High Rebaudioside M Content Stevia Plant

Abstract

The present invention provides a non-genetically modified stevia plant with a high rebaudioside M content that has a higher content of rebaudioside M than wild-type stevia species. The present invention also provides a method for producing such a non-genetically modified stevia plant with a high rebaudioside M content, and dried leaves obtained from such a plant. [Selection diagram] None

Description

[Technical field]

[0001] The present invention relates to stevia plants having a high content of rebaudioside M. [Background technology]

[0002] In order to meet the diversifying needs of consumers, various beverages have been developed and are commercially available. Sugars such as sucrose are commonly added to beverages for the purpose of sweetening, but the health effects of excessive intake have been pointed out, and the need for lower-calorie, naturally derived sweeteners is increasing. For example, Patent Document 1 discloses a functional sweetener composition containing vitamins, a high-intensity sweetener, and a sweetness improving composition.

[0003] Rebaudioside (hereinafter referred to as "Reb") is known as a sweet component contained in stevia extract. Stevia extract is extracted and purified from stevia leaves. Stevia is a perennial plant of the Asteraceae family native to Paraguay in South America, and its scientific name is Stevia Rebaudiana Bertoni. Stevia contains a component that is about 300 times sweeter than sugar, so it is cultivated to extract this sweet component and use it as a natural sweetener. As for Reb, the existence of various glycosides such as RebA, RebB, RebC, RebD, RebE, and RebM has been reported (Patent Publication 2012-504552). Among the various Rebs, for example, RebA is evaluated as a sweetener with high sweetness and good quality sweetness, and is widely used. It is becoming clear that other Rebs also have their own unique sweetness and accompanying taste.

[0004] In such a situation, a stevia plant containing rebaudioside M and rebaudioside D at 0.38% by weight and 3.28% by weight, respectively, per dry leaf is known (Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Specification of JP2009-517043 [Patent Document 2] Specification of Patent Publication No. 2016-515814 [Patent Document 3] US2016 / 0057955A1 DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0006] Rebaudioside M is said to have the best taste among steviol glycosides, but it cannot be obtained in large quantities from natural stevia plants, making its acquisition a problem. [Means for solving the problem]

[0007] The present invention provides a non-genetically modified stevia plant having a high rebaudioside M content, which contains a higher amount of rebaudioside M than wild-type stevia species, as well as a method for producing and screening the plant.

[0008] Specifically, the present invention provides the following: [1-1] A non-genetically modified stevia plant having a high rebaudioside M content, characterized in that the plant contains 2% or more of rebaudioside M based on the total amount of steviol glycosides contained in the leaves. [1-2] The plant body described in [1-1], further containing 9.5% or more of rebaudioside D based on the total amount of steviol glycosides contained in the leaves. [1-3] A plant body according to [1-1] or [1-2], which has at least one of the following genetic characteristics (1) to (5): (1) The subject is homozygous for an allele in which the base at the position corresponding to position 44 of SEQ ID NO:35 is T. (2) It is homozygous for an allele in which the base at the position corresponding to position 40 of SEQ ID NO:37 is T. (3) SEQ ID NO: 39 48 The individual is homozygous for an allele in which the base at the corresponding position is C. (4) The gene is homozygous for an allele lacking the portion corresponding to positions 55 to 72 of SEQ ID NO:42. (5) The subject is homozygous for an allele in which the base at the position corresponding to position 50 of SEQ ID NO:43 is A. [1-4] The plant body according to any one of [1-1] to [1-3], which is positive for at least one polymorphic marker selected from the group consisting of P01 to P05.

[0009] [1-5] A seed, tissue, tissue culture, or plant cultured cell of the plant according to any one of [1-1] to [1-4]. [1-6] The tissue, tissue culture or plant cultured cell according to [1-5], which is an embryo, a meristematic cell, pollen, a leaf, a root, a root tip, a petal, a protoplast, a leaf slice or a callus. [1-7] A method for producing a stevia plant with a high rebaudioside M content, the method comprising a step of crossing the stevia plant according to any one of [1-1] to [1-4] with a second stevia plant, the stevia plant containing 2% or more of rebaudioside M relative to a total amount of steviol glycosides contained in the leaves. [1-8] The method according to [1-7], wherein the second plant is the stevia plant according to any one of [1-1] to [1-4].

[0010] [1-9] An extract of a plant body according to any one of [1-1] to [1-4], or a seed, tissue, tissue culture or cell according to [1-5]. [1-10] A food, beverage, sweetener composition, flavoring, or pharmaceutical comprising the extract described in [1-9]. [1-11] A method for producing a rebaudioside M-containing extract, comprising a step of obtaining an extract from a plant body described in any one of [1-1] to [1-4], a seed, a tissue, a tissue culture, or a cell described in [1-5]. [1-12] A method for producing rebaudioside M, comprising a step of purifying rebaudioside M from the rebaudioside M-containing extract according to [1-11]. [1-13] A method for producing a food or beverage, a sweetening composition, a flavoring, or a pharmaceutical, comprising a step of mixing an extract obtained by the method described in [1-11] and / or rebaudioside M obtained by the method described in [1-12] with other ingredients.

[0011] [1-14] A method for screening a stevia plant with high rebaudioside M content, comprising a step of detecting the presence and / or absence of at least one of the following genetic features (1) to (5) from the genome of a test plant: (1) The subject is homozygous for an allele in which the base at the position corresponding to position 44 of SEQ ID NO:35 is T. (2) It is homozygous for an allele in which the base at the position corresponding to position 40 of SEQ ID NO:37 is T. (3) SEQ ID NO: 39 48 The individual is homozygous for an allele in which the base at the corresponding position is C. (4) The gene is homozygous for an allele lacking the portion corresponding to positions 55 to 72 of SEQ ID NO:42. (5) The subject is homozygous for an allele in which the base at the position corresponding to position 50 of SEQ ID NO:43 is A. [1-15] The method according to [1-14], comprising a step of detecting at least one polymorphic marker selected from the group consisting of P01 to P05 from the genome of a test plant. [1-16] The method according to [1-14] or [1-15], further comprising a step of measuring the content of rebaudioside M in leaf tissue.

[0012] [1-17] (1) A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 1 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 2; (2) A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 3 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 4; (3) A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 5 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 6; (4) A primer set including a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 7 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 8; or (5) A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 9 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 10; wherein the arbitrary sequence of 15 or more contiguous bases is located at the 3' end of each primer. [1-18] A kit comprising the primer set according to [1-17] and optionally a restriction enzyme, When the primer set includes a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 1, the primer set includes a restriction enzyme XbaI, When the primer set includes a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 3, the primer set includes a restriction enzyme KpnI, When the primer set includes a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO:5, the primer set includes a restriction enzyme AflII, When the primer set includes a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 9, the primer set includes a restriction enzyme PvuI. The kit.

[0013] [1-19] A probe comprising a base sequence shown in any one of SEQ ID NOs: 55 to 64, which may be bound to a detectable label. [1-20] A probe according to [1-19], having a fluorescent label, dye or binding moiety. [1-21] A method for screening a non-genetically modified stevia plant with high rebaudioside M content, comprising: a step of performing PCR amplification on genomic DNA of a test plant using the primer set described in [1-17] for the purpose of detecting at least one polymorphic marker selected from the group consisting of P01 to P05; and a step of treating a PCR product obtained by the PCR amplification with a restriction enzyme and detecting a restriction enzyme-treated product when the polymorphic marker is at least one selected from the group consisting of P01 to P03 and P05. [1-22] The method according to [1-21], wherein the restriction enzyme is at least one selected from the group consisting of XbaI, KpnI, AflII and PvuI. [1-23] The method according to [1-21], wherein the polymorphic markers include P02 and P05. [1-24] The method according to [1-23], wherein the restriction enzymes include KpnI and PvuI. [1-25] The method according to any one of [1-21] to [1-24], wherein the plant obtained by screening contains 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in the leaves.

[0014] The present invention also provides the following: [2-1] A non-genetically modified stevia plant having a high rebaudioside M content, characterized in that the plant contains 2% or more of rebaudioside M based on the total amount of steviol glycosides contained in the dried leaves. [2-2] The plant body according to [2-1], further containing 9.5% or more of rebaudioside D based on the total amount of steviol glycosides contained in the dried leaves. [2-3] The plant body described in [2-1] or [2-2] above, which is positive for at least one polymorphic marker selected from the group consisting of P01, P02, P03, P04 and P05. [2-4] A seed of the plant according to any one of [2-1] to [2-3]. [2-5] Dried leaves of the plant according to any one of [2-1] to [2-3]. [2-6] A tissue culture or a plant culture cell of the plant according to any one of [2-1] to [2-3] above. [2-7] The tissue culture or plant cultured cell according to [2-6] above, which is an embryo, a meristematic cell, pollen, a leaf, a root, a root tip, a petal, a protoplast, a leaf slice, or a callus.

[0015] [2-8] A method for producing a stevia plant with a high rebaudioside M content, the method comprising a step of crossing the stevia plant according to any one of the above [2-1] to [2-3] with a second stevia plant, the stevia plant containing 2% or more of rebaudioside M relative to a total amount of steviol glycosides contained in dried leaves. [2-9] The method according to the above [2-8], wherein the second plant is a stevia plant according to any one of the above [2-1] to [2-3]. [2-10] An extract of the plant body according to any one of the above [2-1] to [2-3], the seed according to the above [2-4], or the dried leaf according to the above [2-5]. [2-11] A food or beverage, a sweetening composition, a flavoring, or a pharmaceutical comprising the extract described in [2-10].

[0016] [2-12] A method for producing an extract containing rebaudioside M, comprising the step of obtaining an extract from the plant body described in any one of [2-1] to [2-3] above, the seed described in [2-4] above, or the dried leaf described in [2-5] above. [2-13] A method for producing rebaudioside M, comprising a step of purifying rebaudioside M from the rebaudioside M-containing extract described in [2-12] above. [2-14] A method for producing a food or beverage, a sweetening composition, a flavoring, or a pharmaceutical, comprising a step of mixing an extract obtained by the method described in [2-12] above and / or rebaudioside M obtained by the method described in [2-13] above with other ingredients. [2-15] A method for screening for a stevia plant with high rebaudioside M content, comprising a step of detecting at least one polymorphic marker selected from the group consisting of P01, P02, P03, P04 and P05 from the genome of a test plant. [2-16] The method according to [2-15] above, further comprising a step of measuring the rebaudioside M content in leaf tissue.

[0017] [2-17] (1) A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 1 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 2; (2) A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 3 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 4; (3) A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 5 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 6; (4) A primer set including a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 7 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 8; or (5) A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 9 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 10; wherein the arbitrary sequence of 15 or more contiguous bases is located at the 3' end of each primer. [2-18] A kit comprising at least one primer set selected from (1) to (5) described in [2-17] above and a restriction enzyme, When the primer set is (1), the restriction enzyme is XbaI; When the primer set is (2), the restriction enzyme is KpnI; When the primer set is (3), the restriction enzyme is AflII; When the primer set is (5), the restriction enzyme is PvuI. The kit.

[0018] [2-19] A probe comprising a base sequence shown in any one of SEQ ID NOs: 1 to 10, which may be bound to a detectable label. [2-20] The probe according to [2-19] above, having a fluorescent label, dye or binding moiety. [2-21] A step of performing PCR amplification on genomic DNA of a test plant using the primer set described in [2-17] above for the purpose of detecting at least one polymorphic marker selected from the group consisting of P01, P02, P03, P04, and P05; and when the polymorphic marker is at least one selected from the group consisting of P01, P02, P03, and P05, treating a PCR product obtained by the PCR amplification with a restriction enzyme and detecting a restriction enzyme treatment product; The method for screening for a non-genetically modified stevia plant having a high rebaudioside M content, comprising: [2-22] The method according to [2-21] above, wherein the restriction enzyme is at least one selected from the group consisting of XbaI, KpnI, AflII and PvuI. [2-23] The method according to [2-21] above, wherein the polymorphic markers are P02 and P05. [2-24] The method according to [2-23] above, wherein the restriction enzymes are KpnI and PvuI. [2-25] The method according to any one of the above [2-21] to [2-24], wherein the plant obtained by screening contains 2% or more of rebaudioside M based on the total amount of steviol glycosides contained in the dried leaves. Effect of the Invention

[0019] The present invention makes it possible to obtain stevia plants containing higher amounts of rebaudioside M, to provide means for producing such plants, to provide leaves from such plants, and to provide foods, beverages, and the like containing rebaudioside M obtained from the leaves. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 shows the electropherogram obtained by marker detection of population I. [Diagram 2] FIG. 2 shows the electropherogram obtained for marker detection of population II. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention will be described in detail below. The following embodiments are merely examples for explaining the present invention, and are not intended to limit the present invention to such embodiments. The present invention can be embodied in various forms without departing from the gist of the present invention. All documents cited in this specification, as well as published patent applications, patent publications, and other patent documents, are incorporated herein by reference. This specification also includes the contents of the specification and drawings of the Japanese patent application (Patent Application No. 2017-198515), which was filed on October 12, 2017 and is the basis for the priority claim of this application.

[0022] 1. Non-genetically modified stevia plant with high rebaudioside M content according to the present invention The present invention provides a rebaudioside M-rich non-genetically modified stevia plant (hereinafter, sometimes referred to as "the plant of the present invention" or "the stevia plant of the present invention") that contains 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in the leaves (e.g., dried leaves or fresh leaves). The stevia plant of the present invention is a species derived from a wild stevia plant, but has a mutation in its gene that increases the amount of rebaudioside M. Furthermore, the mutation in the gene is generated non-recombinantly (described below). In the present invention, among the high rebaudioside M-containing non-genetically modified stevia plants, a stevia plant characterized by containing 2 to 4.6% rebaudioside M relative to the total amount of steviol glycosides contained in the leaves (e.g., dried leaves or fresh leaves) may be referred to as a high rebaudioside M-containing non-genetically modified stevia plant, and a stevia plant characterized by containing 4.7% or more rebaudioside M may be referred to as an ultra-high rebaudioside M-containing non-genetically modified stevia plant.

[0023] Total Steviol Glycoside (TSG) does not include unknown steviol glycosides or steviol glycosides present below the detection limit. Preferably, the total steviol glycosides are any combination of two or more selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside M, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, rubusoside, steviol, steviol monoside, steviol bioside, and stevioside. For example, in one embodiment, the total steviol glycosides consist of rebaudioside A, rebaudioside B, rebaudioside M, rebaudioside D, rebaudioside F, rebaudioside M, and steviol, and in another embodiment, the total steviol glycosides consist of rebaudioside A, rebaudioside B, rebaudioside M, rebaudioside D, rebaudioside F, rebaudioside M, rebaudioside N, rebaudioside O, and steviol. [ka]

[0024] Containing 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in the leaves means that, when the content of rebaudioside M (RebM) is expressed as RebM / TSG% as a ratio to the total amount of steviol glycosides obtained from the leaves (e.g., dried leaves or fresh leaves), the lower limit of the RebM / TSG value is 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 12% or more, 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, or 38% or more. On the other hand, the upper limit of the RebM / TSG value is characterized by being 15% or less, 16% or less, 18% or less, 20% or less, 22% or less, 24% or less, 26% or less, 28% or less, 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, or 40% or less. The combination of the lower limit and the upper limit is not particularly limited as long as the upper limit value exceeds the lower limit value, but a combination of 2% or more and less than 4.7% is sometimes referred to as a high rebaudioside M content phenotype, and a combination of 4.7% or more and 15% or less is sometimes referred to as an ultra-high rebaudioside M content phenotype.

[0025] The plant of the present invention may contain 0.19 g or more of rebaudioside M per 100 g of dried leaves. This means that, when dried leaves are obtained from a plant of the present invention, rebaudioside M is present in an amount of 0.19 g or more, 0.20 g or more, 0.25 g or more, 0.30 g or more, 0.35 g or more, 0.40 g or more, 0.45 g or more, 0.50 g or more, 0.55 g or more, 0.60 g or more, 0.65 g or more, 0.70 g or more, 0.75 g or more, 0.80 g or more, 0.85 g or more, 0.90 g or more, 0.95 g or more, 1.00 g or more, 1.05 g or more, 1.10 g or more, 1.15 g or more, 1.20 g or more, 1.25 g or more, 1.30 g or more, 1.35 g or more, 1.40 g or more, or 1.45 g or more per 100 g of the dried leaves.

[0026] Here, the dried leaves of the plant of the present invention refer to fresh leaves of the Stevia plant of the present invention that have been dried to reduce the moisture content to 3 to 4% by weight.

[0027] Here, the plant body of the present invention has an acid content of 1.00g or more, 1.05g or more, 1.10g or more, 1.15g or more, 1.20g or more, 1.25g or more, 1.30g or more, 1.35g or more, 1.40g or more, 1.45g or more, 1.50g or more, 1.55g or more, 1.60g or more, 1.65g or more, 1.70g or more, 1.75g ​​or more, 1.80g or more, 1.85g or more, 1.90g or more, 1.95g or more, 2.00g or more, 2.05g or more, 2.10g or more, 2.15g or more, 2.20g or more, 2.25g or more, 2.30g or more, or 2.40g or more. g or more, 2.35 g or more, 2.40 g or more, 2.45 g or more, 2.50 g or more, 2.55 g or more, 2.60 g or more, 2.65 g or more, 2.70 g or more, 2.75 g or more, 2.80 g or more, 2.85 g or more, 2.90 g or more, 2.95 g or more, 3.00 g or more, 3.05 g or more, 3.10 g or more, 3.15 g or more, 3.20 g or more, 3.25 g or more, 3.30 g or more, 3.35 g or more, 3.40 g or more, 3.45 g or more, 3.50 g or more, 3.55 g or more, or 3.57 g or more of rebaudioside D. Here, the combination of the contents of rebaudioside M and rebaudioside D is not particularly limited and may be any combination. Preferably, the plant of the present invention contains 1.03 g or more of rebaudioside M and 1.1 g or more of rebaudioside D per 100 g of dried leaves.

[0028] Alternatively, the leaves of the plant of the present invention (e.g., dried leaves or fresh leaves) have a rebaudioside M content of 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, 1100% or more, 1200% or more, compared to wild-type Stevia species, when the amount (g) of rebaudioside M contained per 100 g of leaves of a wild-type Stevia plant is taken as 100%. % or more, 1300% or more, 1400% or more, 1500% or more, 1600% or more, 1700% or more, 1800% or more, 1900% or more, 2000% or more, 2100% or more, 2200% or more, 2300% or more, 2400% or more, 2500% or more, 2600% or more, 2700% or more, 2800% or more, 2900% or more, or 3000% or more higher.

[0029] Furthermore, the stevia plant of the present invention is characterized in that, when the contents of rebaudioside M (RebM) and rebaudioside D (RebD) in the leaves (e.g., dried or fresh leaves) are expressed as a ratio of RebM / RebD, the lower limit of the value of RebM / RebD is 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.8 or more, or 1.0 or more. Meanwhile, the upper limit of the value of RebM / RebD is 0.3 or less, 0.4 or less, 0.5 or less, 0.6 or less, 0.8 or less, 1.0 or less, 1.1 or less, or 1.2 or less. The combination of the lower limit and the upper limit is not particularly limited as long as the upper limit is higher than the lower limit, but is preferably 0.2 or more and 1.2 or less, or 0.6 or more and 1.1 or less. In addition, the stevia plant of the present invention is characterized in that, when the content of rebaudioside M (RebM) and rebaudioside D (RebD) in the leaves (e.g., dried leaves or fresh leaves) is expressed as a ratio to the total amount of steviol glycosides, in terms of (RebD+RebM) / TSG%, the lower limit of the value of (RebD+RebM) / TSG is 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, or 38% or more. On the other hand, the upper limit of the value of (RebD+RebM) / TSG is 18% or less, 20% or less, 22% or less, 24% or less, 26% or less, 28% or less, 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, or 40% or less. The combination of the upper and lower limits is not particularly limited as long as the upper limit is greater than the lower limit, but is preferably 14% or more and 40% or less, or 16% or more and 40% or less.

[0030] In another embodiment, the plant of the present invention may have a lower total amount of steviol glycosides than the wild type. Specifically, the plant may have a total amount of steviol glycosides of less than 19 g per 100 g of dried leaves. This means that when dried leaves are obtained from the plant of the present invention, the total amount of steviol glycosides per 100 g of dried leaves is less than 19 g, less than 18 g, less than 17 g, less than 16 g, less than 15 g, less than 14 g, less than 13 g, less than 12 g, less than 11 g, less than 10 g, less than 9 g, less than 8 g, or less than 7 g.

[0031] In addition, the stevia plant of the present invention is characterized in that, when the content of rebaudioside M (RebM) and rebaudioside A (RebA) in 100 g of leaves (e.g., dried or fresh leaves) is expressed as a ratio of RebM / RebA, the lower limit of the value of RebM / RebA is 0.03 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.08 or more, 0.10 or more, 0.12 or more, or 0.14 or more. Meanwhile, the upper limit of the value of RebM / RebA is 0.08 or less, 0.10 or less, 0.12 or less, 0.14 or less, 0.16 or less, 0.18 or less, 0.20 or less, 0.24 or less, or 0.26 or less. The combination of the lower limit and the upper limit is not particularly limited as long as the upper limit is higher than the lower limit, but is preferably 0.03 or more and 0.26 or less, or 0.10 or more and 0.26 or less.

[0032] Furthermore, the stevia plant of the present invention is characterized in that, when the contents of rebaudioside M (RebM) and rebaudioside A (RebA) in 100 g of leaves (e.g., dried leaves or fresh leaves) are expressed as a ratio to the total amount of steviol glycosides as (RebA+RebM) / TSG, the lower limit of the value of (RebA+RebM) / TSG is 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 12% or more, 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, or 38% or more. On the other hand, the upper limit of the value of (RebA+RebM) / TSG is characterized by being 10% or less, 12% or less, 14% or less, 16% or less, 18% or less, 20% or less, 22% or less, 24% or less, 26% or less, 28% or less, 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, or 40% or less. The combination of the lower limit and the upper limit is not particularly limited as long as the upper limit value exceeds the lower limit value, but is preferably 4% or more and 40% or less, or 16% or more and 40% or less.

[0033] As described above, the stevia plant of the present invention has a mutation that increases the amount of rebaudioside M. This mutation has at least one of the following genetic features (1) to (5) (hereinafter, sometimes referred to as the "genetic feature of the present invention"). (1) The subject is homozygous for an allele in which the base at the position corresponding to position 44 of SEQ ID NO:35 is T. (2) It is homozygous for an allele in which the base at the position corresponding to position 40 of SEQ ID NO:37 is T. (3) SEQ ID NO: 39 48 The individual is homozygous for an allele in which the base at the corresponding position is C. (4) The gene is homozygous for an allele lacking the portion corresponding to positions 55 to 72 of SEQ ID NO:42. (5) The subject is homozygous for an allele in which the base at the position corresponding to position 50 of SEQ ID NO:43 is A.

[0034] When a sequence identical to a reference sequence (e.g., SEQ ID NO: 35, 37, 39, 42, 43, etc.) exists in the genome, the "position (or portion) corresponding to ..." refers to a position or portion (e.g., 44th position, 40th position, 48 The term "reference sequence" refers to a position or portion of a sequence in the genome that corresponds to the reference sequence (position 1, 55-72, 50, etc.), and when no sequence identical to the reference sequence exists in the genome, it refers to a position or portion of a sequence in the genome that corresponds to the reference sequence. Whether or not a sequence identical to or corresponding to the reference sequence exists in the genome can be determined, for example, by amplifying the genomic DNA of a target stevia plant with primers that can amplify the reference sequence by PCR, sequencing the amplified product, and performing an alignment analysis of the obtained sequence with the reference sequence. Non-limiting examples of sequences corresponding to the reference sequence include base sequences having 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. The position or portion corresponding to the position or portion in the reference sequence in the sequence corresponding to the reference sequence in the genome can be determined in consideration of the base sequence before and after the position or portion in the reference sequence. For example, by performing alignment analysis between a reference sequence and a sequence corresponding to the reference sequence in a genome, a position or portion in the sequence corresponding to the reference sequence in a genome that corresponds to the position or portion in the reference sequence can be determined.

[0035] For example, taking the "position corresponding to position 44 of SEQ ID NO: 35" of genetic feature (1) as an example, if the genome of a stevia plant has a portion consisting of the same nucleotide sequence as SEQ ID NO: 35, the "position corresponding to position 44 of SEQ ID NO: 35" is the 44th position from the 5' side of the portion consisting of the same nucleotide sequence as SEQ ID NO: 35 in the genome. On the other hand, if the genome of a stevia plant has a portion consisting of a nucleotide sequence that is not identical to SEQ ID NO: 35 but corresponds to it, the genome does not have a portion consisting of the same nucleotide sequence as SEQ ID NO: 35, so the "position corresponding to position 44 of SEQ ID NO: 35" does not necessarily correspond to the 44th position from the 5' side of the portion corresponding to SEQ ID NO: 35. However, by considering the nucleotide sequences around position 44 of SEQ ID NO: 35, the "position corresponding to position 44 of SEQ ID NO: 35" in the genome of such a stevia plant can be specified. For example, the "position corresponding to position 44 of SEQ ID NO: 35" in the genome of a stevia plant can be specified by performing an alignment analysis between the nucleotide sequence of the portion corresponding to SEQ ID NO: 35 in the genome of a stevia plant and the nucleotide sequence of SEQ ID NO: 35.

[0036] A "portion consisting of a nucleotide sequence corresponding to SEQ ID NO:35" means, for example, a portion consisting of a nucleotide sequence having 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 to the nucleotide sequence of SEQ ID NO:35.

[0037] In some embodiments, the "portion consisting of a base sequence corresponding to SEQ ID NO:35" includes a portion of the genome of a Stevia plant that can be amplified by PCR using a forward primer that hybridizes to a complementary sequence of a portion 15 to 25 bases long from the 5' end of SEQ ID NO:35 and a reverse primer that hybridizes to a portion 15 to 25 bases long from the 3' end of SEQ ID NO:35. For simplicity, the genetic characteristic (1) has been explained as an example here, but the same applies to the genetic characteristics (2) to (5).

[0038] In a specific embodiment, the "portion consisting of a nucleotide sequence corresponding to SEQ ID NO: 35" includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using a forward primer containing the nucleotide sequence of SEQ ID NO: 45 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 46. In a specific embodiment, the "portion consisting of a nucleotide sequence corresponding to SEQ ID NO: 37" includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using a forward primer containing the nucleotide sequence of SEQ ID NO: 47 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 48. In a particular embodiment, the "portion consisting of a nucleotide sequence corresponding to SEQ ID NO: 39" includes, for example, a portion of the genome of a Stevia plant that can be amplified by PCR using a forward primer containing the nucleotide sequence of SEQ ID NO: 49 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 50. In a particular embodiment, the "portion consisting of a nucleotide sequence corresponding to SEQ ID NO: 42" includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using a forward primer containing the nucleotide sequence of SEQ ID NO: 51 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 52. In a specific embodiment, the "portion consisting of a nucleotide sequence corresponding to SEQ ID NO: 43" includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using a forward primer containing the nucleotide sequence of SEQ ID NO: 53 and a reverse primer containing the nucleotide sequence of SEQ ID NO: 54.

[0039] In a specific embodiment, the "allele in which the base at the position corresponding to position 44 of SEQ ID NO:35 is T" includes the base sequence of SEQ ID NO:55, 65, or 75. In a specific embodiment, the "allele in which the base at the position corresponding to position 40 of SEQ ID NO:37 is T" includes the base sequence of SEQ ID NO:57, 67, or 77. In certain embodiments, "SEQ ID NO: 39" 48"Alleles in which the base at the position corresponding to the nucleotide position is C" include the base sequences of SEQ ID NO: 59, 69, or 79. In a specific embodiment, the “allele lacking a portion corresponding to positions 55 to 72 of SEQ ID NO: 42” comprises the base sequence of SEQ ID NO: 61, 71, or 81. In a specific embodiment, the "allele in which the base at the position corresponding to position 50 of SEQ ID NO:43 is A" includes the base sequence of SEQ ID NO:63, 73, or 83.

[0040] Here, (1) the position corresponding to position 44 of SEQ ID NO:35, (2) the position corresponding to position 40 of SEQ ID NO:37, (3) the position corresponding to position 41 of SEQ ID NO:39 48 A position selected from the group consisting of (1) a portion corresponding to positions 55 to 72 of SEQ ID NO:42, and (2) a portion corresponding to position 50 of SEQ ID NO:43 may be referred to as the "polymorphic site of the present invention" or the "mutation site of the present invention". In addition, (1) an A to T mutation at the position corresponding to position 44 of SEQ ID NO:35, (2) a C to T mutation at the position corresponding to position 40 of SEQ ID NO:37, (3) a C to T mutation at the position corresponding to position 41 of SEQ ID NO:39, 48 A mutation selected from the group consisting of (1) a G to C mutation at a position corresponding to position 55 of SEQ ID NO:42, (2) a deletion of a portion corresponding to positions 55 to 72 of SEQ ID NO:42, and (3) a G to A mutation at a position corresponding to position 50 of SEQ ID NO:43 may be referred to as a "polymorphism of the present invention" or a "mutation of the present invention".

[0041] The above genetic characteristics were determined using PCR, TaqMan PCR, sequencing, microarray, Invader, TILLING, random amplified polymorphic DNA (RAD), restriction fragment length polymorphism (RFLP), PCR-SSCP, amplified fragment length polymorphism (AFLP), simple sequence length polymorphism (SSLP), cleaved amplified polymorphic sequence (CAPS), derived cleaved amplified polymorphic sequence (dCAPS), allele-specific oligonucleotide (ASO), ARMS, denaturing gradient gel electrophoresis (DGGE), chemical cleavage of mismatch (CCM), DOL, MALDI-TOF / MS, TDI, padlock probe, molecular beacon, dynamic allele specific hybridization (DASH), UCAN, ECA, PINPOINT, primer oligo base extension (PROBE), very short extension (VSET), and Survivor Detection can be performed by, but is not limited to, methods such as PCR assay, Sniper assay, Luminex assay, GOOD method, LCx method, SNaPshot method, Mass ARRAY method, pyrosequencing method, SNP-IT method, melting curve analysis method, etc.

[0042] In a particular embodiment, the above genetic characteristics can be detected as "polymorphic marker positive" using polymorphic markers developed by the present inventors. Here, the polymorphic marker is at least one selected from the group consisting of P01 to P05.

[0043] Positive for P01 means that when PCR amplification is performed on the genomic DNA of a candidate plant using a forward primer having the nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 2, and the resulting PCR product (approximately 383 bp long: for example, SEQ ID NO: 21 or 22) is treated with XbaI restriction enzyme, only a band of approximately 383 bp long (for example, SEQ ID NO: 21) is obtained. Conversely, when the PCR product is treated with XbaI restriction enzyme and a restriction enzyme treatment product of approximately 344 bp (for example, SEQ ID NO: 23) is generated, the candidate plant is negative for P01. Positive for P02 means that when PCR amplification is performed on the genomic DNA of a candidate plant using a forward primer having the nucleotide sequence shown in SEQ ID NO: 3 and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 4, and the resulting PCR product (297 bp long) (e.g., SEQ ID NO: 24 or 25) is treated with KpnI restriction enzyme, only a band of about 297 bp long (e.g., SEQ ID NO: 24) is obtained. Conversely, when a restriction enzyme treatment product of about 258 bp (e.g., SEQ ID NO: 26) is obtained, the candidate plant is negative for P02. Positive for P03 means that when PCR amplification is performed on the genomic DNA of a candidate plant using a forward primer having the nucleotide sequence shown in SEQ ID NO:5 and a reverse primer having the nucleotide sequence shown in SEQ ID NO:6, and the resulting PCR product (about 390 bp long) (e.g., SEQ ID NO:27 or 28) is treated with AflII restriction enzyme, only a band of about 390 bp long (e.g., SEQ ID NO:27) is obtained. Conversely, when a restriction enzyme treatment product of about 347 bp (e.g., SEQ ID NO:29) is obtained, the candidate plant is negative for P03.

[0044] Positive for P04 means that when PCR amplification is performed on the genomic DNA of a candidate plant using a forward primer having the base sequence shown in SEQ ID NO:7 and a reverse primer having the base sequence shown in SEQ ID NO:8, only a PCR product of approximately 140 bp (e.g., SEQ ID NO:30) is produced, whereas negative means that PCR products of 140 bp (e.g., SEQ ID NO:30) and 158 bp (e.g., SEQ ID NO:34) are produced. Positive for P05 means that when PCR amplification is performed on the genomic DNA of a candidate plant using a forward primer having the nucleotide sequence shown in SEQ ID NO:9 and a reverse primer having the nucleotide sequence shown in SEQ ID NO:10, and the resulting PCR product (about 288 bp long) (e.g., SEQ ID NO:31 or 32) is treated with PvuI restriction enzyme, only a band about 288 bp long (e.g., SEQ ID NO:31) is obtained. Conversely, when a restriction enzyme treatment product of about 240 bp (e.g., SEQ ID NO:33) is obtained, the candidate plant is negative for P05. With respect to the above bp length, "about" means ±5 bp. Restriction enzyme treatment can be performed according to the conditions recommended by the vendor of each restriction enzyme used.

[0045] For details of the screening method for plants of the present invention, please refer to the section "3. Screening method for plants of the present invention" described later. It has been confirmed in the Examples that the above genetic characteristics (e.g., the positive polymorphisms of the above polymorphic markers) have a statistical correlation with the phenotype of high rebaudioside M content and / or high rebaudioside D content.

[0046] Rebaudioside M and D can be extracted in the form of an extract by reacting fresh or dried leaves of the plant of the present invention with an appropriate solvent (aqueous solvent such as water or organic solvent such as alcohol, ether, and acetone). The extraction conditions can be referenced to the methods described in Ohta et al., J. Appl. Glycosci., Vol. 57, No. 3 (2010) or WO2010 / 038911, or the methods described in the Examples below. Furthermore, rebaudioside M can be purified from the extract thus obtained by using a known method such as a gradient of ethyl acetate or other organic solvent:water, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), ultra (high) performance liquid chromatography (UPLC), etc.

[0047] The rebaudioside M content of the present invention can be measured by the method described in Ohta et al., J. Appl. Glycosci., Vol. 57, No. 3 (2010) or WO2010 / 038911, or the method described in the Examples below. Specifically, the rebaudioside M content can be measured by sampling fresh leaves from the stevia plant of the present invention and performing LC / MS-MS.

[0048] The plant body of the present invention may include not only the whole plant body, but also plant organs (e.g., leaves, petals, stems, roots, seeds, etc.), plant tissues (e.g., epidermis, phloem, parenchyma, xylem, vascular bundles, palisade tissue, spongy tissue, etc.), or various forms of plant cells (e.g., suspension culture cells), protoplasts, leaf slices, callus, etc. Furthermore, the leaves may be dried leaves as described above.

[0049] The plant body of the present invention may also include tissue cultures or plant cultured cells. This is because a plant body can be regenerated by culturing such tissue cultures or plant cultured cells. Examples of the plant body tissue cultures or plant cultured cells of the present invention include, but are not limited to, embryos, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf slices, and callus.

[0050] 2. Method for Producing the Plant of the Present Invention In another embodiment, the present invention provides a method for producing a stevia plant with high rebaudioside M content, characterized in that the stevia plant contains 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in its leaves (e.g., dried leaves or fresh leaves), comprising a step of crossing the stevia plant of the present invention with a second stevia plant (hereinafter, sometimes referred to as the "production method of the present invention"). The "high rebaudioside M content stevia plant characterized by containing 2% or more of rebaudioside M based on the total amount of steviol glycosides contained in the leaves" produced by this method has the same phenotype and genetic properties as the plant of the present invention.

[0051] Specifically, when leaves (e.g., dried or fresh leaves) are obtained from a plant obtained by the production method of the present invention, the leaves contain 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in the leaves. This is characterized in that when the content of rebaudioside M (RebM) is expressed as a ratio to the total amount of steviol glycosides obtained from the leaves, RebM / TSG%, the lower limit of the value of RebM / TSG is 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 12% or more, 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, or 38% or more. On the other hand, the upper limit of the RebM / TSG value means 15% or less, 16% or less, 18% or less, 20% or less, 22% or less, 24% or less, 26% or less, 28% or less, 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, or 40% or less. The combination of the lower limit and the upper limit is not particularly limited as long as the upper limit value exceeds the lower limit value, but is preferably 2% or more and 20% or less, or 7% or more and 15% or less.

[0052] Furthermore, the plant obtained by the production method of the present invention may contain 0.19 g or more of rebaudioside M per 100 g of dried leaves. This means that, when dried leaves are obtained from a plant of the present invention, rebaudioside M is present in an amount of 0.19 g or more, 0.20 g or more, 0.25 g or more, 0.30 g or more, 0.35 g or more, 0.40 g or more, 0.45 g or more, 0.50 g or more, 0.55 g or more, 0.60 g or more, 0.65 g or more, 0.70 g or more, 0.75 g or more, 0.80 g or more, 0.85 g or more, 0.90 g or more, 0.95 g or more, 1.00 g or more, 1.05 g or more, 1.10 g or more, 1.15 g or more, 1.20 g or more, 1.25 g or more, 1.30 g or more, 1.35 g or more, 1.40 g or more, or 1.45 g or more per 100 g of the dried leaves.

[0053] Here, the plant obtained by the production method of the present invention has a yield of 1.00g or more, 1.05g or more, 1.10g or more, 1.15g or more, 1.20g or more, 1.25g or more, 1.30g or more, 1.35g or more, 1.40g or more, 1.45g or more, 1.50g or more, 1.55g or more, 1.60g or more, 1.65g or more, 1.70g or more, 1.75g ​​or more, 1.80g or more, 1.85g or more, 1.90g or more, 1.95g or more, 2.00g or more, 2.05g or more, 2.10g or more, 2.15g or more, 2.20g or more, 2.25g or more per 100g of dry leaves. or 2.30 g or more, 2.35 g or more, 2.40 g or more, 2.45 g or more, 2.50 g or more, 2.55 g or more, 2.60 g or more, 2.65 g or more, 2.70 g or more, 2.75 g or more, 2.80 g or more, 2.85 g or more, 2.90 g or more, 2.95 g or more, 3.00 g or more, 3.05 g or more, 3.10 g or more, 3.15 g or more, 3.20 g or more, 3.25 g or more, 3.30 g or more, 3.35 g or more, 3.40 g or more, 3.45 g or more, 3.50 g or more, 3.55 g or more, or 3.57 g or more of rebaudioside D. Here, the combination of the contents of rebaudioside M and rebaudioside D is not particularly limited and may be any combination. Preferably, the plant obtained by the production method of the present invention contains 1.03 g or more of rebaudioside M and 1.1 g or more of rebaudioside D per 100 g of dried leaves.

[0054] Alternatively, the leaves of the plant obtained by the production method of the present invention have rebaudioside M content of 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, 1100% or more compared to wild-type Stevia species, when the amount (g) of rebaudioside M contained in 100 g of leaves (e.g., dried leaves or fresh leaves) of a wild-type Stevia plant is taken as 100%. or more, 1200% or more, 1300% or more, 1400% or more, 1500% or more, 1600% or more, 1700% or more, 1800% or more, 1900% or more, 2000% or more, 2100% or more, 2200% or more, 2300% or more, 2400% or more, 2500% or more, 2600% or more, 2700% or more, 2800% or more, 2900% or more, or 3000% or more higher.

[0055] Furthermore, the plant obtained by the production method of the present invention is characterized in that, when the contents of rebaudioside M (RebM) and rebaudioside D (RebD) in the leaves (e.g., dried leaves or fresh leaves) are expressed as a ratio of RebM / RebD, the lower limit of the value of RebM / RebD is 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.8 or more, or 1.0 or more. Meanwhile, the upper limit of the value of RebM / RebD is 0.3 or less, 0.4 or less, 0.5 or less, 0.6 or less, 0.8 or less, 1.0 or less, 1.1 or less, or 1.2 or less. The combination of the lower limit and the upper limit is not particularly limited as long as the upper limit exceeds the lower limit, but is preferably 0.2 or more and 1.2 or less, or 0.6 or more and 1.1 or less. Furthermore, the plant obtained by the production method of the present invention is characterized in that, when the content of rebaudioside M (RebM) and rebaudioside D (RebD) in the leaves (e.g., dried leaves or fresh leaves) is expressed as a ratio to the total amount of steviol glycosides, in terms of (RebD+RebM) / TSG%, the lower limit of the value of (RebD+RebM) / TSG is 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, or 38% or more. On the other hand, the upper limit of the value of (RebD+RebM) / TSG is 18% or less, 20% or less, 22% or less, 24% or less, 26% or less, 28% or less, 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, or 40% or less. The combination of the upper and lower limits is not particularly limited as long as the upper limit is greater than the lower limit, but is preferably 14% or more and 40% or less, or 16% or more and 40% or less.

[0056] In another embodiment, the plant obtained by the production method of the present invention may have a lower total amount of steviol glycosides than the wild type. Specifically, the plant may contain less than 19 g of total steviol glycosides per 100 g of dried leaves. This means that when dried leaves are obtained from the plant of the present invention, the total amount of steviol glycosides per 100 g of dried leaves is less than 19 g, less than 18 g, less than 17 g, less than 16 g, less than 15 g, less than 14 g, less than 13 g, less than 12 g, less than 11 g, less than 10 g, less than 9 g, less than 8 g, or less than 7 g.

[0057] Furthermore, the plant obtained by the production method of the present invention is characterized in that, when the contents of rebaudioside M (RebM) and rebaudioside A (RebA) in 100 g of leaves (e.g., dried leaves or fresh leaves) are expressed as a ratio to the total amount of steviol glycosides as (RebA+RebM) / TSG, the lower limit of the value of (RebA+RebM) / TSG is 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 12% or more, 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, 38% or more, or 40% or more. On the other hand, the upper limit of the value of (RebA+RebM) / TSG is 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, 40% or less, 42% or less, 44% or less, 46% or less, 48% or less, 50% or less, 52% or less, 54% or less, 56% or less, 58% or less, 60% or less, 62% or less, 64% or less, 66% or less, 68% or less, 70% or less, 72% or less, 74% or less, 76% or less, 78% or less, or 80% or less. The combination of the lower limit and the upper limit is not particularly limited as long as the upper limit value exceeds the lower limit value, but is preferably 4% or more and 80% or less, or 40% or more and 80% or less.

[0058] The plants obtained by the production methods of the present invention have at least one of the following genetic characteristics (1) to (5) that result in high rebaudioside M content. (1) The subject is homozygous for an allele in which the base at the position corresponding to position 44 of SEQ ID NO:35 is T. (2) It is homozygous for an allele in which the base at the position corresponding to position 40 of SEQ ID NO:37 is T. (3) SEQ ID NO: 39 48 The individual is homozygous for an allele in which the base at the corresponding position is C. (4) The gene is homozygous for an allele lacking the portion corresponding to positions 55 to 72 of SEQ ID NO:42. (5) The subject is homozygous for an allele in which the base at the position corresponding to position 50 of SEQ ID NO:43 is A. Such genetic characteristics can be detected as "polymorphic marker positive" using the polymorphic markers developed by the present inventors. Here, the polymorphic marker is at least one selected from the group consisting of P01 to P05.

[0059] Positive for P01 means that when PCR amplification is performed on the genomic DNA of a candidate plant using a forward primer having the nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 2, and the resulting PCR product (approximately 383 bp long: for example, SEQ ID NO: 21 or 22) is treated with XbaI restriction enzyme, only a band of approximately 383 bp long (for example, SEQ ID NO: 21) is obtained. Conversely, when the PCR product is treated with XbaI restriction enzyme and a restriction enzyme treatment product of approximately 344 bp (for example, SEQ ID NO: 23) is generated, the candidate plant is negative for P01. Positive for P02 means that when PCR amplification is performed on the genomic DNA of a candidate plant using a forward primer having the nucleotide sequence shown in SEQ ID NO: 3 and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 4, and the resulting PCR product (297 bp long) (e.g., SEQ ID NO: 24 or 25) is treated with KpnI restriction enzyme, only a band of about 297 bp long (e.g., SEQ ID NO: 24) is obtained. Conversely, when a restriction enzyme treatment product of about 258 bp (e.g., SEQ ID NO: 26) is obtained, the candidate plant is negative for P02. Positive for P03 means that when PCR amplification is performed on the genomic DNA of a candidate plant using a forward primer having the nucleotide sequence shown in SEQ ID NO:5 and a reverse primer having the nucleotide sequence shown in SEQ ID NO:6, and the resulting PCR product (about 390 bp long) (e.g., SEQ ID NO:27 or 28) is treated with AflII restriction enzyme, only a band of about 390 bp long (e.g., SEQ ID NO:27) is obtained. Conversely, when a restriction enzyme treatment product of about 347 bp (e.g., SEQ ID NO:29) is obtained, the candidate plant is negative for P03.

[0060] Positive for P04 means that when PCR amplification is performed on the genomic DNA of a candidate plant using a forward primer having the base sequence shown in SEQ ID NO:7 and a reverse primer having the base sequence shown in SEQ ID NO:8, only a PCR product of approximately 140 bp (e.g., SEQ ID NO:30) is produced, whereas negative means that PCR products of 140 bp (e.g., SEQ ID NO:30) and 158 bp (e.g., SEQ ID NO:34) are produced. Positive for P05 means that when PCR amplification is performed on the genomic DNA of a candidate plant using a forward primer having the nucleotide sequence shown in SEQ ID NO:9 and a reverse primer having the nucleotide sequence shown in SEQ ID NO:10, and the resulting PCR product (about 288 bp long) (e.g., SEQ ID NO:31 or 32) is treated with PvuI restriction enzyme, only a band about 288 bp long (e.g., SEQ ID NO:31) is obtained. Conversely, when a restriction enzyme treatment product of about 240 bp (e.g., SEQ ID NO:33) is obtained, the candidate plant is negative for P05. With respect to the above bp length, "about" means ±5 bp. Restriction enzyme treatment can be performed according to the conditions recommended by the vendor of each restriction enzyme used.

[0061] In the production method of the present invention, "crossing" means crossing a plant of the present invention (first generation (S1)) with a second plant (S1) to obtain a child plant (a plant produced by the production method of the present invention (second generation (S2))). A preferred crossing method is backcrossing. "Backcrossing" is a technique for producing a plant having the genetic characteristics of the present invention by crossing a child plant (S2) born between a plant of the present invention and a second plant with a plant of the present invention (i.e., a plant having the genetic characteristics of the present invention) (S1). When the second plant (S1) used in the production method of the present invention has the same phenotype and genetic properties as the plant of the present invention, it is essentially backcrossing. The genetic polymorphism of the present invention is inherited according to Mendel's law, and the phenotype correlated with the genetic polymorphism, i.e., the phenotype of high rebaudioside M content, is also inherited according to Mendel's law.

[0062] Alternatively, the plant of the present invention can be produced by self-pollination, which can be achieved by self-pollinating the pistil of the plant of the present invention with pollen from the stamen of the plant of the present invention.

[0063] Since the plant produced by the production method of the present invention has the same phenotype and genetic properties as the plant of the present invention, it is possible to produce a high rebaudioside M content stevia plant characterized by containing 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in the leaves (e.g., dried leaves or fresh leaves) by further crossing the plant produced by the production method of the present invention with a third stevia plant.

[0064] In another embodiment, the plant of the present invention can be produced by regenerating the plant by culturing the above-mentioned tissue culture or plant cell culture. The culturing conditions are the same as those for culturing tissue culture or plant cell culture 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 molecular biology, vol. 1391, pp113-123.).

[0065] Alternatively, since the plant of the present invention is a non-genetically modified plant, it can also be produced by incorporating the above polymorphism into a wild-type stevia plant by a non-genetically modified method. An example of a "non-genetically modified method" is a method of inducing a mutation in a gene of a host cell (or a host plant) without introducing a foreign gene. Such a method includes a method of applying a mutagen to a plant cell. Examples of such a mutagen include ethyl methanesulfonate (EMS) and sodium azide. For example, ethyl methanesulfonate (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%, 1.0%, and the like. The treatment time is 1 to 48 hours, 2 to 36 hours, 3 to 30 hours, 4 to 28 hours, 5 to 26 hours, or 6 to 24 hours. The treatment procedure itself is known, and can be carried out by immersing the water-absorbed seeds that have undergone the water absorption process in a treatment solution containing the mutagen at the above concentration for the above treatment time.

[0066] Alternatively, as an example of a non-genetically modified technique, plant cells can be irradiated with radiation or light such as X-rays, gamma rays, ultraviolet rays, etc. In this case, cells irradiated with an appropriate amount of ultraviolet light (ultraviolet lamp strength, distance, time) can be cultured in a selection medium, and then cells, calli, or plants having the desired traits can be selected. The irradiation intensity is 0.01 to 100Gr, 0.03 to 75Gr, 0.05 to 50Gr, 0.07 to 25Gr, 0.09 to 20Gr, 0.1 to 15Gr, 0.1 to 10Gr, 0.5 to 10Gr, and 1 to 10Gr, the irradiation distance is 1cm to 200m, 5cm to 100m, 7cm to 75m, 9cm to 50m, 10cm to 30m, 10cm to 20m, and 10cm to 10m, and the irradiation time is 1 minute to 2 years, 2 minutes to 1 year, 3 minutes to 0.5 years, 4 minutes to 1 month, 5 minutes to 2 weeks, and 10 minutes to 1 week. The irradiation intensity, distance, and time vary depending on the type of radiation and the state of the target of irradiation (cells, callus, plant body), but can be appropriately adjusted by a person skilled in the art.

[0067] In addition, techniques such as cell fusion, anther culture (haploid cultivation), and distant hybridization (haploid cultivation) are also known. In general, plant cells may undergo mutations during cultivation, and therefore it is preferable to reinstate them into an individual plant for more stable maintenance of traits. Although the plant of the present invention is a non-genetically modified stevia plant, a plant obtained by subsequently performing genetic modification (e.g., by genome editing) using the plant of the present invention as a host (e.g., a plant obtained by performing genetic modification using the plant of the present invention as a host to further impart another trait) is not excluded from the scope of the present invention.

[0068] 3. Screening method for plants of the present invention The plant of the present invention and plants having the same phenotype and genetic properties as the plant of the present invention can be screened by detecting the genetic features of the present invention from the tissue of the plant. Here, "screening" means distinguishing the plant of the present invention from other plants and selecting the plant of the present invention. Therefore, as another embodiment, the present invention provides a method for screening for a stevia plant with high rebaudioside M content (hereinafter, may be referred to as the "screening method of the present invention"), which comprises a step of detecting the presence and / or absence of at least one of the following genetic features (1) to (5) from the genome of a test plant: (1) The subject is homozygous for an allele in which the base at the position corresponding to position 44 of SEQ ID NO:35 is T. (2) It is homozygous for an allele in which the base at the position corresponding to position 40 of SEQ ID NO:37 is T. (3) SEQ ID NO: 39 48 The individual is homozygous for an allele in which the base at the corresponding position is C. (4) The gene is homozygous for an allele lacking the portion corresponding to positions 55 to 72 of SEQ ID NO:42. (5) The subject is homozygous for an allele in which the base at the position corresponding to position 50 of SEQ ID NO:43 is A. The screening method of the present invention may further comprise the step of selecting, from the test plants, a plant in which the presence of at least one of the above genetic features (1) to (5) has been detected.

[0069] The presence of the genetic features of the present invention (A) an allele in which the base at the position corresponding to position 44 of SEQ ID NO: 35 is T; (B) an allele in which the base at the position corresponding to position 40 of SEQ ID NO: 37 is T; (C) SEQ ID NO: 39 48 an allele in which the base at the corresponding position is C; (D) an allele in which a portion corresponding to positions 55 to 72 of SEQ ID NO: 42 is deleted; and (E) An allele in which the base at the position corresponding to position 50 of SEQ ID NO: 43 is A and / or detecting the presence of an allele selected from the group consisting of: (a) an allele in which the base at the position corresponding to position 44 of SEQ ID NO:35 is A; (b) an allele in which the base at the position corresponding to position 40 of SEQ ID NO:37 is C; (c) SEQ ID NO: 39 48 an allele in which the base at the corresponding position is G; (d) an allele in which the portion corresponding to positions 55 to 72 of SEQ ID NO: 42 is not deleted; and (e) an allele in which the base at the position corresponding to position 50 of SEQ ID NO: 43 is G; Detection of the absence of an allele selected from the group consisting of: It can be determined by:

[0070] The absence of the genetic features of the present invention may (A) an allele in which the base at the position corresponding to position 44 of SEQ ID NO: 35 is T; (B) an allele in which the base at the position corresponding to position 40 of SEQ ID NO: 37 is T; (C) SEQ ID NO: 39 48 an allele in which the base at the corresponding position is C; (D) an allele in which a portion corresponding to positions 55 to 72 of SEQ ID NO: 42 is deleted; and (E) An allele in which the base at the position corresponding to position 50 of SEQ ID NO: 43 is A and / or detecting the absence of an allele selected from the group consisting of: (a) an allele in which the base at the position corresponding to position 44 of SEQ ID NO:35 is A; (b) an allele in which the base at the position corresponding to position 40 of SEQ ID NO:37 is C; (c) SEQ ID NO: 39 48 an allele in which the base at the corresponding position is G; (d) an allele in which the portion corresponding to positions 55 to 72 of SEQ ID NO: 42 is not deleted; and (e) an allele in which the base at the position corresponding to position 50 of SEQ ID NO: 43 is G; detecting the presence of an allele selected from the group consisting of: It can be determined by:

[0071] Specific examples of methods for detecting genetic characteristics 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, padlock probe, molecular beacon, DASH, UCAN, ECA, PINPOINT, PROBE, VSET, Survivor assay, Sniper assay, Luminex assay, GOOD, LCx, SNaPshot, Mass ARRAY, pyrosequencing, SNP-IT, and melting curve analysis.

[0072] In the case of PCR, it is preferable to prepare a primer whose 3'-end portion has a sequence complementary to the polymorphic site of the present invention. When using a primer designed in this way, if the template sample has a polymorphism, the primer will completely hybridize to the template, and the polymerase extension reaction will proceed, but if the template does not have the mutation of the present invention, the nucleotide at the 3'-end of the primer will mismatch with the template, and the extension reaction will not occur. Therefore, PCR amplification is performed using such a primer, and the amplified product is analyzed by agarose gel electrophoresis, etc. If an amplified product of a predetermined size is confirmed, it means that the template sample has a mutation, and if there is no amplified product, it can be determined that the template does not have a mutation. Alternatively, the genetic characteristic of the present invention can be detected by designing a primer sequence that does not overlap with the polymorphism of the present invention and that enables PCR amplification of the genetic mutation of the present invention, and sequencing the base sequence of the amplified nucleotide fragment. For information on PCR and agarose gel electrophoresis, see Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual" 2nd Edition (1989), Cold Spring Harbor Laboratory Press.

[0073] The TaqMan PCR method uses a PCR reaction using fluorescently labeled allele-specific oligos and Taq DNA polymerase (Livak, KJ Genet. Anal. 14, 143 (1999); Morris T. et al., J. Clin.Microbiol. 34, 2933 (1996)). The sequencing method is a method for analyzing the presence or absence of mutations by amplifying the region containing the mutation by PCR and sequencing the DNA sequence using a dye terminator or the like (Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual" 2nd Edition (1989), Cold Spring Harbor Laboratory Press). A DNA microarray is an array of nucleotide probes fixed on a support, and includes DNA chips, gene chips, microchips, bead arrays, and the like. By using a probe containing a sequence complementary to the polymorphism of the present invention, the presence or absence of the polymorphism of the present invention can be comprehensively detected. An example of a DNA microarray assay such as a DNA chip is the GeneChip assay (Affymetrix; see U.S. Pat. Nos. 6,045,996, 5,925,525, and 5,858,659). The GeneChip technology utilizes a miniaturized high-density microarray of oligonucleotide probes attached to a chip.

[0074] The Invader method combines hybridization of two types of reporter probes specific to each allele of a polymorphism such as SNP and one type of Invader probe to a template DNA, and cleavage of the DNA with a cleavase enzyme that has a special endonuclease activity that recognizes and cleaves the DNA structure (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.). The TILLING (Targeting Induced Local Lesions IN Genomes) method is a method for screening for mutation mismatches in the genome of a population of mutants by PCR amplification and CEL I nuclease treatment.

[0075] In one embodiment, the screening method of the present invention may include a step of detecting the polymorphic marker of the present invention positive from the genome of a test plant. Such a step includes an operation of extracting genomic DNA from the test plant and detecting the polymorphic marker of the present invention from the genomic DNA. As already described, the polymorphic marker is at least one selected from the group consisting of P01 to P05. Moreover, the polymorphic marker positive means that at least one marker selected from the group consisting of P01 to P05 is positive. The method for detecting positive results for P01 to P05 has already been described. In this embodiment, the screening method of the present invention may further comprise the step of selecting, from among the test plants, plants for which a positive result for the polymorphic marker of the present invention has been detected.

[0076] In one embodiment, a step of PCR amplification of genomic DNA of a test plant using at least one of the following primer sets (1) to (5): treating the PCR product obtained by the PCR amplification with a restriction enzyme and detecting the restriction enzyme treatment product; The present invention provides a method for screening for a non-genetically modified stevia plant having high rebaudioside M content, comprising: (1) a primer set comprising a forward primer comprising the nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer comprising the nucleotide sequence shown in SEQ ID NO: 2; (2) a primer set comprising a forward primer comprising the nucleotide sequence shown in SEQ ID NO: 3 and a reverse primer comprising the nucleotide sequence shown in SEQ ID NO: 4; (3) A primer set comprising a forward primer containing the nucleotide sequence shown in SEQ ID NO:5 and a reverse primer containing the nucleotide sequence shown in SEQ ID NO:6; (4) A primer set comprising a forward primer comprising the nucleotide sequence shown in SEQ ID NO: 7 and a reverse primer comprising the nucleotide sequence shown in SEQ ID NO: 8; or (5) A primer set comprising a forward primer containing the nucleotide sequence shown in SEQ ID NO: 9 and a reverse primer containing the nucleotide sequence shown in SEQ ID NO: 10. The conditions for PCR amplification are as described above.

[0077] However, the primer set is not limited to those having the sequences of SEQ ID NOs: 1 to 10, and for example, the forward primer may be one having at its 3' end a sequence 15 bases upstream from the 3' end of SEQ ID NO: 1, 3, 5, 7 or 9 (see the table below), and the reverse primer may be one having at its 3' end a sequence 15 bases upstream from the 3' end of SEQ ID NO: 2, 4, 6, 8 or 10 (see the table below). Such primers may be 15 to 50 bases long, or 20 to 45 bases long. [Table 1]

[0078] The primer set is not limited to those having the sequences of SEQ ID NOs: 1 to 10. For example, the forward primer may have or include any sequence of 15 or more consecutive bases in SEQ ID NO: 1, 3, 5, 7 or 9, and the reverse primer may have or include any sequence of 15 or more consecutive bases in SEQ ID NO: 2, 4, 6, 8 or 10. (1'') A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 1 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 2; (2'') A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 3 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 4; (3'') A primer set comprising a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 5 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 6; (4'') A primer set including a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 7 and a reverse primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 8; or (5'') A primer set comprising a forward primer having or containing any sequence of 15 or more contiguous bases in SEQ ID NO: 9 and a reverse primer having or containing any sequence of 15 or more contiguous bases in SEQ ID NO: 10. Such a primer may be 15 to 50 bases long, 20 to 45 bases long, or 30 to 65 bases long, so long as the any sequence of 15 or more contiguous bases is present at the 3'-end.

[0079] The screening method of the present invention may further include a step of measuring the RebM content in tissues (e.g., leaves) of a test stevia plant in which a genetic feature of the present invention has been detected. The measurement of the RebM content is as described in the section on the plant of the present invention. In this embodiment, an individual with a high RebM content may be selected from among the test stevia plants in which a genetic feature of the present invention has been detected, and crossed with another stevia plant, and the screening method of the present invention may be applied to the resulting offspring plant. Thus, the screening method of the present invention may include one or more of the following steps. (i) detecting the genetic features of the present invention in the genome of a test stevia plant; (ii) measuring the content of RebM in the test stevia plant tissue in which the genetic feature of the present invention has been detected; (iii) selecting individuals having a high RebM content from among the test stevia plants in which the genetic characteristics of the present invention have been detected; (iv) crossbreeding the selected individual with a high RebM content with another stevia plant; (v) detecting the genetic characteristic of the present invention from the genome of the offspring plant obtained by crossing; (vi) measuring the content of RebM in the offspring tissue in which the genetic trait of the present invention has been detected; (vii) A step of selecting individuals having a high RebM content from among the offspring plants in which the genetic characteristics of the present invention have been detected.

[0080] The selected individuals with high RebM content may be, for example, the top 50%, top 40%, top 30%, top 20%, top 10%, top 5%, top 4%, top 3%, top 2%, or top 1% individuals in terms of RebM content among the test stevia plants in which the genetic feature of the present invention has been detected. In addition, the other stevia plants to be crossed may or may not contain the genetic feature of the present invention. In the above embodiment, steps (iv) to (vii) can be repeated multiple times. In this manner, stevia plants with higher RebM content can be screened.

[0081] In the screening method of the present invention, the test stevia plant may be a natural plant or a non-genetically modified plant. The non-genetically modified plant is as described in the section on the plant of the present invention. In the screening method of the present invention, the test stevia plant may include a stevia plant that has been subjected to a mutagenesis treatment and its progeny plants. The mutagenesis treatment is as described in the section on the plant of the present invention, and includes treatment with a mutagen, treatment with radiation or light, etc.

[0082] The present invention also provides the primer sets described above, for example, any one or more primer sets selected from the group consisting of (1) to (5), (1') to (5'), and (1'') to (5'') above. The present invention further provides primer sets capable of amplifying by PCR a region having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35 to 44, for example, a primer set of a forward primer comprising the nucleotide sequence of SEQ ID NO: 45 and a reverse primer comprising the nucleotide sequence of SEQ ID NO: 46, a primer set of a forward primer comprising the nucleotide sequence of SEQ ID NO: 47 and a reverse primer comprising the nucleotide sequence of SEQ ID NO: 48, a primer set of a forward primer comprising the nucleotide sequence of SEQ ID NO: 49 and a reverse primer comprising the nucleotide sequence of SEQ ID NO: 50, a primer set of a forward primer comprising the nucleotide sequence of SEQ ID NO: 51 and a reverse primer comprising the nucleotide sequence of SEQ ID NO: 52, and a primer set of a forward primer comprising the nucleotide sequence of SEQ ID NO: 53 and a reverse primer comprising the nucleotide sequence of SEQ ID NO: 54.

[0083] Furthermore, the present invention provides a probe capable of detecting the presence and / or absence of the genetic feature of the present invention (hereinafter, sometimes referred to as "the probe of the present invention"). The probe of the present invention may have a structure suitable for various detection methods of the presence and / or absence of the genetic feature of the present invention. For example, the probe of the present invention may contain a base sequence having complementarity to a portion of the genome containing the mutation site of the present invention. Non-limiting examples of such probes include those containing a base sequence selected from SEQ ID NOs: 55 to 64. Among these sequences, SEQ ID NOs: 55, 57, 59, 61, and 63 are specific to an allele containing the mutation of the present invention, and SEQ ID NOs: 56, 58, 60, 62, and 64 are specific to an allele not containing the mutation of the present invention. The presence of the genetic feature of the present invention can be detected by detecting an allele containing the mutation of the present invention and / or not detecting an allele not containing the mutation of the present invention, and the absence of the genetic feature of the present invention can be detected by not detecting an allele containing the mutation of the present invention and / or not detecting an allele not containing the mutation of the present invention. The probe of the present invention preferably has a label. Non-limiting examples of such labels include fluorescent labels, luminescent labels, radioactive labels, dyes, enzymes, quenchers, moieties that bind to detectable labels, etc. In a specific embodiment, the probe of the present invention has a base sequence selected from SEQ ID NOs: 55 to 64 and a label.

[0084] The present invention further provides a screening kit comprising one or more primer sets selected from the group consisting of (1) to (5), (1') to (5'), and (1'') to (5'') above, and optionally a restriction enzyme. In the case where the kit uses one or more primer sets selected from the group consisting of (1), (1') and (1''), the restriction enzyme contained in the kit is XbaI. In the case where the kit uses one or more primer sets selected from the group consisting of (2), (2') and (2''), the restriction enzyme contained in the kit is KpnI. In the case where the kit uses one or more primer sets selected from the group consisting of (3), (3') and (3''), the restriction enzyme contained in the kit is AflII. In the kit, when one or more primer sets selected from the group consisting of (5), (5') and (5'') are used, the restriction enzyme contained in the kit is PvuI.

[0085] In another embodiment of the kit, When the primer set includes a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 1, the restriction enzyme includes XbaI, When the primer set includes a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO: 3, the restriction enzyme includes KpnI, When the primer set includes a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO:5, the restriction enzyme includes AflII, When the primer set includes a forward primer having or including any sequence of 15 or more consecutive bases in SEQ ID NO:9, the restriction enzyme includes PvuI.

[0086] The present invention also provides a screening kit comprising a primer set capable of amplifying by PCR a region having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35 to 44, and the probe of the present invention.

[0087] These primer sets, probes, and kits can be used to detect the genetic features of the present invention, can be used in the screening method of the present invention, etc. Furthermore, these primer sets and kits may include instructions including explanations regarding the detection of the genetic features of the present invention or the screening method of the present invention, such as a user's manual, and a medium on which information regarding the method of use is recorded, such as a flexible disk, CD, DVD, Blu-ray disk, memory card, USB memory, etc.

[0088] 5. Manufacturing method of plant-derived extract and products using said extract In a further aspect of the present invention, there is provided a method for producing an extract containing rebaudioside M (hereinafter, may be referred to as "a method for producing an extract of the present invention"), which comprises a step of obtaining an extract from a plant of the present invention, or seeds or leaves (e.g., dried leaves or fresh leaves) of the plant. Furthermore, there is provided a method for producing rebaudioside M (hereinafter, may be referred to as "a method for producing rebaudioside M of the present invention"), which comprises a step of purifying rebaudioside M from an extract obtained by the method for producing an extract of the present invention. Specifically, there is provided a method for producing rebaudioside M, rebaudioside D, or both, comprising a step of obtaining an extract containing rebaudioside M, rebaudioside D, or both, from the rebaudioside M high content stevia plant of the present invention, a rebaudioside M high content stevia plant selected by the screening method of the present invention, or a rebaudioside M high content stevia plant produced by the method of the present invention. An extract containing rebaudioside M, rebaudioside D, or both can be obtained by reacting fresh or dried leaves of the plant of the present invention with an appropriate solvent (aqueous solvent such as water, or organic solvent such as alcohol, ether, and acetone). The extraction conditions can be referenced to the methods described in Ohta et al., J. Appl. Glycosci., Vol. 57, No. 3 (2010) or WO2010 / 038911, or the methods described in the Examples below. Alternatively, rebaudioside M, rebaudioside D, or both can be purified from an extract containing rebaudioside M, rebaudioside D, or both, using a known method such as ethyl acetate or other organic solvent:water gradient, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), ultra (high) performance liquid chromatography (UPLC), or the like.

[0089] The extract obtained by the method for producing an extract of the present invention (hereinafter referred to as "the extract of the present invention") contains higher contents of rebaudioside M, rebaudioside D, or both, as compared to wild-type Stevia species. The extract of the present invention has 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, 1100% or more, 1200% or more, 1300% or more, 1400% or more, 1500% or more, 1600% or more, 1700% or more, 1800% or more, 1900% or more, 2000% or more, 2100% or more, 2200% or more, 2300% or more, 2400% or more, 250% or more of rebaudioside M, rebaudioside D, or both, compared to an extract obtained from a wild-type Stevia species. 0% or more, 2600% or more, 2700% or more, 2800% or more, 2900% or more, 3000% or more, 3100% or more, 3200% or more, 3300% or more, 3400% or more, 3500% or more, 3600% or more, 3700% or more, 3800% or more, 3900% or more, 4000% or more, 4100% or more, 4200% or more, 4300% or more, 4400% or more, 4500% or more, 4600% or more, 4700% or more, 4800% or more, 4900% or more, or 5000% or more high content. Here, the extract of the present invention and the extract obtained from a wild-type Stevia species may be obtained by the same method.

[0090] By mixing the extract of the present invention thus obtained and / or rebaudioside M, rebaudioside D, or both obtained by the method of the present invention for producing rebaudioside M, rebaudioside D, or both with other ingredients, a novel medicine, flavor, or food or drink having an increased content of rebaudioside M, rebaudioside D, or both can be produced. Thus, as another embodiment, the present invention provides a method for producing a medicine, flavor, or food or drink, comprising a step of mixing rebaudioside M, rebaudioside D, or both obtained by the extract of the present invention and / or the method of the present invention for producing rebaudioside M, rebaudioside D, or both with other ingredients. Furthermore, the present invention provides a novel medicine, flavor, or food or drink having an increased content of rebaudioside M, rebaudioside D, or both obtained by the above-mentioned production method. Here, food or drink means beverages and foods. Thus, in one embodiment, the present invention provides a novel pharmaceutical product, flavoring, beverage or food product, and a method for producing the pharmaceutical product, flavoring, beverage or food product. EXAMPLES

[0091] Experimental examples and working examples relating to the present invention are described below, but the present invention is not limited to these specific embodiments.

[0092] [Example 1] Creation of high-RebM stevia plants 1. Introduction of breeding materials and initial selection Commercially available stevia seeds were sown and raised in August 2014, and approximately 3,000 individuals were selected based on their growth status and leaf morphology between October of the same year and March 2015. The content and ratio of each steviol glycoside was measured for 115 individuals obtained in April 2015 by quantitative analysis using LC-MS / MS, and three individuals with high RebD and RebM contents (individuals with a total of 20% or more of TSG) were selected. At the same time, selection was also performed based on total steviol glycoside (TSG) content, and 14 individuals with a TSG yield (total amount of measurable steviol glycosides obtained from unit leaf dry weight) of 20% or more were obtained.

[0093] 2. Creation and selection of inbred lines From the 115 individuals obtained above, five individuals with high RebA type that showed good growth conditions were selected, and artificial pollination was performed between or within individuals from January 2015. In March of the same year, mass seed collection was performed to obtain the first self-pollination generation (S1 seeds). These S1 seeds were then sown and raised to obtain an S1 population of 105 individuals. In June 2015, these S1 populations were grown individually, and the second self-pollination generation (S2 seeds) was obtained by artificial pollination within the individual. The obtained S2 seeds were classified as lineages for each S1 individual, and an S2 population of 105 lineages was obtained. The growth conditions of the obtained S2 individuals were then confirmed, and four leaves from each individual with good growth were sampled and quantitative analysis of the amount of steviol glycosides was performed using LC-MS / MS. From the analysis results, the RebD content, RebM content and TSG content were calculated, and individuals with a combined RebD content + RebM content of more than 2g (=2%) per 100g of leaves were selected as superior individuals.

[0094] 3. Creation and selection of hybrid lines Three high-RebD and high-RebM plants and six high-TSG plants selected by component analysis were used as breeding mother plants for a total of 53 cross-breeding tests. The selected plants were propagated vegetatively by cuttings in April 2015, seedlings were established in June of the same year, and they were nursed until November of the same year to establish multiple parent plants. Artificial cross-breeding was started in January 2016, and about 1,000 hybrid seeds (F1 seeds) were obtained for each combination. The F1 seeds were then sown and raised in March of the same year. The growth status of the obtained F1 plants was then confirmed, and four leaves from each well-grown plant were sampled and quantitatively analyzed for the amount of steviol glycosides using LC-MS / MS. From the analysis results, the RebD content, RebM content and TSG content were calculated, and individuals with a combined RebD content and RebM content exceeding 2 g / 100 g (=2%) were selected as superior individuals.

[0095] 4. Creation of genetic markers Genetic markers were created using 10 individuals obtained from the initial selection. In June 2015, the plants were classified into two groups, high (29.16% or more) and low (6.06% or less), based on the combined RebD+RebM content. A search for polymorphisms common to each group was conducted in the WRKY coding region and WD40 coding region, which are known to activate the biosynthetic pathway of secondary metabolites, and specific single nucleotide polymorphisms (SNPs) were obtained. Primers and restriction enzymes were selected based on the obtained SNPs, and polymorphic markers (1)-(5) that enable the identification of specific SNPs were established.

[0096] (1) P01 To detect marker P01, PCR was performed using the following primers, a restriction enzyme (XbaI) was added to the PCR product, and the enzyme reaction was carried out at 37°C to treat with the restriction enzyme. After restriction enzyme treatment, electrophoresis was performed using a microchip electrophoresis device, LabChip GX Touch HT, and the markers were identified based on the band pattern after electrophoresis. The primer sequences are as follows: Fw primer: 5'-AAGGTTCTTTATTTTTAAACTTATGTTAATTTATTGTATCTAG-3' (SEQ ID NO: 1) Rv primer: 5'-CCTTATGTACACATGCTACAC-3' (SEQ ID NO: 2) The resulting PCR product (approximately 383 bp in length) was treated with XbaI restriction enzyme, and those that did not produce a restriction enzyme product of approximately 344 bp (for example, SEQ ID NO: 23) were determined to be P01 positive.

[0097] (2)P02 To detect marker P02, PCR was performed using the following primers, a restriction enzyme (KpnI) was added to the PCR product, and an enzyme reaction was carried out at 37°C to treat with the restriction enzyme. After restriction enzyme treatment, electrophoresis was performed using a microchip electrophoresis device, LabChip GX Touch HT, and the markers were identified based on the band pattern after electrophoresis. The primer sequences are as follows: Fw primer: 5'-TAATCATCCAAACCCTAATCTCGCCAAACAACCGGGTAC-3' (SEQ ID NO: 3) Rv primer: 5'-GAGGAAGACATTGGCAACTC-3' (SEQ ID NO: 4) The resulting PCR product (approximately 297 bp in length) was treated with KpnI restriction enzyme, and those that did not produce a restriction enzyme product of approximately 260 bp (for example, SEQ ID NO: 26) were determined to be P02 positive.

[0098] (3) P03 To detect marker P03, PCR was performed using the following primers, a restriction enzyme (AflII) was added to the PCR product, and the enzyme reaction was carried out at 37°C to treat with the restriction enzyme. After restriction enzyme treatment, electrophoresis was performed using a microchip electrophoresis device, LabChip GX Touch HT, and the markers were identified based on the band pattern after electrophoresis. The primer sequences are as follows: Fw primer: 5'-CGATGGTTTTTGCTACATGAAAACCCTAGAAGACGAAACCCGCTTAA-3' (SEQ ID NO: 5) Rv primer: 5'-ACCAGCAATAATCCTTGAATTAG-3' (SEQ ID NO: 6) The resulting PCR product (approximately 390 bp in length) was treated with AflII restriction enzyme, and those that did not produce a restriction enzyme product of approximately 347 bp (for example, SEQ ID NO: 29) were determined to be P03 positive.

[0099] (4)P04 To detect marker P04, PCR was performed using the following primers. The PCR products were electrophoresed using a microchip electrophoresis device, LabChip GX Touch HT, and the markers were identified based on the band patterns after electrophoresis. The primer sequences are as follows: Fw primer: 5'-CGCAAACACGTATACTAATC-3' (SEQ ID NO: 7) Rv primer: 5'-TTTAGCATGGTATGTACAAC-3' (SEQ ID NO: 8) Those that only produced a PCR product of about 140 bp (eg, SEQ ID NO: 30) were considered P04 positive.

[0100] (5)P05 To detect marker P05, PCR was performed using the following primers, a restriction enzyme (PvuI) was added to the PCR product, and the enzyme reaction was carried out at 37°C to treat with the restriction enzyme. After restriction enzyme treatment, electrophoresis was performed using a microchip electrophoresis device, LabChip GX Touch HT, and the markers were identified based on the band pattern after electrophoresis. The primer sequences are as follows: Fw primer: 5'-ATACAAAAACACAACCCATATGGTCAAATCAACCCATTCATGAGCGATC-3' (SEQ ID NO: 9) Rv primer: 5'-CCCTTGTAAATCCCATATGTAG-3' (SEQ ID NO: 10) The resulting PCR product (approximately 288 bp in length) was treated with PvuI restriction enzyme, and those that did not produce a restriction enzyme product of approximately 240 bp (for example, SEQ ID NO: 33) were determined to be P05 positive.

[0101] 5. Genetic marker validation Using the polymorphic markers established above and population I, a marker validation experiment was carried out. A component analysis of 192 individuals from population I was carried out, and the top 8 individuals (0.35% or more) and bottom 8 individuals (0.12% or less) were selected based on the RebM content value, and marker testing was carried out as described above. As a result, only the top 8 individuals with high RebM content were selected as individuals positive for the target polymorphism (Figure 1). A similar verification experiment was also performed for Population II. A component analysis was performed on 137 individuals from Population II, and the top 8 individuals (0.24% or more) and bottom 8 individuals (0.01% or less) were selected based on the RebM content value, and marker testing was performed. As a result, only the top 8 individuals with high RebM content were selected as individuals with the target polymorphism (Figure 2). Figures 1 and 2 show that a band of the target size was generated only in individuals with a high RebM phenotype.

[0102] 6. Increasing the sample population For further validation, the number of individuals was increased for the two segregating populations, populations I and II, used in validating the genetic markers. Including the above numbers of individuals, 62 and 109 individuals, respectively, were used. Based on the RebM content, the populations were divided into three groups: 0.2% or more, 0.1% to less than 0.2%, and 0% to less than 0.1%. Marker testing was performed, and it was found that the 0.2% or more group was preferentially detected by this marker, proving that the frequency of positive individuals differed statistically significantly between groups (goodness-of-fit test using chi-square test; null hypothesis is that the marker test results are not linked to the phenotype and the frequency distribution is uniform. See the table below for the test results). [Table 2] [Table 3] [Table 4] [Table 5]

[0103] 7. Selection of high-RebM plants When high-RebM plants were selected using the genetic marker obtained above, it was possible to select individuals with a RebM ratio of 2% or more even in segregating populations other than the validation population, as shown in the table below, confirming that the marker can be used as a practical selection marker. The selection results for high-RebM plants are shown in the table below. "○" in the table indicates that the marker test result was positive. [Table 6] [Industrial Applicability]

[0104] Since the present invention makes it possible to provide rebaudioside M and D more efficiently, it is possible to provide medicines, flavorings, foods and beverages, and the like that contain sufficient amounts of rebaudioside M and D and have a high-quality taste.

Claims

1. A non-genetically modified stevia plant having a high rebaudioside M content, the plant containing 1.19% or more rebaudioside M per dry leaf and having all of the following genetic characteristics (1) to (5): (1) It is homozygous for an allele in which the base at the position corresponding to position 44 of SEQ ID NO:35 is T. (2) It is homozygous for an allele in which the base at the position corresponding to position 40 of SEQ ID NO:37 is T. (3) being homozygous for an allele in which the base at the position corresponding to position 48 of SEQ ID NO:39 is C; (4) The gene is homozygous for an allele that contains a deletion corresponding to positions 55 to 72 of SEQ ID NO:

42. (5) being homozygous for an allele in which the base at the position corresponding to position 50 of SEQ ID NO:43 is A;

2. The plant body according to claim 1, having all of the following characteristics (i) to (v). (i) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:1 and a reverse primer having the base sequence shown in SEQ ID NO:2, and the resulting PCR product is treated with XbaI restriction enzyme, only a band approximately 383 bp in length is obtained. (ii) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:3 and a reverse primer having the base sequence shown in SEQ ID NO:4, and the resulting PCR product is treated with the KpnI restriction enzyme, only a band approximately 297 bp in length is obtained. (iii) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:5 and a reverse primer having the base sequence shown in SEQ ID NO:6, and the resulting PCR product is treated with the AflII restriction enzyme, only a band of approximately 390 bp in length is obtained. (iv) When genomic DNA is subjected to PCR amplification using a forward primer having the nucleotide sequence shown in SEQ ID NO:7 and a reverse primer having the nucleotide sequence shown in SEQ ID NO:8, only a PCR product of about 140 bp is generated. (v) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:9 and a reverse primer having the base sequence shown in SEQ ID NO:10, and the resulting PCR product is treated with the PvuI restriction enzyme, only a band approximately 288 bp in length is obtained.

3. A seed, tissue, tissue culture or cultured plant cell of the plant according to claim 1 or 2.

4. 4. The tissue, tissue culture or plant cultured cell according to claim 3, which is an embryo, a meristematic cell, a pollen, a leaf, a root, a root tip, a petal, a protoplast, a leaf slice or a callus.

5. 3. A method for producing a stevia plant with a high rebaudioside M content, comprising a step of crossing the stevia plant of claim 1 or 2 with a second stevia plant, the method comprising the step of crossing the stevia plant of claim 1 or 2 with a second stevia plant, the second stevia plant containing 1.19% or more rebaudioside M per dry leaf.

6. 6. The method of claim 5, wherein the second plant is a Stevia plant according to claim 1 or 2.

7. 3. A rebaudioside M-containing extract from the leaves of the plant according to claim 1 or 2.

8. A food or drink, a sweetening composition, a flavoring, or a medicine comprising the extract according to claim 7.

9. A method for producing an extract containing rebaudioside M, comprising the step of obtaining an extract from the plant body according to claim 1 or 2.

10. 10. A method for producing rebaudioside M, comprising purifying rebaudioside M from the extract obtained by the method of claim 9.

11. A method for producing a food or beverage, a sweetening composition, a flavoring, or a pharmaceutical, comprising a step of mixing the extract obtained by the method according to claim 9 with other ingredients.

12. 10. A method for producing a food or beverage, a sweetening composition, a flavoring, or a pharmaceutical, comprising: purifying rebaudioside M from the extract obtained by the method according to claim 9; and mixing the purified rebaudioside M with other ingredients.

13. A method for screening for a high rebaudioside M content stevia plant containing 1.19% or more rebaudioside M per dry leaf, the method comprising the steps of detecting the presence of all of the following genetic characteristics (1) to (5) from the genome of a test plant: (1) It is homozygous for an allele in which the base at the position corresponding to position 44 of SEQ ID NO:35 is T. (2) It is homozygous for an allele in which the base at the position corresponding to position 40 of SEQ ID NO:37 is T. (3) being homozygous for an allele in which the base at the position corresponding to position 48 of SEQ ID NO:39 is C; (4) The gene is homozygous for an allele that contains a deletion corresponding to positions 55 to 72 of SEQ ID NO:

42. (5) being homozygous for an allele in which the base at the position corresponding to position 50 of SEQ ID NO:43 is A;

14. The method according to claim 13, comprising the step of detecting all of the following characteristics (i) to (v) from the genome of a test plant: (i) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:1 and a reverse primer having the base sequence shown in SEQ ID NO:2, and the resulting PCR product is treated with XbaI restriction enzyme, only a band approximately 383 bp in length is obtained. (ii) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:3 and a reverse primer having the base sequence shown in SEQ ID NO:4, and the resulting PCR product is treated with the KpnI restriction enzyme, only a band approximately 297 bp in length is obtained. (iii) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:5 and a reverse primer having the base sequence shown in SEQ ID NO:6, and the resulting PCR product is treated with the AflII restriction enzyme, only a band of approximately 390 bp in length is obtained. (iv) When genomic DNA is subjected to PCR amplification using a forward primer having the nucleotide sequence shown in SEQ ID NO:7 and a reverse primer having the nucleotide sequence shown in SEQ ID NO:8, only a PCR product of about 140 bp is generated. (v) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:9 and a reverse primer having the base sequence shown in SEQ ID NO:10, and the resulting PCR product is treated with the PvuI restriction enzyme, only a band approximately 288 bp in length is obtained.

15. 15. The method of claim 13 or 14, further comprising the step of measuring the content of Rebaudioside M in leaf tissue.

16. (1) A primer set comprising a forward primer comprising any sequence of 15 or more consecutive bases in SEQ ID NO: 1 and a reverse primer comprising any sequence of 15 or more consecutive bases in SEQ ID NO: 2; (2) A primer set comprising a forward primer comprising any sequence of 15 or more consecutive bases in SEQ ID NO: 3 and a reverse primer comprising any sequence of 15 or more consecutive bases in SEQ ID NO: 4; (3) A primer set comprising a forward primer comprising any sequence of 15 or more consecutive bases in SEQ ID NO: 5 and a reverse primer comprising any sequence of 15 or more consecutive bases in SEQ ID NO: 6; (4) A primer set comprising a forward primer comprising any sequence of 15 or more consecutive bases in SEQ ID NO: 7 and a reverse primer comprising any sequence of 15 or more consecutive bases in SEQ ID NO: 8; or (5) A primer set comprising a forward primer comprising any sequence of 15 or more consecutive bases in SEQ ID NO: 9 and a reverse primer comprising any sequence of 15 or more consecutive bases in SEQ ID NO: 10; wherein the arbitrary sequence of 15 or more consecutive bases is located at the 3' end of each primer.

17. A kit comprising the primer set according to claim 16 and, optionally, a restriction enzyme, When the primer set includes a forward primer including any sequence of 15 or more consecutive bases in SEQ ID NO: 1, the primer set includes a restriction enzyme XbaI, When the primer set includes a forward primer including any sequence of 15 or more consecutive bases in SEQ ID NO: 3, the primer set includes a restriction enzyme KpnI, When the primer set includes a forward primer including any sequence of 15 or more consecutive bases in SEQ ID NO:5, the primer set includes a restriction enzyme AflII, When the primer set includes a forward primer including any sequence of 15 or more consecutive bases in SEQ ID NO: 9, the primer set includes a restriction enzyme PvuI. The kit.

18. A probe comprising a base sequence shown in any one of SEQ ID NOs:55 to 64, which may be bound to a detectable label.

19. 20. The probe of claim 18, comprising a fluorescent label, dye or binding moiety.

20. 17. A method for screening for a non-genetically modified stevia plant with a high rebaudioside M content containing 1.19% or more rebaudioside M per dry leaf, the method comprising the steps of: performing PCR amplification on genomic DNA of a test plant using the primer set according to claim 16 for the purpose of detecting all of the following characteristics (i) to (v); and, in detecting characteristics (i) to (iii) and (v), treating a PCR product obtained by the PCR amplification with a restriction enzyme and detecting a restriction enzyme-treated product, wherein the restriction enzyme is XbaI for characteristic (i), KpnI for characteristic (ii), AflII for characteristic (iii), and PvuI for characteristic (v). (i) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:1 and a reverse primer having the base sequence shown in SEQ ID NO:2, and the resulting PCR product is treated with XbaI restriction enzyme, only a band approximately 383 bp in length is obtained. (ii) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:3 and a reverse primer having the base sequence shown in SEQ ID NO:4, and the resulting PCR product is treated with the KpnI restriction enzyme, only a band approximately 297 bp in length is obtained. (iii) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:5 and a reverse primer having the base sequence shown in SEQ ID NO:6, and the resulting PCR product is treated with the AflII restriction enzyme, only a band of approximately 390 bp in length is obtained. (iv) When genomic DNA is subjected to PCR amplification using a forward primer having the nucleotide sequence shown in SEQ ID NO:7 and a reverse primer having the nucleotide sequence shown in SEQ ID NO:8, only a PCR product of about 140 bp is generated. (v) When genomic DNA is amplified by PCR using a forward primer having the base sequence shown in SEQ ID NO:9 and a reverse primer having the base sequence shown in SEQ ID NO:10, and the resulting PCR product is treated with the PvuI restriction enzyme, only a band approximately 288 bp in length is obtained.

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