Single Nucleotide Polymorphism (SNP) Markers for Stevia

• US 20190330648A1
• Filed: 05/21/2019
• Published: 10/31/2019
• Est. Priority Date: 09/26/2014
• Status: Active Grant

• Current Assignee: PureCircle Sdn Bhd (Ingredion Incorporated)
• Original Assignee: PureCircle (S.E.A) Sdn. Bhd. (Ingredion Incorporated)
• Inventors: Markosyan, Avetik, Ong, Seong Siang, Wong, Yeen Yee, Bu, Yu Cheng, Chen, Jian Ning
• Granted Patent: US 10,975,381 B2
• Time in Patent Office: Days
• CPC Class Codes: A01H 5/12 Leaves
• CPC Class Codes: A23L 27/36 Terpene glycosides
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: 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
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/8245 involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• CPC Class Codes: 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
• CPC Class Codes: C12N 5/04 Plant cells or tissues culture media C12N5/0025
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae

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

1. A stevia plant, wherein the leaves of said stevia plant have a rebaudioside D/total steviol glycoside percentage between 8% and 38% of dry weight and a rebaudioside M/total steviol glycoside percentage between 0% and 14% of dry weight, and wherein said stevia plant further comprises three SNPs in homozygous form, wherein said SNPs comprise:

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, and SNP 20, which comprises a G to A nucleotide substitution at position number 221 in SEQ ID NO;

6.

2. The stevia plant of claim 1, wherein said rebaudioside D/total steviol glycoside percentage is between 8% and 17%, and wherein said rebaudioside M/total steviol glycoside percentage is between 0% and 4%.

3. The stevia plant of claim 1, wherein said rebaudioside D/total steviol glycoside percentage is between 17.1% and 27%, and wherein said rebaudioside M/total steviol glycoside percentage is between 4.1% and 8%.

4. The stevia plant of claim 1, wherein said rebaudioside D/total steviol glycoside percentage is between 27.1% and 38%, and wherein said rebaudioside M/total steviol glycoside percentage is between 8.1% and 14%.

5. A method for producing a stevia plant comprising:

(a) screening a population of stevia plants for at least one of the following SNPs;

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;

(b) selecting a first stevia plant having at least one said SNP;

(c) crossing said first selected stevia plant having at least one said SNP with a second stevia plant having at least one said SNP;

(d) repeating steps (b) and (c) to obtain stevia plants homozygous for all said SNPs; and

(e) 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.

6. A stevia plant produced by the method of claim 5, wherein said rebaudioside D content of the leaves is at least 0.6%.

7. A stevia plant produced by the method of claim 5, wherein said rebaudioside M content of the leaves is at least 0.5%.

8. A stevia plant produced by the method of claim 5, wherein said rebaudioside D content of the leaves is at least 0.6% and wherein said rebaudioside M content of the leaves is at least 0.5%.

9. The method of claim 5, wherein said screening comprises a PCR reaction followed by DNA sequencing, wherein said PCR reaction uses at least one of the following primer sequences:

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, and SEQ ID NO;

138.