Stevia plants rich in steviol glycosides and their screening methods

Abstract

This invention provides a stevia plant rich in steviol glycosides with the following genetic traits (1) and (2), its preparation method, screening method, etc. (1) It exhibits homozygosity for the allele with a base of T at position 290 of sequence number 1. (2) It exhibits homozygosity or heterozygosity for the allele with a base of A at position 40 of sequence number 2.

Description

Technical Field

[0001] This invention relates to stevia plants with high stevioside content and their screening methods. Background Technology

[0002] To meet diverse consumer demands, a wide variety of beverages have been developed and marketed. Sugars such as sucrose are commonly added to beverages to provide sweetness; however, it has been pointed out that excessive consumption of sugars can have health effects, leading to a growing demand for lower-calorie, naturally derived sweeteners. For example, Patent Document 1 discloses a functional sweetener composition containing vitamins, high-sweetness sweeteners, and sweetness-improving compositions.

[0003] Stevia glycosides are well-known as the sweet components found in stevia extract. Stevia extract is primarily obtained by extraction and purification from the dried leaves of stevia. Stevia is a perennial plant of the Asteraceae family, native to Paraguay, South America, with the scientific name Stevia rebaudiana Bertoni. Because stevia contains components with a sweetness approximately 300 times greater than that of sugar, it is cultivated for extraction and use as a natural sweetener. Various glycosides, such as Rebaudioside A (hereinafter sometimes abbreviated as "Reb"), RebB, RebC, RebD, RebE, and RebM, have been reported as stevia glycosides (Patent Document 2). Among these various stevia glycosides, RebA, for example, is widely used as a sweetener with high sweetness and excellent flavor. The unique sweetness and accompanying flavors of other stevia glycosides are also being continuously clarified.

[0004] In such cases, a method for screening stevia plants with high levels of sweet components is known (Patent Document 3).

[0005] Patent documents

[0006] Patent Document 1: International WO2007 / 070224

[0007] Patent Document 2: International WO2010 / 038911

[0008] Patent Document 3: International WO2020 / 027155 Summary of the Invention

[0009] Stevia plants with high stevioside content are sought.

[0010] In one aspect, the present invention provides the following invention.

[0011] [1] A method for screening stevia plants rich in steviol glycosides, characterized in that it includes the steps of detecting the presence and / or absence of the following genetic traits (1) and the presence and / or absence of the following genetic traits (2) from the genome of the tested stevia plant.

[0012] (1) Alleles with a base of T at position 290 of sequence number 1 exhibit isotype conjugation;

[0013] (2) Alleles with base A at position 40 of sequence number 2 exhibit homozygous or heterozygous conjugation.

[0014] [2] The method according to [1] is characterized by further comprising a step of determining the content of steviol glycosides in the tested stevia plant tissue after detecting the presence and / or absence of the genetic trait.

[0015] [3] The method according to [1] or [2] is characterized in that the steviol glycoside content of the stevia plant rich in steviol glycosides is more than 3% higher than the steviol glycoside content of the stevia plant selected by a screening method that includes a step of detecting the presence and / or absence of the hereditary trait (2) but does not include a step of detecting the presence and / or absence of the hereditary trait (1).

[0016] [4] The method according to any one of [1] to [3] is characterized in that the step of detecting the presence and / or absence of hereditary traits is carried out using the dCAPS method or the TaqMan PCR method.

[0017] [5] A screening kit for stevia plants rich in steviol glycosides, characterized in that it comprises reagents for detecting the presence and / or absence of the following genetic trait (1), and reagents for detecting the presence and / or absence of the following genetic trait (2).

[0018] (1) Alleles with a base of T at position 290 of sequence number 1 exhibit isotype conjugation;

[0019] (2) Alleles with base A at position 40 of sequence number 2 exhibit homozygous or heterozygous conjugation.

[0020] [6] The kit according to [5] is characterized in that the reagent contains primers and / or probes used in the CAPS method, dCAPS method or TaqMan PCR method.

[0021] [7] A stevia plant rich in steviol glycosides, characterized by having the following genetic traits (1) and (2),

[0022] (1) Alleles with a base of T at position 290 of sequence number 1 exhibit isotype conjugation;

[0023] (2) Alleles with base A at position 40 of sequence number 2 exhibit homozygous or heterozygous conjugation.

[0024] [8] The plant body according to [7] is characterized in that it is a non-transgenic plant body.

[0025] [9] The plant body according to [7] or [8] is characterized in that it comprises a stevia plant body and its progeny plants that have been induced by mutation.

[0026]

[10] A seed, tissue, dried leaf, tissue culture or cell, characterized in that it is a seed, tissue, dried leaf, tissue culture or cell of any one of [7] to [9].

[0027]

[11] The tissue, tissue culture or cell according to

[10] is characterized in that it is selected from embryos, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf sections and callus.

[0028]

[12] A method for producing stevia plant body rich in steviol glycosides, characterized in that it includes a step of hybridizing the stevia plant body described in any one of [7] to [9] with a second stevia plant body.

[0029]

[13] The method according to

[12] is characterized in that the second plant body is the stevia plant body described in any one of [7] to [9].

[0030]

[14] A method for producing stevia plant rich in steviol glycosides, characterized in that it includes a step of adding alterations to the genome of the stevia plant in such a way that the genome has the following genetic traits (1) and (2).

[0031] (1) Alleles with a base of T at position 290 of sequence number 1 exhibit isotype conjugation;

[0032] (2) Alleles with base A at position 40 of sequence number 2 exhibit homozygous or heterozygous conjugation.

[0033]

[15] The method according to

[14] is characterized in that the genomic alteration is implemented by a mutation-inducing treatment.

[0034]

[16] An extract, which is an extract of any one of the plant bodies, seeds, tissues, dried leaves, tissue cultures or cells described in any one of [7] to [9],

[10] or

[11] , characterized in that it contains steviol glycosides.

[0035]

[17] A method for manufacturing an extract containing steviol glycosides, characterized in that it includes a step of obtaining the extract from any of the plants, seeds, tissues, dried leaves, tissue cultures or cells described in any of [7] to [9],

[10] or

[11] .

[0036]

[18] A method for manufacturing steviol glycosides, characterized in that it includes a step of purifying steviol glycosides from the extract described in

[16] .

[0037]

[19] A method for manufacturing a food product, sweetener composition, flavoring, or pharmaceutical product, characterized by comprising the step of providing an extract of a plant body as described in any one of [7] to [9], an extract of seeds, tissues, dried leaves, tissue cultures, or cells as described in

[10] or

[11] , or an extract as described in

[16] , and,

[0038] The process of adding the extract to the raw materials of food, beverages, sweetener compositions, flavorings or pharmaceuticals.

[0039] According to the present invention, stevia plant body containing more steviol glycosides can be obtained, and methods for producing such plant body, leaves obtained from such plant body, and food or beverages containing steviol glycosides obtained from such leaves can be provided. Attached Figure Description

[0040] Figure 1 A diagram showing the positions of the bases involved in the genetic trait (1) in the base sequence of sequence number 1. The bases enclosed in boxes are the bases involved in the genetic trait (1).

[0041] Figure 2 A diagram showing the positions of the bases involved in the genetic trait (2) in the base sequence of sequence number 2. The bases enclosed in boxes are the bases involved in the genetic trait (2).

[0042] Figure 3 This is a chart showing the average (%) of TSG content in the dried leaves of individuals with various genetic traits in Group A. "All" represents all individuals in Group A, "(2)" represents individuals with genetic trait (2), "(1)+(2)" represents individuals with the genetic trait of the present invention, and "Not(1)+(2)" represents individuals without the genetic trait of the present invention. Detailed Implementation

[0043] The present invention will now be described in detail. The following embodiments are illustrative of the invention, and the invention is not limited to these embodiments. The invention can be implemented in various ways without departing from its spirit.

[0044] Furthermore, all documents, publications, patent gazettes, and other patent documents cited in this specification are incorporated herein by reference. In addition, this specification includes the contents of the specification and drawings of Japanese patent application (Japanese Patent Application No. 2020-084133), filed on May 12, 2020, which forms the basis of this application's priority claim.

[0045] 1. Stevia plants rich in steviol glycosides

[0046] The present invention provides, on one side, a stevia plant body rich in steviol glycosides (hereinafter, sometimes referred to as "the plant body of the present invention"), characterized in that it has the following genetic traits (1) and (2),

[0047] (1) Alleles with a base of T at position 290 of sequence number 1 exhibit isotype conjugation;

[0048] (2) Alleles with base A at position 40 of sequence number 2 exhibit homozygous or heterozygous conjugation.

[0049] The plant of the present invention is a species derived from wild stevia plants, and is a species that obtains the above-mentioned genetic traits (1) and (2) with increased steviol glycoside content (hereinafter, the above-mentioned genetic traits (1) and (2) are sometimes collectively referred to as "the genetic traits of the present invention").

[0050] The term "position equivalent to" refers to the location within the genome of a sequence identical to the standard sequence (e.g., sequence number 1) when such a sequence exists in the genome (e.g., position 290, position 40, etc.). Conversely, it refers to the position within a sequence in the genome that is equivalent to the standard sequence when no identical sequence exists. The existence of a sequence identical to or equivalent to the standard sequence in the genome can be determined, for example, by amplifying the genomic DNA of the stevia plant using primers suitable for PCR amplification of the standard sequence, sequencing the amplified products, and comparing the resulting sequence with the standard sequence. Non-limiting examples of sequences equivalent to the standard sequence include, for instance, base sequences that have a sequence identity of 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.1% or more, 98.4% or more, 98.7% or more, 99% or more, 99.2% or more, 99.5% or more, or 99.8% or more relative to the standard sequence. The position in the genome that corresponds to the position in the standard sequence can be determined by considering the base sequences before and after that position in the standard sequence. For example, by comparing and analyzing a standard sequence with sequences in the genome that are equivalent to the standard sequence, the position in the genome that is equivalent to the standard sequence can be determined.

[0051] For example, taking the "position corresponding to position 290 of sequence number 1" of the genetic trait (1) of the present invention as an example, when the genome of the stevia plant has a portion consisting of the same base sequence as sequence number 1, the "position corresponding to position 290 of sequence number 1" is 290 positions starting from the 5' side of the portion of the genome consisting of the same base sequence as sequence number 1. On the other hand, when the genome of the stevia plant has a portion consisting of a base sequence different from but corresponding to sequence number 1, since the genome does not have a portion consisting of the same base sequence as sequence number 1, the "position corresponding to position 290 of sequence number 1" does not necessarily correspond to 290 positions starting from the 5' side of the portion corresponding to sequence number 1. Instead, the base sequences before and after position 290 of sequence number 1 can be considered to determine the "position corresponding to position 290 of sequence number 1" in the genome of the stevia plant in question. For example, by comparing the base sequence of the portion of the stevia genome corresponding to sequence number 1 with the base sequence of sequence number 1, the "position corresponding to position 290 of sequence number 1" in the stevia genome can be determined.

[0052] "The portion consisting of a base sequence equivalent to Serial No. 1" refers to, for example, a portion consisting of a base sequence that has a sequence identity of 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.1% or more, 98.4% or more, 98.7% or more, 99% or more, 99.2% or more, 99.5% or more, or 99.8% or more, relative to the base sequence of Serial No. 1.

[0053] In one embodiment, the “part consisting of a base sequence corresponding to sequence number 1” includes a portion of the stevia plant genome that can be amplified by PCR using primers that hybridize with the complementary sequence of the portion from position 1 to 289 (i.e., from the 5' end of sequence number 1 to the 290th base on the 5' end of the position involved in trait (1)) using primers that hybridize with the complementary sequence of the portion from position 1 to 289 (i.e., from the 5' end of sequence number 1 to the 290th base on the 5' end of the position involved in trait (1)) using primers that hybridize with the portion from position 1 to 36 (i.e., from the 3' end of sequence number 1 to the 290th base on the 3' end of the position involved in trait (1)) using primers that hybridize with the sequence of sequence number 4.

[0054] In one embodiment, the “part consisting of a base sequence corresponding to sequence number 2” includes a portion of the stevia plant genome that can be amplified by PCR using primers that hybridize with the complementary sequence of the portion from position 1 to 39 (i.e., from the 5' end of sequence number 2 to the 40th base on the 5' end of the position involved in trait (2)) using primers that hybridize with the complementary sequence of the portion from position 1 to 105 (i.e., from the 3' end of sequence number 2 to the 40th base on the 3' end of the position involved in trait (2)) using primers that hybridize with the portion from position 1 to 105 (i.e., from the 3' end of sequence number 2 to the 40th base on the 3' end of the position involved in trait (2)).

[0055] In a particular manner, "the portion consisting of a base sequence corresponding to sequence number 1" may, for example, contain a portion of the stevia genome that can be amplified by PCR using primers containing the base sequence of sequence number 3 and the reverse primer containing the base sequence of sequence number 4.

[0056] In a particular manner, "the portion consisting of the base sequence corresponding to sequence number 2" may, for example, contain a portion of the stevia genome that can be amplified by PCR using primers containing the base sequence of sequence number 5 and the reverse primer containing the base sequence of sequence number 6.

[0057] In a particular manner, the “allele with a base of T at position 290 of sequence number 1” (hereinafter, sometimes referred to as the “allele involved in the hereditary trait (1)”) contains the base sequence of sequence number 1, 7, 8 or 9.

[0058] In a particular manner, the “allelicity with an A base at position 40 of sequence number 2” (hereinafter, sometimes referred to as the “allelicity involved in hereditary trait (2)”) contains the base sequence of sequence number 2, 10, 11 or 12.

[0059] Here, the positions (1) corresponding to position 290 of sequence number 1 and (2) corresponding to position 40 of sequence number 2 are sometimes collectively referred to as "the polymorphic sites of the present invention" or "the sites involved in the hereditary traits of the present invention".

[0060] The genetic traits can be detected by the following methods: PCR, TaqMan PCR, sequencing, microarray, Invader method, TILLING method, RAD (random amplified polymorphic DNA) method, restriction enzyme fragment length polymorphism (RFLP) method, PCR-SSCP method, AFLP (amplified fragment length polymorphism) method, SSLP (simple sequence length polymorphism) method, CAPS (cleaved amplified polymorphic sequence) method, dCAPS (derived cleaved amplified polymorphic sequence) method, allele-specific oligonucleotide (ASO) method, ARMS method, denaturing gradient gel electrophoresis (DGGE) method, CCM (chemical cleavage of mismatch) method, DOL method, MALDI-TOF / MS method, TDI method, lock probe method, molecular beacon method, DASH (dynamic allele specific hybridization) method, UCAN method, ECA method, PINPOINT method, PROBE (primer oligobase) method. Methods for detecting gene variations include the VSET (very short extension) method, Survivor assay, Sniper assay, Luminex assay, GOOD method, LCx method, SNaPshot method, Mass ARRAY method, Pyrosequences method, SNP-IT method, melting curve analysis, etc., but the detection methods are not limited to these. Details of the detection methods for gene variations are described later.

[0061] In a particular manner, the genetic traits of the present invention can be detected by the dCAPS method based on the following primer set and restriction enzyme combination.

[0062] When a candidate plant possesses the genetic trait (1), for example, PCR amplification is performed on the genomic DNA of the candidate plant using a forward primer with the base sequence shown in Serial No. 21 and a reverse primer with the base sequence shown in Serial No. 22. When the resulting PCR product (approximately 326 bp long, for example, Serial No. 30) is treated with the restriction enzyme RsaI, a band of approximately 290 bp long (for example, Serial No. 31) and a band of approximately 36 bp long (for example, Serial No. 32) are obtained. On the other hand, when a candidate plant does not possess the genetic trait (1), PCR amplification is performed in the same manner as described above, generating a PCR product of approximately 326 bp long (for example, Serial No. 33 or Serial No. 30 and 33). However, even with restriction enzyme treatment, only the band of the uncuttered PCR product of approximately 326 bp long (for example, Serial No. 33) can be identified.

[0063] When a candidate plant possesses the genetic trait (2), for example, PCR amplification is performed on the genomic DNA of the candidate plant using a forward primer with the base sequence shown in SEQ ID NO. 25 and a reverse primer with the base sequence shown in SEQ ID NO. 28. When the resulting PCR product (approximately 367 bp long, for example, SEQ ID NO. 34 or SEQ ID NO. 34 and 35) is treated with the restriction enzyme RsaI, a band of approximately 46 bp long (for example, SEQ ID NO. 36) and a band of approximately 320 bp long (for example, SEQ ID NO. 37) are obtained. On the other hand, when a candidate plant does not possess the genetic trait (2), PCR amplification is performed in the same manner as described above, generating a PCR product of approximately 367 bp long (for example, SEQ ID NO. 35). However, even with restriction enzyme treatment, only the band of the uncuttered PCR product of approximately 367 bp long (for example, SEQ ID NO. 35) can be confirmed.

[0064] The stevia plant of the present invention is a species derived from wild-type stevia plants, possessing the aforementioned genetic trait of increased steviol glycoside content. This genetic trait can be produced by transgenic methods or by non-transgenic methods. Therefore, the plant of the present invention can be a plant obtained through transgenic methods or its offspring (hereinafter, sometimes referred to as a "transgenic plant"), or a plant obtained through non-transgenic methods or its offspring (hereinafter, sometimes referred to as a "non-transgenic plant").

[0065] In this specification, as an example of a "non-GMO method," methods for inducing gene mutations in host cells (or host plants) without introducing foreign genes can be listed. Methods for inducing the action of mutagens in plant cells can be listed as such methods. Examples of such mutagens include ethyl methanesulfonate (EMS) and sodium azide. For example, EMS can be used to treat plant cells at concentrations of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1.0%. Treatment times are approximately 1 to approximately 48 hours, approximately 2 to approximately 36 hours, approximately 3 to approximately 30 hours, approximately 4 to approximately 28 hours, approximately 5 to approximately 26 hours, and approximately 6 to approximately 24 hours. The treatment process itself is well known and can be carried out by immersing water-absorbing seeds, which have undergone a water absorption process, in a treatment solution containing mutagens at the above-mentioned concentrations for the above-mentioned treatment times.

[0066] Other examples of non-GMO methods include irradiating plant cells with radiation or light such as X-rays, gamma rays, and ultraviolet rays. When irradiated with ultraviolet light, cells irradiated with an appropriate amount of ultraviolet light (ultraviolet lamp intensity, distance, and time) are cultured in a selective culture medium to select cells, callus tissue, or plant bodies with the target traits. The irradiation intensity at this time is 0.01–100 Gr, 0.03–75 Gr, 0.05–50 Gr, 0.07–25 Gr, 0.09–20 Gr, 0.1–15 Gr, 0.1–10 Gr, 0.5–10 Gr, and 1–10 Gr; the irradiation distance is 1 cm–200 m, 5 cm–100 m, 7 cm–75 m, 9 cm–50 m, 10 cm–30 m, 10 cm–20 m, and 10 cm–10 m; and the irradiation time is 1 minute–2 years, 2 minutes–1 year, 3 minutes–0.5 years, 4 minutes–1 month, 5 minutes–2 weeks, and 10 minutes–1 week. The irradiation intensity, distance, and time vary depending on the type of radiation or light or the state of the irradiated object (cells, callus tissue, plant body), and can be appropriately adjusted by those skilled in the art.

[0067] In addition, cell fusion, anther culture (haploid culture), and distant hybridization (haploid culture) are also well-known methods.

[0068] Generally speaking, plant cells sometimes undergo variation during culture, so it is preferable to restore them to individual plants in order to maintain more stable traits.

[0069] Plants obtained by transgenic modification (e.g., through genome editing) using non-transgenic stevia plants as hosts are also not excluded from the scope of this invention.

[0070] The plant of the present invention is a steviol glycoside-rich type. A steviol glycoside-rich stevia plant refers to one with a higher steviol glycoside content compared to stevia plants without the genetic trait of the present invention. A higher steviol glycoside content means, for example, that the average or median steviol glycoside content in a group of stevia plants of the present invention is higher than the average or median steviol glycoside content in a group of stevia plants without the genetic trait of the present invention, and / or higher than the average or median steviol glycoside content in a group of stevia plants with genetic trait (2).

[0071] In some embodiments, the average content of steviol glycosides in the group of plants of the present invention is about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, higher than the average content of steviol glycosides in the group of stevia plants that do not have the genetic traits of the present invention. 26% or more, approximately 27% or more, approximately 28% or more, approximately 29% or more, approximately 30% or more, approximately 31% or more, approximately 32% or more, approximately 33% or more, approximately 34% or more, approximately 35% or more, approximately 36% or more, approximately 37% or more, approximately 38% or more, approximately 39% or more, approximately 40% or more, approximately 41% or more, approximately 42% or more, approximately 43% or more, approximately 43.5% or more, approximately 44% or more, approximately 45% or more, approximately 46% or more, approximately 47% or more, approximately 48% or more, approximately 49% or more, or approximately 50% or more.

[0072] Furthermore, in some embodiments, the average content of steviol glycosides in the plant body group of the present invention is about 1.0% or more, about 1.3% or more, about 1.5% or more, about 1.8% or more, about 2.0% or more, about 2.3% or more, about 2.5% or more, about 2.8% or more, about 3.0% or more, about 3.3% or more, about 3.5% or more, about 3.8% or more, about 4.0% or more, about 4.3% or more, about [missing information] higher than the average content of steviol glycosides in the stevia plant body group having the genetic trait (2). 4.5% or more, approximately 4.8% or more, approximately 5.0% or more, approximately 5.3% or more, approximately 5.5% or more, approximately 5.8% or more, approximately 6.0% or more, approximately 6.3% or more, approximately 6.5% or more, approximately 6.8% or more, approximately 7.0% or more, approximately 7.2% or more, approximately 7.5% or more, approximately 7.8% or more, approximately 8.0% or more, approximately 8.3% or more, approximately 8.5% or more, approximately 8.8% or more, approximately 9.0% or more, approximately 9.3% or more, approximately 9.5% or more, approximately 9.8% or more, or approximately 10.0% or more.

[0073] Steviosides are a general term for compounds with sugars such as glucose, rhamnose, and xylose bonded to the steviol skeleton. Examples include RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebI, RebJ, RebK, RebN, RebM, RebO, RebQ, RebR, Duke glycoside A, raspberry glycoside, steviol monosaccharide glycoside, steviol disaccharide glycoside, and steviol glycoside. In some embodiments, steviol glycosides contain one or more glycosides selected from RebA, RebB, RebC, RebD, RebE, RebF, RebI, RebJ, RebK, RebN, RebM, RebO, RebQ, RebR, Duke glycoside A, raspberry glycoside, steviol monosaccharide glycoside, steviol disaccharide glycoside, and steviol glycoside. In certain embodiments, steviol glycosides include RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebM, RebN, and steviol glycosides, or are selected from these steviol glycosides.

[0074] Total Steviol Glycoside (TSG) is a general term for measurable steviol glycosides, excluding unknown steviol glycosides or steviol glycosides present in amounts below the detection limit. TSG is preferably selected from any combination of two or more of RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebI, RebJ, RebK, RebM, RebN, RebO, RebQ, RebR, Duke glycoside A, raspberry glycoside, steviol monosaccharide glycoside, steviol disaccharide glycoside, and steviol glycoside. In a specific embodiment, TSG is composed of a combination of RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebM, RebN, and steviol glycoside.

[0075] Steviosides can be extracted by reacting fresh or dried leaves of the plant of the present invention with a suitable solvent (aqueous solvents such as water or organic solvents such as alcohols, ethers, and acetone) to form an extract. Extraction conditions, etc., can be found in the methods described in Ohta et al., J. Appl. Glycosci., Vol. 57, No. 3, 199-209 (2010) or WO2010 / 038911, and in the examples described later. Dried leaves refer to leaves whose moisture content has been reduced to 10% by weight or less, 7% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, or 1% by weight or less by drying fresh leaves. The moisture content of the dried leaves of the plant of the present invention is preferably 3 to 4% by weight.

[0076] Furthermore, the extract thus obtained can be purified by known methods such as using ethyl acetate and other organic solvents, water gradients, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), and ultra-high performance liquid chromatography (UPLC).

[0077] The steviol glycoside content involved in this invention can be determined by the method described in Ohta et al. or WO2010 / 038911 above, or by the method described in the examples described below. Specifically, for example, fresh leaves can be collected from the stevia plant of this invention as samples and determined by performing LC / MS-MS or the like.

[0078] The plant body of the present invention includes not only the whole plant body, but also plant organs (e.g., leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g., epidermis, phloem, soft tissue, xylem, vascular bundles, palisade tissue, spongy tissue, etc.) or plant cells of various forms (e.g., suspended cultured cells), protoplasts, leaf sections, callus tissue, etc. In addition, the leaves can be dried leaves.

[0079] Furthermore, the plant body of the present invention may also include tissue cultures or plant cultured cells. This is because the plant body can be regenerated by culturing such tissue cultures or plant cultured cells. Examples of regenerative methods of the plant body of the present invention include embryos, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf sections, and callus tissue, but are not limited thereto.

[0080] 2. The method for preparing the plant body according to the present invention

[0081] In other embodiments of the present invention, a method for producing stevia plant material rich in steviol glycosides (hereinafter referred to as "the method of the present invention") is provided, characterized in that it includes a step of hybridizing the stevia plant material of the present invention with a second stevia plant material.

[0082] The "stevia plant body rich in steviol glycosides" produced by this method can have the same phenotype and genetic traits as the plant body of the present invention.

[0083] Specifically, the phenotype of the plant produced according to the method of the present invention is the steviol glycoside-rich phenotype described in the plant section of the present invention. The genetic traits of the plant produced according to the method of the present invention possess the genetic traits of the present invention. Methods for detecting these genetic traits are as described above and below.

[0084] In the manufacturing method of the present invention, "hybridization" refers to obtaining daughter plants (plants produced according to the manufacturing method of the present invention) by mating the plant of the present invention with a second plant. Backcrossing is preferred as a hybridization method. "Backcrossing" is a method of further hybridizing the daughter plant produced between the plant of the present invention and the second plant with the plant of the present invention (i.e., a plant having the genetic traits of the present invention) to produce a plant having the genetic traits of the present invention. Backcrossing is essentially performed when the second plant used in the manufacturing method of the present invention has the same phenotype and genetic traits as the plant of the present invention. Hybridization is preferably performed for two or more generations; when the genetic traits are heterozygous, etc., sometimes a plant with a combination of desired genetic traits can be obtained even in one generation.

[0085] Alternatively, the plant of the present invention can also be produced by self-fertilization. Self-fertilization can be carried out by pollinating the stamens of the plant of the present invention to the pistils of the plant of the present invention.

[0086] The phenotype and genetic traits of the plant produced by the method of the present invention are the same as those of the plant produced by the present invention. Therefore, by further hybridizing the plant produced by the method of the present invention with a third stevia plant, a stevia plant rich in steviol glycosides can also be produced.

[0087] Alternatively, the plant body of the present invention can also be produced by regenerating the plant body through culturing the aforementioned tissue culture or plant culture cells. Regarding the culture conditions, it is known that they are the same as those for the tissue culture or plant culture cells of wild-type stevia (Protocols for In Vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, Method in molecular biology, vol. 1391, pp. 113-123).

[0088] Furthermore, as another method, the plant of the present invention can be produced by altering the genome of the stevia plant to obtain the genetic traits of the present invention. The acquisition of the genetic traits of the present invention can be carried out by transgenic methods or by the non-transgenic methods described above. Non-transgenic methods, as described in the plant section of the present invention, include mutation-inducing treatments such as treatment based on mutagens, treatment based on radiation or light irradiation, etc. Specifically, for example, for an individual having an allele at position 290 of sequence number 1 where the base is not T (e.g., an allele where the base at that position is C), the genetic traits of the present invention are obtained by substituting the base at that position for T; and for an individual having an allele at position 40 of sequence number 2 where the base is not A (e.g., an allele where the base at that position is C), the genetic traits of the present invention are obtained by substituting the base at that position for A.

[0089] 3. The plant screening method of the present invention

[0090] The plant organisms of the present invention, and plant organisms having the same phenotype and / or genetic traits as those of the present invention, can be screened by detecting the genetic traits of the present invention in the tissues of such plant organisms. Here, "screening" means identifying and selecting the plant organisms of the present invention from those other than those of the present invention.

[0091] Therefore, in other aspects, the present invention provides a method for screening stevia plants rich in steviol glycosides (hereinafter, sometimes referred to as "the screening method of the present invention"), characterized by comprising the steps of detecting the presence and / or absence of the genetic trait of the present invention (1) and the presence and / or absence of the genetic trait of the present invention (2) from the genome of the tested stevia plant.

[0092] The screening method of the present invention may further include the step of selecting plants from the tested plants that are found to have at least one of the above-mentioned genetic traits.

[0093] The presence of the heritable traits of the present invention can be determined, for example, by detecting the following results:

[0094] • Only alleles with a T base at position 290 corresponding to sequence number 1 exist (e.g., alleles containing the base sequence of sequence number 1, 7, 8, or 9).

[0095] • There exists an allele with an A base at position 40 corresponding to sequence number 2 (e.g., an allele containing the base sequence of sequence numbers 2, 10, 11, or 12), and / or,

[0096] There is no C allele at position 290 of sequence number 1 (e.g., an allele containing the sequence number 13, 14, 15, or 16).

[0097] The absence of the heritable trait of the present invention can be determined, for example, by detecting the following results:

[0098] The following alleles were not detected.

[0099] • Alleles with a T base at position 290 corresponding to sequence number 1 (e.g., alleles containing the base sequence of sequence number 1, 7, 8, or 9), and,

[0100] • Alleles whose base at position 40 corresponding to sequence number 2 is A (e.g., alleles containing the base sequence of sequence number 2, 10, 11, or 12), and / or,

[0101] • An allele with a C base at position 290 of sequence number 1 was detected (e.g., an allele containing the base sequence of sequence number 13, 14, 15, or 16).

[0102] Specific examples of the mutation detection methods of the present invention include PCR, TaqMan PCR, sequencing, microarray, Invader, TILLING, RAD, RFLP, PCR-SSCP, AFLP, SSLP, CAPS, dCAPS, ASO, ARMS, DGGE, CCM, DOL, MALDI-TOF / MS, TDI, lock-probe, molecular beacon, DASH, UCAN, ECA, PINPOINT, PROBE, VSET, Survivor assay, Sniper assay, Luminex assay, GOOD, LCx, SNaPshot, Mass ARRAY, Pyrosequences, SNP-IT, melting curve analysis, etc., but are not limited to these.

[0103] In the case of PCR, primers with a 3' end sequence complementary to the site of the hereditary trait involved in the present invention are preferably prepared. When primers designed in this way are used, if the template sample contains the allele involved in the hereditary trait of the present invention, polymerase extension occurs because the primers completely hybridize with the template; if the template does not contain the allele involved in the hereditary trait of the present invention, extension does not occur because the nucleotides at the 3' end of the primers do not match the template. Therefore, by performing PCR amplification using such primers and analyzing the amplification products by agarose gel electrophoresis or the like, if an amplification product of a certain size can be confirmed, the template sample contains the allele involved in the hereditary trait of the present invention; if the amplification product is absent, it can be determined that the template does not contain the allele involved in the hereditary trait of the present invention.

[0104] Alternatively, the primer sequence may be designed to avoid duplication of the sites involved in the hereditary traits of the present invention with the primer sequence, and the primer sequence may be designed to enable PCR amplification of nucleotide fragments containing alleles involved in the hereditary traits of the present invention. The hereditary traits of the present invention can be detected by sequencing the base sequence of the amplified nucleotide fragments.

[0105] For information on PCR and agarose gel electrophoresis, please refer to: Sambrook, Fritsch and Maniatis, “Molecular Cloning: A Laboratory Manual” 2nd Edition (1989), Cold Spring Harbor Laboratory Press.

[0106] The so-called TaqMan PCR method is a PCR reaction based on fluorescently labeled allele-specific oligonucleotides and Taq DNA polymerase (Livak, KJ Genet. Anal. 14, 143 (1999); Morris T. et al., J. Clin. Microbiol. 34, 2933 (1996)).

[0107] The so-called sequencing method is a method that uses PCR to amplify the region containing the part involved in the hereditary trait, and uses DyeTerminator or similar instruments to sequence the DNA sequence to analyze the presence or absence of the hereditary trait (Sambrook, Fritsch and Maniatis, “Molecular Cloning: A Laboratory Manual” 2nd Edition (1989), Cold SpringHarbor Laboratory Press).

[0108] DNA microarrays are materials in which one end of nucleotide probes is fixed in an array on a support, including DNA chips, gene chips, microchips, bead arrays, etc. The presence or absence of the heritable traits of the present invention can be detected in detail by using probes containing sequences complementary to the sequences containing the heritable traits of the present invention. Examples of DNA microarray assays, such as DNA chips, include gene chip assays (Affymetrix, Inc.; see U.S. Patent Nos. 6,045,996, 5,925,525, and 5,858,659). Gene chip technology utilizes miniaturized, high-density microarrays of oligonucleotide probes attached to a chip.

[0109] The Invader method is a combination of two reporter probes and one Invader probe that are specific to alleles such as SNPs that have or do not have heritable traits, hybridizing to template DNA, and DNA cutting based on Cleavase enzymes with special endonuclease activity that recognize DNA structure and cut DNA (Livak, KJ Biomol. Eng. 14, 143-149 (1999); Morris T. et al., J. Clin. Microbiol. 34, 2933 (1996); Lyamichev, V. et al., Science, 260, 778-783 (1993), etc.).

[0110] The so-called TILLING (Targeting Induced Local Lesions IN Genomes) method is a screening method that uses PCR amplification and CELI nuclease treatment to screen for variant mismatches in the genome of a mutant group with introduced variants.

[0111] In one manner, the genetic trait (1) of the present invention can be detected, for example, by using the following primer set with restriction enzymes via the dCAPS method.

[0112] Primer set:

[0113] A primer set comprising: a forward primer containing any consecutive sequence of 15 or more bases on the 5' side starting from position 289 of sequence number 1 (e.g., sequence number 21), and a reverse primer containing a sequence of 15 to 36 bases in length starting from the 3' end of a sequence selected from sequence numbers 22 to 24.

[0114] • Restriction enzymes:

[0115] The restriction enzyme corresponding to the primer set based on sequence number 22 is RsaI, the restriction enzyme corresponding to the primer set based on sequence number 23 is SnaI, and the restriction enzyme corresponding to the primer set based on sequence number 24 is AluI.

[0116] In one manner, the genetic trait (2) of the present invention can be detected, for example, by using the following primer set with restriction enzymes via the dCAPS method.

[0117] Primer set:

[0118] A primer set comprising: a forward primer containing a sequence of 15 to 48 bases in length from the 3' end of a sequence selected from Serial Numbers 25 to 27, and a reverse primer containing any sequence of 15 or more bases in length (e.g., Serial Number 28) located on the 3' side starting from position 49 of Serial Number 29.

[0119] • Restriction enzymes:

[0120] The restriction enzymes corresponding to the primer set based on sequence number 25 include SpeI or MaeI, the restriction enzymes corresponding to the primer set based on sequence number 26 include AflII / MseI, and the restriction enzymes corresponding to the primer set based on sequence number 27 include BspHI.

[0121] The primer sequences can be optimized within the range that meets the above conditions. For optimization of primer design, refer to, for example, Sambrook and Russell, “Molecular Cloning: A Laboratory Manual” 3rd Edition (2001), Cold Spring Harbor Laboratory Press. The primers mentioned above can be 15–50 bases long, 18–48 bases long, 20–45 bases long, 30–40 bases long, etc. The restriction enzymes corresponding to each primer set also include other enzymes that recognize the same sequence as the enzymes mentioned above, cleaved at the same position, or isolytic restriction enzymes of the enzymes mentioned above. Furthermore, primer sets other than those mentioned above can be designed based on the genetic traits of this invention, and corresponding restriction enzymes can be selected.

[0122] In a particular manner, the genetic traits of the present invention can be detected, for example, by using a primer set with a restriction enzyme via the dCAPS method.

[0123] [Table 1]

[0124] Table 1. Examples of primer sets and restriction enzyme combinations used for detecting hereditary traits (1).

[0125] forward primer reverse primer restriction enzymes Serial Number 21 Serial Number 22 RsaI Serial Number 21 Serial Number 23 SnaI Serial Number 21 Serial Number 24 AluI

[0126] [Table 2]

[0127] Table 2 shows examples of primer sets and restriction enzyme combinations used for detecting hereditary trait (2).

[0128] forward primer reverse primer restriction enzymes Serial Number 25 Serial Number 28 SpeI / MaeI Serial Number 26 Serial Number 28 AflII / MseI Serial Number 27 Serial Number 28 BspHI

[0129] Furthermore, the above-described primer set and restriction enzyme combination is merely an example. Anyone skilled in the art can find other primer sets and restriction enzyme combinations that can detect the genetic traits of the present invention.

[0130] The screening method of the present invention may further include a step of determining the content of steviol glycosides (e.g., TSG content) in the tissues (e.g., leaves) of the tested stevia plant that exhibits the genetic trait of the present invention. The determination of steviol glycoside content is as described in the plant section of the present invention. Furthermore, in this manner, individuals with high steviol glycoside content are selected from the tested stevia plants exhibiting the genetic trait of the present invention, and these individuals are crossbred with other stevia plants; the resulting offspring plants can also be screened using the screening method of the present invention. Thus, the screening method of the present invention may include one or more of the following steps.

[0131] (i) The step of detecting the genetic traits of the present invention from the genome of the tested stevia plant.

[0132] (ii) A step of determining the content of steviol glycosides in the tested stevia plant tissue that exhibits the genetic trait of the present invention.

[0133] (iii) The step of selecting individuals with high levels of steviol glycosides from the tested stevia plants that exhibit the genetic traits of the present invention.

[0134] (iv) The process of mating selected individuals with high levels of steviol glycosides with other stevia plants.

[0135] (v) The step of detecting the genetic traits of the present invention from the genome of the offspring obtained through mating.

[0136] (vi) A step of determining the content of steviol glycosides in the seed plant tissues that detect the genetic traits of the present invention.

[0137] (vii) The step of selecting individuals with high levels of steviol glycosides from the daughter plants in which the genetic traits of the present invention have been detected.

[0138] Individuals selected with high steviol glycoside content can be, for example, individuals among the tested stevia plants that exhibit the genetic trait of the present invention, whose steviol glycoside content ranks in the top 50%, top 40%, top 30%, top 20%, top 10%, top 5%, top 4%, top 3%, top 2%, or top 1%. Furthermore, other stevia plants used for mating may or may not contain the genetic trait of the present invention. In the above method, steps (iv) to (vii) can be repeated several times. In this way, stevia plants with even higher steviol glycoside content can be screened.

[0139] In the screening method of this invention, the stevia plant being tested can be a natural plant or a non-GMO plant. Regarding non-GMO plants, see the description in the plant section of this invention.

[0140] In the screening method of this invention, the stevia plants to be tested may also include stevia plants and their progeny plants that have undergone mutation induction treatment. Regarding mutation induction treatment, as described in the plant section of this invention, it includes treatment based on mutagens, treatment based on radiation or light irradiation, etc.

[0141] Furthermore, the present invention provides primer sets and combinations thereof as described above, for example, the primer sets described in Tables 1-2 above, and combinations of primer sets for detecting the genetic trait (1) described in Table 1 above and primer sets for detecting the genetic trait (2) described in Table 2 above. The present invention further provides primer sets that can amplify regions having base sequences selected from sequence numbers 1, 2, 13, and 17 by PCR, for example, primer sets containing a forward primer of sequence number 3 and a reverse primer of sequence number 4, and primer sets containing a forward primer of sequence number 5 and a reverse primer of sequence number 6. Furthermore, the present invention provides a combination of primer sets that can amplify regions having a base sequence of sequence number 1 or 13 by PCR and primer sets that can amplify regions having a base sequence of sequence number 2 or 17 by PCR. For example, a primer set containing a forward primer containing a base sequence of sequence number 3 and a reverse primer containing a base sequence of sequence number 4, and a primer set containing a forward primer containing a base sequence of sequence number 5 and a reverse primer containing a base sequence of sequence number 6.

[0142] Furthermore, the present invention provides probes (hereinafter, sometimes referred to as "probes of the present invention") capable of detecting the presence and / or absence of the hereditary traits of the present invention. The probes of the present invention may have a structure suitable for various methods of detecting the presence and / or absence of the hereditary traits of the present invention (e.g., Realtime PCR methods such as TaqMan PCR). For example, the probes of the present invention may contain a base sequence complementary to a portion of the genome containing the site of the hereditary trait of the present invention. As a non-limiting example of said probe, probes containing sequences complementary to base sequences selected from sequence numbers 7-12, 14-16, and 18-20 are listed. Among these sequences, sequence numbers 7-12 are specific for alleles related to the hereditary traits of the present invention, and sequence numbers 14-16 and 18-20 are specific for alleles not related to the hereditary traits of the present invention. Furthermore, sequence numbers 7-9 are specific for alleles related to the hereditary trait (1) of the present invention, and sequence numbers 10-12 are specific for alleles related to the hereditary trait (2) of the present invention. Furthermore, sequence numbers 14-16 are specific for alleles of alleles involved in the hereditary trait (1) not of the present invention, and sequence numbers 18-20 are specific for alleles of alleles involved in the hereditary trait (2) not of the present invention. The presence of the hereditary trait of the present invention can be detected by detecting the alleles involved in the hereditary trait of the present invention and / or by not detecting the alleles of alleles involved in the hereditary trait not of the present invention, and the absence of the hereditary trait of the present invention can be detected by not detecting the alleles involved in the hereditary trait of the present invention and / or by detecting the alleles of alleles involved in the hereditary trait not of the present invention. The probe of the present invention preferably has a label. As non-limiting examples of the label involved, fluorescent labels, luminescent labels, radioactive labels, pigments, enzymes, quenchers, and portions that bind to detectable labels may be listed. In a particular embodiment, the probe of the present invention has a polynucleotide comprising a base sequence complementary to the sequence selected from sequence numbers 7-12, 14-16, and 18-20 and a label.

[0143] The present invention also provides a kit comprising the primer set described above and the corresponding restriction enzyme. In a specific embodiment, the kit of the present invention comprises: a primer set comprising a combination of the forward and reverse primers described in Tables 1-2 above and the corresponding restriction enzyme.

[0144] Furthermore, the kit of the present invention comprises a set of primers for amplifying regions having base sequences selected from sequence numbers 1, 2, 13 and 17 by PCR, and the corresponding probes of the present invention described above.

[0145] These primer sets, probes, and kits can be used to detect the genetic traits of the present invention, and for the screening methods of the present invention, etc. Furthermore, these primer sets and kits may also include: instructions containing descriptions related to the detection of the genetic traits of the present invention or the screening methods of the present invention, such as user manuals or website information (e.g., URLs, QR codes) containing information related to the method of use, and media containing information related to the method of use, such as floppy disks, CDs, DVDs, Blu-ray discs, memory cards, USB storage devices, etc.

[0146] In one embodiment, the present invention provides a screening kit for stevia plants rich in steviol glycosides, characterized in that it comprises reagents for detecting the presence and / or absence of a hereditary trait (1), and reagents for detecting the presence and / or absence of a hereditary trait (2). The reagents may comprise primers and / or probes used in CAPS, dCAPS, or TaqMan PCR methods. In a particular embodiment, the reagents for detecting the presence and / or absence of the hereditary trait (1) comprise: a combination of primer sets and restriction enzymes for detecting the aforementioned hereditary trait (1) by dCAPS, for example, the combination of primer sets and restriction enzymes described in Table 1, or a combination of primer sets that can be used in TaqMan PCR and the like to amplify the site involved in the hereditary trait (1) (e.g., a site containing sequences selected from sequence numbers 7-9), and a combination of probes complementary to the site involved in the hereditary trait (1). In a particular manner, the reagent used to detect the presence and / or absence of the hereditary trait (2) comprises: a combination of primer sets and restriction enzymes for detecting the hereditary trait (2) by the dCAPS method, such as the combination of primer sets and restriction enzymes described in Table 2, or a set of primers that can be used in the TaqMan PCR method to amplify the site involved in the hereditary trait (2) (e.g., the site containing sequences selected from sequence numbers 10 to 12), and a combination of probes complementary to the site involved in the hereditary trait (2).

[0147] 4. Methods for manufacturing plant extracts and products using the extracts

[0148] In a further embodiment of the invention, a method for manufacturing an extract containing steviol glycosides (e.g., RebD and / or RebM) is provided (hereinafter, sometimes referred to as "the method for manufacturing the extract of the invention"), characterized by comprising a step of obtaining the extract from the plant of the invention, stevia plant selected by the screening method according to the invention, or stevia plant manufactured by the manufacturing method according to the invention, or the seeds, leaves (e.g., dried or fresh leaves), tissues, tissue cultures, or cells of the plant.

[0149] Furthermore, an extract containing steviol glycosides (e.g., RebD and / or RebM) obtained from the plant of the present invention, stevia plant selected according to the screening method of the present invention, or stevia plant manufactured according to the method of the present invention, or seeds, leaves (e.g., dried or fresh leaves), tissues, tissue cultures, or cells of the plant is provided (hereinafter, sometimes referred to as "the extract of the present invention"). The extract of the present invention is preferably an extract manufactured according to the method of manufacturing the extract of the present invention. Furthermore, a method for manufacturing steviol glycosides (hereinafter, sometimes referred to as "the method for manufacturing steviol glycosides of the present invention") is provided, characterized by comprising a step of purifying steviol glycosides (e.g., RebD and / or RebM) from the extract of the present invention. The method for manufacturing steviol glycosides of the present invention may further comprise a step of obtaining an extract containing steviol glycosides from the stevia plant of the present invention, stevia plant selected according to the screening method of the present invention, or stevia plant manufactured according to the method of the present invention.

[0150] Extracts containing steviol glycosides can be obtained by reacting fresh or dried leaves of the plant of the present invention with a suitable solvent (aqueous solvents such as water or organic solvents such as alcohols, ethers, and acetone). Extraction conditions, etc., can be referred to the methods described in Ohta et al. or WO2010 / 038911 above or the methods described in the examples described later.

[0151] Furthermore, the extracts containing steviol glycosides can be purified by known methods such as gradients of ethyl acetate and other organic solvents: water, high-performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), and ultra-high-performance liquid chromatography (UPLC). Examples of steviol glycosides are described in the plant section of this invention.

[0152] The extract obtained by the method of producing the extract according to the present invention (hereinafter referred to as "the extract of the present invention") contains a higher content of steviol glycosides compared with the extract of stevia plants that do not have the genetic traits of the present invention.

[0153] The extract of the present invention, compared with extracts obtained from stevia plants lacking the genetic traits of the present invention, may contain 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, and 35% or more of steviol glycosides. Here, the extract of the present invention and the extract obtained from stevia plants lacking the genetic traits of the present invention can be obtained by the same method.

[0154] By mixing the extract of the present invention thus obtained and / or the steviol glycoside refined product obtained by the method of manufacturing steviol glycoside refined products according to the present invention (e.g., RebD and / or RebM) with other ingredients, food and beverage products, sweetener compositions, flavorings, or pharmaceutical products containing steviol glycosides can be manufactured. Thus, as another embodiment, the present invention provides a method for manufacturing a food and beverage product, sweetener composition, flavoring, or pharmaceutical product, characterized by comprising a step of mixing the extract of the present invention and / or the steviol glycoside refined product obtained by the method of manufacturing steviol glycoside refined products according to the present invention with other ingredients. Further, the present invention provides a food and beverage product, sweetener composition, flavoring, or pharmaceutical product, characterized by containing steviol glycosides obtained according to the manufacturing method. Here, food and beverage products include beverages and food products. Therefore, in some embodiments, the present invention provides a beverage, food product, sweetener composition, flavoring, or pharmaceutical product, and also provides a method for manufacturing such a beverage, food product, sweetener composition, flavoring, or pharmaceutical product.

[0155] 5. The base sequence involved in the plant body of this invention.

[0156] In other ways, the present invention provides the base sequence involved in the stevia plant body of the present invention.

[0157] The stevia plant with genetic trait (1) involves a base sequence comprising or consisting of a base sequence selected from or consisting of sequence numbers 1, 7, 8, and 9. The stevia plant with genetic trait (2) involves a base sequence comprising or consisting of a base sequence selected from or consisting of sequence numbers 2, 10, 11, and 12. The stevia plant with the genetic trait of the present invention involves a base sequence comprising or consisting of a combination of a base sequence selected from or consisting ...

[0158] Example

[0159] The following describes experimental examples and embodiments related to the present invention, but the present invention is not limited to these specific methods.

[0160] (1) Creation of a group rich in steviol glycosides

[0161] Wild-type stevia seeds (commercially available varieties) were treated with ethyl methanesulfonate (EMS) and sown in a greenhouse at Suntory International Research Centre for cultivation. Fresh leaves were collected from each mature individual as samples, and the concentration of steviol glycosides was quantified using LC-MS / MS (Shimadzu LCMS8050). Specifically, 0.25 g of fresh leaves were freeze-dried, and 0.05 g of the dried powder was added to 100 times its volume (5 mL) of pure water. Extraction was performed by ultrasonic treatment for 20 minutes, followed by centrifugation and filtration. The liquid was diluted 60 times with 32% acetonitrile as the sample solution. 1 mL of this sample solution was analyzed by LC-MS / MS in MRM mode on the LCMS8050 to quantify the concentrations of RebA, RebB, RebC, RebD, RebF, RebM, RebN, RebO, and steviol glycosides. Individuals with a combined concentration of approximately 5–20% were selected for mating to obtain seeds. This selection process is repeated for four generations to obtain Group A.

[0162] (2) Genetic analysis of individuals rich in steviol glycosides

[0163] Fresh leaves were collected from individuals in Group A as samples, and the concentrations of RebA, RebB, RebC, RebD, RebE, RebF, RebG, RebM, RebN, and TSG were quantified using LC-MS / MS (Shimadzu LCMS8050), similar to (1) above. Furthermore, genomic DNA was extracted from fresh leaves of a subset of individuals and analyzed using a sequencer (HiSeq 2500, Illumina). The results showed that the average TSG content of individuals possessing the genetic traits of this invention tended to be higher than that of individuals without the genetic traits of this invention, all individuals in Group A, and individuals possessing genetic trait (2). Therefore, to improve the efficiency of genetic trait detection, dCAPS primers were prepared for detecting genetic traits (1) and (2), and the remaining individuals were evaluated for the presence of these genetic traits using the dCAPS method.

[0164] The following primers and restriction enzymes are used for dCAPS.

[0165] [Table 3]

[0166] Table 3. Sequences and restriction enzymes of dCAPS primers

[0167]

[0168] The detection of each hereditary trait based on the dCAPS method was performed as follows. First, genomic DNA was extracted from fresh leaves of each individual, and PCR was performed using the dCAPS primers corresponding to each hereditary trait. The restriction enzymes corresponding to each hereditary trait were added to the PCR products, and the enzymatic reaction was carried out at 37°C. Electrophoresis of the restriction enzyme-treated products was performed using a microchip electrophoresis device, LabChip GXTouch HT (PerkinElmer), and the presence or absence of the hereditary trait was determined based on the band patterns obtained. Specifically, for hereditary traits exhibiting isoconjugation (1), individuals with bands that only show decomposition products were identified as having the hereditary trait; for hereditary traits exhibiting isoconjugation or heteroconjugation (2), individuals with bands that show no decomposition products were identified as having the hereditary trait.

[0169] As shown in Table 4 and Figure 3 The results shown confirm the tendency identified in the sequencing. That is, the average TSG content of the population of individuals with the genetic trait of the present invention is 43.5%, 27.4%, and 7.2% higher than that of individuals without the genetic trait of the present invention, all individuals in group A, and individuals with genetic trait (2), respectively. Furthermore, genetic trait (2) is well known as a marker for selecting stevia plants rich in sweet components (Patent Document 3), and it is known that by combining it with the genetic trait (1) of the present invention, individuals with higher TSG content can be selected.

[0170] [Table 4]

[0171] Table 4 Relationship between TSG content and hereditary traits

[0172]

[0173] Industrial applicability

[0174] According to the present invention, steviol glycosides can be provided more effectively, thereby providing superior-tasting food and beverage, sweetener compositions, flavorings or pharmaceuticals, etc., by containing sufficient amounts of steviol glycosides. sequence list <110> Suntory Holdings, Inc. <120> Stevia plants rich in steviol glycosides and their screening methods <130> G2603 <150> JP 2020-084133 <151> 2020-05-12 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 326 <212> DNA <213> Stevia <400> 1 ggcagccatt gatgatgttg ttgaatgtga ttaatttgaa tgttataaag aatttggaaa 60 agaaaaagga ggggacaaag ttgatgaaat taggggagtt atgattatga tggccatggt 120 gattgtgatg agtggcacta tgtaatctaa tatttgaaga tatgagacca cttgaccatg 180 ttataatctt atacaaaata attaatccct cacggtaatt tttttctaat ccttaaactg 240 aaatttgaaa gtaatttgag atagtgtttc ccctaattta tgcttttagt atgcatttat 300 tctatcatat tttctatgag aattgg 326 <210> 2 <211> 145 <212> DNA <213> Stevia <400> 2 gatccaatgg agggggtgat tcaggtaata aaaggcatta gtatggaata taccaaaaca 60 ttgcgattcg ttattagcat ggatctttca agtaataaac ttatcggaga aataccagtt 120 gagttaactg cccttcatgc cttgg 145 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 3 ggcagccatt gatgatgttg ttgaa 25 <210> 4 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 4 ccaattctca tagaaaatat gatagaataa atgcat 36 <210> 5 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 5 gatccaatgg agggggtgat t 21 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 6 ccaaggcatg aagggcagtt a 21 <210> 7 <211> twenty one <212> DNA <213> Stevia <400> 7 atgcttttag tatgcattta t 21 <210> 8 <211> 41 <212> DNA <213> Stevia <400> 8 cccctaattt atgcttttag tatgcattta ttctatcata t 41 <210> 9 <211> 61 <212> DNA <213> Stevia <400> 9 gatagtgttt cccctaattt atgcttttag tatgcattta ttctatcata ttttctatga 60 g 61 <210> 10 <211> 21 <212> DNA <213> Stevia <400> 10 aaaaggcatt agtatggaat a 21 <210> 11 <211> 41 <212> DNA <213> Stevia <400> 11 ttcaggtaat aaaaggcatt agtatggaat ataccaaaac a 41 <210> 12 <211> 61 <212> DNA <213> Stevia <400> 12 gagggggtga ttcaggtaat aaaaggcatt agtatggaat ataccaaaac attgcgattc 60 ​​​​​​​​​​​​​​​​gattgtgatg agtggcacta tgtaatctaa tatttgaaga tatgagacca cttgaccatg 180 ttataatctt atacaaaata attaatccct cacggtaatt tttttctaat ccttaaactg 240 aaatttgaaa gtaatttgag atagtgtttc ccctaattta tgcttttagc atgcatttat 300 tctatcatat tttctatgag aattgg 326 <210> 14 <211> 21 <212> DNA <213> Stevia <400> 14 atgcttttag catgcattta t 21 <210> 15 <211> 41 <212> DNA <213> Stevia <400> 15 cccctaattt atgcttttag catgcattta ttctatcata t 41 <210> 16 <211> 61 <212> DNA <213> Stevia <400> 16 gatagtgttt cccctaattt atgcttttag catgcattta ttctatcata ttttctatga 60 g 61 <210> 17 <211> 145 <212> DNA <213> Stevia <400> 17 gatccaatgg agggggtgat tcaggtaata aaaggcattc gtatggaata taccaaaaca 60 ttgcgattcg ttattagcat ggatctttca agtaataaac ttatcggaga aataccagtt 120 gagttaactg cccttcatgc cttgg 145 <210> 18 <211> 21 <212> DNA <213> Stevia <400> 18 aaaaggcatt cgtatggaat a 21 <210> 19 <211> 41 <212> DNA <213> Stevia <400> 19 ttcaggtaat aaaaggcatt cgtatggaat ataccaaaac a 41 <210> 20 <211> 61 <212> DNA <213> Stevia <400> 20 gagggggtga ttcaggtaat aaaaggcatt cgtatggaat ataccaaaac attgcgattc 60 g 61 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 21 ggcagccatt gatgatgttg ttgaa 25 <210> 22 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 22 ccaattctca tagaaaatat gatagaataa atgcgt 36 <210> twenty three <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> twenty three ccaattctca tagaaaatat gatagaataa atgtat 36 <210> twenty four <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> twenty four ccaattctca tagaaaatat gatagaataa atgcaa 36 <210> 25 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 25 ttatttaatg atccaatgga gggggtgatt caggtaataa aaggcact 48 <210> 26 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 26 ttatttaatg atccaatgga gggggtgatt caggtaataa aaggctta 48 <210> 27 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 27 ttatttaatg atccaatgga gggggtgatt caggtaataa aagtcatg 48 <210> 28 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 28 tgagggttct caattgattt ccgattgg 28 <210> 29[[ID=२४]] <211> 367 <212> DNA <213> Stevia <400> 29 ttatttaatg atccaatgga gggggtgatt caggtaataa aaggcattag tatggaatat 60 accaaaacat tgcgattcgt tattagcatg gatctttcaa gtaataaact tatcggagaa 120 ataccagttg agttaactgc ccttcatgcc ttggtgagtc tcaatttgtc taataatcat 180 cttattggac acattccgaa tagcattgga aacatgaaag ctttaaattc tctagatttc 240 tcgagaaacg agttaaatgg gttgatccct ccaagcattg gagctttgaa ttttttgagt 300 catttaaatt tgtcaaacaa caacttatca ggaccaattc caatcggaaa tcaattgaga 360 accctca 367 <210> 30 <211> 326 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 30 ggcagccatt gatgatgttg ttgaatgtga ttaatttgaa tgttataaag aatttggaaa 60 agaaaaagga ggggacaaag ttgatgaaat taggggagtt atgattatga tggccatggt 120 gattgtgatg agtggcacta tgtaatctaa tatttgaaga tatgagacca cttgaccatg 180 ttataatctt atacaaaata attaatccct cacggtaatt tttttctaat ccttaaactg 240 aaatttgaaa gtaatttgag atagtgtttc ccctaattta tgcttttagt acgcatttat 300 tctatcatat tttctatgag aattgg 326 <210> 31 <211> 290 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 31 ggcagccatt gatgatgttg ttgaatgtga ttaatttgaa tgttataaag aatttggaaa 60 agaaaaagga ggggacaaag ttgatgaaat taggggagtt atgattatga tggccatggt 120 gattgtgatg agtggcacta tgtaatctaa tatttgaaga tatgagacca cttgaccatg 180 ttataatctt atacaaaata attaatccct cacggtaatt tttttctaat ccttaaactg 240 aaatttgaaa gtaatttgag atagtgtttc ccctaattta tgcttttagt 290 <210> 32 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 32 acgcatttat tctatcatat tttctatgag aattgg 36 <210> 33 <211> 326 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA [[ID=三十二]]<400> 33 ggcagccatt gatgatgttg ttgaatgtga ttaatttgaa tgttataaag aatttggaaa 60 agaaaaagga ggggacaaag ttgatgaaat taggggagtt atgattatga tggccatggt 120 gattgtgatg agtggcacta tgtaatctaa tatttgaaga tatgagacca cttgaccatg 180 ttataatctt atacaaaata attaatccct cacggtaatt tttttctaat ccttaaactg 240 aaatttgaaa gtaatttgag atagtgtttc ccctaattta tgcttttagc acgcatttat 300 tctatcatat tttctatgag aattgg 326 <210> 34 <211> 367 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 34 ttatttaatg atccaatgga gggggtgatt caggtaataa aaggcactag tatggaatat 60 accaaaacat tgcgattcgt tattagcatg gatctttcaa gtaataaact tatcggagaa 120 ataccagttg agttaactgc ccttcatgcc ttggtgagtc tcaatttgtc taataatcat 180 cttattggac acattccgaa tagcattgga aacatgaaag ctttaaattc tctagatttc 240 tcgagaaacg agttaaatgg gttgatccct ccaagcattg gagctttgaa ttttttgagt 300 catttaaatt tgtcaaacaa caacttatca ggaccaattc caatcggaaa tcaattgaga 360 accctca 367 <210> 35 <211> 367 <212> DNA [[ID=3l]]<213> Artificial Sequence <220> <223> Synthetic DNA <400> 35 [[ID=}39]]ttatttaatg atccaatgga gggggtgatt caggtaataa aaggcactcg tatggaatat 60 accaaaacat tgcgattcgt tattagcatg gatctttcaa gtaataaact tatcggagaa 120 ataccagttg agttaactgc ccttcatgcc ttggtgagtc tcaatttgtc taataatcat 180 cttattggac acattccgaa tagcattgga aacatgaaag ctttaaattc tctagatttc 240 tcgagaaacg agttaaatgg gttgatccct ccaagcattg gagctttgaa ttttttgagt 300 catttaaatt tgtcaaacaa caacttatca ggaccaattc caatcggaaa tcaattgaga 360 accctca 367 <210> 36 <211> 46 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 36 ttatttaatg atccaatgga gggggtgatt caggtaataa aaggca 46 <210> 37 <211> 320 <212> DNA <213> Artificial sequence <220> <223> Synthetic DNA <400> 37 ctagtatgga atataccaaa acattgcgat tcgttattag catggatctt tcaagtaata 60 aacttatcgg agaaatacca gttgagttaa ctgcccttca tgccttggtg agtctcaatt 120 tgtctaataa tcatcttatt ggacacattc cgaatagcat tggaaacatg aaagctttaa 180 attctctaga tttctcgaga aacgagttaa atgggttgat ccctccaagc attggagctt 240 tgaatttttt gagtcattta aatttgtcaa acaacaactt atcaggacca attccaatcg 300 gaaatcaatt gagaaccctc 320

Claims

1. A method for screening stevia plants rich in steviol glycosides, characterized in that, The procedure includes the steps of detecting the presence of the following genetic traits (1) and the following genetic traits (2) from the genome of the tested stevia plant. (1) Alleles containing the base sequence of sequence number 7 exhibit isomorphic conjugation; (2) Alleles containing sequence number 10 exhibit homozygous or heterozygous conjugation.

2. The method according to claim 1, characterized in that, The procedure further includes determining the content of steviol glycosides in the tested stevia plant tissue after detecting the presence of the genetic trait.

3. The method according to claim 1 or 2, characterized in that, The stevioside content of stevioside-rich stevioside plants is more than 3% higher than that of stevioside plants selected by a screening method that includes a step of detecting the presence of hereditary traits (2) but does not include a step of detecting the presence of hereditary traits (1).

4. The method according to any one of claims 1 to 3, characterized in that, The detection of heritable traits is performed using the dCAPS method or the TaqMan PCR method.

5. A screening kit for stevia plants rich in steviol glycosides, characterized in that, It contains reagents for detecting the presence of the following hereditary trait (1), and reagents for detecting the presence of the following hereditary trait (2). (1) Alleles containing the base sequence of sequence number 7 exhibit isomorphic conjugation; (2) Alleles containing sequence number 10 exhibit homozygous or heterozygous conjugation.

6. The reagent kit according to claim 5, characterized in that, The reagents contain primers and / or probes used in the CAPS, dCAPS, or TaqMan PCR methods.

7. A dried leaf of a stevia plant rich in steviol glycosides, characterized in that, The plant has the following genetic traits (1) and (2), (1) Alleles containing the base sequence of sequence number 7 exhibit isomorphic conjugation; (2) Alleles containing sequence number 10 exhibit homozygous or heterozygous conjugation.

8. The dried leaf according to claim 7, characterized in that, The plant body is a non-GMO plant body.

9. The dried leaf according to claim 7 or 8, characterized in that, The plant body comprises stevia plants treated with mutation induction and their progeny plants.

10. A method for preparing stevia plant material rich in steviol glycosides, characterized in that, Includes (i) the step of hybridizing a stevia plant having the genetic traits (1) and (2) as defined in claim 7 with a stevia plant having the genetic traits (1) and (2) as defined in claim 7; or (ii) The step of hybridizing a stevia plant having the genetic traits (1) and (2) as defined in claim 7 with a second stevia plant, and the step of hybridizing the offspring plant obtained by the above steps with a stevia plant having the genetic traits (1) and (2) as defined in claim 7.

11. The method according to claim 10, characterized in that, The second plant body is a stevia plant body having the genetic traits (1) and (2) as defined in claim 7.

12. A method for preparing stevia plant material rich in steviol glycosides, characterized in that, The process includes adding alterations to the genome of a stevia plant in such a way that the genome has the following genetic traits (1) and (2). (1) Alleles containing the base sequence of sequence number 7 exhibit isomorphic conjugation; (2) Alleles containing sequence number 10 exhibit homozygous or heterozygous conjugation.

13. A method for producing an extract containing steviol glycosides, characterized in that, The process includes obtaining extracts from stevia plants having the genetic traits (1) and (2) as defined in claims 7, their seeds, tissues, dried leaves, tissue cultures or cells.

14. A method for manufacturing steviol glycosides, characterized in that, The process includes obtaining extracts from stevia plants having the genetic traits (1) and (2) as defined in claim 7, their seeds, tissues, dried leaves, tissue cultures, or cells, and The process of purifying steviol glycosides from the extract.

15. A method for manufacturing a food or beverage, characterized in that, The process includes obtaining extracts from stevia plants having the genetic traits (1) and (2) as defined in claim 7, their seeds, tissues, dried leaves, tissue cultures, or cells, and, The process of adding the extract to the raw materials of a food or beverage.

16. A method for manufacturing a sweetener composition, characterized in that, The process includes obtaining extracts from stevia plants having the genetic traits (1) and (2) as defined in claim 7, their seeds, tissues, dried leaves, tissue cultures, or cells, and, The process of adding the extract to the raw materials of the sweetener composition.

17. A method for manufacturing a spice, characterized in that, The process includes obtaining extracts from stevia plants having the genetic traits (1) and (2) as defined in claim 7, their seeds, tissues, dried leaves, tissue cultures, or cells, and, The process of adding the extract to the raw materials of the fragrance.

18. A method for manufacturing a pharmaceutical product, characterized in that, The process includes obtaining extracts from stevia plants having the genetic traits (1) and (2) as defined in claim 7, their seeds, tissues, dried leaves, tissue cultures, or cells, and, The process of adding the extract to the raw materials of a pharmaceutical product.

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