A method for identifying cherry tomato varieties and its specific SNP primer combination
By using competitive allele-specific PCR technology and SNP primer combinations at ToSNP01 and ToSNP07 sites, the problem of rapid identification of cherry tomato varieties has been solved, achieving efficient, accurate, and low-cost cherry tomato variety identification, and supporting agricultural production and variety breeding.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JINGYAN YINONG (BEIJING) SEED TECH CO LTD
- Filing Date
- 2025-07-14
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies are insufficient for quickly and accurately identifying cherry tomato varieties, and traditional field phenotypic identification is time-consuming and labor-intensive, failing to meet current needs for cherry tomato variety identification.
A rapid method for identifying cherry tomato varieties was developed by using competitive allele-specific PCR (KASP) technology combined with SNP primer combinations at ToSNP01 and ToSNP07 loci to identify the genotype of cherry tomatoes through fluorescence signals.
It enables high-throughput, accurate, low-cost, and simple cherry tomato variety identification, saving manpower and resources, and is suitable for agricultural production and variety breeding.
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Figure CN120796546B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for identifying cherry tomato varieties and its dedicated SNP primer combination. Background Technology
[0002] Tomatoes are one of the world's most important vegetables and the most widely cultivated greenhouse vegetable in my country. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), my country's tomato planting area reached 1.1692 million hectares, with a total agricultural output value of 323.98 billion yuan. Furthermore, tomatoes are highly nutritious, rich in lycopene, vitamin C, flavonoids, and carotene—all beneficial antioxidants that promote collagen synthesis and improve iron absorption. During tomato cultivation, different types and varieties not only differ in morphology but also in flavor and quality. According to publicly available data from the China Seed Industry Big Data Platform, tomatoes are mainly categorized as large-fruited tomatoes (150-300 grams per fruit), medium-fruited tomatoes (50-150 grams per fruit), and cherry tomatoes (10-30 grams per fruit). With the continuous improvement of people's living standards, the demand for fresh tomatoes is gradually increasing. The application scenarios for cherry tomatoes have expanded from traditional fresh consumption and cooking to areas such as snacks and light meals. Currently, the widespread adoption of innovative technologies such as vertical farming and smart greenhouses has brought new breakthroughs in the yield and economic benefits of cherry tomatoes. The cherry tomato industry is experiencing rapid growth globally, and the planting area and yield of cherry tomatoes in my country are also increasing year by year.
[0003] Because cherry tomatoes differ significantly from other tomato varieties in terms of cultivation environment and commercial use, it is crucial to clearly define tomato types during variety selection and agricultural production. However, with the increasing crossbreeding and gene exchange between different tomato types, cherry tomatoes can no longer be easily identified using only seeds or seedlings. Statistics show that over 3,600 tomato varieties have been registered in my country, and traditional field phenotypic identification methods are time-consuming and labor-intensive, failing to meet the current surge in identification demands. Therefore, there is an urgent need to establish a simple and rapid method for distinguishing cherry tomato varieties, providing technical support for cherry tomato variety selection and identification.
[0004] The large dataset of tomato variants lays the foundation for screening population-specific loci in cherry tomatoes from the whole genome. SNPs, as third-generation molecular markers, are widely distributed throughout the genome, with an average of one SNP per 1000 bp. They are genetically stable and easily detected automatically. KASP (Kompetitive Allele Specific PCR) is a commonly used method for SNP genotyping, characterized by high stability, accuracy, and low cost, and has been widely applied in high-throughput molecular-assisted breeding and variety identification. Summary of the Invention
[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for identifying cherry tomato varieties and a specific SNP primer set thereof. This primer set can be used to rapidly identify whether a tomato variety is a cherry tomato.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] The first aspect of the present invention provides an SNP primer set, which may include primer set 1 for amplifying the ToSNP01 site of the tomato genome and / or primer set 2 for amplifying the ToSNP07 site of the tomato genome.
[0008] The ToSNP01 site is nucleotide 44955024 on chromosome 2;
[0009] The ToSNP07 site is nucleotide 46612819 on chromosome 2;
[0010] The positions of ToSNP01 and ToSNP07 on the chromosome were determined based on alignment with the tomato Heinz 1706 reference genome sequence, version number V3.0.
[0011] In the SNP primer combination, primer set 1 includes a first forward primer, a second forward primer, and a first reverse primer, wherein the nucleotide sequence of the first forward primer is shown as positions 22 to 48 from the 5' end of SEQ ID NO: 1, the nucleotide sequence of the second forward primer is shown as positions 22 to 47 from the 5' end of SEQ ID NO: 2, and the nucleotide sequence of the first reverse primer is shown as SEQ ID NO: 3. Primer set 2 includes a third forward primer, a fourth forward primer, and a second reverse primer, wherein the nucleotide sequence of the third forward primer is shown as positions 22 to 46 from the 5' end of SEQ ID NO: 4, the nucleotide sequence of the fourth forward primer is shown as positions 22 to 47 from the 5' end of SEQ ID NO: 5, and the nucleotide sequence of the second reverse primer is shown as SEQ ID NO: 6.
[0012] To facilitate the identification of SNP locus genotypes via fluorescence signals, a fluorescent tag sequence is added to the 5' end of the forward primers. The fluorescent tag sequences of the first and second forward primers exhibit different fluorescence signal colors. The fluorescent tag sequences of the third and fourth forward primers also exhibit different fluorescence signal colors.
[0013] Any of the SNP primer combinations described above can be primer set 1 alone, primer set 2 alone, or a combination of primer set 1 and primer set 2.
[0014] In the above text, the nucleotide sequence shown in SEQ ID NO: 1 from position 1 to 21 starting from the 5' end is a fluorescent tag sequence (i.e., a FAM fluorescent tag sequence), and the fluorescence signal is specifically blue. The nucleotide sequence shown in SEQ ID NO: 2 from position 1 to 21 starting from the 5' end is also a fluorescent tag sequence (i.e., a HEX fluorescent tag sequence), and the fluorescence signal is specifically red.
[0015] In the SNP primer combination, as a preferred embodiment, primer set 1 may consist of forward primer 1F1 shown in SEQ ID NO: 1, forward primer 1F2 shown in SEQ ID NO: 2, and reverse primer 1R shown in SEQ ID NO: 3. Primer set 2 may consist of forward primer 2F1 shown in SEQ ID NO: 4, forward primer 2F2 shown in SEQ ID NO: 5, and reverse primer 2R shown in SEQ ID NO: 6.
[0016] In any of the primer sets described above, the molar ratio of the primer containing "F1" in its name, the primer containing "F2" in its name, and the primer containing "R" in its name can specifically be 2:2:5.
[0017] Kits containing any of the SNP primer combinations described above are also within the scope of protection of this invention. Therefore, a second aspect of this invention provides a kit comprising the SNP primer combinations described in the first aspect.
[0018] The kit may also include other reagents for competitive allele-specific PCR, such as DNA polymerase and buffer.
[0019] The preparation method of the kit is also within the scope of protection of this invention. The preparation method of the kit includes the step of individually packaging each primer from any of the primer sets described above.
[0020] The application of the reagent kit also falls within the scope of protection of this invention. The reagent kit can be used to identify whether a tomato variety to be tested is a cherry tomato or a non-cherry tomato.
[0021] The third aspect of the present invention provides the application of the SNP primer combination described in the first aspect above, wherein the application is either of the following two: (A) in the preparation of a kit for identifying whether a tomato variety to be tested is a cherry tomato or a non-cherry tomato; (B) in the identification of whether a tomato variety to be tested is a cherry tomato or a non-cherry tomato.
[0022] The fourth aspect of this invention provides the application of the reagent kit described in the second aspect above in identifying whether a tomato variety to be tested is a cherry tomato or a non-cherry tomato.
[0023] The fifth aspect of this invention provides a method for identifying whether a tomato variety to be tested is a cherry tomato or a non-cherry tomato, comprising the following steps: detecting the genotype of the tomato variety to be tested based on the ToSNP01 locus and / or the ToSNP07 locus, and then making the following judgments: if the genotype based on the ToSNP01 locus is GG homozygous or AG heterozygous, and / or the genotype based on the ToSNP07 locus is TT homozygous or CT heterozygous, then the tomato variety to be tested is identified as or suspected to be a cherry tomato; if the genotype based on the ToSNP01 locus is AA homozygous and / or the genotype based on the ToSNP07 locus is CC homozygous, then the tomato variety to be tested is identified as or suspected to be a non-cherry tomato.
[0024] The ToSNP01 site is nucleotide 44955024 on chromosome 2;
[0025] The ToSNP07 site is nucleotide 46612819 on chromosome 2;
[0026] The positions of the ToSNP01 and ToSNP07 sites on the chromosome were determined based on the alignment of the tomato HEINZ 1706 reference genome sequence, version number V3.0.
[0027] In the above method, the step of detecting the genotype of the tomato variety to be tested based on the ToSNP01 and / or ToSNP07 loci can be as follows:
[0028] (1) Using the genomic DNA of the tomato variety to be tested as a template, PCR amplification was performed using the primer set 1 and / or the primer set 2 described in the first aspect above to obtain PCR amplification products.
[0029] (2) After completing step (1), the fluorescence signal of the PCR amplification product is detected by an instrument, and the genotype of the tomato variety to be tested based on the ToSNP01 site and / or ToSNP07 site is obtained according to the color of the fluorescence signal.
[0030] Alternatively, in the above method, the step of detecting the genotype of the tomato variety to be tested based on the ToSNP01 and / or ToSNP07 loci is as follows:
[0031] (1) Using the genomic DNA of the tomato variety to be tested as a template, PCR amplification was performed using primer set 1 and / or primer set 2 described in the first aspect above to obtain PCR amplification products;
[0032] (2) Take the PCR amplification product obtained in step (1) and sequence it;
[0033] (3) Based on the sequencing results obtained in step (2), obtain the genotype of the tomato variety to be tested based on the ToSNP01 site and / or the ToSNP07 site.
[0034] In any of the methods described above, the reaction procedure for "PCR amplification using primer set 1 and / or primer set 2 described in the first aspect" can be as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touch-down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, continued amplification for 26 cycles. If the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20 s, 55℃ annealing and extension for 1 min, 5 cycles) until satisfactory results are obtained.
[0035] Beneficial effects of this invention:
[0036] With the development of genomics technology, the tomato genome has been published, and whole-genome resequencing of over 1000 tomato resources has been completed. This rich dataset of variant data provides marker resources for screening population-specific genetic loci in tomatoes. Using resequencing data from cherry tomato and non-cherry tomato germplasm resources, population-specific genetic loci are screened from the whole genome, leading to the development of SNP primer combinations for identifying cherry and non-cherry tomatoes. The SNP primer combinations provided in this invention can be used to identify whether a tomato variety is a cherry or non-cherry tomato, which has significant application value in clarifying the population attributes of tomatoes during agricultural production and variety breeding. The method provided in this invention has advantages such as high throughput, accuracy, low cost, simple operation, and saving manpower and resources, and has a very broad application prospect. Attached Figure Description
[0037] Figure 1 PCA clustering results for resequencing data of 96 representative tomato resources.
[0038] Figure 2 This represents the variation in genome-wide SNP frequencies between cherry tomatoes and other types of tomatoes.
[0039] Figure 3 The distribution of eight cherry tomato-specific SNP sites located on chromosome 2.
[0040] Figure 4 The results of partial SNP typing of the tested tomato varieties were obtained using primer set 1.
[0041] Figure 5To identify some SNP typing results of the tested tomato varieties using primer set 2.
[0042] Figure 6 The results of partial SNP typing of the tomato varieties to be tested were obtained using primer set 1.
[0043] Figure 7 The results of partial SNP typing of the tomato varieties to be tested were obtained using primer set 2. Detailed Implementation
[0044] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0046] Example 1: Obtaining the SNP primer combination for identifying the tomato variety as cherry tomato.
[0047] I. Discovery of 8 SNP sites
[0048] This invention utilizes resequencing data from 96 representative tomato resources in a vegetable variant SNP variation database (http: / / www.vegsnpdb.cn). It screens for genome-specific perfect SNP loci with no other variations within 50 bp flanking each SNP, ultimately obtaining 31 SNP loci. These 96 representative tomato resources represent broad tomato genetic diversity, including 68 non-cherry tomato varieties and 28 cherry tomato varieties. The PCA clustering results of the resequencing data from these 96 representative tomato resources are shown below. Figure 1 .
[0049] Specifically, the screening criteria for SNP sites are as follows: First, SNP sites with MAF > 0.3, heterozygosity < 0.1, deletion rate < 0.1, and conserved 50bp sequences on both sides (without other variations) were selected from the whole genome, resulting in 33,656 perfect SNPs for tomato; then, the SNP frequency difference between cherry tomatoes and other types of tomatoes was calculated, revealing a significant peak on chromosome 2 in cherry tomato materials (…). Figure 2 ), and a total of 8 sites with a SNP frequency difference of 1 between the two populations were obtained in this interval. Figure 3The basic information of the 8 SNP sites is detailed in Table 1. The positions of the SNP sites on the chromosome were determined based on the alignment of the tomato HEINZ 1706 reference genome sequence, version V3.0 (downloadable from: https: / / plants.ensembl.org / Solanum_lycopersicum).
[0050] Table 1. Basic information of 8 SNP sites
[0051]
[0052] II. Obtaining the SNP primer combination for identifying the tomato variety as cherry tomato
[0053] Based on the eight SNP loci discovered in step one, the inventors of this invention designed and synthesized eight SNP primer combinations and performed SNP genotyping on 24 cherry tomato varieties. According to the SNP genotype results, two SNP loci with a high proportion of homozygous genotypes and 100% linkage to cherry tomatoes were obtained, namely ToSNP01 and ToSNP07.
[0054] SNP primer sets consist of primer set 1 and / or primer set 2. Primer set 1 is used to amplify ToSNP01. Primer set 2 is used to amplify ToSNP07. Each primer set consists of 3 primer sequences used to amplify one SNP site. The nucleotide sequences of each primer in primer set 1 and primer set 2 are shown in column 4 of Table 2.
[0055] Table 2. SNP primer combinations used to identify the tomato variety as cherry tomato.
[0056]
[0057] Note: A single underscore indicates a FAM fluorescent tag sequence, and a double underscore indicates a HEX fluorescent tag sequence.
[0058] Example 2: Validation of the SNP primer combinations developed in Example 1
[0059] Basic information on the 159 tested tomato varieties in this embodiment is shown in columns 1 to 4 of Table 3. All 159 tested tomato varieties are common market varieties. Based on phenotype, 46 tested tomato varieties are cherry tomatoes, and 113 tested tomato varieties are non-cherry tomatoes.
[0060] Table 3. Basic information of 3159 tested tomato varieties
[0061]
[0062]
[0063]
[0064]
[0065]
[0066] 1. Obtaining genomic DNA from the tested tomato varieties
[0067] Genomic DNA was extracted from leaves of 159 tested tomato varieties using the SDS method. The quality and concentration of the genomic DNA from the tested tomato varieties had to meet PCR requirements, with the following criteria: agarose gel electrophoresis showed a single DNA band without significant diffusion; UV spectrophotometer Nanodrop 2000 (Thermo) showed an A260 / A280 ratio of approximately 1.8 and an A260 / A230 ratio greater than 1.8; and the concentration of the genomic DNA from the tested tomato varieties was between 10 and 30 ng / μL.
[0068] 2. Using genomic DNA from 159 tested tomato varieties as templates, PCR amplification was performed using primer set 1 or primer set 2 to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2", and primers containing "R" was 2:2:5.
[0069] The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touch down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, and continued amplification for 26 cycles.
[0070] 3. After completing step 2, when the temperature of each PCR amplification product drops below 40℃, the fluorescence value is read by scanning with the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is read at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is read at an excitation wavelength of 528nm and an emission wavelength of 560nm). The genotype of the 159 tested tomato varieties based on each SNP locus is determined according to the fluorescence signal color. The specific judgment principles are as follows: If a tested tomato variety shows a blue fluorescent signal based on a certain SNP site, then the genotype of the tested tomato variety based on that SNP site is homozygous, meaning it is the 3' terminal first base of the primer that amplifies the SNP site and whose name contains "F1"; if a tested tomato variety shows a red fluorescent signal based on a certain SNP site, then the genotype of the tested tomato variety based on that SNP site is homozygous, meaning it is the 3' terminal first base of the primer that amplifies the SNP site and whose name contains "F2"; if a tested tomato variety shows a green fluorescent signal based on a certain SNP site, then the genotype of the tested tomato variety based on that SNP site is heterozygous, with one base being the 3' terminal first base of the primer that amplifies the SNP site and whose name contains "F1", and the other base being the 3' terminal first base of the primer that amplifies the SNP site and whose name contains "F2".
[0071] It should be noted that if the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20s, 55℃ annealing and extension for 1min, 5 cycles) until the results are satisfactory.
[0072] Partial SNP genotyping results of primer set 1 are shown below Figure 4 The statistical results are shown in column 5 of Table 3.
[0073] Partial SNP genotyping results of primer set 2 are shown below Figure 5 The statistical results are shown in column 6 of Table 3.
[0074] The results showed that primer set 1 and primer set 2 could both achieve good typing results in 159 tested tomato varieties.
[0075] 4. Efficiency evaluation of using primer set 1 or primer set 2 to identify 159 tested tomato varieties as cherry tomatoes.
[0076] (1) The genotypes of 159 tested tomato varieties based on the ToSNP01 locus were statistically analyzed.
[0077] The results showed that 27 tomato varieties with the GG homozygous genotype based on the ToSNP01 locus and 19 varieties with the AG heterozygous genotype were identified as cherry tomatoes. All 46 tomatoes exhibited the cherry tomato phenotype, with a 100% uniformity. 113 tomato varieties with the AA homozygous genotype based on the ToSNP01 locus were identified as large-fruited tomatoes. All 113 tomatoes exhibited the non-cherry tomato phenotype, with a 100% uniformity.
[0078] (2) The genotypes of 159 tested tomato varieties based on the ToSNP07 locus were statistically analyzed.
[0079] The results showed that 37 tomato varieties with the TT homozygous genotype and 9 varieties with the CT heterozygous genotype based on the ToSNP07 locus were identified as cherry tomatoes. All 46 of these tomatoes exhibited the cherry tomato phenotype, with a 100% similarity. 113 tomato varieties with the CC homozygous genotype based on the ToSNP07 locus were identified as non-cherry tomatoes. All 113 of these tomatoes exhibited the non-cherry tomato phenotype, with a 100% similarity.
[0080] Therefore, it can be seen that the SNP primer combination developed in Example 1 can identify the tomato variety to be tested as cherry tomato.
[0081] Example 3: The accuracy of using the SNP primer combination developed in Example 1 to identify the tested tomato variety as cherry tomato.
[0082] The tomato varieties to be tested were tomato variety 1 to tomato variety 12, a total of 12.
[0083] 1. The SNP primer combinations developed in Example 1 were used to identify each tested tomato variety as a cherry tomato. The specific steps are as follows:
[0084] (1) Plant the seeds of the tomato variety to be tested to obtain tomato seedlings; take the leaves or roots of the tomato seedlings to be tested and extract genomic DNA using the SDS method to obtain the genomic DNA of the tomato variety to be tested.
[0085] (2) Using the genomic DNA of the tomato variety to be tested as a template, PCR amplification was performed using primer set 1 or primer set 2 to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2", and primers containing "R" in their names was 2:2:5.
[0086] The reaction procedure is the same as in Part 2 of Example 2.
[0087] (3) After completing step (2), the fluorescence analysis of each PCR product is performed using the same method as in Part 3 of Example 2, and the genotype of a certain SNP site is determined based on the same judgment principle as in Part 3 of Example 2.
[0088] It should be noted that if the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20s, 55℃ annealing and extension for 1min, 5 cycles) until the results are satisfactory.
[0089] Partial SNP genotyping results of primer set 1 are shown below Figure 6 And column 2 in Table 4.
[0090] Partial SNP genotyping results of primer set 2 are shown below Figure 7 And column 3 in Table 4.
[0091] (4) After completing step (3), make the following judgment: if the genotype based on the ToSNP01 site is GG homozygous or AG heterozygous, and / or the genotype based on the ToSNP07 site is TT homozygous or CT heterozygous, then it is identified as a cherry tomato; if the genotype based on the ToSNP01 site is AA homozygous and / or the genotype based on the ToSNP07 site is CC homozygous, then it is identified as a non-cherry tomato.
[0092] Table 4. Identification results of 12 tomato varieties tested
[0093] Tomato varieties to be tested Genotyping based on ToSNP01 Genotyping based on ToSNP07 type Tomato variety 1 to be tested GG TT cherry tomatoes Tomato variety 2 to be tested AG TT cherry tomatoes Tomato variety 3 to be tested GG TT cherry tomatoes Tomato varieties to be tested 4 GG TT cherry tomatoes 5 tomato varieties to be tested AG CT cherry tomatoes 6 tomato varieties to be tested AA CC Non-cherry tomatoes 7 tomato varieties to be tested AA CC Non-cherry tomatoes 8 tomato varieties to be tested AA CC Non-cherry tomatoes 9 tomato varieties to be tested AA CC Non-cherry tomatoes 10 tomato varieties to be tested AA CC Non-cherry tomatoes 11 tomato varieties to be tested AA CC Non-cherry tomatoes 12 tomato varieties to be tested AA CC Non-cherry tomatoes
[0094] 2. Twelve tomato varieties were planted for testing. Based on their phenotypes, the 12 tomato varieties were determined to be either cherry tomatoes or non-cherry tomatoes.
[0095] The phenotypic statistics are shown in column 4 of Table 4.
[0096] The results showed that the identification results of the SNP primer combination developed in Example 1 were completely consistent with the phenotypic identification results.
[0097] Therefore, it can be seen that the SNP primer combination developed in Example 1 can completely identify whether a tomato is a cherry tomato or a non-cherry tomato.
[0098] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. The application of SNP primer combinations in the preparation of kits for identifying whether a tomato variety is a cherry tomato or a non-cherry tomato. The SNP primer set includes primer set 1 for amplifying the ToSNP01 site of the tomato genome and / or primer set 2 for amplifying the ToSNP07 site of the tomato genome. The SNP primer set is used to determine the genotype of the ToSNP01 site and / or the ToSNP07 site. The ToSNP01 site is nucleotide 44955024 on chromosome 2; The ToSNP07 site is nucleotide 46612819 on chromosome 2; The positions of ToSNP01 and ToSNP07 on the chromosome were determined based on the alignment of the tomato HEINZ 1706 reference genome sequence, version number V3.
0. The primer set 1 includes a first forward primer, a second forward primer, and a first reverse primer, wherein the nucleotide sequence of the first forward primer is shown as SEQ ID NO: 1 from position 22 to 48 from the 5' end, the nucleotide sequence of the second forward primer is shown as SEQ ID NO: 2 from position 22 to 47 from the 5' end, and the nucleotide sequence of the first reverse primer is shown as SEQ ID NO: 3; The primer set 2 includes a third forward primer, a fourth forward primer, and a second reverse primer, wherein the nucleotide sequence of the third forward primer is shown as SEQ ID NO: 4 from position 22 to 46 from the 5' end, the nucleotide sequence of the fourth forward primer is shown as SEQ ID NO: 5 from position 22 to 47 from the 5' end, and the nucleotide sequence of the second reverse primer is shown as SEQ ID NO:
6.
2. The application according to claim 1, characterized in that, The primer set 1 consists of the forward primer 1F1 shown in SEQ ID NO: 1, the forward primer 1F2 shown in SEQ ID NO: 2, and the reverse primer 1R shown in SEQ ID NO: 3; The primer set 2 consists of the forward primer 2F1 shown in SEQ ID NO: 4, the forward primer 2F2 shown in SEQ ID NO: 5, and the reverse primer 2R shown in SEQ ID NO:
6.
3. Application of SNP primer combinations in identifying whether a tomato variety is a cherry tomato or a non-cherry tomato, among which, The SNP primer set includes primer set 1 for amplifying the ToSNP01 site of the tomato genome and / or primer set 2 for amplifying the ToSNP07 site of the tomato genome. The SNP primer set is used to determine the genotype of the ToSNP01 site and / or the ToSNP07 site. The ToSNP01 site is nucleotide 44955024 on chromosome 2; The ToSNP07 site is nucleotide 46612819 on chromosome 2; The positions of ToSNP01 and ToSNP07 on the chromosome were determined based on the alignment of the tomato HEINZ 1706 reference genome sequence, version number V3.
0. The primer set 1 includes a first forward primer, a second forward primer, and a first reverse primer, wherein the nucleotide sequence of the first forward primer is shown as SEQ ID NO: 1 from position 22 to 48 from the 5' end, the nucleotide sequence of the second forward primer is shown as SEQ ID NO: 2 from position 22 to 47 from the 5' end, and the nucleotide sequence of the first reverse primer is shown as SEQ ID NO: 3; The primer set 2 includes a third forward primer, a fourth forward primer, and a second reverse primer, wherein the nucleotide sequence of the third forward primer is shown as SEQ ID NO: 4 from position 22 to 46 from the 5' end, the nucleotide sequence of the fourth forward primer is shown as SEQ ID NO: 5 from position 22 to 47 from the 5' end, and the nucleotide sequence of the second reverse primer is shown as SEQ ID NO: 6; Based on the genotypes at the ToSNP01 and / or ToSNP07 loci, the following method is used to determine whether the tomato variety being tested is a cherry tomato or a non-cherry tomato: If the genotype at the ToSNP01 locus is GG homozygous or AG heterozygous, and / or the genotype at the ToSNP07 locus is TT homozygous or CT heterozygous, then the tomato variety being tested is identified as or suspected to be a cherry tomato; if the genotype at the ToSNP01 locus is AA homozygous and / or the genotype at the ToSNP07 locus is CC homozygous, then the tomato variety being tested is identified as or suspected to be a non-cherry tomato.
4. The application according to claim 3, characterized in that, The primer set 1 consists of the forward primer 1F1 shown in SEQ ID NO: 1, the forward primer 1F2 shown in SEQ ID NO: 2, and the reverse primer 1R shown in SEQ ID NO: 3; The primer set 2 consists of the forward primer 2F1 shown in SEQ ID NO: 4, the forward primer 2F2 shown in SEQ ID NO: 5, and the reverse primer 2R shown in SEQ ID NO:
6.
5. A method for identifying whether a tomato variety to be tested is a cherry tomato or a non-cherry tomato, comprising the following steps: detecting the genotype of the tomato variety to be tested based on the ToSNP01 locus and / or the ToSNP07 locus, and then making the following judgments: if the genotype based on the ToSNP01 locus is GG homozygous or AG heterozygous, and / or the genotype based on the ToSNP07 locus is TT homozygous or CT heterozygous, then the tomato variety to be tested is identified as or suspected to be a cherry tomato; if the genotype based on the ToSNP01 locus is AA homozygous and / or the genotype based on the ToSNP07 locus is CC homozygous, then the tomato variety to be tested is identified as or suspected to be a non-cherry tomato; The ToSNP01 site is nucleotide 44955024 on chromosome 2; The ToSNP07 site is nucleotide 46612819 on chromosome 2; The positions of ToSNP01 and ToSNP07 on the chromosome were determined based on the alignment of the tomato HEINZ 1706 reference genome sequence, version number V3.
0. The steps for detecting the genotype of the tomato variety under test based on the ToSNP01 and / or ToSNP07 loci are as follows: (1) Using the genomic DNA of the tomato variety to be tested as a template, PCR amplification was performed using primer set 1 and / or primer set 2 to obtain the PCR amplification products; among which, The primer set 1 consists of the forward primer 1F1 shown in SEQ ID NO: 1, the forward primer 1F2 shown in SEQ ID NO: 2, and the reverse primer 1R shown in SEQ ID NO: 3; The primer set 2 consists of the forward primer 2F1 shown in SEQ ID NO: 4, the forward primer 2F2 shown in SEQ ID NO: 5, and the reverse primer 2R shown in SEQ ID NO: 6; (2) After completing step (1), the fluorescence signal of the PCR amplification product is detected by an instrument, and the genotype of the tomato variety to be tested is obtained based on the color of the fluorescence signal at the ToSNP01 site and / or ToSNP07 site.