# US 12,215,394 B2 · Molecular marker nicotine associated SNP 1 for identifying high or low nicotine content of tobacco and its kit as well as use thereof

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- Number: US 12,215,394 B2
- Document ID: US12215394B2
- Status: Granted
- Kind: B2
- Application number: 17/534,204
- Filed: 2021-11-23
- Published: 2023-06-29
- Granted: 2025-02-04
- Earliest claimed: 2021-11-10
- Applicants: Yunan Academy of Tobacco Agricultural Science
- Inventors: He Xie; Ge Bai; Yong Li; Aiguo Yang; Tao Pang; Dahai Yang; Bingguang Xiao; Yongping Li; Min Ren; Mingliang Fei
- Web page: https://patentgrep.com/patent/US12215394B2

## CPC

- C12Q 1/6895: for plants, fungi or algae
- A01H 1/04: Processes of selection {involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection}
- A01H 1/045: {using molecular markers}
- A01H 5/12: Leaves
- A01H 6/823: {Nicotiana, e.g. tobacco}
- G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
- 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
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

The present invention “A Molecular marker Nicotine Associated SNP 1 for identifying high or nicotine content of tobacco and its kit as well as use thereof” belongs to field of molecular biology technology. The molecular marker Nicotine Associated SNP 1 is a SNP Nitab4.5_0002539:95304 A/G at base No. 95304 of Genomic segment No. 0002539 in tobacco genome version of Nitab v4.5 Genome Scaffolds Edwards2017. The present invention can accurately identify and screen tobacco germplasm resources with high or low nicotine content, and the screened tobacco can be directly used for breeding new tobacco varieties without transgenic methods. At the same time, gene sequence where the SNP site is located can also significantly activate promoters of key enzyme genes in nicotine synthesis pathway.

## Claims

### Claim 1

A kit for identifying high or low nicotine content of tobacco, comprising a molecular marker Nicotine Associated SNP 1, wherein the molecular marker Nicotine Associated SNP 1 is a single nucleotide polymorphism SNP site Nitab4.5_0002539:95304 A/G at base No. 95304 of Genomic segment No. 0002539 in tobacco genome version of Nitab v4.5 Genome Scaffolds Edwards2017; the SNP site is located at the 324th nucleotide of the amplicon sequence set forth in SEQ ID NO: 13, which is derived from the tobacco genome using primers set forth in SEQ ID NO:1 and SEQ ID NO:2, and wherein the kit further comprises a pair of specific primers for the molecular marker Nicotine Associated SNP 1 comprising SEQ ID NO:1 and SEQ ID NO:2.

### Claim 2 (depends on 1)

The kit for identifying high or low nicotine content of tobacco according to claim 1, further comprising: reagents for PCR, reagents for sequencing, and/or reagents for a genotyping assay.

### Claim 3

A method for breeding tobacco varieties with high or low nicotine content, wherein tobaccos with high or low nicotine content from candidate tobaccos are screened by using a molecular marker Nicotine Associated SNP 1 for identifying high or low nicotine content of tobacco comprising a single nucleotide polymorphism (SNP) site Nitab4.5_0002539:95304 A/G at base No. 95304 of Genomic segment No. 0002539 in tobacco genome version of Nitab v4.5 Genome Scaffolds Edwards2017; the SNP site is located at the 324th nucleotide of the amplicon sequence set forth in SEQ ID NO:13, which is derived from the tobacco genome using primers set forth in SEQ ID NO:1 and SEQ ID NO:2.

### Claim 4 (depends on 3)

The method for breeding tobacco varieties with high or low nicotine content according to claim 3, wherein tobacco with high nicotine content is selected as a female parent or a male parent, and tobacco required to be improved is selected as the male parent or the female parent, wherein a F1 generation is obtained by crossing the female parent and the male parent.

### Claim 5 (depends on 4)

The method for breeding tobacco varieties with high or low nicotine content according to claim 4, wherein F2 generation plants are obtained by inbred F1 generation plants, and F2 generation plants are backcrossed with selected tobaccos with high or low nicotine content or tobaccos required to be improved.

### Claim 6 (depends on 5)

The method for breeding tobacco varieties with high or low nicotine content according to claim 5, wherein tobaccos with high or low nicotine content are screened from the backcrossed population through a method for identifying high or low nicotine content of tobacco, and wherein a molecular marker Nicotine Associated SNP 1 is used to screen candidate tobaccos; the molecular marker Nicotine Associated SNP 1 is a SNP site Nitab4.5_0002539:95304 A/G at base No. 95304 of Genomic segment No. 0002539 in tobacco genome version of Nitab v4.5 Genome Scaffolds Edwards2017; the SNP site is located at the 324th nucleotide of the amplicon sequence set forth in SEQ ID NO: 13, which is derived from the tobacco genome using primers set forth in SEQ ID NO: 1 and SEQ ID NO:2.