JP2008096317A - Method for identifying variety by composition ratio of flavonoid that plants contain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a variety identification method which is developed over a development period which is shorter than a conventional method and which requires lower development cost. <P>SOLUTION: Since the types of flavonoids and the relative amounts of various types of flavonoids do not change by plant tissues or their development stages, but will change by varieties, as a result of the preparation, comparison, and examination of flavonoid-containing extracts at various periods from plant tissues, it is possible to use the relative amounts of flavonoids in the flavonoid-containing extracts for the identification of varieties of plants. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention belongs to a plant variety identification method.

  Many plants have hundreds to thousands of varieties, and as a result of the development of new varieties by breeding, the number of varieties is increasing year by year.

  For breeding excellent varieties, techniques such as crossing are used, but for efficient and appropriate breeding, a cultivar identification method is necessary as a prerequisite.

  While breeding of useful varieties by growers has become popular, infringement of breeder rights has become a problem in recent years. In order to sufficiently protect growers from infringement of rights, a simple and accurate method for identifying varieties is required.

  In conventional variety identification, analysis of genomic DNA, which is genetic information of a variety, has been performed exclusively. This is because genomic DNA is not affected by the plant growth time or the breeding environment, and an accurate variety identification result can be obtained.

  Examples of the method for identifying a variety using genomic DNA include a genomic fingerprint method (for example, AFLP (Amplified Fragment Length Polymorphism)). However, this method requires a large amount of DNA necessary for the reaction, has a complicated process compared to PCR, and cannot determine a change in the level of one base other than the restriction enzyme recognition site. Has drawbacks.

  As a method for identifying a variety using genomic DNA, there is a method using PCR. Examples of the variety identification method using PCR include the microsatellite method, the RAPD method (random amplified polymorphic DNA method), and the RAPD-derived STS (sequence tagged site) method. The microsatellite method is a method of amplifying a several base repetitive sequence (microsatellite) present in genomic DNA and discriminating a polymorphism such as a difference in the number of repetitive sequences. Since this method detects a slight PCR amplification fragment length difference, that is, a difference in the number of repeats of a repetitive sequence of several bases, a high-concentration gel is prepared and carefully analyzed when analyzing the amplification product. It is necessary to perform electrophoresis for a long time. Moreover, it has the fault that single nucleotide polymorphism cannot be discriminated. The RAPD method is a method for discriminating polymorphisms by amplifying genomic DNA using a random primer of generally about 12 bases and detecting the amplified band pattern by electrophoresis. In the RAPD method, although primer preparation is simple, it is necessary to set PCR conditions precisely, and different results occur depending on slight differences in conditions (for example, temperature). Therefore, in the RAPD method, it is difficult to set PCR conditions, and even if the conditions are set, reproducibility is poor and stable discrimination is difficult. In addition, the RAPD method is affected by the purity of the template DNA, and further has a drawback that multiple PCRs and electrophoresis are required. The RAPD-derived STS method is a method in which a specific band identified by the RAPD method is sequenced, primers are designed based on the sequence information, and PCR is performed. Since this method is based on a band identified by the RAPD method, it can be used only in a genomic portion having a polymorphism with a considerably different base sequence, and it is difficult to discriminate single base substitution. The SNPs method is a method capable of discriminating genotypes of single nucleotide polymorphisms, but for stable discrimination, an expensive apparatus such as a multi-label counter of PerkinElmer (Boston, MA, USA) is used. Need to use.

  As described above, the conventional method for identifying varieties uses genomic DNA, but has the disadvantage that it takes time and effort to develop a primer design suitable for the cultivar to be identified.

On the other hand, although there has been a report on the content ratio of tulip flower color and anthocyanin (Non-Patent Documents 1 to 6), teaching and suggesting the use of the content ratio of anthocyanins for the identification of varieties has been made. Absent. Moreover, although there are few reports on flavonoids other than anthocyanins (Non-Patent Documents 7 to 8), many of them have been found in tulip tissues for the first time in the present invention.

Prior art document information relating to the invention of this application includes the following.
Torskangerpoll et al., Biochemical Sytematics and Ecology, (2005) 33, pp499-510 Torskangerpoll et al., Phytochemistry, (1999) 52, pp1687-1692 Niewhof et al., Biochemical Systematics and Ecology, (1990) 18, pp399-404 Niewhof et al., Netherlands Journal of Agricultural Science, (1989) 37, pp365-370 Van Eijk et al., Euphytica, (1987) 36, pp855-862 Haleny andAsen, Plant Physiology, 34, (1959), pp494-499 Budzianowski, Phytochemistry, 30, (1991), pp1679-1682 Budzianowskiand Skrzypczakowa, Phytochemistry, (1978), 17, p2044

  It is a problem to be solved by the present invention to overcome these disadvantages of the conventional method and to develop a variety identification method that has a shorter development period and requires less development cost than the conventional method.

  As a result of preparing and comparing flavonoid-containing extracts from plant tissues at various times, the present inventors have found that the types of flavonoids and the relative amounts of various flavonoids are constant depending on the plant tissues and the growth stage. And it discovered that it changed with varieties, Therefore, the relative amount of the flavonoid in a flavonoid containing extract could be used for the plant | cultivar identification, and solved the said subject.

Thus, according to the present invention, the following is provided:
(Item 1) A method for determining whether or not a first plant and a second plant are of the same variety, the method comprising:
a) preparing a flavonoid-containing extract from the first plant and determining its composition;
b) preparing a flavonoid-containing extract from a second plant and determining its composition; and c) comparing the composition of the flavonoid of the first plant with the composition of the flavonoid of the second plant, Determining whether the first plant and the second plant are of the same variety,
Including the method.

  (Item 2) The flavonoid is selected from the group consisting of anthocyanins, flavonols, flavones, isoflavones, flavanones, flavanols, flavan-3,4-diols, flavan-3-ols, anthocyanidins, aurones, chalcones, and dihydrochalcones. The method according to item 1.

  (Item 3) The method according to item 2, wherein the flavonoid is selected from the group consisting of anthocyanins and flavonols.

  (Item 4) The method according to item 1, wherein the plant is selected from the group consisting of tulip, chrysanthemum, rose, carnation, orchid, petunia, torenia, spinach, lettuce, rice, and wheat.

  (Item 5) The method according to item 4, wherein the plant is a tulip.

  (Item 6) The method according to item 1, wherein the flavonoid-containing extract is prepared from an organ selected from the group consisting of petals, leaves, fruits, seeds, stems, and roots of the plant.

  (Item 7) The method according to item 6, wherein the flavonoid-containing extract is prepared from petals of the plant.

  (Item 8) The method according to item 7, wherein the flavonoid-containing extract is prepared at the time of flowering or when a bud is formed.

  (Item 9) The method according to item 8, wherein the flavonoid-containing extract is prepared at the time of flowering.

(Item 10) A method for identifying the variety of a sample plant, comprising:
a) preparing a flavonoid-containing extract from a sample plant and determining its composition; and
b) comparing the composition determined in step (a) with the flavonoid composition of a known variety to identify the variety of the sample plant;
Including the method.

  (Item 11) The flavonoid is selected from the group consisting of anthocyanins, flavonols, flavones, isoflavones, flavanones, flavanols, flavan-3,4-diols, flavan-3-ols, anthocyanidins, aurones, chalcones, and dihydrochalcones. The method according to item 10.

  (Item 12) The method according to item 11, wherein the flavonoid is selected from the group consisting of anthocyanins and flavonols.

  (Item 13) The method according to item 10, wherein the plant is selected from the group consisting of tulip, chrysanthemum, rose, carnation, orchid, petunia, torenia, spinach, lettuce, rice, and wheat.

  (Item 14) The method according to item 13, wherein the plant is a tulip.

  (Item 15) The method according to item 10, wherein the flavonoid-containing extract is prepared from an organ selected from the group consisting of petals, leaves, fruits, seeds, stems, and roots of the plant.

  (Item 16) The method according to item 15, wherein the flavonoid-containing extract is prepared from petals of the plant.

(Item 17) The method according to item 16, wherein the flavonoid-containing extract is prepared at the time of flowering or when a bud is formed. (Item 18) The flavonoid-containing extract is prepared at the time of flowering. The method described.

(Item 19) The method according to Item 10, further comprising:
c) Sample plant and known plant:
Plant type, plant height, stem thickness, stem color, leaf shape, leaf size, leaf color, leaf gloss, leaf number, leaf formation angle, presence of white powder on leaves, bulb shape, bulb size , Bulb color, bulb hull color and bulbous three bud shape, flower shape, flower size, flower color, petal shape, petal color, petal number, number of stamens, flower color, yak Selected from the group consisting of color, pollen color, floral pattern length, floral pattern thickness, floral pattern color, flowering period, aboveground withering period, branch bloom, disease resistance, and insect resistance Comparing traits;
Including the method.

  (Item 20) Compound having the following structure Quercetin 3-rhamnosyl- (1 → 6) -galactoside-7-glucronide

(Item 21) Compound having the following structure Kaempferol 3-rhamnosyl- (1 → 6) -galactoside-7-glucronide:

(Item 22) A method for determining whether or not a first plant and a second plant are of the same variety, the method comprising:
a) preparing a flavonoid-containing extract from the first plant and determining the composition of the compound of item 20 and the compound of item 21;
a) preparing a flavonoid-containing extract from the second plant and determining the composition of the compound according to item 20 and the compound according to item 21; and c) the compound according to item 20 and the compound according to item 21 Regarding the composition of the compound, the composition of the flavonoid of the first plant and the composition of the flavonoid of the second plant are compared to determine whether the first plant and the second plant are of the same variety. The process of
Including the method.

  (Item 23) The method according to item 22, wherein the plant is a tulip.

  (Item 24) The method according to item 22, wherein the flavonoid-containing extract is prepared from petals of the plant.

(Item 25) A method for identifying a variety of a sample plant, the following:
a) preparing a flavonoid-containing extract from a sample plant and determining the composition of the compound of item 20 and the compound of item 21;
b) comparing the composition determined in step (a) with the composition of the compound according to item 20 and the compound according to item 21 in a known variety to identify the variety of the sample plant;
Including the method.

  (Item 26) The method according to item 25, wherein the plant is a tulip.

  (Item 27) The method according to item 25, wherein the flavonoid-containing extract is prepared from petals of the plant.

(Item 28) A method for identifying a plant variety from which a flavonoid-containing mixture isolated from nature is derived, comprising the following:
a) determining a flavonoid composition in a flavonoid-containing mixture isolated from nature; and
b) comparing the composition determined in step (a) with the flavonoid composition of known varieties to identify the plant variety from which the flavonoid-containing mixture is derived;
Including the method.

  (Item 29) The flavonoid is selected from the group consisting of anthocyanins, flavonols, flavones, isoflavones, flavanones, flavanols, flavan-3,4-diols, flavan-3-ols, anthocyanidins, aurones, chalcones, and dihydrochalcones. 29. The method according to item 28.

  According to the present invention, plant varieties can be identified and compared easily and rapidly as compared with cultivar identification and cultivar comparison methods based on genomic information. Further, according to the present invention, it is possible to easily and quickly identify and compare plant varieties that have heretofore been difficult to distinguish from morphological features.

  The present invention will be described below. Throughout this specification, it should be understood that expression in the singular includes the concept of the plural unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  As used herein, the term “flavonoid” means a phenyl compound having a skeleton (phenylchroman skeleton) in which two benzene rings (A ring and B ring in the figure below) are connected by three carbon atoms in the basic structure. It is a generic name for the group, and refers to flavan derivatives and glycosides thereof. Flavan is free of double bonds or carbonyl groups in the C ring and has the following basic structure:

Flavonoids include, for example, flavonols, flavones, isoflavones, flavanones, flavonols, flavan-3,4-diols, flavan-3-ols (flavanols), anthocyanidins, aurones, chalcones, and dihydrochalcones. It is not limited. In addition to these basic skeletons, diversity exists in hydroxylation, condensation, and prenylation at different positions, and there are various structures. Many anthocyanins exist as glycosides, such as glycosides containing anthocyanidins as aglycones.

As used in the present invention, the term “flavonol” is a compound in which a hydroxyl group is bonded to the 3-position of the C ring of flavone, and a compound having the following general formula and a skeleton of a compound having the following formula have a hydroxyl group, Substituted compounds such as methoxyl groups or other atomic groups, as well as glycosides of these compounds:

As used in the present invention, the term “flavone” is a compound having one double bond in the C ring and a carbonyl group at the 4-position of the C ring, and having the following general formula: A compound in which a hydroxyl group, a methoxyl group or other atomic group is substituted on the skeleton of a compound having the formula: and a glycoside of these compounds:

When used in the present invention, the term “isoflavone” is a flavone in which the binding position of the B ring is replaced from the 2nd position to the 3rd position of the C ring, and the compound having the following general formula: A compound in which the skeleton of the compound has a hydroxyl group, a methoxyl group or other atomic group substituted, and a glycoside of these compounds:

As used in the present invention, the term “flavanone” means a compound having a carbonyl group at the 4-position of the C ring, a compound having the following general formula, and a hydroxyl group or a methoxyl group in the skeleton of the compound having the following formula: Alternatively, it refers to substituted compounds such as other atomic groups, and glycosides of these compounds:

When used in the present invention, the term “flavanonol” is a compound in which a hydroxyl group is bonded to the 3-position of the C-ring of flavan, a compound having the following general formula, and a skeleton of a compound having the following formula, Substituted compounds such as methoxyl groups or other atomic groups, as well as glycosides of these compounds:

When used in the present invention, the term “flavan-3,4-diol” is a compound in which a hydroxyl group is bonded to the 3rd and 4th positions of the C ring of a flavan, a compound having the following general formula, and the following formula: A compound in which a hydroxyl group, a methoxyl group, or other atomic group is substituted on the skeleton of a compound having a compound, and a glycoside of these compounds:

When used in the present invention, the term “flavan-3-ol” is a compound in which a hydroxyl group is bonded to the 3-position of the C ring of flavan, a compound having the following general formula, and a skeleton of the compound having the following formula: And a substituted compound such as hydroxyl group, methoxyl group or other atomic group, and glycosides of these compounds:

As used in the present invention, the term “anthocyanidin” means a compound having two double bonds in the C ring, wherein the oxygen at the 1-position of the C ring is sufficiently charged, and having the following general formula: , A compound in which the skeleton of a compound having the following formula is substituted with a hydroxyl group, a methoxyl group or other atomic group, and a glycoside of these compounds:

As used in the present invention, the term “aurone” means a compound having the following general formula, and a compound having a hydroxyl group, a methoxyl group or other atomic group substituted on the skeleton of the compound having the following formula, and A glycoside of these compounds:

As used in the present invention, the term “chalcone” means a compound having the following general formula, and a compound having the following formula, which is obtained by reducing the oxygen at the 1-position of the C ring of flavone and opening the ring at the 2-position. A compound in which the skeleton is substituted with a hydroxyl group, a methoxyl group or other atomic group, and a glycoside of these compounds:

As used in the present invention, the term “dihydrochalcone” is a compound in which the oxygen at the 1-position of ring C of flavanone is reduced and opened at the 2-position, and has the following general formula and the following formula: A compound in which a hydroxyl group, a methoxyl group or other atomic group is substituted on the skeleton of the compound, and a glycoside of these compounds:

As used herein, the term “plant” refers to not only plant bodies but also plant cells, seeds, tissues, organs, or parts thereof, unless specifically limited. Plant tissues or organs include, but are not limited to, petals, leaves, fruits, seeds, stems, and roots.

  As used herein, the term “flavonoid-containing extract” refers to an extract prepared from a plant and containing a flavonoid.

  As used herein, the term “composition” of flavonoids means the relative proportions of the various compound species encompassed by the flavonoids. Accordingly, in this specification, the term “composition” is used interchangeably with “composition ratio”.

(Preparation of flavonoid-containing extract)
All kinds of plant species can be used as the plant species to be the subject of the present invention. Examples of the plant species that are the subject of the present invention include flowers, plants other than flowers, and flower garden plants. Such plant species include, but are not limited to, for example, tulips, chrysanthemums, roses, carnations, orchids, petunias, torenia, spinach, lettuce, rice, and wheat. In the present invention, when preparing a flavonoid-containing extract from a plant, for example, plant petals, leaves, fruits, seeds, stems, and roots can be used. Any kind of plant species can be used as the plant species. When the flavonoid-containing extract is prepared from plant petals, the flavonoid-containing extract is prepared at the time of flowering or when a bud is formed.

  As a flavonoid extraction method, any method available in this field can be used. Examples of the flavonoid extraction method include, but are not limited to, water extraction, organic solvent extraction, and aqueous solution extraction of an organic solvent and water. As an organic solvent used for organic solvent extraction, an organic solvent generally used for organic solvent extraction in this field can be used. Examples of such organic solvents include, but are not limited to, methanol, ethanol, butanol, acetonitrile, dimethyl sulfoxide, and aqueous solutions thereof. The temperature and time for organic solvent extraction can be appropriately selected by those skilled in the art. In the present invention, it is possible to compare and identify plant varieties using the types and composition ratios of flavonoids in the flavonoid-containing extract without considering the yield of flavonoids per plant weight. Even if the flavonoid extraction yield changes due to differences in flavonoid extraction methods and conditions, it does not affect the implementation of the present invention.

(Analysis of flavonoid-containing extracts)
Various kinds of analytical methods known in the art can be used for the type and composition ratio (relative amount) of the flavonoid in the flavonoid-containing extract prepared in the present invention. Examples of such analytical methods include, but are not limited to, HPLC, gas chromatography, column chromatography, mass spectrometry, and thin layer chromatography.

  As a typical analysis method, a known type of a known amount of flavonoid is analyzed using the above analysis method. When a substance that exhibits the same behavior (for example, mobility, retention time, etc.) as a known flavonoid is present in the sample, the known flavonoid is present in the sample. Furthermore, the amount of the flavonoid in the sample can be determined by evaluating the amount of the flavonoid in the sample as a relative value of the amount of the known amount of the flavonoid.

  In addition, when a plurality of flavonoids exhibiting the same behavior exist in a certain analysis method, different analysis methods and / or analysis conditions are used to appropriately adjust the plurality of flavonoids to behave differently.

(error)
The type and composition ratio of the flavonoid in the flavonoid-containing extract calculated in the method of the present invention includes an error. Such errors vary with experimental conditions and can be, for example, 5%, 4%, 3%, 2%, and 1%. For example, when the composition ratio of a specific flavonoid “A” in all flavonoids is 50%, the composition ratio of flavonoid “B” is 20%, and the measurement error is 10%, the composition ratio of flavonoid “A” Is 45% to 55%, and the composition ratio of the flavonoid “B” is 18% to 22%.

(Variety)
In accordance with the present invention, a method is provided for determining whether a first plant and a second plant are of the same variety. The method is
a) preparing a flavonoid-containing extract from the first plant, determining its composition;
b) preparing a flavonoid-containing extract from a second plant, determining its composition, and
c) by comparing the composition of the flavonoid of the first plant with the composition of the flavonoid of the second plant and determining whether the first plant and the second plant are of the same variety Done. In this variety identification, not only the flavonoid composition, but also the plant type, plant height, stem thickness, stem color, leaf shape, leaf size, leaf color, leaf gloss, number of leaves, leaf formation angle White leaf powder, bulb shape, bulb size, bulb color, bulb hull color and bulbous three bud shape, flower shape, flower size, flower color, petal shape, petal color, Number of petals, number of stamens, flower color, yak color, pollen color, flower pattern length, flower pattern thickness, flower pattern color, flowering period, above-ground death period, branch bloom, disease resistance And insect resistance can also be considered. In comparing the flavonoid composition and determining the identity, measurement errors need to be taken into account. Two species having only differences within the error range are considered to be the same species.

  Various substances can be used as the flavonoid. For example, flavonoids include, but are not limited to, anthocyanins, flavonols, flavones, isoflavones, flavanones, flavanols, flavan-3,4-diols, flavan-3-ols, anthocyanidins, aurones, chalcones, and dihydrochalcones. . In particular, anthocyanins and flavonols are preferable.

Furthermore, in this invention, the method of identifying the kind of sample plant is provided. This method
a) preparing a flavonoid-containing extract from the sample plant, determining its composition; and
b) by comparing the composition determined in step (a) with the flavonoid composition of known varieties to identify the varieties of the sample plants.

  In the above method, it is necessary to accumulate data of flavonoid compositions of a plurality of varieties in advance. The accumulated flavonoid composition data is compared and compared with the experimental results, and the variety of the strain that is the subject of the experiment is determined. In this variety identification, not only the flavonoid composition, but also the plant type, plant height, stem thickness, stem color, leaf shape, leaf size, leaf color, leaf gloss, number of leaves, leaf formation angle White leaf powder, bulb shape, bulb size, bulb color, bulb hull color and bulbous three bud shape, flower shape, flower size, flower color, petal shape, petal color, Number of petals, number of stamens, flower color, yak color, pollen color, flower pattern length, flower pattern thickness, flower pattern color, flowering period, above-ground death period, branch bloom, disease resistance And insect resistance can also be considered. In comparing the flavonoid composition and variety identification, it is necessary to consider measurement errors. Two species having only differences within the error range are considered to be the same species.

  Various substances can be used as the flavonoid. For example, flavonoids include, but are not limited to, anthocyanins, flavonols, flavones, isoflavones, flavanones, flavanols, flavan-3,4-diols, flavan-3-ols, anthocyanidins, aurones, chalcones, and dihydrochalcones. . In particular, anthocyanins and flavonols are preferable.

  Furthermore, in the present invention, a novel flavonol is provided. This novel substance is useful as a marker for variety identification / identification. These new substances, alone or in combination with known flavonoids, enable the identification of plant varieties by determining their composition.

  Further provided herein are methods for identifying plant varieties from which a flavonoid-containing mixture (eg, a flavonoid-containing mixture isolated from nature) is derived. That is, a method for identifying from which plant variety the flavonoid-containing mixture of interest has been prepared is provided. Flavonoid-containing mixtures include, but are not limited to, foods, beverages, food supplements, food supplements, supplements, and pharmaceutical compositions. In this method, (a) determining a flavonoid composition in a flavonoid-containing mixture (eg, a flavonoid-containing mixture isolated from nature); and (b) a composition determined in step (a); Comparing the flavonoid composition of each of the varieties and identifying the cultivar of the plant from which the flavonoid-containing mixture is derived.

  In the above method, it is necessary to accumulate data of flavonoid compositions of a plurality of varieties in advance. The accumulated flavonoid composition data is compared with experimental results and the experimental plant variety is determined.

  Various substances can be used as the flavonoid. For example, flavonoids include, but are not limited to, anthocyanins, flavonols, flavones, isoflavones, flavanones, flavanols, flavan-3,4-diols, flavan-3-ols, anthocyanidins, aurones, chalcones, and dihydrochalcones. . In particular, anthocyanins and flavonols are preferable.

  In the present invention, the first flavonoid-containing mixture (for example, a flavonoid-containing mixture isolated from nature) and the second flavonoid-containing mixture (for example, a flavonoid-containing mixture isolated from nature) are derived from the same variety. A method is provided for determining whether or not. That is, a method is provided for determining whether the first flavonoid-containing mixture and the second flavonoid-containing mixture are prepared from plants of the same variety. In this method, (a) the flavonoid composition of the first flavonoid-containing mixture is compared with the flavonoid composition of the second flavonoid-containing mixture, and the first flavonoid-containing mixture and the second flavonoid-containing mixture are derived from the same variety. The step of determining whether or not.

  The present invention is illustratively described using the following examples, which are merely illustrative of the invention and should not be construed as limiting the invention in any way.

(Example 1: Isolation and analysis of flavonoids from tulips)
(Flavonoid extraction)
One tulip petal immediately after flowering was cut into 1 cm squares with scissors and placed in a 15 mL test tube. Next, extraction was performed with 3 mL of an aqueous solution of 10% acetonitrile and 0.5% trifluoroacetic acid for 1 to 3 hours. 1 mL of the extract was placed in a 1.5 mL microtube and centrifuged at 15000 rpm, and the supernatant was transferred to another tube and centrifuged again. 10 μL of the supernatant is analyzed by HPLC.

(Separation by HPLC)
Waters Alliance 2695 as the HPLC device, PDA (photodiode array) detector as the detector, solution (A) 10% acetonitrile, 0.5% trifluoroacetic acid, and solution (B) 30% as the mobile phase Acetonitrile, 0.5% trifluoroacetic acid was used. The column used is Mightysil RP-18 GP Aqua (150-4.6 mm) (Kanto Chemical Co., Ltd.), the column temperature was 40 ° C., and the flow rate was 1 mL / min, and the detection was performed at 530 nm (for anthocyanin detection). And 360 nm (for flavonol detection).

  Elution was performed under the conditions of 0-3 minutes: 0% B, 3-20 minutes: 0-100% B linear gradient, 20-30 minutes: 0% B.

  Examples of HPLC chromatograms of flavonols and anthocyanins for each tulip variety are shown in FIGS.

(Example 2: Flavonoids contained in tulips)
(Anthocyanin)
As a result of Example 1, the following seven types were identified as anthocyanin pigments contained in the petals of tulips.
(1) Delphinidin 3-O- (6-O-α-rhamnopyranosyl-β-glucopyranoside) (Delphinidin 3-O- (6-O-α-rhamnopyranosyl-β-glucopyranoside))
(2) Pelargonidin 3-O- (6-O-α-rhamnopyranosyl-β-glucopyranoside) (Pelargonidin 3-O- (6-O-α-rhamnopyranosyl-β-glucopyranoside))
(3) Cyanidin 3-O- (6-O-α-rhamnopyranosyl-β-glucopyranoside) (Cyanidin 3-O- (6-O-α-rhamnopyranosyl-β-glucopyranoside))
(4) Cyanidin 3-O- [6-O- (2-O-acetyl-α-rhamnopyranosyl) -β-glucopyranoside] (Cyanidin 3-O- [6-O- (2-O-acetyl-α-rhamnopyranosyl) ) -β-glucopyranoside])
(5) Pelargonidin 3-O- [6-O- (2-O-acetyl-α-rhamnopyranosyl) -β-glucopyranoside] (Pelargonidin 3-O- [6-O- (2-O-acetyl-α-rhamnopyranosyl) ) -β-glucopyranoside])
(6) Cyanidin 3-O- [6-O- (3-O-acetyl-α-rhamnopyranosyl) -β-glucopyranoside] (Cyanidin 3-O- [6-O- (3-O-acetyl-α-rhamnopyranosyl) ) -β-glucopyranoside])
(7) Delphinidin 3-O- [6-O- (3-O-acetyl-α-rhamnopyranosyl) -β-glucopyranoside] (Delphinidin 3-O- [6-O- (3-O-acetyl-α-rhamnopyranosyl) ) -β-glucopyranoside])
(8) Delphinidin 3-O- [6-O- (2-O-acetyl-α-rhamnopyranosyl) -β-glucopyranoside] (Delphinidin 3-O- [6-O- (2-O-acetyl-α-rhamnopyranosyl) ) -β-glucopyranoside])
These structures (1) to (8) are shown in FIG.

  Of these, (1) (2) (3) (4) and (5) are Torskangerpoll et al., (1999) Phytochemistry 52, 1687-1692. And Torskangerpoll et al., (2005) Biochemical Systematics and Ecology 33, (7) and (8) are described in Nakayama et al., (1999) Biosci. Biotechnol. Biochem., 63, 1509-1511., And Nakayama et al., (2004) JARQ 38, As reported by 185-190, (6) was a new compound.

(Flavonol)
Twenty-two flavonols contained in tulips have been reported in the paper (one of which overlaps).
(Leaf) Harborne (1965) Phytochemistry 4, 107.
(1) Kaempferol 3-rutinoside-7-glucoside
(2) Quercetin 3-rutinoside-7-glucoside
(3) Quercetin 3-rhamnoside (rutin)
(Pollen 'Apeldoorn') Stack, et. Al., (1981) Pflanzenphysiol. 103, 291.
(4) Kaempferol 3-rhamosylglucoside
(5) Kaempferol 3-xylosylrhamnosylglucoside
(6) = (3) Quercetin 3-rhamnosylglucoside (rutin)
(7) Quercetin 3-glucosylrhamnosylglucoside
(8) Isorhamnetin 3-rhamnosylglucoside
(9) Isorhamnetin 3-xylosylrhamnosylglucoside
(Flower 'Parade') Budzianowski and Skrzypczak (1979) Pol. J. Chem. 53. 1489.
(10) Kaempferol 3-rhamnoside
(11) Kaempferol 7-glucoside
(12) Kaempferol 3-rutinoside-7-diglycoside
(13) Quercetin 3-rutinoside-7-diglycoside
(14) Kaempferol 3,7-diglycoside
(15) Quercetin 3,7-diglycoside
(16) C-glucosylflavanone (hemiphloin)
(Flower 'Parade') Budzianowski (1991) Phytochemistry 30, 1679.
(17) Quercetin 3-O-gentiobioside-7-O-glucuronide
(18) Kaempferol 3-O-gentiobioside-7-O-glucuronide Kaempferol 3-O-gentiobioside-7-O-glucuronide
(19) Quercetin 3-O-rutinoside-7-O-glucuronide
(20) Kaempferol 3-O-rutinoside-7-O-glucuronide Kaempferol 3-O-rutinoside-7-O-glucuronide
(21) Kaempferol 3-O-glucoside-7-O-glucuronide
(22) Quercetin 3-O-glucoside-7-O-glucuronide
In the present invention, the following 11 types of flavonols were identified.
F1: Quercetin 3-rhamnosyl- (1 → 2) [rhamnosyl- (1 → 6) -glucoside] (mangusrin) (Quercetin 3-rhamnosyl- (1 → 2) [rhamnosyl- (1 → 6) -glucoside] ( manghaslin))
F2: Kaempferol 3-rhamnosyl- (1 → 2) [rhamnosyl- (1 → 6) -glucoside] (Kaempferol 3-rhamnosyl- (1 → 2) [rhamnosyl- (1 → 6) -glucoside])
F3 = (6): Quercetin 3-rhamnosyl- (1 → 6) -glucoside (rutin)
F5 = (19): Quercetin 3-rhamnosyl- (1 → 6) -glucoside-7-glucuronide (Quercetin 3-rhamnosyl- (1 → 6) -glucoside-7-glucuronide)
BF0: Quercetin 3-rhamnosyl- (1 → 6) -galactoside-7-glucuronide (new compound)
BF1: Kaempferol 3-rhamnosyl- (1 → 6) -galactoside-7-glucuronide (new compound)
BF2 = (20): kaempferol 3-rhamnosyl- (1 → 6) -glucoside-7-glucuronide (Kaempferol 3-rhamnosyl- (1 → 6) -glucoside-7-glucuronide)
BF3 = (21): Kaempferol 3-glucoside-7-glucuronide
BF6 = (4): Kaempferol 3-rhamnosyl- (1 → 6) -glucoside
BF7: Kaempferol 3-rhamnosyl- (1 → 6) -galactoside (Kaempferol 3-rhamnosyl- (1 → 6) -galactoside)
MF1: kaempferol 3-rhamnosyl- (1 → 2) [rhamnosyl- (1-6) -galactoside] (Mauritianin) (Kaempferol 3-rhamnosyl- (1 → 2) [rhamnosyl- (1-6) -galactoside] (mauritianin) ))
These structures are shown in FIGS. As described above, BF0 and BF1 are novel compounds.

(Example 3: The composition ratio of flavonoids prepared from petals is constant regardless of the difference in petal patterns)
In order to use the flavonoid composition for variety identification, even if the petal pattern is different, it must show a certain flavonoid composition without reflecting the difference in the pattern. Since flavonoids are substances that can affect the color of petals, whether or not the flavonoid composition is affected by individual differences in varieties with different petal patterns, such as the Washington species Is a problem.

  Therefore, it was proved by using Washington as an example that there is no difference between petals of variegated patterns between petals of anthocyanins and flavonols. The results are shown in FIG. The photograph of each petal used as a sample and the result of flavonol detected at 360 nm at the top of each panel and the result of anthocyanin detected at 520 nm at the bottom of each panel are shown on the right.

  As shown in the right panel, a constant flavonoid composition was shown regardless of petal pattern.

(Example 4: cultivation temperature does not affect flavonoid composition)
In order to use the flavonoid composition for variety identification, it is necessary to show a constant flavonoid composition regardless of the cultivation conditions. Therefore, flavonoids were isolated from tulips grown at different cultivation temperatures, analyzed, and the effect of cultivation temperature on flavonoid composition was confirmed. The results are shown in FIG.

  As the tulip, purple crystal was used, and the tulip was cultured at a culture temperature of A: 20 ° C. (appropriate temperature), B: 12 ° C. (color becomes darker), and C: 28 ° C. (color becomes lighter). As shown in the photographs of each panel, the color became darker at 12 ° C and lighter at 28 ° C than the appropriate temperature at 20 ° C. The results of flavonoid analysis by HPLC are shown on the right. The upper part of each panel is the result of flavonol detected at 360 nm, and the lower part of each panel is the result of anthocyanin detected at 520 nm.

  As is clear from the results, the cultivation temperature did not affect the flavonoid composition. Therefore, the flavonoid composition can be used for variety identification regardless of cultivation conditions such as cultivation temperature.

(Example 5: Identification of various varieties)
Using the method of the present invention, it was confirmed that various varieties can be identified.

  Flavonoids were isolated and analyzed from the various tulip varieties shown in FIGS. 9 and 10, and the composition ratios were shown as graphs. As is clear from the results, it was possible to identify and identify various varieties by combining the composition ratios of flavonol and anthocyanin. It has been demonstrated that the method of the present invention can be applied to purple varieties that are difficult to identify and identify.

(Example 6: Identification of rose varieties)
In order to demonstrate that the application range of the present invention is not limited to tulip variety identification, rose variety identification and identification were performed. The results are shown in FIG.

  It was possible to identify and identify cultivars even among red cultivars that were predicted to have similar pigment compositions.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, it becomes possible to easily and easily identify and identify plant varieties.

FIG. 1 shows the results showing a flavonol analysis of tulip petals. FIG. 2 shows the results of anthocyanin analysis of tulip petals. 3A to H show the results of HPLC chromatogram examples of flavonols and anthocyanins for each tulip variety. 3A to H show the results of HPLC chromatogram examples of flavonols and anthocyanins for each tulip variety. 3A to H show the results of HPLC chromatogram examples of flavonols and anthocyanins for each tulip variety. 3A to H show the results of HPLC chromatogram examples of flavonols and anthocyanins for each tulip variety. 3A to H show the results of HPLC chromatogram examples of flavonols and anthocyanins for each tulip variety. 3A to H show the results of HPLC chromatogram examples of flavonols and anthocyanins for each tulip variety. 3A to H show the results of HPLC chromatogram examples of flavonols and anthocyanins for each tulip variety. 3A to H show the results of HPLC chromatogram examples of flavonols and anthocyanins for each tulip variety. FIG. 4 is a diagram showing chemical formulas of eight kinds of anthocyanins in tulip petals. FIG. 5 is a diagram showing the chemical formula of flavonol of tulip. FIG. 6 is a diagram showing the chemical formula of flavonol of tulip. FIGS. 7A to E show the results showing Washington as an example that there is no difference between petals of variegated patterns for each petal of anthocyanins and flavonols. FIGS. 7A to E show the results showing Washington as an example that there is no difference between petals of variegated patterns for each petal of anthocyanins and flavonols. FIGS. 7A to E show the results showing Washington as an example that there is no difference between petals of variegated patterns for each petal of anthocyanins and flavonols. FIGS. 7A to E show the results showing Washington as an example that there is no difference between petals of variegated patterns for each petal of anthocyanins and flavonols. FIGS. 7A to E show the results showing Washington as an example that there is no difference between petals of variegated patterns for each petal of anthocyanins and flavonols. 8A to 8C are results showing purple crystal as an example that the difference in cultivation temperature does not affect the flavonoid composition. 8A to 8C are results showing purple crystal as an example that the difference in cultivation temperature does not affect the flavonoid composition. 8A to 8C are results showing purple crystal as an example that the difference in cultivation temperature does not affect the flavonoid composition. FIG. 9 shows the results showing the composition ratio of anthocyanins and flavonols for each tulip variety. FIG. 9 shows the results showing the composition ratio of anthocyanins and flavonols for each tulip variety. FIG. 10-1 is a result showing the composition ratio of anthocyanins and flavonols in a purple variety. FIG. 10-2 is a result showing the composition ratio of anthocyanins and flavonols in a purple variety. FIGS. 11A to 11E show the results showing the anthocyanin and flavonol composition in red rose cultivars. FIGS. 11A to 11E show the results showing the anthocyanin and flavonol composition in red rose cultivars. FIGS. 11A to 11E show the results showing the anthocyanin and flavonol composition in red rose cultivars. FIGS. 11A to 11E show the results showing the anthocyanin and flavonol composition in red rose cultivars. FIGS. 11A to 11E show the results showing the anthocyanin and flavonol composition in red rose cultivars.

Claims (29)

A method for determining whether a first plant and a second plant are of the same variety, the method comprising:
a) preparing a flavonoid-containing extract from the first plant and determining its composition;
b) preparing a flavonoid-containing extract from a second plant and determining its composition; and c) comparing the composition of the flavonoid of the first plant with the composition of the flavonoid of the second plant, Determining whether the first plant and the second plant are of the same variety,
Including the method. The flavonoid is selected from the group consisting of anthocyanins, flavonols, flavones, isoflavones, flavanones, flavanols, flavan-3,4-diols, flavan-3-ols, anthocyanidins, aurones, chalcones, and dihydrochalcones. The method according to 1. The method of claim 2, wherein the flavonoid is selected from the group consisting of anthocyanins and flavonols. The method of claim 1, wherein the plant is selected from the group consisting of tulip, chrysanthemum, rose, carnation, orchid, petunia, torenia, spinach, lettuce, rice, and wheat. The method according to claim 4, wherein the plant is a tulip. The method of claim 1, wherein the flavonoid-containing extract is prepared from an organ selected from the group consisting of petals, leaves, fruits, seeds, stems, and roots of the plant. 7. The method of claim 6, wherein a flavonoid-containing extract is prepared from the petals of the plant. The method according to claim 7, wherein the flavonoid-containing extract is prepared at the time of flowering or when a bud is formed. 9. The method of claim 8, wherein the flavonoid-containing extract is prepared at flowering time. A method for identifying the variety of a sample plant, comprising:
a) preparing a flavonoid-containing extract from a sample plant and determining its composition; and
b) comparing the composition determined in step (a) with the flavonoid composition of a known variety to identify the variety of the sample plant;
Including the method. The flavonoid is selected from the group consisting of anthocyanins, flavonols, flavones, isoflavones, flavanones, flavanols, flavan-3,4-diols, flavan-3-ols, anthocyanidins, aurones, chalcones, and dihydrochalcones. 10. The method according to 10. The method of claim 11, wherein the flavonoid is selected from the group consisting of anthocyanins and flavonols. 11. The method of claim 10, wherein the plant is selected from the group consisting of tulips, chrysanthemums, roses, carnations, orchids, petunias, torenia, spinach, lettuce, rice, and wheat. The method according to claim 13, wherein the plant is a tulip. 11. The method of claim 10, wherein the flavonoid-containing extract is prepared from an organ selected from the group consisting of petals, leaves, fruits, seeds, stems, and roots of the plant. 16. The method of claim 15, wherein a flavonoid-containing extract is prepared from the petals of the plant. The method according to claim 16, wherein the flavonoid-containing extract is prepared at the time of flowering or at the time of formation of a bud. 18. A method according to claim 17, wherein the flavonoid-containing extract is prepared at flowering time. The method according to claim 10, further comprising:
c) Sample plant and known plant:
Plant type, plant height, stem thickness, stem color, leaf shape, leaf size, leaf color, leaf gloss, leaf number, leaf formation angle, presence of white powder on leaves, bulb shape, bulb size , Bulb color, bulb hull color and bulbous three bud shape, flower shape, flower size, flower color, petal shape, petal color, petal number, number of stamens, flower color, yak Selected from the group consisting of color, pollen color, floral pattern length, floral pattern thickness, floral pattern color, flowering period, aboveground withering period, branch bloom, disease resistance, and insect resistance Comparing traits;
Including the method. A compound having the following structure:
A compound having the following structure:
A method for determining whether a first plant and a second plant are of the same variety, the method comprising:
a) preparing a flavonoid-containing extract from the first plant and determining the composition of the compound of claim 20 and the compound of claim 21;
a) preparing a flavonoid-containing extract from a second plant and determining the composition of the compound of claim 20 and the compound of claim 21; and c) the compound of claim 20 and the claim. 21. Compare the composition of the flavonoid of the first plant and the composition of the flavonoid of the second plant with respect to the composition of the compound according to 21, and determine whether the first plant and the second plant are of the same variety Determining whether or not,
Including the method. 23. A method according to claim 22, wherein the plant is a tulip. 23. The method of claim 22, wherein a flavonoid-containing extract is prepared from the petals of the plant. A method for identifying the variety of a sample plant, comprising:
a) preparing a flavonoid-containing extract from a sample plant and determining the composition of the compound of claim 20 and the compound of claim 21; and
b) comparing the composition determined in step (a) with the composition of the compound of claim 20 and the compound of claim 21 in a known variety to identify the variety of the sample plant;
Including the method. 26. The method of claim 25, wherein the plant is a tulip. 26. The method of claim 25, wherein a flavonoid-containing extract is prepared from the petals of the plant. A method for identifying a plant variety from which a flavonoid-containing mixture isolated from nature is derived, comprising:
a) determining a flavonoid composition in a flavonoid-containing mixture isolated from nature; and
b) comparing the composition determined in step (a) with the flavonoid composition of known varieties to identify the plant variety from which the flavonoid-containing mixture is derived;
Including the method. The flavonoid is selected from the group consisting of anthocyanins, flavonols, flavones, isoflavones, flavanones, flavanols, flavan-3,4-diols, flavan-3-ols, anthocyanidins, aurones, chalcones, and dihydrochalcones. 28. The method according to 28.