High rebaudioside M content stevia plant

Abstract

To provide a stevia plant having a high content of rebaudioside M. [Solution] 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.

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 diversifying consumer needs, various beverages have been developed and are commercially available. While sugars such as sucrose are commonly added to beverages to provide sweetness, the health effects of excessive intake have been pointed out, and there is a growing need for lower-calorie, naturally derived sweeteners. 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 sweetening component contained in stevia extract. Stevia extract is extracted and purified from stevia leaves. Stevia, a perennial plant in the Asteraceae family native to Paraguay in South America, is known as Stevia Rebaudiana Bertoni. Stevia contains compounds that are approximately 300 times sweeter than sugar, and is cultivated for their extraction and use as a natural sweetener. Various Reb glycosides, including RebA, RebB, RebC, RebD, RebE, and RebM, have been reported (Patent Publication No. 2012-504552). Among the various Reb glycosides, RebA, for example, is widely used and highly valued for its high sweetness and high-quality sweetness. Other Reb glycosides are also increasingly being discovered to have their own unique sweetness and associated flavors.

[0004] In such a situation, a stevia plant containing rebaudiosides M and D in an amount of 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 Patent Publication No. 2009-517043 [Patent Document 2] Specification of Patent Publication No. 2016-515814 [Patent Document 3] US2016 / 0057955A1 DISCLOSURE OF THE INVENTION [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 high-rebaudioside M content non-genetically modified stevia plant that contains a higher rebaudioside M content than wild-type stevia species, as well as methods for producing and screening for said plant.

[0008] Specifically, the present invention provides the following: [1-1] A non-genetically modified stevia plant with a high rebaudioside M content, characterized by containing 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in the leaves. [1-2] The plant body according to [1-1], further containing rebaudioside D at 9.5% or more of the total amount of steviol glycosides contained in the leaves. [1-3] A plant according to [1-1] or [1-2], which has at least one of the following genetic characteristics (1) to (5): (1) The individual 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 48The patient is homozygous for the allele in which the base at the position corresponding to position 1 is C. (4) The allele is homozygous for a deletion corresponding to positions 55 to 72 of SEQ ID NO: 42. (5) It 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 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, a 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, 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, comprising a step of crossbreeding the stevia plant according to any one of [1-1] to [1-4] with a second stevia plant. [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 the plant body according to any one of [1-1] to [1-4], or the seed, tissue, tissue culture or cell according to [1-5]. [1-10] A food or drink, a sweetening composition, a flavoring, or a pharmaceutical containing the extract according to [1-9]. [1-11] A method for producing a rebaudioside M-containing extract, comprising the step of obtaining an extract from the plant body according to any one of [1-1] to [1-4], or the seed, tissue, tissue culture, or cell according to [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 for a stevia plant with a high rebaudioside M content, comprising the step of detecting the presence and / or absence of at least one of the following genetic characteristics (1) to (5) from the genome of a test plant: (1) The individual 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 patient is homozygous for the allele in which the base at the position corresponding to position 1 is C. (4) The allele is homozygous for a deletion corresponding to positions 55 to 72 of SEQ ID NO: 42. (5) It 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], which comprises the 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 comprising 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 consecutive 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 the base sequence shown in any one of SEQ ID NOs: 55 to 64, which may be bound to a detectable label. [1-20] The probe according to [1-19], having a fluorescent label, dye or binding moiety. [1-21] A method for screening for non-genetically modified stevia plants with a 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 the PCR product obtained by the PCR amplification with a restriction enzyme and detecting the 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 with a high rebaudioside M content, characterized by containing 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in the dried leaves. [2-2] The plant body according to [2-1] above, further containing rebaudioside D at 9.5% or more of the total amount of steviol glycosides contained in the dried leaves. [2-3] A plant body according to [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] above. [2-5] Dried leaves of the plant according to any one of [2-1] to [2-3] above. [2-6] A tissue culture of a plant or a cultured plant cell according to any one of [2-1] to [2-3] above. [2-7] The tissue culture or plant cultured cells according to [2-6] above, which are embryos, meristematic cells, pollen, leaves, roots, root tips, petals, protoplasts, leaf slices, and calluses.

[0015] [2-8] A method for producing a stevia plant with a 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 the dried leaves, comprising a step of crossbreeding the stevia plant according to any one of [2-1] to [2-3] above with a second stevia plant. [2-9] The method according to [2-8] above, wherein the second plant is a stevia plant according to any one of [2-1] to [2-3] above. [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 drink, a sweetening composition, a flavoring, or a pharmaceutical comprising the extract described in [2-10] above.

[0016] [2-12] A method for producing a rebaudioside M-containing extract, comprising a 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 the 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 a high rebaudioside M content, comprising the 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 content of rebaudioside M 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 comprising 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 consecutive 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, which has a fluorescent label, a dye or a 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 a step of treating the PCR product obtained by the PCR amplification with a restriction enzyme and detecting the restriction enzyme-treated product when the polymorphic marker is at least one selected from the group consisting of P01, P02, P03, and P05; A method for screening for a non-genetically modified stevia plant with 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 [2-21] to [2-24] above, wherein the plant obtained by screening contains 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in the dried leaves. [Effects 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 obtained from such plants, and to provide foods, beverages, and the like containing rebaudioside M obtained from the leaves. [Brief explanation of the drawings]

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

[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 incorporates the contents of the specification and drawings of Japanese Patent Application No. 2017-198515, filed on October 12, 2017, from which the present application claims priority.

[0022] 1. Non-genetically modified stevia plant with high rebaudioside M content according to the present invention The present invention provides a non-genetically modified stevia plant with a high rebaudioside M content, characterized in that the plant contains 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in its leaves (e.g., dried or fresh leaves) (hereinafter, this may be referred to as the "plant of the present invention" or "stevia plant of the present invention"). The stevia plant of the present invention is a species derived from a wild-type stevia plant, but has a genetic mutation that increases the amount of rebaudioside M. The genetic mutation is generated without genetic modification (described below). In the present invention, among non-genetically modified stevia plants with a high rebaudioside M content, stevia plants 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 non-genetically modified stevia plants with a high rebaudioside M content, and stevia plants characterized by containing 4.7% or more rebaudioside M may be referred to as non-genetically modified stevia plants with an ultra-high rebaudioside M content.

[0023] Total steviol glycosides (TSG) do not include unknown steviol glycosides or steviol glycosides present below the detection limit. Preferably, 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, steviolmonosides, steviolbioside, 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; 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 rebaudioside M (RebM) content 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 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 water content to 3 to 4% by weight.

[0027] Here, the plant body of the present invention has an amount 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 per 100g of dried leaves. 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, or more than 1500%, when the amount (g) of rebaudioside M contained in 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 leaves (e.g., dried or fresh leaves) are expressed as a RebM / RebD ratio, the lower limit of the RebM / RebD ratio 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. On the other hand, the upper limit of the RebM / RebD ratio 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 stevia plant of the present invention is characterized in that, when the content of rebaudioside M (RebM) and rebaudioside D (RebD) in leaves (e.g., dried or fresh leaves) is expressed as a ratio of (RebD+RebM) / TSG% to the total amount of steviol glycosides, the lower limit of the (RebD+RebM) / TSG value 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 (RebD+RebM) / TSG value 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 higher 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 plant. Specifically, the plant may contain less than 19 g of 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.

[0031] 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 or fresh leaves) are expressed as the RebM / RebA ratio, the lower limit of the RebM / RebA value 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 RebM / RebA value 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 exceeds 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 content of rebaudioside M (RebM) and rebaudioside A (RebA) in 100 g of leaves (e.g., dried leaves or fresh leaves) is 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 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 upper and lower limits is not particularly limited as long as the upper limit exceeds the lower limit, 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 individual 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 patient is homozygous for the allele in which the base at the position corresponding to position 1 is C. (4) The allele is homozygous for a deletion corresponding to positions 55 to 72 of SEQ ID NO: 42. (5) It 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 NOs: 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 If a sequence identical to the reference sequence does not exist in the genome, the term "reference sequence" refers to a position or portion of a sequence in the genome that corresponds to the position or portion of the reference sequence. Whether 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 alignment analysis of the obtained sequence with the reference sequence. Non-limiting examples of sequences corresponding to a reference sequence include nucleotide sequences that have 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 sequence identity to the reference sequence. A position or portion in a sequence corresponding to a reference sequence in a genome that corresponds to a position or portion in the reference sequence can be determined by taking into account the nucleotide 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 position 44 from the 5' side of the portion in the genome consisting of the same nucleotide sequence as SEQ ID NO: 35. 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 is equivalent to SEQ ID NO: 35, the genome does not have a portion consisting of the same nucleotide sequence as SEQ ID NO: 35. Therefore, the "position corresponding to position 44 of SEQ ID NO: 35" does not necessarily correspond to position 44 from the 5' side of the portion corresponding to SEQ ID NO: 35. However, by taking into account the nucleotide sequences around position 44 of SEQ ID NO: 35, etc., it is possible to identify the "position corresponding to position 44 of SEQ ID NO: 35" in the genome of such a stevia plant. For example, the "position corresponding to position 44 of SEQ ID NO: 35" in the genome of a stevia plant can be identified by alignment analysis of the nucleotide sequence of the portion in the genome of a stevia plant that corresponds to SEQ ID NO: 35 with the nucleotide sequence of SEQ ID NO: 35.

[0036] The term "portion consisting of a nucleotide sequence corresponding to SEQ ID NO: 35" refers to, for example, a portion consisting of a nucleotide sequence that has 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.1% or more, 98.4% or more, 98.7% or more, 99% or more, 99.2% or more, 99.5% or more, or 99.8% or more sequence identity to the nucleotide sequence of SEQ ID NO: 35.

[0037] In some embodiments, the "portion consisting of a nucleotide 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 used as an example for the explanation here, but the same applies to the genetic characteristics (2) to (5).

[0038] In a specific embodiment, the "portion consisting of the 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 the 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 specific embodiment, the "portion consisting of the 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 specific embodiment, the "portion consisting of the 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" comprises 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 position is C" include the base sequences of SEQ ID NO: 59, 69, or 79. In a specific embodiment, the "allele lacking the 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, and (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, (2) a portion corresponding to positions 55 to 72 of SEQ ID NO: 42, and (3) 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) a mutation from A to T at the position corresponding to position 44 of SEQ ID NO: 35, (2) a mutation from C to T at the position corresponding to position 40 of SEQ ID NO: 37, (3) a mutation from C to T 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 analyzed 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, 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, if treatment of the PCR product with XbaI restriction enzyme produces a restriction enzyme treatment product of approximately 344 bp (for example, SEQ ID NO: 23), 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 in length) (e.g., SEQ ID NO: 24 or 25) is treated with the KpnI restriction enzyme, only a band of approximately 297 bp in length (e.g., SEQ ID NO: 24) is obtained. Conversely, if a restriction enzyme treatment product of approximately 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 (approximately 390 bp in length) (e.g., SEQ ID NO:27 or 28) is treated with the AflII restriction enzyme, only a band of approximately 390 bp in length (e.g., SEQ ID NO:27) is obtained. Conversely, if a restriction enzyme treatment product of approximately 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 the 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 (approximately 288 bp in length) (e.g., SEQ ID NO:31 or 32) is treated with the PvuI restriction enzyme, only a band of approximately 288 bp in length (e.g., SEQ ID NO:31) is obtained. Conversely, if a restriction enzyme treatment product of approximately 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 carried out according to the conditions recommended by the vendor of each restriction enzyme used.

[0045] For details of the method for screening plants of the present invention, please refer to the section "3. Screening method for plants of the present invention" described below. It has been confirmed in the Examples that the above genetic characteristics (e.g., the 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] Rebaudiosides 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, or acetone). The extraction conditions can be determined by reference 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 known methods such as ethyl acetate or other organic solvent:water gradient, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), and ultra (high) performance liquid chromatography (UPLC).

[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 plants of the present invention may include not only whole plants 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 of the present invention may also include tissue cultures or plant cultured cells, since plants can be regenerated by culturing such tissue cultures or plant cultured cells. Examples of plant 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 a 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), which method comprises a step of crossing the stevia plant of the present invention with a second stevia plant (hereinafter, this may be referred to as the "production method of the present invention"). The "high rebaudioside M stevia plant characterized by containing 2% or more of rebaudioside M relative to 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 rebaudioside M (RebM) content is expressed as RebM / TSG% as a ratio to the total amount of steviol glycosides obtained from the 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 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 hydroxyl group 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 per 100g of dried leaves. or 2.30g or more, 2.35g or more, 2.40g or more, 2.45g or more, 2.50g or more, 2.55g or more, 2.60g or more, 2.65g or more, 2.70g or more, 2.75g or more, 2.80g or more, 2.85g or more, 2.90g or more, 2.95g or more, 3.00g or more, 3.05g or more, 3.10g or more, 3.15g or more, 3.20g or more, 3.25g or more, 3.30g or more, 3.35g or more, 3.40g or more, 3.45g or more, 3.50g or more, 3.55g or more, or 3.57g 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 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, plants obtained by the production method of the present invention are characterized in that, when the contents of rebaudioside M (RebM) and rebaudioside D (RebD) in leaves (e.g., dried or fresh leaves) are expressed as a RebM / RebD ratio, the lower limit of the RebM / RebD value 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. On the other hand, the upper limit of the RebM / RebD value 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, when the content of rebaudioside M (RebM) and rebaudioside D (RebD) in leaves (e.g., dried or fresh leaves) is expressed as a ratio of (RebD+RebM) / TSG% to the total amount of steviol glycosides, the plant obtained by the production method of the present invention is characterized in that the lower limit of the (RebD+RebM) / TSG value 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 (RebD+RebM) / TSG value 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 higher 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, a plant obtained by the production method of the present invention may have a lower total amount of steviol glycosides than a wild-type plant. Specifically, the plant may contain less than 19 g of steviol glycosides per 100 g of dried leaves. This means that when dried leaves are obtained from a 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 plants obtained by the production method of the present invention are characterized in that, when the content of rebaudioside M (RebM) and rebaudioside A (RebA) in 100 g of leaves (e.g., dried leaves or fresh leaves) is expressed as the 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 upper and lower limits is not particularly limited as long as the upper limit exceeds the lower limit, 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 method 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 individual 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 patient is homozygous for the allele in which the base at the position corresponding to position 1 is C. (4) The allele is homozygous for a deletion corresponding to positions 55 to 72 of SEQ ID NO: 42. (5) It 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, if treatment of the PCR product with XbaI restriction enzyme produces a restriction enzyme treatment product of approximately 344 bp (for example, SEQ ID NO: 23), 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 in length) (e.g., SEQ ID NO: 24 or 25) is treated with the KpnI restriction enzyme, only a band of approximately 297 bp in length (e.g., SEQ ID NO: 24) is obtained. Conversely, if a restriction enzyme treatment product of approximately 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 (approximately 390 bp in length) (e.g., SEQ ID NO:27 or 28) is treated with the AflII restriction enzyme, only a band of approximately 390 bp in length (e.g., SEQ ID NO:27) is obtained. Conversely, if a restriction enzyme treatment product of approximately 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 the 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 (approximately 288 bp in length) (e.g., SEQ ID NO:31 or 32) is treated with the PvuI restriction enzyme, only a band of approximately 288 bp in length (e.g., SEQ ID NO:31) is obtained. Conversely, if a restriction enzyme treatment product of approximately 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 carried out according to the conditions recommended by the vendor of each restriction enzyme used.

[0061] In the production method of the present invention, "crossing" refers to crossing a plant of the present invention (first generation (S1)) with a second plant (S1) to obtain a progeny plant (a plant (second generation (S2)) produced by the production method of the present invention). Backcrossing is a preferred method of crossing. "Backcrossing" refers to a technique in which a progeny plant (S2) born between a plant of the present invention and a second plant is further crossed with a plant of the present invention (i.e., a plant having the genetic characteristics of the present invention) (S1) to produce a plant having the genetic characteristics of the present invention. 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, this is essentially backcrossing. The genetic polymorphisms of the present invention are inherited according to Mendel's laws, and therefore the phenotype correlated with the genetic polymorphism, i.e., the phenotype of high rebaudioside M content, is also inherited according to Mendel's laws.

[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 further crossbreed a plant produced by the production method of the present invention with a third stevia plant to produce a stevia plant with a high rebaudioside M content 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).

[0064] In another embodiment, the plant of the present invention can be produced by regenerating the plant through culturing the tissue culture or plant cell culture described above. 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, pp. 113-123).

[0065] Alternatively, since the plant of the present invention is non-genetically modified, it can also be produced by incorporating the above polymorphism into a wild-type stevia plant using non-genetic techniques. An example of a "non-genetic technique" is a method of inducing mutations in a gene in a host cell (or host plant) without introducing a foreign gene. Such a method includes the treatment of plant cells with a mutagen. Examples of such mutagen include ethyl methanesulfonate (EMS) and sodium azide. For example, plant cells can be treated with ethyl methanesulfonate (EMS) at concentrations of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%. 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 method, plant cells can be irradiated with radiation or light such as X-rays, gamma rays, or ultraviolet rays. In this case, cells irradiated with an appropriate amount of ultraviolet light (ultraviolet lamp intensity, distance, and time) can be cultured on a selective medium, and then cells, calli, or plants with the desired traits can be selected. The irradiation intensity is 0.01 to 100 Gr, 0.03 to 75 Gr, 0.05 to 50 Gr, 0.07 to 25 Gr, 0.09 to 20 Gr, 0.1 to 15 Gr, 0.1 to 10 Gr, 0.5 to 10 Gr, and 1 to 10 Gr, the irradiation distance is 1 cm to 200 m, 5 cm to 100 m, 7 cm to 75 m, 9 cm to 50 m, 10 cm to 30 m, 10 cm to 20 m, and 10 cm to 10 m, 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, or 10 minutes to 1 week. The irradiation intensity, distance, and time vary depending on the type of radiation and the state of the target (cells, callus, plant body), but can be adjusted appropriately by one skilled in the art.

[0067] In addition, techniques such as cell fusion, anther culture (haploid rearing), and distant crossing (haploid rearing) are also known. Generally, plant cells may undergo mutations during cultivation, and therefore it is preferable to return them to individual plants in order to maintain traits more stably. Although the plant of the present invention is a non-genetically modified stevia plant, plants obtained by subsequently genetically modifying (for example, by genome editing) the plant of the present invention as a host (for example, a plant obtained by genetically modifying the plant of the present invention as a host to further impart another trait) are not excluded from the scope of the present invention.

[0068] 3. Screening method for plants of the present invention The plants of the present invention and plants having the same phenotype and genetic properties as the plants of the present invention can be screened by detecting the genetic features of the present invention from the tissues of the plants. Here, "screening" means distinguishing the plants of the present invention from other plants and selecting the plants of the present invention. Therefore, as another embodiment, the present invention provides a method for screening for a stevia plant with a high rebaudioside M content (hereinafter, sometimes 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 individual 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 patient is homozygous for the allele in which the base at the position corresponding to position 1 is C. (4) The allele is homozygous for a deletion corresponding to positions 55 to 72 of SEQ ID NO: 42. (5) It 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 genetic characteristics (1) to (5) above has been detected.

[0069] The presence of the genetic trait 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 position corresponding to the position is C, (D) an allele in which the 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 position corresponding to the 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 absence of an allele selected from the group consisting of: can be determined by

[0070] The absence 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 position corresponding to the position is C, (D) an allele in which the 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 position corresponding to the 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: 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 has a sequence complementary to the polymorphic site of the present invention. When a primer designed in this way is used, if the template sample contains a polymorphism, the primer will completely hybridize to the template, and the polymerase extension reaction will proceed. However, if the template does not contain 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 primers, and the amplified product is analyzed by agarose gel electrophoresis or the like. If an amplified product of a predetermined size is confirmed, it means that the sample template contains a mutation. If an amplified product is not present, it can be determined that the template does not contain a mutation. Alternatively, the genetic characteristics of the present invention can be detected by designing primer sequences that do not overlap with the polymorphisms of the present invention and that enable PCR amplification of the genetic mutations of the present invention, and sequencing the base sequences of the amplified nucleotide fragments. 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 is a method that 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 involves amplifying the region containing the mutation by PCR and sequencing the DNA sequence using a dye terminator or similar to analyze the presence or absence of the mutation (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 to a support, and includes DNA chips, gene chips, microchips, bead arrays, and the like. The presence or absence of the polymorphisms of the present invention can be comprehensively detected by using probes containing sequences complementary to the polymorphisms of the present invention. Examples of DNA microarray assays, such as DNA chips, include the GeneChip assay (Affymetrix; see U.S. Patent Nos. 6,045,996, 5,925,525, and 5,858,659). 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 an SNP and one type of Invader probe to template DNA, and cleavage of the DNA with a cleavase enzyme that has 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 that have undergone mutation introduction 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 positive polymorphic markers of the present invention from the genome of a test plant. Such a step includes extracting genomic DNA from the test plant and detecting polymorphic markers of the present invention in the genomic DNA. As already mentioned, the polymorphic marker is at least one selected from the group consisting of P01 to P05. Furthermore, positive polymorphic markers means positive for at least one marker selected from the group consisting of P01 to P05. 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 the test plants, plants that have been detected as positive for the polymorphic marker of the present invention.

[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-treated product; The present invention provides a method for screening for a non-genetically modified stevia plant with a high rebaudioside M content, comprising: (1) A primer set comprising a forward primer containing the nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer containing the nucleotide sequence shown in SEQ ID NO: 2; (2) A primer set comprising a forward primer containing the nucleotide sequence shown in SEQ ID NO: 3 and a reverse primer containing 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 containing the nucleotide sequence shown in SEQ ID NO: 7 and a reverse primer containing 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 PCR amplification conditions are as described above.

[0077] However, 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 at its 3' end the sequence of SEQ ID NO: 1, 3, 5, 7, or 9, extending 15 bases upstream from the 3' end (see the table below), and the reverse primer may have at its 3' end the sequence of SEQ ID NO: 2, 4, 6, 8, or 10, extending 15 bases upstream from the 3' end (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 comprising 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 consecutive bases in SEQ ID NO: 9 and a reverse primer having or containing any sequence of 15 or more consecutive 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, as long as the any sequence of 15 or more consecutive bases is present at the 3' end.

[0079] The screening method of the present invention may further comprise 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 the test stevia plants in which a genetic feature of the present invention has been detected, and this may be crossed with another stevia plant, and the screening method of the present invention may be applied to the resulting offspring plants. Therefore, the screening method of the present invention may comprise 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 with 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 trait of the present invention from the genome of the offspring plant obtained by crossing; (vi) measuring the content of RebM in the offspring plant tissue in which the genetic trait of the present invention has been detected; (vii) A step of selecting individuals with 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, those in the top 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the tested stevia plants in which the genetic feature of the present invention has been detected, in terms of RebM content. Furthermore, 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"). 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 probes capable of detecting the presence and / or absence of the genetic features of the present invention (hereinafter, sometimes referred to as "probes of the present invention"). The probes of the present invention may have a structure suitable for various detection methods for the presence and / or absence of the genetic features of the present invention. For example, the probes of the present invention may contain a nucleotide sequence complementary to a portion of the genome containing the mutation site of the present invention. Non-limiting examples of such probes include those containing a nucleotide sequence selected from SEQ ID NOs: 55 to 64. Of these sequences, SEQ ID NOs: 55, 57, 59, 61, and 63 are specific to alleles containing the mutation of the present invention, and SEQ ID NOs: 56, 58, 60, 62, and 64 are specific to alleles not containing the mutation of the present invention. The presence of the genetic features 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 features of the present invention can be detected by not detecting an allele containing the mutation of the present invention and / or detecting an allele not containing the mutation of the present invention. The probes of the present invention preferably contain 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 kit, when one or more primer sets selected from the group consisting of (1), (1') and (1'') are used, the restriction enzyme contained in the kit is XbaI. In the kit, when one or more primer sets selected from the group consisting of (2), (2') and (2'') are used, the restriction enzyme contained in the kit is KpnI. In the kit, when one or more primer sets selected from the group consisting of (3), (3') and (3'') are used, 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 characteristics of the present invention, can be used in the screening methods of the present invention, etc. Furthermore, these primer sets and kits may include instructions including explanations regarding the detection of the genetic characteristics of the present invention or the screening methods of the present invention, such as instruction manuals, or media on which information regarding the methods of use is recorded, such as flexible disks, CDs, DVDs, Blu-ray disks, memory cards, 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 a rebaudioside M-containing extract (hereinafter sometimes referred to as a "method for producing an extract of the present invention"), which comprises the 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 sometimes referred to as a "method for producing rebaudioside M of the present invention"), which comprises the step of purifying rebaudioside M from the 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, which includes a step of obtaining an extract containing rebaudioside M, rebaudioside D, or both from the rebaudioside M-rich stevia plant of the present invention, a rebaudioside M-rich stevia plant selected by the screening method of the present invention, or a rebaudioside M-rich 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 (an aqueous solvent such as water, or an organic solvent such as alcohol, ether, or acetone). The extraction conditions can be determined by reference 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), or ultra (high) performance liquid chromatography (UPLC).

[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 a higher content of rebaudioside M, rebaudioside D, or both than 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. The extract of the present invention and the extract obtained from wild-type Stevia species may contain 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 higher. Here, the extract of the present invention and the extract obtained from 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, novel pharmaceuticals, flavors, or foods and beverages having increased contents of rebaudioside M, rebaudioside D, or both can be produced. Therefore, in another embodiment, the present invention provides a method for producing a pharmaceutical, flavor, or food and beverage, comprising the step of mixing the extract of the present invention 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. Furthermore, the present invention provides novel pharmaceuticals, flavors, or foods and beverages having increased contents of rebaudioside M, rebaudioside D, or both obtained by the above-mentioned production methods. Here, "food and beverage" means beverages and foods. Thus, in one embodiment, the present invention provides novel pharmaceuticals, flavorings, beverages, or foods, and methods for producing the pharmaceuticals, flavorings, beverages, or foods. [Example]

[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. Approximately 3,000 individuals were selected based on growth status and leaf morphology between October 2014 and March 2015. The content and percentage of each steviol glycoside were measured for 115 individuals obtained in April 2015 using quantitative analysis with LC-MS / MS, and three individuals with high RebD and RebM contents (those two components together accounted for 20% or more of TSG) were selected. Simultaneously, selection was also conducted based on total steviol glycoside (TSG) content, and 14 individuals with a TSG yield (the total amount of measurable steviol glycosides obtained per 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 good growth conditions and high RebA were selected. Artificial pollination between and within individuals was performed from January 2015, and mass seed collection was conducted in March of the same year to obtain the first selfed 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 individually cultivated and artificial pollination within individuals was performed to obtain the second selfed generation (S2 seeds). The resulting S2 seeds were then classified as lineages for each S1 individual, resulting in an S2 population of 105 lineages. The growth status of the resulting S2 individuals was then confirmed, and four leaves from each well-growing individual were sampled and quantitatively analyzed for steviol glycoside content 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 of more than 2 g (= 2%) per 100 g 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 through component analysis were used as breeding mother plants for a total of 53 cross-breeding experiments. These selected plants were propagated vegetatively by cuttings in April 2015, and seedlings were established in June of the same year. They were then nursed until November of the same year to establish multiple parent plants. Artificial cross-breeding began in January 2016, yielding approximately 1,000 hybrid seeds (F1 seeds) for each combination. The F1 seeds were then sown and raised in March of the same year. The growth status of the resulting F1 plants was then monitored, and four leaves from each well-growing plant were sampled and quantitatively analyzed for steviol glycoside content using LC-MS / MS. From the analysis results obtained, the RebD content, RebM content, and TSG content were calculated, and individuals with a combined RebD content and RebM content of more than 2 g / 100 g (= 2%) were selected as superior individuals.

[0095] 4. Genetic Marker Creation Genetic markers were developed using 10 individuals obtained through initial selection. In June 2015, the plants were classified into two groups: high (29.16% or higher) and low (6.06% or lower) based on the combined RebD+RebM content. A search for polymorphisms common to each group was conducted in the WRKY and WD40 coding regions, which are known to activate the secondary metabolite biosynthetic pathway, and specific single nucleotide polymorphisms (SNPs) were identified. Based on the SNPs identified, primers were designed and restriction enzymes were selected, establishing polymorphic markers (1)–(5) that enable the identification of specific SNPs.

[0096] (1)P01 To detect marker P01, PCR was performed using the following primers, and a restriction enzyme (XbaI) was added to the PCR product. The PCR product was then subjected to an enzymatic reaction at 37°C, followed by restriction enzyme treatment. 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, and a restriction enzyme (KpnI) was added to the PCR product. The PCR product was then treated with the restriction enzyme at 37°C. 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, and a restriction enzyme (AflII) was added to the PCR product. The PCR product was then subjected to an enzymatic reaction at 37°C, followed by restriction enzyme treatment. 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 produced only a PCR product of approximately 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, and a restriction enzyme (PvuI) was added to the PCR product. The PCR product was then subjected to an enzymatic reaction at 37°C, followed by restriction enzyme treatment. 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 Verification Marker validation experiments were conducted using the polymorphic markers established above and Population I. A component analysis of 192 individuals from Population I was performed, and the top 8 individuals (over 0.35%) and bottom 8 individuals (below 0.12%) were selected based on their RebM content. Marker testing was then performed as described above. As a result, only the top 8 individuals with the highest RebM content were selected as individuals positive for the target polymorphism (Figure 1). A similar validation experiment was performed on Population II. A component analysis of 137 individuals from Population II was performed, and the top eight individuals (over 0.24%) and bottom eight individuals (below 0.01%) were selected based on their RebM content values, followed by marker testing. As a result, only the top eight individuals with the highest RebM content were selected as individuals with the target polymorphism (Figure 2). Figures 1 and 2 show that the target size band appears only in individuals with a high RebM phenotype.

[0102] 6. Increasing the sample population For further validation, we increased the number of individuals in the two segregating populations, Populations I and II, used in the validation of the genetic markers. Including the above numbers, 62 and 109 individuals were used, respectively. Based on RebM content, the populations were divided into three groups: 0.2% or higher, 0.1% to less than 0.2%, and 0% to less than 0.1%. Marker testing revealed that the 0.2% or higher group was preferentially detected by the marker, demonstrating that the frequency of positive individuals differed statistically significantly between groups (goodness-of-fit test using a chi-square test; the null hypothesis was that the marker test results were not linked to phenotypes and that the frequency distribution was uniform. See the table below for 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, individuals with a RebM ratio of 2% or more could be selected 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 results of the selection of high-RebM plants are shown in the table below. A "○" in the table indicates a positive marker test result. [Table 6] [Industrial Applicability]

[0104] According to the present invention, rebaudiosides M and D can be provided more efficiently, and by containing sufficient amounts of rebaudioside M and D, it is possible to provide medicines, flavorings, foods and beverages, etc. with a high-quality taste.

Claims

1. A stevia plant having at least one of the following genetic characteristics (1) to (2): (1) It is homozygous for an allele in which the base at the position corresponding to position 40 of SEQ ID NO: 37 is T. (2) It is homozygous for an allele in which the base at the position corresponding to position 50 of SEQ ID NO:43 is A.

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

3. 10. A method for producing a stevia plant with a high rebaudioside M content, the method comprising the step of crossing the stevia plant of claim 1 with a second stevia plant, the stevia plant containing 2% or more of rebaudioside M relative to the total amount of steviol glycosides contained in the leaves.

4. 10. A method for producing a rebaudioside M-containing extract, comprising the step of obtaining an extract from the plant body of claim 1 or the seed, tissue, tissue culture, or cell of claim 2.

5. A process for obtaining a rebaudioside M-containing extract from the plant body of claim 1 or the seed, tissue, tissue culture, or cell of claim 2; and and purifying rebaudioside M from the extract. (i) obtaining a rebaudioside M-containing extract from the plant body of claim 1 or the seed, tissue, tissue culture, or cell of claim 2; and and / or mixing the extract with other ingredients. (ii) obtaining a rebaudioside M-containing extract from the plant body of claim 1 or the seed, tissue, tissue culture, or cell of claim 2; purifying rebaudioside M from the extract; and mixing the rebaudioside M with other ingredients A method for producing a food or drink, a sweetening composition, a flavoring agent, or a pharmaceutical, comprising the steps of:

7. A method for screening for a stevia plant with a high rebaudioside M content, comprising the step of detecting the presence and / or absence of at least one of the following genetic characteristics (1) to (2) from the genome of a test plant: (1) It is homozygous for an allele in which the base at the position corresponding to position 40 of SEQ ID NO: 37 is T. (2) It is homozygous for an allele in which the base at the position corresponding to position 50 of SEQ ID NO:43 is A.

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