# US 11,993,779 B2 · Single nucleotide polymorphism (SNP) markers for stevia

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- Number: US 11,993,779 B2
- Document ID: US11993779B2
- Status: Granted
- Kind: B2
- Application number: 17/205,889
- Filed: 2021-03-18
- Published: 2021-07-08
- Granted: 2024-05-28
- Earliest claimed: 2014-09-26
- Assignees: PureCircle Sdn Bhd
- Applicants: PureCircle SDN BHD
- Inventors: Avetik Markosyan; Seong Siang Ong; Yeen Yee Wong; Yu Cheng Bu; Jian Ning Chen
- Web page: https://patentgrep.com/patent/US11993779B2

## 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 method for producing a Stevia plant comprising:
(a) screening a population of Stevia plants for at least one SNP comprising: SNP2 in SEQ ID NO:1, SNP10 in SEQ ID NO:2, SNP12 in SEQ ID NO:3, SNP17 in SEQ ID NO:4, SNP19 in SEQ ID NO:5, SNP20 in SEQ ID NO:6, SNP22 in SEQ ID NO:7, and SNP24 in SEQ ID NO:8;
(b) selecting a first Stevia plant having at least one said SNP;
(c) selecting a second Stevia plant having at least one said SNP, wherein the SNP of the second Stevia plant is different from the SNP of the first Stevia plant of step (b);
(d) crossing said first selected Stevia plant with said second selected Stevia plant, wherein said second Stevia plant is genetically distinct from said first selected Stevia plant;
(e) repeating steps (b), (c) and (d) on plants resulting from said crossing to obtain Stevia plants homozygous for all said SNPs; and
(f) screening said Stevia plants homozygous for all said SNPs to confirm the presence of all said SNPs in homozygous form to produce a Stevia plant, wherein the leaves of said Stevia plant have a high rebaudioside D content, a high rebaudioside M content, or a high rebaudioside D and high rebaudioside M content.

### Claim 2 (depends on 1)

The method of claim 1, wherein said screening comprises a PCR reaction followed by DNA sequencing, wherein said PCR reaction uses at least one of the following primers: SEQ ID NO: 117, SEQ, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, or SEQ ID NO:138.