# US 10,370,673 B2 · Single nucleotide polymorphism (SNP) markers for stevia

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- Number: US 10,370,673 B2
- Document ID: US10370673B2
- Status: Granted
- Kind: B2
- Application number: 15/512,271
- Filed: 2015-09-25
- Published: 2017-10-05
- Granted: 2019-08-06
- Earliest claimed: 2014-09-26
- Assignees: PureCircle Sdn Bhd
- Applicants: PureCircle USA Inc.
- Inventors: Avetik Markosyan; Seong Siang Ong; Yeen Yee Wong; Yu Cheng Bu; Jian Ning Chen
- Web page: https://patentgrep.com/patent/US10370673B2

## CPC

- C12N 15/8245: {involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis}
- A01H 5/12: Leaves
- A23L 27/36: {Terpene glycosides}
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12N 5/04: Plant cells or tissues {(culture media C12N5/0025)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12Q 1/68: involving nucleic acids
- C12Q 1/6895: for plants, fungi or algae
- C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00)
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Stevia varieties with high a content of RebD, a high content of RebM, and a high content of RebD and RebM containing various SNP markers and UGT isoforms, are disclosed. Methods of screening for the SNPs are also disclosed as well as for using the SNPs in marker assisted breeding. Further provided are methods for introgressing the disclosed SNPs associated with high RebD and high RebM into stevia plants by selecting plants comprising for one or more SNPs and breeding with such plants to confer such desirable agronomic phenotypes to plant progeny.

## Claims

### Claim 1

A stevia plant comprising eight single nucleotide polymorphisms (SNPs), wherein said SNPs are found in homozygous form, wherein said eight SNPs comprise: SNP2, which comprises a G to C nucleotide substitution at position number 225 in SEQ ID NO:1, SNP10, which comprises a G to A nucleotide substitution at position number 187 in SEQ ID NO:2, SNP12, which comprises a C to T nucleotide exchange at position number 345 in SEQ ID NO:3, SNP17, which comprises an A to C nucleotide substitution at position number 129 in SEQ ID NO:4, SNP19, which comprises an A to T nucleotide substitution at position number 173 in SEQ ID NO:5, SNP20, which comprises a G to A nucleotide substitution at position number 221 in SEQ ID NO:6, SNP22, which comprises a T to G nucleotide substitution at position number 160 in SEQ ID NO:7, and SNP24, which comprises a G to A nucleotide substitution at position number 325 in SEQ ID NO:8.

### Claim 2 (depends on 1)

The stevia plant of claim 1, wherein the leaves of said stevia plant have a rebaudioside D content of between 0.6% and 3.3% of dry weight.

### Claim 3 (depends on 1)

The stevia plant of claim 1, wherein the leaves of said stevia plant have a rebaudioside M content of between 0.5% and 1.15% of dry weight.

### Claim 4 (depends on 1)

The stevia plant of claim 1, wherein the leaves of said stevia plant have a rebaudioside D content of between 0.6% and 3.3% of dry weight, and a rebaudioside M content of between 0.5% and 1.15% of dry weight.

### Claim 5 (depends on 1)

The stevia plant of claim 1, further comprising isoforms of UDP-glucosyltransferase 76G1 (UGT76G1), wherein said isoforms correspond to SEQ ID NOs: 19-23, and isoforms of UDP-glucosyltransferase 91D2 (UGT91D2), wherein said isoforms correspond to SEQ ID NOs: 24-54, and wherein said stevia plant has high rebaudioside D content.

### Claim 6 (depends on 1)

The stevia plant of claim 1, further comprising isoforms of UDP-glucosyltransferase 76G1 (UGT76G1), wherein said isoforms correspond to SEQ ID NOs: 9-13, and isoforms of UDP-glucosyltransferase 91D2 (UGT91D2), wherein said isoforms correspond to SEQ ID NOs: 86-116, and wherein said stevia plant has high rebaudioside M content.

### Claim 7 (depends on 1)

The stevia plant of claim 1, further comprising isoforms of UDP-glucosyltransferase 76G1 (UGT76G1), wherein said isoforms correspond to SEQ ID NOs: 14-18, and isoforms of UDP-glucosyltransferase 91D2 (UGT91D2), wherein said isoforms correspond to SEQ ID NOs: 55-85, and wherein said stevia plant has high rebaudioside M content.

### Claim 8 (depends on 6)

The stevia plant of claim 6, wherein said stevia plant further comprises a high rebaudioside D content.

### Claim 9 (depends on 7)

The stevia plant of claim 7, wherein said stevia plant further comprises a high rebaudioside D content.

### Claim 10 (depends on 1)

A method of producing a commodity plant product, comprising obtaining the plant of claim 1, or a part thereof, and producing the commodity plant product from the plant or part thereof, wherein the commodity plant product is a composition of glycosides.