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KR-20260067516-A - SNP marker for identifying of tomato 'TY CAROL' cultivar and its use

KR20260067516AKR 20260067516 AKR20260067516 AKR 20260067516AKR-20260067516-A

Abstract

The present invention relates to an SNP marker composition for purity testing of the cherry tomato variety 'TY Carol', a kit for purity testing of the cherry tomato variety 'TY Carol' using the composition, and a purity testing method. Specifically, based on existing SNP information related to the cherry tomato variety 'TY Carol', five SNPs for purity testing of the 'TY Carol' variety were discovered. After preparing a primer set for HRM to amplify the SNPs and performing HRM analysis, it was confirmed that for each SNP, the parental line with a homozygous genotype and the F1 individuals ('TY Carol') with a heterozygous genotype were distinguished. This enables efficient purity testing and can be usefully utilized as an efficient molecular marker for the protection and seed production of the 'TY Carol' variety.

Inventors

  • 오영재
  • 장윤정
  • 조주성
  • 구창림
  • 신원영
  • 윤다빈

Assignees

  • 충북대학교 산학협력단

Dates

Publication Date
20260513
Application Date
20241105

Claims (10)

  1. (a) a polynucleotide or a polynucleotide complementary thereto in which the 401st base in the CBNU-01 base sequence represented by SEQ ID NO. 1 is T or G, and which consists of 10 to 400 consecutive bases including the 401st base; (b) a polynucleotide or a polynucleotide complementary thereto in which the 401st base in the CBNU-02 base sequence indicated by SEQ ID NO. 2 is G or A, and which consists of 10 to 400 consecutive bases including the 401st base; (c) a polynucleotide or a polynucleotide complementary thereto in which the 401st base in the CBNU-03 base sequence indicated by SEQ ID NO. 3 is G or C, and which consists of 10 to 400 consecutive bases including the 401st base; (d) a polynucleotide or a polynucleotide complementary thereto in which the 401st base in the CBNU-04 base sequence indicated by SEQ ID NO. 4 is C or T and the 401st base is included in 10 to 400 consecutive bases; and (e) an SNP marker composition for purity testing of the cherry tomato variety 'TY Carol', comprising at least one nucleic acid molecule selected from the group consisting of a polynucleotide composed of 10 to 400 consecutive bases including the 401st base in the CBNU-05 base sequence indicated by SEQ ID NO. 5, where the 401st base is G or C, or a polynucleotide complementary thereto.
  2. In paragraph 1, If the 401st base in the CBNU-01 sequence indicated by SEQ ID NO. 1 is a T/G heterozygous pair; or If the 401st base in the CBNU-02 sequence indicated by SEQ ID NO. 2 is a G/A heterozygous form; or If the 401st base in the CBNU-03 sequence indicated by SEQ ID NO. 3 is a G/C heterozygous pair; or If the 401st base in the CBNU-04 sequence indicated by SEQ ID NO. 4 is a C/T heterozygosity; or An SNP marker composition for purity testing of the cherry tomato variety 'TY Carol', wherein if the 401st base in the CBNU-05 base sequence indicated by SEQ ID NO. 5 is a G/C heterozygous, it is determined to be the cherry tomato variety 'TY Carol'.
  3. (a) A preparation capable of detecting or amplifying an SNP in which the 401st base is T or G in the CBNU-01 base sequence indicated by SEQ ID NO. 1; (b) A preparation capable of detecting or amplifying an SNP in which the 401st base is G or A in the CBNU-02 base sequence indicated by SEQ ID NO. 2; (c) A preparation capable of detecting or amplifying an SNP in which the 401st base is G or C in the CBNU-03 base sequence indicated by SEQ ID NO. 3; (d) a preparation capable of detecting or amplifying an SNP in which the 401st base is C or T in the CBNU-04 base sequence indicated by SEQ ID NO. 4; and A composition for testing the purity of the cherry tomato variety 'TY Carol', comprising at least one SNP selected from the group consisting of: (e) a preparation capable of detecting or amplifying an SNP in which the 401st base in the CBNU-05 base sequence indicated by SEQ ID NO. 5 is G or C.
  4. In paragraph 3, A composition for testing the purity of the cherry tomato variety 'TY Carol', characterized in that the agent capable of detecting or amplifying the above SNP is a primer or probe.
  5. In paragraph 4, The above primer is a primer set composed of the polynucleotide of SEQ ID NO. 6 and the polynucleotide of SEQ ID NO. 7; A primer set composed of the polynucleotide of SEQ ID NO. 8 and the polynucleotide of SEQ ID NO. 9; A primer set composed of the polynucleotide of SEQ ID NO. 10 and the polynucleotide of SEQ ID NO. 11; A primer set composed of the polynucleotide of SEQ ID NO. 12 and the polynucleotide of SEQ ID NO. 13; and A composition for testing the purity of the cherry tomato variety 'TY Carol', characterized by comprising a primer set composed of the polynucleotide of SEQ ID NO. 14 and the polynucleotide of SEQ ID NO. 15.
  6. A kit for testing the purity of the cherry tomato variety 'TY Carol', characterized by including the composition of claim 3.
  7. 1) A polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO. 1 or its complementary polynucleotide, a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO. 2 or its complementary polynucleotide, a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO. 3 or its complementary polynucleotide, obtained from DNA isolated from an individual. A step of amplifying a site containing each SNP present in a polynucleotide composed of a nucleotide sequence represented by SEQ ID NO. 4 or a polynucleotide complementary thereof, and a polynucleotide composed of a nucleotide sequence represented by SEQ ID NO. 5 or a polynucleotide complementary thereof; and 2) A method for testing the purity of the cherry tomato variety 'TY Carol', comprising the step of determining the type of base of the SNP corresponding to the 401st base in an amplification product containing the above SNP.
  8. In Paragraph 7, In the case where the 401st base in the CBNU-01 sequence indicated by SEQ ID NO. 1 is a T/G heterozygosity; In the case where the 401st base in the CBNU-02 sequence indicated by SEQ ID NO. 2 is a G/A heterozygous pair; In the case where the 401st base in the CBNU-03 sequence indicated by SEQ ID No. 3 is a G/C heterozygosity; In the case where the 401st base in the CBNU-04 sequence indicated by SEQ ID NO. 4 is a C/T heterozygosity; and A method for determining the purity of the cherry tomato variety 'TY Carol', wherein the 401st base of the CBNU-05 gene sequence indicated by SEQ ID NO. 5 is a G/C heterozygous case; and at least one selected from the group consisting of
  9. In Paragraph 7, A method for testing the purity of the cherry tomato 'TY Carol' variety, characterized in that the step of amplifying the region containing the SNP in step 1) above involves performing PCR using a primer set comprising: a primer set composed of the polynucleotide of SEQ ID NO. 6 and the polynucleotide of SEQ ID NO. 7; a primer set composed of the polynucleotide of SEQ ID NO. 8 and the polynucleotide of SEQ ID NO. 9; a primer set composed of the polynucleotide of SEQ ID NO. 10 and the polynucleotide of SEQ ID NO. 11; a primer set composed of the polynucleotide of SEQ ID NO. 12 and the polynucleotide of SEQ ID NO. 13; and a primer set composed of the polynucleotide of SEQ ID NO. 14 and the polynucleotide of SEQ ID NO. 15.
  10. In Paragraph 7, A method for testing the purity of the cherry tomato variety 'TY Carol', characterized in that the sample separated from the above-mentioned organism is a tomato leaf, root, stem, flower, fruit, separated tissue, or separated cell.

Description

SNP marker for identifying of tomato 'TY CAROL' cultivar and its use The present invention relates to an SNP marker composition for purity testing of the cherry tomato variety 'TY Carol', a kit for purity testing of the cherry tomato variety 'TY Carol' using said composition, and a purity testing method. Tomatoes are plants of the Solanaceae family native to Latin America. The current tomato market size increased from $174.7 billion in 2023 to $186.4 billion in 2024, and the domestic tomato cultivation area has also been continuously increasing from 6,058 hectares in 2018. Furthermore, as tomatoes contain various functional substances such as lycopene, flavonoids, glutamic acid, beta-carotene, and vitamins C and E, consumer interest is also continuously rising. 'TY CAROL', a tomato F1 variety utilizing hybrid vigor, is created by crossing parents with superior horticultural traits such as yield, resistance, and homogeneity. Since F1 variety seeds cannot be reused, when seed companies mass-produce F1 varieties for commercialization, problems may arise regarding the purity of the parents, contamination by other pollen during fertilization, and the deterioration of the quality of F1 varieties resulting from the pollination of the mother plant. For seeds produced from F1 varieties, a purity test must be conducted to verify whether the cross between the parents is established, that is, heterozygosity, and to indicate the degree of this as purity (%). Conventional purity testing is carried out through field testing based on morphological observation, but this method is ineffective because it requires a large cultivation area and labor, as well as a considerable amount of time to harvest the tomatoes and compare their morphological characteristics with those of the parents. Furthermore, since it relies on the subjective judgment of the tester, the accuracy of the purity test is low, which can lead to a lack of reliability. With the recent advancement of molecular biology, technologies that explore polymorphisms in specific DNA base sequences and utilize them as molecular markers are being applied in various fields, such as gene mapping, mapping of disease resistance genes, and variety identification, because they not only inherently reflect genetic characteristics but are also unaffected by environmental factors and exhibit minimal year-to-year variation. Molecular markers can be effectively utilized because they allow for the rapid differentiation of varieties without being affected by the crop's cultivation environment or growth stage. Molecular markers are a method that identifies polymorphisms between individuals based on differences in DNA base sequences, which are the essence of genetic phenomena; they primarily utilize microsatellites composed of simple repetitive base sequences, markers based on gene base sequences, or Single Nucleotide Polymorphism (SNP) markers. In particular, the Random-Amplified Polymorphic DNA (RAPD) method is the most commonly used among various analysis methods because it is easy and convenient to perform. However, the RAPD method has the disadvantage that it is difficult to expect reproducible experimental results because the results can vary depending on the reaction conditions. To solve this problem, a method can be used to perform PCR by constructing new single nucleotide polymorphism (SNP) markers that represent polymorphisms between varieties using DNA sequencing technology, which has recently developed rapidly. Since SNP markers are not affected by environmental factors such as reaction conditions compared to other PCR markers, they can be used as more accurate and detailed molecular markers, and the High Resolution Melting Curve (HRM) method can be used to omit the procedure for verifying PCR products. HRM-PCR (High-Resolution Melting Curve-PCR) is one of the PCR methods based on real-time PCR techniques, and it is a PCR method that detects differences in the melting temperature unique to PCR products in real time and distinguishes even differences in the sequence of PCR products by a single difference. When a PCR product is produced, it acquires a unique Tm value based on its DNA sequence. Intercalating dyes that insert into the double strands are incorporated into the PCR product and emit fluorescence wavelengths. If the temperature is gradually raised from a low to a high level, the PCR product undergoes denaturation—that is, it changes from a double strand to a single strand—at its unique Tm value (specific temperature), causing the fluorescence value to drop sharply. This temperature is called the melting temperature. The HRM analysis method is utilized to detect differences in melting temperatures, enabling the identification of even single-base sequence differences within PCR products. As prior art for the present invention, Bae Jung-hwan et al. (Development of SNP molecular markers for tomato variety differentiation, 2010) disclosed an SNP marker composition selected