sweetener stevia plant enriched in rebaudioside m

Abstract

The present invention provides at least one stevia plant with genetic characteristics (1) to (5).

Description

Technical Field

[0001] This invention relates to a stevia plant with high content of useful steviol glycosides such as riboside M, and a screening method thereof. 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, excessive consumption has been identified as having 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 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. As for steviol glycosides, various glycosides have been reported, including Rebaudioside A (hereinafter sometimes abbreviated as "Reb"), RebB, RebC, RebD, RebE, RebM, and RebN (Patent Document 2).

[0004] In such cases, it is known that stevia plants contain 1.53% RebM per unit of dried leaf (Patent Document 3).

[0005] Patent documents Patent Document 1: WO2007 / 070224 Patent Document 2: WO2010 / 038911 Patent Document 3: WO2021 / 230256 Summary of the Invention

[0006] RebM cannot be obtained in large quantities from the natural stevia plant, so efficient acquisition is sought.

[0007] This invention provides a stevia plant containing a higher content of useful steviol glycosides such as RebM compared to wild-type stevia, as well as a method for preparing and screening the plant.

[0008] In one embodiment, the present invention provides the following. [1] A stevia plant body, characterized in that it has at least one of the following genetic characteristics (1) to (5), (1) For the portion of the sequence corresponding to positions 150-151 of sequence number 1, the alleles with the sequence name T exhibit homozygous or heterozygous conjugation. (2) Alleles with the base TAGGTGGTGACACTGTAGC at position 151 of sequence number 2 exhibit homozygous or heterozygous binding. (3) Alleles with an A base at position 151 of sequence number 3 exhibit homozygous or heterozygous binding. (4) Alleles with an A base at position 151 of sequence number 4 exhibit homozygous or heterozygous binding. (5) Alleles with base A at position 151 of sequence number 5 exhibit isomorphic conjugation. [2] A stevia plant body, characterized in that it has at least one of the following chemical characteristics (i) to (v), (i) The content of RebM (rebodiin M) per unit mass of dried leaves is 1.8% or more. (ii) The difference between the RebM content and the steviol glycoside (ST) content per unit mass of dried leaves is greater than 0.9%. (iii) The difference between the RebM content and the RebE content per unit mass of dried leaves is more than 1.3%. (iv) The ratio of RebM content to RebN content per unit mass of dried leaves (RebM / RebN) is 1.52 or higher. (v) The RebM content per unit mass of dried leaves relative to the content of stevioside is 1.83 or higher. [3] The plant body according to [1] or [2] above is characterized in that it comprises a stevia plant body subjected to mutation induction treatment and its progeny plant bodies. [4] A dead tissue or dead cell, characterized in that it is a dead tissue or dead cell of a plant body as described in any one of [1] to [3] above. [5] 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 the above [1] to [3]. [6] The tissue, tissue culture or cell described in [5] above is characterized in that it is selected from embryos, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf slices and callus tissue. [7] A method for producing stevia plant material according to any one of [1] to [3] above, characterized in that it includes a step of hybridizing the plant material according to any one of [1] to [3] above with a second stevia plant material. [8] A method for producing stevia plant material according to any one of [1] to [3] above, characterized in that it includes a step of introducing at least one of the following variations into the genome of stevia plant material. (1) A variation that makes the portion of the sequence corresponding to positions 150-151 of sequence number 1 a variant of T. (2) A variation that makes the base at position 151 of sequence number 2 TAGGTGGTGACACTGTAGC. (3) A variation that makes the base at position 151 of sequence number 3 be A. (4) A variation that makes the base at position 151 of sequence number 4 be A. (5) A variation that makes the base at position 151 of sequence number 5 be A. [9] An extract, characterized in that it is an extract of the plant body described in any one of [1] to [3] above, the dead tissue or dead cells described in [4] above, the seeds, tissues, dried leaves, tissue cultures or cells described in [5] above, or the tissues, tissue cultures or cells described in [6] above.

[10] 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 described in any of [1] to [3] above, dead tissues or dead cells described in [4] above, seeds, tissues, dried leaves, tissue cultures or cells described in [5] above, or tissues, tissue cultures or cells described in [6] above.

[11] A method for manufacturing steviol glycosides, characterized in that it includes a step of purifying steviol glycosides from the extract described above [9].

[12] A method for manufacturing a food product, sweetener composition, flavoring, or pharmaceutical product, characterized in that it comprises the steps of providing an extract of a plant body as described in any one of [1] to [3] above, an extract of dead tissue or dead cells as described in [4] above, an extract of seeds, tissues, dried leaves, tissue cultures, or cells as described in [5] above, an extract of tissues, tissue cultures, or cells as described in [6] above, or an extract as described in [9] above, and, The process of adding the extract to the raw materials of food, beverages, sweetener compositions, flavorings or pharmaceuticals.

[13] A food, beverage, sweetener composition, flavoring or pharmaceutical product, characterized in that it contains an extract of plant body as described in any one of [1] to [3] above, an extract of dead tissue or dead cells as described in [4] above, an extract of seeds, tissues, dried leaves, tissue cultures or cells as described in [5] above, an extract of tissues, tissue cultures or cells as described in [6] above, or an extract as described in [9] above.

[14] A method for manufacturing a food product, sweetener composition, flavoring or pharmaceutical product, characterized in that it includes a step of purifying steviol glycosides from the extract described above [9], and, The process of adding purified steviol glycosides to the raw materials of food and beverage, sweetener compositions, flavorings or pharmaceuticals.

[15] A method for screening stevia plants having at least one of the chemical characteristics (i) to (v) described above [2], characterized in that it includes a step of detecting the presence and / or absence of at least one of the genetic characteristics (1) to (5) described above [1] in the tested stevia plants.

[16] A screening kit for stevia plants having at least one of the chemical characteristics (i) to (v) described above [2], characterized in that it comprises a reagent for detecting the presence and / or absence of at least one of the genetic characteristics (1) to (5) described above [1].

[0009] Through this invention, stevia plant body containing more useful steviol glycosides can be obtained, and methods for producing such plant body, leaves obtained from such plant body, and foods or beverages containing useful steviol glycosides obtained from such leaves can be provided. Attached Figure Description

[0010] Figure 1 A diagram showing the location of the variation involved in the genetic characteristic (1) in the base sequence of sequence number 1. The left side of the slash in parentheses represents the wild-type sequence, and the right side represents the variant sequence. Figure 2 A diagram showing the location of the variation involved in the genetic characteristic (2) in the base sequence of sequence number 2. The left side of the slash in parentheses represents the wild-type sequence, and the right side represents the variant sequence. Figure 3 A diagram showing the location of the variation involved in the genetic characteristic (3) in the base sequence of sequence number 3. The left side of the slash in parentheses represents the wild-type sequence, and the right side represents the variant sequence. Figure 4 A diagram showing the location of the variation involved in the genetic characteristic (4) in the base sequence of sequence number 4. The left side of the slash in parentheses represents the wild-type sequence, and the right side represents the variant sequence. Figure 5 A diagram showing the location of the variation involved in the genetic characteristic (5) in the base sequence of sequence number 5. The left side of the slash in parentheses represents the wild-type sequence, and the right side represents the variant sequence. Figure 6 The results of the analysis on whether the stevia plant has genetic characteristics (2'), (4') or (5) are shown. Detailed Implementation

[0011] 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. 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. 2023-204266), filed on December 1, 2023, which forms the basis of this application's priority claim.

[0012] 1. The plant body of the present invention 1-1. A stevia plant body of at least one species possessing genetic characteristics (1) to (5) In one embodiment, the present invention provides at least one stevia plant body having the following genetic characteristics (1) to (5) (hereinafter, sometimes referred to as "plant body A of the present invention" or "stevia plant body A of the present invention"). (1) Alleles of the sequence T corresponding to the part of sequence number 150-151 are homozygous or heterozygous. (2) Alleles with the base TAGGTGGTGACACTGTAGC at position 151 of sequence number 2 are homozygous or heterozygous. (3) Alleles with base A at position 151 of sequence number 3 exhibit homozygous or heterozygous binding. (4) Alleles with base A at position 151 of sequence number 4 exhibit homozygous or heterozygous conjugation. (5) Alleles with base A at position 151 of sequence number 5 exhibit isomorphic conjugation.

[0013] In the preferred embodiment, the genetic characteristics (1) to (4) are heterozygous as shown below. (1') Alleles of T corresponding to the portion of sequence 150-151 of sequence number 1 are heterozygous. (2') The alleles of TAGGTGGTGACACTGTAGC at position 151 of sequence number 2 are heteroconjugated. (3') Alleles with base A at position 151 of sequence number 3 exhibit heterozygous conjugation. (4') Alleles with base A at position 151 of sequence number 4 exhibit heterozygous conjugation.

[0014] The term "position / part equivalent to" refers to the location / part of the genome that corresponds to the standard sequence (e.g., sequence numbers 1-5) when such a sequence exists in the genome. Conversely, it refers to the location / part of the genome that corresponds to the standard sequence when no such 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 more than 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.4%, 98.7%, 99%, 99.2%, 99.5%, or 99.8% sequence identity with the standard sequence. The position / part of a sequence in the genome that is equivalent to the position / part of the standard sequence can be determined by considering the base sequences before and after that position / part in the standard sequence. For example, by comparing a standard sequence with sequences in the genome that are equivalent to the standard sequence, one can determine the position / part of the sequence in the genome that is equivalent to the position / part of the standard sequence.

[0015] For example, taking the "part corresponding to positions 150-151 of sequence number 1" in the genetic feature (1) of the present invention as an example, when the genome of the stevia plant has a part consisting of the same base sequence as sequence number 1, the "part corresponding to positions 150-151 of sequence number 1" is positions 150-151 starting from the 5' side of the part of the genome consisting of the same base sequence as sequence number 1. On the other hand, when the genome of the stevia plant is different from sequence number 1 but has a part consisting of a base sequence corresponding to it, since the genome does not have a part consisting of the same base sequence as sequence number 1, the "part corresponding to positions 150-151 of sequence number 1" is not necessarily positions 150-151 starting from the 5' side of the part corresponding to sequence number 1. Instead, the base sequences before and after positions 150-151 of sequence number 1 can be considered to determine the "part corresponding to positions 150-151 of sequence number 1" in the genome of the stevia plant involved. For example, by comparing the base sequence of the part of the stevia genome corresponding to sequence number 1 with the base sequence of sequence number 1, the part of the stevia genome corresponding to positions 150-151 of sequence number 1 can be determined.

[0016] Here, the positions selected from (1) the portion corresponding to the 150th to 151st position of serial number 1, (2) the position corresponding to the 151st position of serial number 2, (3) the position corresponding to the 151st position of serial number 3, (4) the position corresponding to the 151st position of serial number 4, and (5) the position corresponding to the 151st position of serial number 5 are sometimes collectively referred to as "the polymorphic part of the present invention" or "the variant part of the present invention". In addition, the positions / parts of (1) to (5) above are sometimes referred to as "polymorphic part (1)", "polymorphic part (2)" or "variant part (1)", "variant part (2)", etc.

[0017] "The portion of the sequence corresponding to Serial Number 1" refers to, for example, a portion of the sequence that has 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 of the sequence identity with respect to Serial Number 1.

[0018] 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 the following primers: a forward primer for hybridization with the complementary sequence of the portion from position 1 to 149 (i.e., from the 5' end of sequence number 1 to the base on the 5' end side of the variant site (1)) and a reverse primer for hybridization with the portion from position 1 to 150 (i.e., from the 3' end of sequence number 1 to the base on the 3' end side of the variant site (1)). For the sake of brevity, the genetic feature (1) of the present invention is used as an example for explanation, but the same applies to the genetic features (2) to (5) of the present invention.

[0019] 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 6 and the reverse primer containing the base sequence of sequence number 7. 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 8 and the reverse primer containing the base sequence of sequence number 9. In a particular manner, "the portion consisting of the base sequence corresponding to sequence number 4" 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 10 and the reverse primer containing the base sequence of sequence number 11. In a particular manner, "the portion consisting of the base sequence corresponding to sequence number 5" 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 12 and the reverse primer containing the base sequence of sequence number 13.

[0020] In a specific manner, "the portion of the sequence corresponding to positions 150-151 of sequence number 1 is the T allele", including the base sequence of sequence number 14, 15, 16 or 17. In a specific manner, "the allele of TAGGTGGTGACACTGTAGC at the position corresponding to position 151 of sequence number 2" includes the base sequence of sequence number 18, 19, 20, or 21. In a specific manner, "the allele with an A base at position 151 of sequence number 3" includes the base sequence of sequence numbers 22, 23, 24, or 25. In a specific manner, "the allele with an A base at position 151 of sequence number 4" includes the base sequence of sequence number 26, 27, 28, or 29. In a specific manner, "the allele with an A base at position 151 of sequence number 5" includes the base sequence of sequence numbers 30, 31, 32, or 33.

[0021] Furthermore, the variations selected from (1) the variation from TG to T at the position corresponding to position 150-151 of serial number 1, (2) the variation from T to TAGGTGGTGACACTGTAGC at the position corresponding to position 151 of serial number 2, (3) the variation from G to A at the position corresponding to position 151 of serial number 3, (4) the variation from T to A at the position corresponding to position 151 of serial number 4, and (5) the variation from T to A at the position corresponding to position 151 of serial number 5, are collectively referred to as "polymorphisms of the present invention" or "variations of the present invention". In addition, the variations of (1) to (5) above are sometimes referred to as "polymorphism (1) of the present invention", "polymorphism (2) of the present invention", or "variation (1) of the present invention", "variation (2) of the present invention", etc.

[0022] The above genetic characteristics 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. The methods include (extension) method, VSET (very short extension) method, Survivor assay, Sniper assay, Luminex assay, GOOD method, LCx method, SNaPshot method, Mass ARRAY method, Pyrosequencing method, SNP-IT method, melting curve analysis method, etc., but the detection methods are not limited to these.

[0023] In a specific manner, the genetic characteristics of the present invention can be detected by the dCAPS method based on the following primer set and restriction enzyme combination. When a candidate plant exhibits variation (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 SEQ ID NO. 34 and a reverse primer with the base sequence shown in SEQ ID NO. 35. Even after treating the resulting PCR product (approximately 144 bp long, e.g., SEQ ID NO. 36) with the restriction enzyme Hyp188I, only a band of approximately 144 bp long (e.g., SEQ ID NO. 36) is obtained. On the other hand, when a candidate plant does not exhibit variation (1'), PCR amplification is performed in the same manner as described above. When the resulting PCR product (approximately 145 bp long, e.g., SEQ ID NO. 37) is treated with the restriction enzyme Hyp188I, a band of approximately 117 bp long (e.g., SEQ ID NO. 38) and a band of approximately 28 bp long (e.g., SEQ ID NO. 39) are obtained. Therefore, by confirming the approximately 144 bp long band using the dCAPS method described above, the genetic characteristics of the present invention (1') can be determined.

[0024] When a candidate plant exhibits variation (4'), for example, PCR amplification is performed on the genomic DNA of the candidate plant using a forward primer with the base sequence shown in sequence number 40 and a reverse primer with the base sequence shown in sequence number 41. When the resulting PCR product (approximately 103 bp long, for example, sequence number 42) is treated with the restriction enzyme NdeI, a band of approximately 78 bp long (for example, sequence number 43) and a band of approximately 25 bp long (for example, sequence number 44) are obtained. On the other hand, when a candidate plant does not exhibit variation (4'), PCR amplification is performed in the same manner as described above. Even when the resulting PCR product (approximately 103 bp long, for example, sequence number 45) is treated with the restriction enzyme NdeI, only a band of approximately 103 bp long (for example, sequence number 45) is obtained. Thus, by using the dCAPS method described above, when a band of approximately 78 bp long and / or a band of approximately 25 bp long from the decomposed product and a band of approximately 103 bp long from the non-decomposed product are confirmed, the genetic characteristics of the present invention (4') can be determined. An example of detecting genetic traits (4') using the above method is shown below. Figure 6 .

[0025] When a candidate plant exhibits variation (5'), for example, PCR amplification is performed on the genomic DNA of the candidate plant using a forward primer with the base sequence shown in sequence number 46 and a reverse primer with the base sequence shown in sequence number 47. When the resulting PCR product (approximately 161 bp long, for example, sequence number 48) is treated with the restriction enzyme MfeI, a band of approximately 130 bp (for example, sequence number 49) and a band of approximately 31 bp (for example, sequence number 50) are obtained. Conversely, when a candidate plant does not exhibit variation (5'), PCR amplification is performed in the same manner as described above. Even when the resulting PCR product (approximately 161 bp long, for example, sequence number 51) is treated with the restriction enzyme MfeI, only a band of approximately 161 bp (for example, sequence number 51) is obtained. Therefore, by using the dCAPS method described above, when a band of approximately 130 bp and / or approximately 31 bp from the decomposition product and a band of approximately 161 bp from sources other than the decomposition product are identified, the genetic characteristics of the present invention can be determined (5'). An example of detecting genetic traits (5') using the above method is shown below. Figure 6 .

[0026] Furthermore, when the candidate plant has a variation (2'), for example, when PCR amplification is performed on the genomic DNA of the candidate plant using a forward primer with the base sequence shown in sequence number 52 and a reverse primer with the base sequence shown in sequence number 53, a PCR product (approximately 206 bp long, for example, sequence number 54) is obtained. On the other hand, when the candidate plant does not have a variation (2'), PCR amplification is performed in the same manner as described above, and a PCR product (approximately 188 bp long, for example, sequence number 55) is obtained. Thus, when a band of approximately 206 bp long and a band of approximately 188 bp long are confirmed, the genetic characteristic (2') of the present invention can be determined. An example of detecting the genetic characteristic (2') by the above method is shown below. Figure 6 .

[0027] In one embodiment, the plant body A of the present invention has a higher characteristic than that of a stevia plant body (hereinafter sometimes referred to as a control plant body) that does not have the genetic characteristics (1'), (2'), (4') and (5) than that of a control plant body (hereinafter sometimes referred to as a control plant body). The characteristics are selected from at least one of RebM content, the ratio of RebM content to RebN content (RebM / RebN), the ratio of RebM content to ST content (RebM / ST), RebM-ST (the difference between RebM content and ST content, hereinafter the same), and RebM-RebE. The term "higher than control plants" means, for example, that when cultivated under the same cultivation conditions, the content (e.g., the content in dried leaves), the content ratio, or the content difference of the above-mentioned components is greater than that of control plants, preferably statistically significantly greater than that of control plants. More specifically, this refers to situations where, under the same cultivation conditions, the content, ratio, or difference of the aforementioned components in the dried leaves is more than 1 time, approximately 1.1 times, more than 1.2 times, more than 1.3 times, more than 1.4 times, more than 1.5 times, more than 1.6 times, more than 1.7 times, more than 1.8 times, more than 1.9 times, more than 2 times, more than 2.1 times, more than 2.2 times, more than 2.3 times, more than 2.4 times, more than 2.5 times, more than 2.6 times, more than 2.7 times, more than 2.8 times, more than 2.9 times, more than 3 times, more than 3.1 times, more than 3.2 times, more than 3.3 times, more than 3.4 times, more than 3.5 times, or more than 3.6 times that of the control plant. The following are listed: approximately 3.7 times or more, approximately 3.8 times or more, approximately 3.9 times or more, approximately 4 times or more, approximately 4.1 times or more, approximately 4.2 times or more, approximately 4.3 times or more, approximately 4.4 times or more, approximately 4.5 times or more, approximately 4.6 times or more, approximately 4.7 times or more, approximately 4.8 times or more, approximately 4.9 times or more, approximately 5 times or more, approximately 6 times or more, approximately 7 times or more, approximately 8 times or more, approximately 9 times or more, approximately 10 times or more, approximately 15 times or more, approximately 20 times or more, approximately 25 times or more, approximately 30 times or more, approximately 35 times or more, approximately 40 times or more, approximately 45 times or more, approximately 50 times or more, approximately 55 times or more, approximately 60 times or more, approximately 65 times or more, approximately 70 times or more, approximately 75 times or more, approximately 80 times or more, approximately 85 times or more, approximately 90 times or more, approximately 95 times or more, or approximately 100 times or more, etc. Furthermore, for example, when comparing the content, content ratio, or content difference of the aforementioned components in the dried leaves of the population of plant A of the present invention with the content, content ratio, or content difference of the aforementioned components in the dried leaves of the population of control plants using Welch's t-test, the average value of the former is larger than the average value of the latter, and the p-value of both sides is less than 0.05, preferably less than 0.01, and particularly preferably less than 0.005. The content, etc., being compared can be the average value of multiple individuals.

[0028] 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 consists of RebA, RebB, RebC, RebD, RebE, RebF, RebI, RebJ, RebM, RebN, and steviol glycoside.

[0029] In one embodiment, the plant A of the present invention may also have multiple of the above-mentioned genetic characteristics (1) to (5). In this embodiment, the number of genetic characteristics may be any one of 2 to 5, that is, 2, 3, 4 or 5. In a specific embodiment, the plant A of the present invention has genetic characteristics (1'), (2'), (4') and (5).

[0030] 1-2. Stevia plants possessing at least one of the chemical characteristics (i) to (v) In one embodiment, the present invention provides a stevia plant body having at least one of the following chemical characteristics (i) to (v) (hereinafter, sometimes collectively referred to as "plant body B of the present invention" or "stevia plant body B of the present invention"). (i) The RebM content per unit mass of dried leaves is 1.8% or more. (ii) The difference between the RebM content and the stevioside content per unit mass of dried leaves is more than 0.9%. (iii) The difference between the RebM content and the RebE content per unit mass of dried leaves is more than 1.3%. (iv) The ratio of RebM content to RebN content per unit mass of dried leaves (RebM / RebN) is 1.52 or higher. (v) The RebM content per unit mass of dried leaves relative to the content of stevioside is 1.83 or higher.

[0031] In the plant body B of the present invention, which has chemical characteristic (i), the RebM content per unit mass of the so-called dried leaf is 1.8% or more, for example, meaning that a specified mass of dried leaves (e.g., 50 mg) contains a proportion of RebM of 1.8% or more by mass (e.g., 0.9 mg or more). The proportion of RebM per unit mass of dried leaves in this manner is not limited, and can be, for example, 1.8% or more, 1.9% or more, 1.95% or more, 2.0% or more, 2.1% or more, 2.2% or more, 2.3% or more, 2.4% or more, 2.5% or more, etc. The upper limit of the proportion of RebM per unit mass of dried leaves is not particularly limited, and can be, for example, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, etc. Here, dried leaves refer to leaves whose moisture content has been reduced to 3-4% by weight by drying the fresh leaves of the stevia plant of the present invention.

[0032] In the plant body B of the present invention, which possesses chemical characteristic (ii), the difference between the RebM content and the steviol glycoside content per unit mass of the so-called dried leaf is 0.9% or more. For example, this means that the difference between the amount of RebM and the amount of steviol glycoside contained in a specified mass of dried leaves (e.g., 50 mg) is 0.9% by mass or more. The difference between the RebM content and the steviol glycoside content per unit mass of the dried leaves in this manner is not limited, and can be, for example, 0.9% or more, 1.0% or more, 1.1% or more, 1.2% or more, 1.3% or more, 1.4% or more, 1.5% or more, 1.6% or more, 1.7% or more, 1.8% or more, 1.9% or more, 2.0% or more, etc. The upper limit of the difference between the RebM content and the steviol glycoside content per unit mass of the dried leaves is not particularly limited, and can be, for example, 5%, 4%, 3%, 2.5%, 2%, etc.

[0033] In the plant body B of the present invention, which possesses chemical characteristic (iii), the difference between the RebM content and the RebE content per unit mass of the so-called dried leaf is 1.3% or more. For example, this means that the difference between the amount of RebM and the amount of RebE contained in a specified mass of dried leaves (e.g., 50 mg) is 1.3% by mass or more. The difference between the RebM content and the RebE content per unit mass of the dried leaf in this manner is not limited, and can be, for example, 1.3% or more, 1.4% or more, 1.5% or more, 1.6% or more, 1.7% or more, 1.8% or more, 1.9% or more, 2.0% or more, 2.1% or more, 2.2% or more, 2.3% or more, 2.4% or more, etc. The upper limit of the difference between the RebM content and the RebE content per unit mass of the dried leaf is not particularly limited, and can be, for example, 5%, 4%, 3%, 2.5%, 2%, etc.

[0034] In the plant body B of the present invention, which has chemical characteristic (iv), the ratio of RebM content to RebN content per unit mass of the so-called dried leaf (RebM / RebN) is 1.52 or more. For example, this means that the ratio of RebM to RebN content in a specified mass of dried leaf (e.g., 50 mg) is 1.52 or more. The RebM / RebN ratio per unit mass of dried leaf in this manner is not limited, and can be, for example, 1.52 or more, 1.6 or more, 1.7 or more, 1.75 or more, 1.8 or more, 1.83 or more, 1.85 or more, 1.90 or more, 1.95 or more, 2.0 or more, 2.1 or more, etc. The upper limit of the ratio of RebM to RebN per unit mass of dried leaf is not particularly limited, and can be, for example, 20.0, 10.0, 9.0, 8.0, 7.0, 6.0, 5.0, etc.

[0035] In the plant body B of the present invention having chemical characteristic (v), the ratio of RebM content per unit mass of the so-called dried leaf to the content of steviol glycoside, RebM / ST, is 1.83 or more. For example, this means that the ratio of RebM content per unit mass of dried leaf (e.g., 50 mg) to steviol glycoside is 1.83 or more. The RebM / ST per unit mass of dried leaf in this manner is not limited, and may be, for example, 1.83 or more, 1.9 or more, 2.0 or more, 2.1 or more, 2.4 or more, 2.5 or more, 2.8 or more, 3.0 or more, 3.1 or more, 3.3 or more, 3.5 or more, 3.7 or more, 3.9 or more, 4.0 or more, 4.5 or more, 5.0 or more, 5.5 or more, 6.0 or more, etc. There is no particular upper limit to the ratio of RebM to stevioside per unit mass of dried leaves, such as 20.0, 10.0, 9.0, 8.0, 7.0, 6.0, 5.0, etc.

[0036] In some embodiments, the plant body B of the present invention may also possess multiple of the aforementioned chemical characteristics. In this embodiment, the number of chemical characteristics may be any one of 2 to 5, i.e., 2, 3, 4, or 5.

[0037] In a particular manner, the present invention provides a stevia plant body having at least one of the genetic characteristics (1) to (5) and at least one of the chemical characteristics (i) to (v) (hereinafter, sometimes collectively referred to as "plant body C of the present invention" or "stevia plant body C of the present invention"). In addition, the plant bodies A to C of the present invention are sometimes collectively referred to as "the plant bodies of the present invention".

[0038] Steviosides such as RebD, RebE, RebM, and RebN 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. Furthermore, the extract thus obtained can be purified using known methods such as 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) to obtain various steviol glycosides, such as RebD, RebE, RebM, and RebN.

[0039] The contents of steviol glycosides such as RebD, RebE, RebM, and RebN can be determined by the methods described in Ohta et al. or WO2010 / 038911 above, or by the methods described in the examples described later. Specifically, for example, fresh leaves can be collected from stevia plants A to C of the present invention as samples, and the contents can be determined by performing LC-MS / MS or the like.

[0040] The plant material of the present invention can be a plant material or its offspring obtained by transgenic methods (hereinafter, sometimes referred to as "transgenic plant material"), or a plant material or its offspring obtained by non-transgenic methods (hereinafter, sometimes referred to as "non-transgenic plant material"). Examples of "non-transgenic methods" include, in addition to hybridization or self-pollination, methods that induce gene variation in host cells (or host plants) without introducing foreign genes. Methods that induce the action of mutagens in plant cells can be listed as examples of 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 1–48 hours, 2–36 hours, 3–30 hours, 4–28 hours, 5–26 hours, and 6–24 hours. The treatment process itself is well known, for example, it can be carried out by immersing the water-absorbing seeds that have undergone the water absorption process in a treatment solution containing a mutagen at the above concentration for the above treatment time.

[0041] Alternatively, as an example of a non-GMO method, plant cells can be irradiated with radiation or light such as X-rays, gamma rays, ion beams (e.g., heavy ion beams), or ultraviolet light. In this case, after irradiating the cells with an appropriate amount of ultraviolet light (ultraviolet lamp intensity, distance, and time), and culturing them in a selective medium, cells, callus tissue, or plant bodies with the target traits can be selected. The irradiation intensity at this time is 0.01~100Gr, 0.03~75Gr, 0.05~50Gr, 0.07~25Gr, 0.09~20Gr, 0.1~15Gr, 0.1~10Gr, 0.5~10Gr, and 1~10Gr; the irradiation distance is 1cm~200m, 5cm~100m, 7cm~75m, 9cm~50m, 10cm~30m, 10cm~20m, and 10cm~10m; 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 the state of the irradiated object (cells, callus tissue, plant body), and can be appropriately adjusted by those skilled in the art.

[0042] In addition, cell fusion, anther culture (haploid culture), and distant hybridization (haploid culture) are also well-known methods. 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. Plants obtained by transgenic modification (e.g., through genome editing) using the plant of the present invention as a host are also not excluded from the scope of the present invention.

[0043] The plant body of the present invention includes not only the whole plant body, but also its parts or derivatives. Therefore, the plant body of the present invention includes 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.

[0044] 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.

[0045] 2. The method for preparing the plant body according to the present invention In other embodiments, the present invention provides a method for producing stevia plants of the present invention (hereinafter, sometimes referred to as "method A of the present invention"), characterized by comprising a step of hybridizing the stevia plants of the present invention with a second stevia plant. Stevia plants produced by this method can have the same phenotype and genetic characteristics as the plants of the present invention. The content and mass ratio range of each component in the plant obtained by method A of the present invention, the combination of each characteristic (chemical characteristics and / or genetic characteristics), etc., are as described above regarding the plant of the present invention.

[0046] In method A of the present invention, "hybridization" refers to obtaining a daughter plant (a plant produced according to the method of the present invention, i.e., a second generation (S2)) by mating any one of the plant bodies of the present invention (first generation (S1)) and a second plant body (S1). Backcrossing is preferred as a hybridization method. "Backcrossing" is, for example, a method of further hybridizing a daughter plant (S2) born between the plant body of the present invention and the second plant body with the plant body of the present invention (i.e., a plant body having the genetic characteristics of the present invention) (S1) to produce a plant body having the genetic characteristics of the present invention. When the second plant body (S1) used in the method of the present invention has the same phenotype and genetic characteristics as the plant body of the present invention, it is essentially a backcross. Hybridization is preferably carried out for two or more generations; when the genetic characteristics are heterozygous, etc., sometimes a plant body with a combination of desired genetic characteristics can be obtained even in a single generation. Alternatively, the plant of the present invention can also be produced by self-pollination. Self-pollination can be carried out by pollinating the pollen of the stamens of the plant of the present invention to the pistil of the plant of the present invention.

[0047] The phenotype and genetic characteristics 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 with the same phenotype as the plant produced by the present invention can also be produced.

[0048] 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. The culture conditions are the same as those for tissue cultures or plant culture cells of wild-type stevia plants, and are well known (Protocols for In Vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, Method in molecularbiology, vol. 1391, pp113-123).

[0049] Furthermore, as another method, the plant of the present invention can be produced by introducing the variants of the present invention into the genome of a stevia plant. Thus, the present invention provides a method for producing a stevia plant of the present invention (hereinafter, sometimes referred to as "method B of the present invention"), characterized by comprising the step of introducing at least one of the following variants into the genome of a stevia plant. (1) A variation of the sequence that corresponds to the portion of the sequence number 150-151. (2) A variation that makes the base at the position corresponding to position 151 of sequence number 2 TAGGTGGTGACACTGTAGC. (3) A variation that makes the base at position 151 of sequence number 3 be A. (4) A variation that makes the base at position 151 of sequence number 4 be A. (5) A variation that makes the base at position 151 of sequence number 5 be A. The introduction of variations can be carried out through transgenic methods or non-transgenic methods. Examples of transgenic methods include introducing the desired variation into the genome of a host cell (or host plant) through genome editing, etc. Non-transgenic methods are described above in the section on plants in this invention.

[0050] 3. The plant screening method of the present invention The plant organisms of the present invention, and plant organisms having the same phenotype and / or genetic characteristics as those of the present invention, can be screened by detecting the genetic characteristics of the present invention in the tissues of such plant organisms. Here, "screening" means identifying the plant organisms of the present invention and other plant organisms, and selecting the plant organisms of the present invention. Thus, in other aspects, the present invention provides a method for screening stevia plants having at least one of the chemical characteristics (i) to (v) (hereinafter, sometimes referred to as "the screening method of the present invention"), characterized in that it includes a step of detecting the presence and / or absence of at least one of the genetic characteristics (1) to (5) in the stevia plant being tested. The screening method of the present invention may further include a step of selecting plants from the tested plants that are found to have at least one of the above-mentioned genetic characteristics.

[0051] The presence of the genetic characteristics of the present invention can be determined, for example, by detecting the following results: (I) There is an allele with a sequence of T corresponding to positions 150-151 of sequence number 1 (e.g., an allele containing the base sequence of sequence number 14, 15, 16, or 17, hereinafter sometimes referred to as "allele i"), (II) An allele exists at position 151 of sequence number 2 with the base sequence TAGGTGGTGACACTGTAGC (e.g., an allele containing the base sequence of sequence number 18, 19, 20, or 21, hereinafter sometimes referred to as "allele ii"). (III) The presence of an allele with an A base at position 151 of sequence number 3 (e.g., an allele containing the sequence of sequence numbers 22, 23, 24, or 25, hereinafter sometimes referred to as "allele iii") (IV) The presence of an allele with an A base at position 151 of sequence number 4 (e.g., an allele containing the sequence of sequence numbers 26, 27, 28, or 29, hereinafter sometimes referred to as "allele iv"), and / or (V) There is no allele other than the allele with an A base at position 151 of sequence number 5 (e.g., the allele containing the base sequence of sequence numbers 30, 31, 32 or 33, hereinafter sometimes referred to as "allele v").

[0052] The genetic characteristics of this invention are absent, for example, this can be determined by detecting the following results: (i) There is no allele i. (ii) No alleles exist. (iii) No alleles iii, (iv) No alleles present, and / or (v) There are alleles other than allele v.

[0053] Specific examples of the genetic characteristic detection methods of the present invention include, but are not limited to, 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, Sniperassay, Luminex assay, GOOD, LCx, SNaPshot, Mass ARRAY, Pyrosequencing, SNP-IT, melting curve analysis, etc.

[0054] In one manner, the genetic characteristics (1) of the present invention can be detected, for example, by using the dCAPS method with the following primer set and restriction enzyme. Primer set: A primer set comprising: a forward primer containing a sequence of 15 to 25 bases from the 3' end of sequence number 34, and a reverse primer containing a sequence complementary to any sequence of 15 or more bases on the 3' side starting from position 26 of sequence number 36 or 37 (e.g., sequence number 35). • Restriction enzyme: Hyp188I or its isoschizase

[0055] In one manner, the genetic characteristics (4) of the present invention can be detected, for example, by using the dCAPS method with the following primer set and restriction enzyme. Primer set: A primer set comprising: a forward primer containing any consecutive sequence of 15 or more bases on the 5' side starting from position 78 of sequence number 42 or 45 (e.g., sequence number 40), and a reverse primer containing a consecutive sequence of 15 to 25 bases from the 3' end of sequence number 41. • Restriction enzyme: NdeI or its isoschizase

[0056] In one manner, the genetic characteristics (5) of the present invention can be detected, for example, by using the dCAPS method with the following primer set and restriction enzyme. Primer set: A primer set comprising: a forward primer containing a sequence of 15 to 28 bases from the 3' end of sequence number 46, and a reverse primer containing a sequence complementary to any sequence of 15 or more bases on the 3' side starting from position 29 of sequence number 48 or 51 (e.g., sequence number 47). • Restriction enzyme: MfeI or its isoschizase

[0057] In one approach, the genetic characteristics (2) of the present invention can be detected, for example, using the following set of primers. Primer set: A primer set comprising: a forward primer containing a sequence of 15 to 25 bases from the 3' end of sequence number 52, and a reverse primer containing a sequence complementary to any sequence of 15 or more bases on the 3' side starting from position 26 of sequence number 54 or 55 (e.g., sequence number 53).

[0058] 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. Furthermore, primer sets other than those mentioned above can be designed based on the genetic characteristics of this invention, and corresponding restriction enzymes can be selected.

[0059] In a particular manner, the genetic characteristics (1), (4) or (5) of the present invention can be detected, for example, by using a primer set with a restriction enzyme and the dCAPS method. [Table 1] 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 characteristics of the present invention.

[0060] The screening method of the present invention may further include a step of determining the content of RebM, steviol glycosides, RebE and / or RebN in tissues (e.g., leaves) of the tested stevia plant that has detected at least one of the genetic characteristics (1) to (5) of the present invention. The determination of the content of RebM, steviol glycosides, RebE and / or RebN is as described in the plant section of the present invention. In addition, in this manner, from the tested stevia plants that have detected at least one of the genetic characteristics (1) to (5) of the present invention, individuals with high RebM content, the difference between RebM content and steviol glycoside content, the difference between RebM content and RebE content, the ratio of RebM content to RebN content and / or the ratio of RebM content to steviol glycoside content are selected, and these individuals are mated with other stevia plants. The screening method of the present invention can also be applied to the resulting offspring plants. Thus, the screening method of the present invention may include one or more of the following steps. (i) The step of detecting at least one of the genetic characteristics (1) to (5) of the present invention from the genome of the tested stevia plant. (ii) A step of determining the content of RebM, steviol glycoside, RebE and / or RebN in the tested stevia plant tissue that detects at least one of the genetic characteristics (1) to (5) of the present invention. (iii) The step of selecting individuals from at least one tested stevia plant that exhibits the genetic characteristics (1) to (5) of the present invention, and having a high RebM content, the difference between the RebM content and the steviol glycoside content, the difference between the RebM content and the RebE content, the ratio of the RebM content to the RebN content, and / or the ratio of the RebM content to the steviol glycoside content. (iv) A process of mating individuals with high RebM content, the difference between RebM content and steviol glycoside content, the difference between RebM content and RebE content, the ratio of RebM content to RebN content, and / or the ratio of RebM content to steviol glycoside content with other stevia plants. (v) The step of detecting at least one of the genetic characteristics (1) to (5) of the present invention from the genome of the offspring obtained through mating. (vi) A step of determining the content of RebM, stevioside, RebE and / or RebN in the seed plant tissue that contains at least one of the genetic characteristics (1) to (5) of the present invention. (vii) A step of selecting individuals with high RebM content, the difference between RebM content and steviol glycoside content, the difference between RebM content and RebE content, the ratio of RebM content to RebN content and / or the ratio of RebM content to steviol glycoside content from at least one of the seed plants in which the genetic characteristics (1) to (5) of the present invention are detected.

[0061] The selected individuals with high RebM content, differences between RebM and steviol glycoside content, differences between RebM and RebE content, the ratio of RebM content to RebN content, and / or the ratio of RebM content to steviol glycoside content, may be individuals from the tested stevia plants that exhibit at least one of the genetic characteristics (1) to (5) of the present invention, and whose RebM content, differences between RebM and steviol glycoside content, differences between RebM and RebE content, the ratio of RebM content to RebN content, and / or the ratio of RebM content to steviol glycoside content are up to 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%. Furthermore, other stevia plants used for mating may or may not contain the genetic characteristics (1) to (5) of the present invention. In the above method, steps (iv) to (vii) can be repeated several times. In this way, individual stevia plants with higher RebM content, the difference between RebM content and steviol glycoside content, the difference between RebM content and RebE content, the ratio of RebM content to RebN content, and / or the ratio of RebM content to steviol glycoside content can be screened.

[0062] 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. 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 the induction of mutation, as described in the plant section of this invention, it includes treatment based on mutagens, treatment based on radiation or light irradiation, etc.

[0063] Furthermore, the present invention provides primer sets and combinations thereof as described above, such as the primer sets described in Table 1 above and combinations thereof. The present invention further provides primer sets that can be used by PCR to amplify regions having base sequences selected from sequence numbers 14-33 and 56-70, such as primer sets containing a forward primer containing the base sequence of sequence number 6 and a reverse primer containing the base sequence of sequence number 7, a primer set containing a forward primer containing the base sequence of sequence number 8 and a reverse primer containing the base sequence of sequence number 9, a primer set containing a forward primer containing the base sequence of sequence number 10 and a reverse primer containing the base sequence of sequence number 11, a primer set containing a forward primer containing the base sequence of sequence number 12 and a reverse primer containing the base sequence of sequence number 13, etc.

[0064] Furthermore, the present invention provides a probe (hereinafter, sometimes referred to as "the probe of the present invention") capable of detecting the presence and / or absence of the genetic characteristics of the present invention. The probe of the present invention may have a structure suitable for various methods of detecting the presence and / or absence of the genetic characteristics of the present invention (e.g., Real-time PCR methods such as TaqMan PCR). For example, the probe of the present invention may contain a base sequence complementary to a portion of the genome containing the variant site of the present invention. As a non-limiting example of the probe, probes containing sequences complementary to base sequences selected from sequence numbers 14-16, 18-20, 22-24, 26-28, 30-32, and 56-70 are listed. Among these sequences, sequence numbers 14-16, 18-20, 22-24, 26-28, and 30-32 are specific for alleles containing the variant of the present invention, and sequence numbers 56-70 are specific for alleles not containing the variant of the present invention. The presence of the genetic characteristics of the present invention can be detected by detecting both alleles containing the variants of the present invention and alleles not containing the variants of the present invention. The absence of the genetic characteristics of the present invention can be detected by detecting only alleles containing the variants of the present invention or only alleles not containing the variants of the present invention. The probe of the present invention preferably has a label. Examples of non-limiting labels include fluorescent labels, luminescent labels, radioactive labels, pigments, enzymes, quenchers, and portions that bind to detectable labels. In a particular embodiment, the probe of the present invention comprises a polynucleotide containing a base sequence complementary to a base sequence selected from sequences 14-16, 18-20, 22-24, 26-28, 30-32, and 56-70, and a label.

[0065] The present invention further 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 the primer set and the corresponding restriction enzyme described in Table 1 above. Furthermore, the present invention provides a kit comprising a primer set for amplifying regions having base sequences selected from the above-described sequence numbers 14-33 and 56-70 by PCR, and the corresponding probes of the present invention described above.

[0066] These primer sets, probes, and kits can be used to detect the genetic characteristics (1) to (5) of the present invention, and for the screening methods of the present invention, etc. In addition, these primer sets and kits may contain media containing the following: instructions containing descriptions related to the detection of the genetic characteristics (1) to (5) of the present invention or the screening methods of the present invention, such as website information (e.g., URL, QR code) containing information related to the instructions for use or the method of use, media containing information related to the method of use (e.g., floppy disk, CD, DVD, Blu-ray disc, memory card, USB memory), etc.

[0067] In one embodiment, the present invention provides a screening kit for stevia plants, characterized in that it comprises at least one reagent for detecting the presence and / or absence of genetic characteristics (1) to (5). The reagent may comprise primers and / or probes used in the CAPS method, dCAPS method, or TaqMan PCR method. In a particular manner, the reagent used to detect the presence and / or absence of at least one of the genetic features (1) to (5) comprises: a combination of a primer set and a restriction enzyme for detecting at least one of the genetic features (1) to (5) by the dCAPS method, such as the combination of the primer set and restriction enzyme described in Table 1, or a combination of a primer set for amplifying the variant sites of the present invention (e.g., sites containing sequences selected from sequence numbers 14 to 33) in the TaqMan PCR method and the like, and a probe having a base sequence complementary to at least one of the sites involved in the genetic features (1) to (5) (e.g., sites containing sequences selected from sequence numbers 14 to 16, 18 to 20, 22 to 24, 26 to 28, 30 to 32).

[0068] 4. The dead tissue or dead cells of the present invention In a further embodiment of the present invention, a substance comprising the dead tissue or cells of a stevia plant body of the present invention, a stevia plant body selected by the screening method of the present invention, or a stevia plant body manufactured by the production method of the present invention is provided (hereinafter, sometimes referred to as "dead tissue or dead cells of the present invention"). "Dead" refers to a state in which the plant lacks the ability to reproduce, proliferate, regenerate, and grow. This includes natural death without human intervention, as well as death caused by human intervention such as cutting, crushing, heating (including heating via gases such as air, liquids such as water, or steam such as water vapor), freezing, drying, and freeze-drying. Dead tissue refers to tissue in which all its cells have died, and also includes dead plant bodies (plant bodies in which all their cells have died). Specific examples of dead tissue or dead cells of the present invention include substances comprising the dead tissue or cells other than seeds. More specifically, substances comprising the dead embryo, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf sections, and callus tissue are examples. Dried leaves are a preferred example. The dead tissues or cells of the present invention can be used as raw materials for extracts, steviol glycosides, pharmaceuticals, fragrances, food and beverages, etc., as described later.

[0069] 5. Methods for manufacturing extracts from plants and products using the extracts In a further embodiment of the present invention, a method for manufacturing an extract containing steviol glycosides (hereinafter, sometimes referred to as "the method for manufacturing the extract of the present invention") is provided, characterized in that it includes a step of obtaining an extract from the plant of the present invention, a stevia plant selected by the screening method of the present invention, or a stevia plant manufactured by the manufacturing method of the present invention, or the dead tissue or dead cells of the plant, the seeds, leaves (e.g., dried leaves or fresh leaves), tissues, tissue cultures, or cells of the plant.

[0070] Furthermore, an extract containing steviol glycosides obtained from the plant of the present invention, stevia plant selected by the screening method of the present invention, or stevia plant manufactured by the manufacturing method of the present invention, seeds, leaves (e.g., dried or fresh leaves), tissues, tissue cultures, or cells of the plant, or dead tissues or dead cells of the present invention (hereinafter, sometimes referred to as "the extract of the present invention") is provided. The extract of the present invention is preferably an extract manufactured by the manufacturing method of 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 including a step of purifying steviol glycosides from the extract of the present invention. The method for manufacturing steviol glycosides of the present invention may further include a step of obtaining an extract containing steviol glycosides from the plant of the present invention, stevia plant selected by the screening method of the present invention, or stevia plant manufactured by the manufacturing method of the present invention, or dead tissues or dead cells of the present invention.

[0071] 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. In addition, extracts containing steviol glycosides can be purified by known methods such as gradients of ethyl acetate and other organic solvents: water, chromatography (e.g., high performance liquid chromatography, ultra-high performance liquid chromatography, gas chromatography, etc.), and time-of-flight mass spectrometry (TOF-MS).

[0072] The types of steviol glycosides are not particularly limited, and include 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, steviol glycoside, etc. In a preferred embodiment, steviol glycosides include RebD, RebE, RebM, RebN, steviol glycoside, or combinations thereof.

[0073] In one embodiment, the extract of the present invention has a higher RebM content, a higher RebM content than any of the stevia species that do not possess the genetic characteristics (2'), (4'), and (5) of the present invention, a higher RebM content than the difference between the RebM content and the steviol glycoside content, a higher RebM content than the RebE content, a higher RebM content relative to the RebN content, and / or a higher RebM content relative to the steviol glycoside content. Compared with extracts obtained from stevia plants that do not possess the genetic characteristics (2'), (4'), and (5) of this invention, the extracts of this invention may have RebM content, the difference between RebM content and steviol glycoside content, the difference between RebM content and RebE content, the ratio of RebM content to RebN content, and / or the ratio of RebM content to steviol glycoside content as high as approximately 50% or more, approximately 100% or more, approximately 150% or more, approximately 200% or more, approximately 250% or more, approximately 300% or more, approximately 350% or more, approximately 400% or more, approximately 450% or more, approximately 500% or more, approximately 550% or more, approximately 600% or more, approximately 650% or more, and approximately 700% or more. Above, approximately 750% or above, approximately 800% or above, approximately 850% or above, approximately 900% or above, approximately 950% or above, approximately 1000% or above, approximately 1050% or above, approximately 1100% or above, approximately 1150% or above, approximately 1200% or above, approximately 1250% or above, approximately 1300% or above, approximately 1350% or above, approximately 1400% or above, approximately 1450% or above, approximately 1500% or above, approximately 1550% or above, approximately 1600% or above, approximately 1650% or above, approximately 1700% or above, approximately 1750% or above, approximately 1800% or above, approximately 1850% or above, approximately 1900% or above, approximately 1950% or above, approximately 2000% or above, approximately 2050% or above, approximately 2100% or above. Here, the extracts of the present invention can be obtained by the same method as extracts obtained from stevia plants that do not have any of the genetic characteristics (2'), (4') and (5) of the present invention.

[0074] By mixing the extract of the present invention thus obtained and / or the steviol glycosides obtained by the method for manufacturing steviol glycosides of the present invention with other ingredients, novel food products, sweetener compositions, flavorings, or pharmaceuticals with increased steviol glycoside content can be manufactured. Therefore, as another embodiment, the present invention provides a method for manufacturing a food product, sweetener composition, flavoring, or pharmaceutical, characterized by a step of mixing the extract of the present invention and / or the steviol glycosides obtained by the method for manufacturing steviol glycosides of the present invention with other ingredients such as raw materials for food products, sweeteners, flavorings, or pharmaceuticals. Further, the present invention provides a novel food product, sweetener composition, flavoring, or pharmaceutical obtained by the said manufacturing method with increased steviol glycoside content. Here, food products include beverages and foods. Thus, in a certain embodiment, the present invention provides a novel beverage, food, sweetener composition, flavoring, or pharmaceutical, and also provides a method for manufacturing such a beverage, food, sweetener composition, flavoring, or pharmaceutical. Furthermore, the present invention provides a method for manufacturing a composition containing steviol glycosides, characterized by a step of mixing the extract of the present invention and / or the steviol glycosides obtained by the method of manufacturing steviol glycosides of the present invention with other ingredients. Furthermore, the present invention provides a composition containing steviol glycosides obtained by the aforementioned manufacturing method, characterized by containing steviol glycosides and other ingredients. The aforementioned "other ingredients" may include ingredients not present in natural stevia plants or non-natural ingredients. Therefore, the above composition may be a non-natural composition. The composition may, for example, be a pharmaceutical composition, a fragrance composition, a beverage composition, or a food composition.

[0075] 6. The base sequence involved in the plant body of this invention. In other embodiments, the present invention provides a base sequence relating to the plant body of the present invention. The stevia plant with genetic characteristic (1) involves a base sequence consisting of or composed of base sequences selected from or composed of sequence numbers 14-17. The stevia plant with genetic characteristic (2) involves a base sequence consisting of or composed of base sequences selected from or composed of sequence numbers 18-21. The stevia plant with genetic characteristic (3) involves a base sequence consisting of or composed of base sequences selected from or composed of sequence numbers 22-25. The stevia plant with genetic characteristic (4) involves a base sequence consisting of or composed of base sequences selected from or composed of sequence numbers 26-29. The stevia plant with genetic characteristic (5) involves a base sequence consisting of or composed of base sequences selected from or composed of sequence numbers 30-33. Example

[0076] The following describes embodiments of the present invention, but the present invention is not limited to these specific methods.

[0077] [Example 1] Identification of variants involved in high RebM expression The steviol glycoside content of the stevia system cultivated by Suntory World Research Centre was determined, and the genome was sequenced. Specifically, 0.25 g of fresh leaves were sampled from individuals of each system, freeze-dried, and 0.05 g of the pulverized dried material was added to 100 times the volume (5 mL) of pure water. After extraction by sonication for 20 minutes, the sample was centrifuged, filtered, and diluted 60 times with 32% acetonitrile to prepare the sample solution. LC-MS / MS analysis of 1 mL of this sample solution was performed using an LCMS8050 (Shimadzu) in MRM mode to quantify the concentrations of RebA, RebB, RebC, RebD, RebE, RebF, RebI, RebJ, RebM, RebN, and ST. Furthermore, genomic DNA was extracted from the sampled fresh leaves and sequenced using a sequencer (HiSeq 2500, Illumina).

[0078] Analysis of the steviol glycoside content data revealed the existence of a system group with high RebM content (high RebM group). The steviol glycoside contents of representative systems in the high RebM group, the low RebM group with relatively low RebM content, and the medium RebM group with lower RebM content than the high RebM group but higher RebM content than the low RebM group are shown in Tables 2 and 3. Furthermore, the differences and ratios of the contents of various steviol glycosides are shown in Table 4.

[0079] In the table, TSG represents total steviol glycosides. Furthermore, the values ​​for each steviol glycoside are expressed as a percentage by weight in the dried leaves, and the TSG value is the sum of the values ​​for all steviol glycosides measured (i.e., RebA, RebB, RebC, RebD, RebE, RebF, RebI, RebJ, RebM, RebN, and ST).

[0080] [Table 2] [Table 3] [Table 4]

[0081] In the high RebM group, medium RebM group and low RebM group, the content of each steviol glycoside was evaluated by Welch's t test (two sides) to determine whether there were statistically significant differences. In the high RebM group, compared with the medium RebM group and the low RebM group, the contents of RebI and RebM were significantly higher, the ratio of RebM content to RebN content (RebM / RebN) and the ratio of RebM content to ST content (RebM / ST) were significantly higher, the RebM-ST (the difference between RebM content and ST content, the same below) and RebM-RebE were significantly higher, and the contents of RebE and ST were significantly lower (Table 5). In addition, significant differences were found in RebM content, RebM-ST, and RebM-RebE between the high RebM group and the medium RebM group using Welch's t-test (both sides) (table omitted).

[0082] In the table, the numerical values ​​represent the average values. "+" indicates that the content in the high RebM group is higher than that in the medium RebM group and the low RebM group, and P < 0.05; "++" indicates that the content in the high RebM group is higher than that in the medium RebM group and the low RebM group, and P < 0.01; "-" indicates that the content in the high RebM group is lower than that in the medium RebM group and the low RebM group, and P < 0.05; "--" indicates that the content in the high RebM group is lower than that in the medium RebM group and the low RebM group, and P < 0.01; "±" indicates that no significant difference was observed between the two groups at the significance level of 0.05. [Table 5]

[0083] Analysis of the differences in genomic sequences among the high-RebM, medium-RebM, and low-RebM systems revealed that the high-RebM system tends to exhibit the following genetic characteristics. (1') The allele (Sequence No. 17) with a sequence corresponding to positions 150-151 of sequence No. 1 is heteroconjugated. (2') The allele (sequence number 21) with the base TAGGTGGTGACACTGTAGC at position 151 of sequence number 2 is heteroconjugated. (3') The allele (sequence number 25) with an A base at position 151 of sequence number 3 exhibits heteroconjugation. (4') The allele (sequence number 29) with an A base at position 151 of sequence number 4 exhibits heteroconjugation. (5) The allele with base A at position 151 of sequence number 5 (sequence number 33) exhibits homozygosity.

[0084] [Example 2] Development of markers for detecting individuals with high RebM levels In order to efficiently detect the genetic characteristics unique to the high RebM system, dCAPS primers were prepared for detecting genetic characteristics (1'), (2'), (4') and (5), and it was investigated whether the individual could be detected by the dCAPS method to determine whether they possess these genetic characteristics.

[0085] The following primers and restriction enzymes are used for dCAPS. [Table 6]

[0086] The detection of each genetic trait based on the dCAPS method was carried out as follows. First, genomic DNA was extracted from fresh leaves of each individual tested in Example 1, and PCR was performed using the dCAPS primers described above corresponding to each genetic trait.

[0087] The restriction enzymes corresponding to each genetic trait (4') and (5) were added to the PCR products, and the enzyme reaction was carried out at 37°C. The restriction enzyme-treated PCR products were subjected to electrophoresis using a microchip electrophoresis device, LabChip GX Touch HT (PerkinElmer), and the presence or absence of genetic traits was determined based on the obtained band patterns.

[0088] Regarding genetic characteristics (4') and (5), individuals confirming only the band of degradation products were classified as 0, individuals confirming only the band of non-degradable products were classified as ×, and individuals confirming both degradation products and non-degradable products were classified as △. Regarding genetic characteristic (2'), individuals confirming only the PCR product of approximately 206 bp were classified as 0, individuals confirming only the PCR product of approximately 188 bp were classified as ×, and individuals confirming both the PCR product of approximately 206 bp and the PCR product of approximately 188 bp were classified as △. The results are shown in the table below. [Table 7]

[0089] Since genetic trait (2') is heteroconjugative, individuals that confirmed both the approximately 206 bp and approximately 188 bp PCR products were identified as having the genetic trait. Since genetic trait (4') is heteroconjugative, individuals that confirmed both the degradation product and non-degradation product bands were identified as having the genetic trait. Since genetic trait (5) is homoconjugative, individuals that confirmed the degradation product band were identified as having the genetic trait. As a result, there was a tendency to detect genetic trait (2'), (4'), and (5) in the high RebM system. On the other hand, there was a tendency not to detect genetic trait (2'), (4'), and (5) in the medium RebM and low RebM systems.

Claims

1. A stevia plant body, characterized in that, Having at least one of the following genetic characteristics (1) to (5), (1) For the portion of the sequence corresponding to positions 150-151 of sequence number 1, the alleles with the sequence name T exhibit homozygous or heterozygous conjugation. (2) Alleles with the base TAGGTGGTGACACTGTAGC at position 151 of sequence number 2 exhibit homozygous or heterozygous binding. (3) Alleles with an A base at position 151 of sequence number 3 exhibit homozygous or heterozygous binding. (4) Alleles with an A base at position 151 of sequence number 4 exhibit homozygous or heterozygous binding. (5) Alleles with base A at position 151 of sequence number 5 exhibit isomorphic conjugation.

2. A stevia plant, characterized in that, Having at least one of the following chemical characteristics (i) to (v), (i) The content of RebM (rebodiin M) per unit mass of dried leaves is 1.8% or more. (ii) The difference between the RebM content and the steviol content per unit mass of dried leaves is 0.9% or more. (iii) The difference between the RebM content and the RebE content per unit mass of dried leaves is more than 1.3%. (iv) The ratio of RebM content to RebN content per unit mass of dried leaves (RebM / RebN) is 1.52 or higher. (v) The RebM content per unit mass of dried leaves relative to the content of stevioside is 1.83 or higher.

3. The plant body according to claim 1 or 2, characterized in that, It includes stevia plants subjected to mutation-induced treatment and their progeny plants.

4. A type of dead tissue or dead cells, characterized in that, The dead tissue or dead cells of the plant body as described in any one of claims 1 to 3.

5. A seed, tissue, dried leaf, tissue culture, or cell, characterized in that, The seed, tissue, dried leaf, tissue culture or cell of the plant as described in any one of claims 1 to 3.

6. The tissue, tissue culture, or cells according to claim 5, characterized in that, Selected from embryos, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf sections, and callus tissue.

7. A method for preparing stevia plant material according to any one of claims 1 to 3, characterized in that, The method includes a step of hybridizing the plant body according to any one of claims 1 to 3 with a second stevia plant body.

8. A method for preparing stevia plant material according to any one of claims 1 to 3, characterized in that, The procedure includes introducing at least one of the following variations into the genome of a stevia plant. (1) A variation that makes the portion of the sequence corresponding to positions 150-151 of sequence number 1 a variant of T. (2) A variation that makes the base at position 151 of sequence number 2 TAGGTGGTGACACTGTAGC. (3) A variation that makes the base at position 151 of sequence number 3 be A. (4) A variation that makes the base at position 151 of sequence number 4 be A. (5) A variation that makes the base at position 151 of sequence number 5 be A.

9. An extract, characterized in that, The plant body as described in any one of claims 1 to 3, the dead tissue or dead cells as described in claim 4, the seeds, tissues, dried leaves, tissue cultures or cells as described in claim 5, or the extracts of the tissues, tissue cultures or cells as described in claim 6.

10. A method for producing an extract containing steviol glycosides, characterized in that, The process includes obtaining an extract from any one of the plant bodies according to claims 1 to 3, the dead tissue or dead cells according to claim 4, the seeds, tissues, dried leaves, tissue cultures or cells according to claim 5, or the tissues, tissue cultures or cells according to claim 6.

11. A method for manufacturing steviol glycosides, characterized in that, The method includes a step of purifying steviol glycosides from the extract of claim 9.

12. A method for manufacturing a food product, sweetener composition, flavoring, or pharmaceutical product, characterized in that, The process includes providing an extract of plant material according to any one of claims 1 to 3, an extract of dead tissue or dead cells according to claim 4, an extract of seeds, tissues, dried leaves, tissue cultures, or cells according to claim 5, an extract of tissues, tissue cultures, or cells according to claim 6, or an extract according to claim 9, and, The process of adding the extract to the raw materials of food, beverages, sweetener compositions, flavorings or pharmaceuticals.

13. A food or beverage, sweetener composition, flavoring, or pharmaceutical product, characterized in that, Extracts containing any one of the plant bodies according to claims 1 to 3, extracts of dead tissue or dead cells according to claim 4, extracts of seeds, tissues, dried leaves, tissue cultures or cells according to claim 5, extracts of tissues, tissue cultures or cells according to claim 6, or extracts according to claim 9.

14. A method for manufacturing a food product, sweetener composition, flavoring, or pharmaceutical product, characterized in that, The process includes purifying steviol glycosides from the extract of claim 9, and, The process of adding purified steviol glycosides to the raw materials of food and beverage, sweetener compositions, flavorings or pharmaceuticals.

15. A method for screening stevia plants having at least one of the chemical characteristics (i) to (v) as described in claim 2, characterized in that, The procedure includes detecting the presence and / or absence of at least one of the genetic characteristics (1) to (5) of claim 1 in the tested stevia plant.

16. A screening kit for at least one stevia plant having the chemical characteristics (i) to (v) of claims 2, characterized in that, The reagent comprises at least one of the following for detecting the presence and / or absence of the genetic characteristics (1) to (5) as described in claim 1.

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