strains possessed by the genetic resources department of the Hokkaido Prefectural Central Agricultural Experiment Station that the present inventor examined. In addition to these, Table 2 shows 89 species of azuki bean cultivars / lines collected and collected from Japan, China, Korea, etc. by the Genetic Resource Department of Hokkaido Central Agricultural Experiment Station.

(2) Extraction of genomic DNA Genomic DNA of azuki bean plants is CTAB (cetyltrimethylammonium bromide) method (Takahiko Hayakawa (1997) Tobacco DNA / RNA isolation method. "New version of PCR experiment protocol" Isao Shimamoto and Takuji Sasaki (Extracted based on Shujunsha, Tokyo, 49-56).

(3) Identification of the insertion site unique to Kitaotome The S-SAP method using the Phre-1 LTR sequence for 26 azuki varieties and strains possessed by the Hokkaido Central Agricultural Experiment Station Genetic Resource Department Analysis was performed. The LTR sequence of Phare-1 is the same by PCR from the genome of the adzuki bean cultivar “Shumari” according to the method of Hirochika et al. Described above, targeting the amino acid conserved sequence present in the reverse transcriptase region of Ty1-Copia type retrotransposon. By isolating the sequence of the enzyme region and isolating the extension sequence of the reverse transcriptase region isolated in this way by the Suppression PCR method, it was identified as the sequence present at the end of Phare-1. is there. As a result of the analysis by the S-SAP method, the Phare-1 retrotransposon insertion site found only in “Kita no Otome” among the azuki bean varieties currently used for cultivation included in these 26 types was identified.

(4) PCR amplification The identified insertion site is cloned and its base sequence (SEQ ID NO: 2) is determined. Based on the base sequence of the Phare-1 retrotransposon and the base sequence of the genomic region adjacent thereto in the sequence The P1-DE-T2-1-D2 / PHARE1-LTR-A1 primer was designed. The fragment length of DNA expected to be amplified by PCR using these primers is 103 bp. The base sequence of the designed P1-DE-T2-1-D2 / PHARE1-LTR-A1 primer is as follows.
PHARE1-LTR-A1 primer (RTP primer): 5'-CAGAATGGGCTAAGCCCAATTAACAG-3 '(SEQ ID NO: 3)
P1-DE-T2-1-D2 primer (adjacent primer): 5'-CATGACTTCAGCAAATGTGCCAGC-3 '(SEQ ID NO: 4)
PCR using PHARE1-LTR-A1 primer and P1-DE-T2-1-D2 primer with 1 μl of genomic DNA solution (20 ng / μl) extracted from azuki bean plants shown in Tables 1 and 2 as a template Went. PCR was performed using Ex Taq (TaKaRa) as a reaction solution with a volume of 10 μl, denaturation at 94 ° C. for 4 minutes, 40 cycles of 94 ° C. for 30 seconds, 59 ° C. for 30 seconds, 72 ° C. for 20 seconds, 72 ° C. for 5 minutes. Cycling conditions for the final extension reaction were performed. The amplified product was subjected to agarose gel electrophoresis, stained with ethidium bromide and observed. The results are shown as an insertion specificity test on the left side of FIG.

  In addition, in order to investigate whether or not the quality of DNA that can be amplified by PCR is maintained for the genomic DNA of azuki bean varieties owned by the Hokkaido Central Agricultural Experiment Station Genetic Resources Department (QC: Quality Control), this is used as a template. PCR was performed using a primer set of P1-DE-T2-1-D2 / PHARE1-LTR-A1 that amplifies the Phare-1 insertion site found in all varieties of azuki bean. The results are shown as DNA quality control on the right side of FIG. Samples with no amplification indicate that the quality was degraded. Tables 1 and 2 summarize the detection results of the Phare-1 retrotransposon insertion site. In Table 1 or Table 2, (I) is the detection result performed by the present inventors, and (II) is the detection result performed at the Hokkaido Central Agricultural Experiment Station.

  As shown in Table 1, as a result of detecting the Phare-1 retrotransposon insertion site in 26 species of azuki bean cultivars and lines owned by the Hokkaido Central Agricultural Experiment Station, the azuki bean variety “Kita no Otome (child)” "And Phala-1 retrotransposon insertion were recognized in" Round (Cutting 63) ".

  As shown in FIG. 1 and Table 2, as a result of PCR using the genomic DNA of azuki bean varieties possessed by the Hokkaido Central Agricultural Experiment Station Genetic Resource Department as a template, the azuki bean cultivar “Kita no Otome” (variety number 4) Phare-1 retrotransposon insertion was observed in "Southern Red" (variety number 33), "Iwate Aya 205" (variety No. 37), and "Ishikawa Aoi 36" (variety No. 40).

  As can be seen from the results in Table 1, Phare-1 retrotransposon insertion was found in “Yenyo (Mori 63)”, so the Phare-1 retrotransposon insertion site was “ It was found that it was derived from “Yenyo (Kori 63)”, the mating parent of the “Kitaotome” mating parent {F5 (cross 5th generation) individual—not existing ”.

  As can be seen from the results in Table 2, Phare-1 retrotransposon insertion sites are two types of native species collected in Japan, except for “Kitaotome”, out of 83 types that could be tested effectively. It was also found that the insertion site was found only in the cultivar grown in Miyagi Agricultural Experiment Station in Taisho 13 by inducing the mutation caused by radiation (southern red).

  Three lines and varieties (“Southern Red” (variety number 33), “Iwate Aya 205” (variety No. 37), and “Ishikawa Ai 36” (variety No. 40)) and “Yenyo (Cutting 63)” in Table 2 Is currently only maintained as a genetic resource at the test site. Therefore, the Phare-1 retrotransposon insertion site is considered to be highly specific for “Kita no Otome”.

  In addition to these varieties, it has been clarified that there are no insertions in 7 breeding parent lines of recently cultivated varieties, and 7 individual lines collected in China recently (see Table 1).

  It is clear that the Phare-1 retrotransposon insertion site is derived from the “male leaf (cutting 63)” that is the parent of the mating parent. In addition, the line selection method is adopted for the breeding method of the azuki bean variety “Kita no Otome”. Specifically, after crossing, self-breeding is repeated to increase genetic homozygosity and then selection is started, and the selected individual or line is further self-breeding and selected to achieve excellent homozygosity. It is a training method for selecting strains.

  In consideration of this breeding method, the Phare-1 retrotransposon insertion site is genetically fixed in the breed in the process of selecting the azuki bean breed “Kita no Otome”, and there are polymorphisms in the breed (there are insertions depending on the individual) It is considered that there is no thing).

  From the above, it was found that the insertion site of Phare-1 retrotransposon is unique to the adzuki cultivar “Kita no Otome” and does not exist in varieties other than “Kita no Otome” currently cultivated.

[Example 2. Detection and breeding of Phare-1 retrotransposon insertion sites among azuki bean varieties in azuki processed foods]
(1) Ingredients Mixed red bean koji mixed with various ratios of red bean koji (manufactured by Hokkaido Central Agricultural Experiment Station) made from azuki bean varieties “Kita no Otome”, “Syumari”, and “Erimo Shozu” was used. . The mixed composition of “Kitaotome” red bean paste, “Syumari” red bean paste, and “Erimoshozu” red bean paste in the mixed red bean paste used in this example is as follows.
Mixed red bean paste (i): “Kita no Otome” red bean paste 1 mg, “Shumari” red bean paste 100 mg, “Erimoshozu” red bean paste 100 mg (mixing ratio 1: 100: 100)
Mixed red bean paste (ii): “Kita no Otome” red bean paste 10 mg, “Shumari” red bean paste 100 mg, “Erimo Shozu” red bean paste 100 mg (mixing ratio 1:10:10)
Mixed Azuki Bean (iii): “Kita no Otome” Azuki Bean 50 mg, “Shumari” Azuki Bean 100 mg, “Erimo Shozu” Azuki Bean 100 mg (mixing ratio 1: 2: 2)
Mixed Azuki Bean (iv): “Kita no Otome” Azuki Bean 100 mg, “Shumari” Azuki Bean 100 mg, “Erimo Shozu” Azuki Bean 100 mg (mixing ratio 1: 1: 1)
Mixed Azuki Bean (v): “Kita no Otome” Azuki Bean 0 mg, “Shumari” Azuki Bean 100 mg, “Erimo Shozu” Azuki Bean 100 mg (mixing ratio 0: 1: 1)
(2) Extraction of genomic DNA Each of the mixed red bean pastes (i) to (v) and “Kitaotome” red bean paste (Gran; Hashimoto Foods Industry (Osaka)) were frozen in liquid nitrogen and then crushed in a mortar. Then, add a 1.5 ml tube containing 600 μl of 2% CTAB (cetyltrimethylammonium bromide) solution (100 mM Tris-HCl pH8.0, 1.4 M NaCl, 20 mM EDTA pH8.0, 2% CTAB) and 10 μl mercaptoethanol in advance. In addition to being heated at 65 ° C, after stirring, the mixture was again heated in a water bath at 65 ° C for 30 minutes. Thereafter, 600 μl of chloroform: isoamyl alcohol (= 24: 1) was added, stirred for 5 minutes, centrifuged at 12000 rpm for 15 minutes, and the supernatant was separated into another tube.

  Then, 800 μl of 1% CTAB solution (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% CTAB) was added to the separated supernatant and mixed well, and then allowed to stand at room temperature for 1 hour. Thereafter, the mixture was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was dissolved in 400 μl of 1M cesium chloride (CsCl). In addition, all the centrifugation so far was performed at room temperature.

  To the dissolved DNA solution, 800 μl of 100% ethanol was added and centrifuged at 12000 rpm for 15 minutes at 4 ° C. Discard the supernatant, add 400 μl of 70% ethanol, shake gently, and centrifuge again at 12000 rpm for 5 minutes at 4 ° C. Discard the supernatant and dry completely using an aspirator in a desiccator. 50 μl TE buffer (10 mM) Tris-HCl pH8.01 mM, EDTA pH8.0).

(3) PCR amplification Under the same primers and PCR conditions as in Example 1 (4), various mixed red bean pastes, “Kitaotome” red bean cakes, and “Kitaotome”, “Sumari”, “Erimo Shozu” PCR was performed using 1 μl of a genomic DNA solution (20 ng / μl) extracted from the plant as a template.

  As a positive control primer, Gr7-LTR-3end / NAR33-F37-A2 primer that can be detected in all varieties of “Kita no Otome”, “Shumari”, and “Erimoshozu” is used, as described above. PCR was performed. The DNA fragment length expected to be amplified by PCR using these primers is 125 bp.

  The results of PCR using P1-DE-T2-1-D2 / PHARE1-LTR-A1 primer using genomic DNA extracted from various mixed red soybean cake as a template are shown in Fig. 2 (A), Gr7-LTR-3end The results of PCR using the / NAR33-F37-A2 primer are shown in FIG.

  As is clear from the figure, not only “Kitaotome” was used as the raw material for the Azuki bean (lane 6), but also the Azuki bean produced from “Kita no Otome”, “Shumari”, and “Erimo Shozu”. 1: 100: 100 (mixed red bean paste (i)), 1:10:10 (mixed red bean paste (ii)), 1: 2: 2 (mixed red bean paste (iii)), 1: 1: 1 ( Even in the mixture at the ratio of the mixed red bean paste (iv)), a fragment (about 100 bp) of the expected length inherent to “Kita no Otome” was amplified (see lanes 1 to 4). On the other hand, the fragment was not amplified in the mixed red bean koji (v) in which the ratio of the red bean koji produced from “Shumari” and “Erimoshozu” was 1: 1. In PCR using Gr7-LTR-3end / NAR33-F37-A2 primer that can be detected in all varieties, a fragment (about 125 bp) of the expected length of all test materials is amplified. In the PCR for the insertion site unique to “Kita no Otome”, amplification was not observed for the test materials that do not contain “Kita Otome”. It can be seen that this is not because the genomic DNA required for amplification of about 100 bp was not included.

  From the above results, it is possible to identify azuki bean varieties to be identified in the red bean koji using a plurality of azuki bean varieties, and the mixing ratio of the azuki bean varieties to be identified can be identified. Was found to be distinguishable even when the value was as low as about 0.5%.

[Example 3. Detection of RLVA retrotransposon insertion site among azuki bean varieties)
The present inventor first examined the insertion site of the RLVA retrotransposon for azuki bean varieties and 26 types of lines owned by the Genetic Resource Department of the Hokkaido Central Agricultural Experiment Station. And the insertion site | part specific to "Sumari" was found among the cultivars of the azuki bean. Furthermore, in addition to these, 32 domestic azuki varieties possessed by the Hokkaido Central Agricultural Experiment Station and 136 genetic resources introduced from overseas were examined for the specificity of the RLVA retrotransposon insertion site.

(1) Material Table 3 shows 26 kinds of azuki bean cultivars / lines possessed by the Genetic Resource Department of the Hokkaido Central Agricultural Experiment Station, which the present inventor examined. Tables 4 and 5 show the 32 domestic azuki bean varieties owned by the Hokkaido Central Agricultural Experiment Station and the 136 genetic resources introduced from overseas.

(2) Extraction of genomic DNA Genomic DNA of azuki bean plants is CTAB (cetyltrimethylammonium bromide) method (Takahiko Hayakawa (1997) Tobacco DNA / RNA isolation method. "New version of PCR experiment protocol" Isao Shimamoto and Takuji Sasaki (Extracted based on Shujunsha, Tokyo, 49-56).

(3) Identification of the insertion site unique to "Shumari" Analyzes by S-SAP using the RLVA LTR sequence for 26 species of azuki bean cultivars and lines possessed by the Genetic Resource Department of Hokkaido Central Agricultural Experiment Station went. The RLVA LTR sequence was originally found as an insertion sequence from the genome of the adzuki bean variety "Erimoshouzu" into another retrotransposon sequence (SGr-7 family) of adzuki bean. As a result of the analysis by the S-SAP method, the RLVA retrotransposon insertion site found only in “Shumari” was identified among the azuki bean varieties currently used for cultivation included in these 26 types.

(4) PCR amplification The identified insertion site is cloned to determine its base sequence (SEQ ID NO: 5). Based on the base sequence of the RLVA retrotransposon and the base sequence of the genomic region adjacent thereto, S8D0 primer (RLVA Shumari) and S8U9_4 primer were designed. The fragment length of DNA expected to be amplified by PCR using these primers is 107 bp. The base sequence of the designed primer is as follows.
S8D0 primer (RLVA Sukumari primer): 5'-GGTGACAGGCGGATATACTC-3 '(SEQ ID NO: 6)
S8U9_4 primer (adjacent primer): 5'-CCTAGAAGTCCTTTGAAATATTCCCTTAG -3 '(SEQ ID NO: 7)
Using S8D0 primer and S8U9_4 primer, PCR was carried out using 1 μl of genomic DNA solution (20 ng / μl) extracted from azuki bean plant shown in Table 3 as a template. PCR was performed using Ex Taq (TaKaRa) as a reaction solution having a volume of 10 μl, denaturation at 94 ° C. for 4 minutes, 30 cycles of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, 72 ° C. for 1 minute, 72 ° C. for 5 minutes. Cycling conditions for the final extension reaction were performed. The amplified product was subjected to agarose gel electrophoresis, stained with ethidium bromide and observed.

  Figure 3 shows the results of PCR using S8D0 and S8U9_4 primers, using the genomic DNA of the adzuki cultivar held by the Hokkaido National Agricultural Experiment Station Genetic Resources Department as a template. Tables 3 to 5 show RLVA retrotransposon insertions. The site detection results were summarized. Table 3 shows detection results performed by the present inventors, and Tables 4 and 5 show detection results performed at the Hokkaido Central Agricultural Experiment Station.

  As shown in Fig. 3 and Table 3, as a result of detecting the RLVA retrotransposon insertion site for 26 kinds of azuki bean cultivars / lines possessed by the Hokkaido National Agricultural Experiment Station Genetic Resource Department, azuki bean cultivar "Sumari" and its The insertion of RLVA retrotransposon was observed only in the azuki genome of the mating parent “Teniku 130”.

  As shown in Table 4, as a result of PCR using the genomic DNA of Azuki domestic varieties possessed by the Hokkaido National Agricultural Experiment Station Genetic Resource Department as a template, among these 32 varieties, the “Sumari” azuki genome Only RLVA retrotransposon was inserted.

  As shown in Table 5, as a result of PCR using the genomic DNA of the Azuki genetic resources from overseas, which is owned by the Hokkaido Central Agricultural Experiment Station, as a template, among these 136 varieties, It was confirmed that none of the cultivars have RLVA retrotransposon insertion sites.

  As can be seen from the results in Table 3, since the insertion of RLVA retrotransposon was found in “Teniku 130”, the RLVA retrotransposon insertion site was the “Shumari” mating parent in the “Shumari” lineage. It turned out to be derived from a certain "Teniku 130".

  Moreover, as can be seen from the results in Table 4, the RLVA retrotransposon insertion site was not found in domestic cultivars other than “Shumari”. Also, from the results in Table 5, it is clear that it is not found in foreign varieties. Therefore, the above-mentioned insertion site of RLVA retrotransposon is considered highly specific for “Sumari”.

  It is clear that the RLVA retrotransposon insertion site is derived from “Jukuiku 130”, the parent of the mating parent. In addition, the line selection method is adopted for the breeding method of the adzuki cultivar “Shumari”. Specifically, after crossing, self-breeding is repeated to increase genetic homozygosity and then selection is started, and the selected individual or line is further self-breeding and selected to achieve excellent homozygosity. It is a training method for selecting strains.

  Considering this breeding method, the RLVA retrotransposon insertion part is genetically fixed in the breed in the process of selecting the azuki bean variety "Shumari", and there is no polymorphism in the breed (there is no insertion depending on the individual) It is considered that there is no such thing.

  INDUSTRIAL APPLICABILITY The present invention can be widely used in the food industry because it can accurately and easily determine the azuki raw material variety of azuki bean processed food. Furthermore, if it is used for illegal labeling of foods and inspection of imported processed foods made from varieties grown illegally overseas, it is expected to greatly contribute to the control of illegal foods.

The left half of the figure attached with the insertion specificity test using the genomic DNA of the adzuki bean held by the Hokkaido Central Agricultural Experiment Station Genetic Resources Department as a template is the P1-DE-T2-1-D2 / PHARE1-LTR-A1 primer The right half of the figure of the PCR amplification product using Gr7-LTR-3end / NAR33-F37-A2 is an image showing the electrophoresis result of the PCR amplification product using Gr7-LTR-3end / NAR33-F37-A2. (A) is an image showing the results of electrophoresis of PCR amplification products using P1-DE-T2-1-D2 / PHARE1-LTR-A1 primers using genomic DNA extracted from mixed red bean paste as a template, B) is an image showing the results of electrophoresis of PCR amplification products using Gr7-LTR-3end / NAR33-F37-A2 primers. It is an image showing the results of electrophoresis of PCR amplification products using S8D0 primer and S8U9_4 primer using the genomic DNA of azuki bean varieties possessed by the Genetic Resource Department of Hokkaido Central Agricultural Experiment Station.

Claims (13)

A method for determining a variety of azuki bean ingredients contained in azuki bean processed food,
By detecting whether or not a retrotransposon is inserted at a specific site of the azuki bean genome, and examining the presence or absence of the Phare-1 retrotransposon insertion site specific to Kitotome, which is a specific azuki bean variety, shown in SEQ ID NO: 2 , A method for determining a variety of azuki bean raw material, comprising a detection step of determining a variety of azuki bean raw material. A method for determining a variety of azuki bean ingredients contained in azuki bean processed food,
By detecting whether or not a retrotransposon is inserted at a specific site in the azuki bean genome, and checking for the presence of the RLVA retrotransposon insertion site specific to the particular azuki bean cultivar, Suzumari, shown in SEQ ID NO: 5, A method for determining a variety of azuki bean raw material, comprising a detection step of determining a variety. The nucleotide sequence of the genomic region adjacent to the Phare-1 retrotransposon at the site of insertion of the Phare-1 retrotransposon unique to the virgin that is the specific adzuki bean cultivar shown in SEQ ID NO: 2 (from the first position in SEQ ID NO: 2) A primer designed based on the 63rd base sequence),
  DNA prepared from azuki bean processed food as a template using a primer set combined with a primer designed based on the base sequence of Phare-1 retrotransposon (base sequence from position 64 to position 203 in SEQ ID NO: 2) The method for determining azuki bean cultivar according to claim 1, wherein a nucleic acid amplification reaction is performed to confirm the presence or absence of an amplification product. The nucleotide sequence of the genomic region adjacent to the RLVA retrotransposon at the site of insertion of the RLVA retrotransposon specific to the particular adzuki bean cultivar shown in SEQ ID NO: 5 (bases 99 to 600 in SEQ ID NO: 5) Primers designed based on the sequence),
Nucleic acids using DNA prepared from azuki bean processed food as a template using a primer set combined with a primer designed based on the nucleotide sequence of RLVA retrotransposon (base sequence 1 to 98 in SEQ ID NO: 5) The method for determining azuki bean raw material varieties according to claim 2, wherein a method of performing an amplification reaction and confirming the presence or absence of an amplification product is used. 5. The method for determining azuki bean cultivar according to claim 3 or 4, wherein a fragment length of the amplification product is 40 bp to 600 bp. In the above detection step, the presence or absence of a Phare-1 retrotransposon insertion site unique to Kitotome, which is a specific adzuki bean variety shown in SEQ ID NO: 2, is detected,
The primer designed based on the base sequence of the genomic region adjacent to the Phare-1 retrotransposon is a primer consisting of the base sequence shown in SEQ ID NO: 4,
The above primers designed based on the Phare-1 retrotransposon nucleotide sequence, adzuki bean raw material varieties determination method according to claim 3, characterized in that a primer comprising the base sequence shown in SEQ ID NO: 3. In the above detection step, the presence or absence of a RLVA retrotransposon insertion site specific to Suzumari, which is a specific azuki bean variety, shown in SEQ ID NO: 5 ,
The primer designed based on the base sequence of the genomic region adjacent to the retrotransposon is a primer consisting of the base sequence shown in SEQ ID NO: 7,
The primer designed based on the base sequence of the retrotransposon is a primer composed of the base sequence shown in SEQ ID NO: 6. A primer used to determine the variety of azuki bean raw material contained in azuki bean processed food,
A primer for determining azuki bean raw material varieties, comprising the base sequences shown in SEQ ID NOs: 3 and 4. A primer used to determine the variety of azuki bean raw material contained in azuki bean processed food,
A primer for determining the varieties of azuki bean, comprising the base sequences shown in SEQ ID NOs: 6 and 7. Azuki raw material type identification kit for determining the type of Azuki raw material contained in azuki processed food,
The nucleotide sequence of the genomic region adjacent to the Phare-1 retrotransposon at the site of insertion of the Phare-1 retrotransposon unique to the virgin that is the specific adzuki bean cultivar shown in SEQ ID NO: 2 (from the first position in SEQ ID NO: 2) And a primer designed based on the base sequence of the Phare-1 retrotransposon ( base sequence from position 64 to position 203 in SEQ ID NO: 2). Azuki raw material variety discriminating kit characterized by that. The kit for discriminating varieties of azuki bean according to claim 10, comprising a primer consisting of the base sequence shown in SEQ ID NO: 3 and a primer consisting of the base sequence shown in SEQ ID NO: 4. Azuki raw material type identification kit for determining the type of Azuki raw material contained in azuki processed food,
The nucleotide sequence of the genomic region adjacent to the RLVA retrotransposon at the site of insertion of the RLVA retrotransposon specific to the particular adzuki bean cultivar shown in SEQ ID NO: 5 (bases 99 to 600 in SEQ ID NO: 5) Sequence) and a primer designed based on the base sequence of RLVA retrotransposon (base sequence Nos. 1 to 98 in SEQ ID NO: 5). Variety identification kit. The kit for discriminating varieties of azuki bean according to claim 12, comprising a primer consisting of the base sequence shown in SEQ ID NO: 6 and a primer consisting of the base sequence shown in SEQ ID NO: 7.