Method for identifying cherry tomato variety and special SNP (Single Nucleotide Polymorphism) primer combination thereof

Abstract

The invention discloses a method for identifying cherry tomato varieties and a special SNP (Single Nucleotide Polymorphism) primer combination thereof. The SNP primer combination is composed of a primer group 1 and / or a primer group 2. Each primer group consists of three primer sequences and is used for amplifying an SNP (Single Nucleotide Polymorphism) site. The nucleotide sequences of the primers in the two primer groups are sequentially shown as a sequence 1 to a sequence 6 in a sequence table. The SNP primer combination provided by the invention can be used for identifying that the tomato variety to be detected is cherry tomato, and has important application value for determining the population attribute of tomato in agricultural production and variety breeding processes. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for identifying cherry tomato varieties and a special SNP primer combination thereof. BACKGROUND

[0002] Tomato is one of the important vegetables in the world, and also the largest area of planting facilities in China. According to the statistics of the Food and Agriculture Organization (FAO), the planting area of tomato in China reaches 116.92 million mu, and the total value of agricultural production reaches 323.98 billion yuan. In addition, the nutritional value of tomato is also very rich, which is rich in lycopene, vitamin C, flavonoids and carotene and other antioxidant substances beneficial to human body, which can promote collagen synthesis and improve iron absorption rate. In the process of tomato planting, different types and varieties of tomatoes not only differ in morphology, but also differ greatly in flavor quality. According to the data published by China Seed Industry Big Data Platform, it is mainly divided into large fruit tomato (single fruit weight 150-300 grams), medium fruit tomato (single fruit weight 50-150 grams) and cherry tomato (single fruit weight 10-30 grams). With the continuous improvement of people's living standards, the demand for fresh tomato is gradually increasing. The application scenarios of cherry tomato have expanded from traditional fresh food and cooking to leisure snacks, light snacks and other fields. At present, the popularization of innovative technologies such as vertical agriculture and intelligent greenhouse has also brought new breakthroughs in the unit yield and economic benefits of cherry tomato. The cherry tomato industry is showing a rapid growth trend in the global range, and the planting area and yield of cherry tomato in China are also increasing year by year.

[0003] Because of the great difference in cultivation environment and commercial use between cherry tomato and other types of tomato varieties, the type of tomato must be identified in the process of variety breeding and agricultural production. However, with the cross breeding and gene exchange between different types of tomatoes, cherry tomato cannot be identified simply by seeds or seedlings. According to statistics, more than 3600 tomato varieties have been applied for registration in China. Traditional field phenotype identification is time-consuming and labor-intensive, and cannot meet the current identification demand of the rapid increase in quantity. Therefore, it is urgent to establish a simple and rapid method for distinguishing cherry tomato varieties, and to provide technical support for cherry tomato variety breeding and identification.

[0004] Tomato variation group big data lays a foundation for screening cherry tomato population specific sites from whole genome. SNP, as the third generation of molecular markers, is widely distributed in the genome, with an average of one SNP per 1000 bp in the genome, which is genetically stable and easy to automate detection. KASP (Kompetitive Allele Specific PCR), i.e. competitive allele specific PCR technology, is a commonly used method for SNP typing, which has the characteristics of high stability, accuracy and low cost, and has been widely used in high-throughput molecular assisted breeding and variety identification. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a method for identifying cherry tomato varieties and a special SNP primer combination thereof. The primer combination can be used to quickly identify whether the tomato variety is cherry tomato.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] The first aspect of the present application provides a SNP primer combination, which can include primer set 1 for amplifying ToSNP01 site of the tomato genome and / or primer set 2 for amplifying ToSNP07 site of the tomato genome;

[0008] The ToSNP01 site is the 44955024th nucleotide on chromosome 2;

[0009] The ToSNP07 site is the 46612819th nucleotide on chromosome 2;

[0010] The positions of the ToSNP01 site and the ToSNP07 site on the chromosome are determined based on the alignment of the tomato Heinz 1706 reference genome sequence, and the version number of the tomato Heinz 1706 reference genome sequence is V3.0.

[0011] In the SNP primer combination, 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 from the 22nd to the 48th from the 5' end of SEQ ID NO: 1, the nucleotide sequence of the second forward primer is shown from the 22nd to the 47th from the 5' end of SEQ ID NO: 2, and the nucleotide sequence of the first reverse primer is shown in 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 from the 22nd to the 46th from the 5' end of SEQ ID NO: 4, the nucleotide sequence of the fourth forward primer is shown from the 22nd to the 47th from the 5' end of SEQ ID NO: 5, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO: 6.

[0012] In order to facilitate the identification of SNP site genotypes by fluorescence signals, the 5' of the forward primer is added with a fluorescent label sequence, wherein the fluorescent signal colors of the fluorescent label sequences of the first forward primer and the second forward primer are different. The fluorescent signal colors of the fluorescent label sequences of the third forward primer and the fourth forward primer are different.

[0013] Any of the above SNP primer combinations can be primer group 1 alone, can be primer group 2 alone, and can also be composed of the primer group 1 and the primer group 2.

[0014] In the above, the nucleotide sequence shown in SEQ ID NO: 1 from 1st to 21st from the 5' end is a fluorescent tag sequence (i.e. FAM fluorescent tag sequence), and the fluorescent signal is specifically blue. The nucleotide sequence shown in SEQ ID NO: 2 from 1st to 21st from the 5' end is also a fluorescent tag sequence (i.e. HEX fluorescent tag sequence), and the fluorescent signal is specifically red.

[0015] In the above SNP primer combination, as a preferred embodiment, the primer group 1 can be composed 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 group 2 can be composed 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.

[0016] In any of the above primer groups, the molar ratio of the primer with "F1" in the name, the primer with "F2" in the name, and the primer with "R" in the name can be specifically 2:2:5.

[0017] The kit containing any of the above SNP primer combinations also belongs to the protection scope of the present application. Therefore, the second aspect of the present application provides a kit, which comprises the SNP primer combination of the first aspect described above.

[0018] The kit can also include other reagents for performing competitive allele-specific PCR, such as DNA polymerase, buffer, etc.

[0019] The preparation method of the kit also belongs to the protection scope of the present application. The preparation method of the kit comprises the step of separately packaging each primer in any of the above primer groups.

[0020] The application of the kit also belongs to the protection scope of the present application. The application of the kit can be to identify whether the tomato variety to be tested is cherry tomato or non-cherry tomato.

[0021] The third aspect of the present application provides the application of the SNP primer combination of the first aspect described above, which is any of the following two: (A) the application in the preparation of a kit for identifying whether the tomato variety to be tested is cherry tomato or non-cherry tomato; (B) the application in identifying whether the tomato variety to be tested is cherry tomato or non-cherry tomato.

[0022] The fourth aspect of the present application provides the use of the kit of the second aspect in identifying whether a tomato variety to be tested is cherry tomato or non-cherry tomato.

[0023] The fifth aspect of the present application provides a method for identifying whether a tomato variety to be tested is cherry tomato or non-cherry tomato, comprising the following steps: detecting the genotype of the tomato variety to be tested based on ToSNP01 locus and / or ToSNP07 locus, and then making the following judgments: if the genotype based on ToSNP01 locus is GG homozygous or AG heterozygous, and / or the genotype based on ToSNP07 locus is TT homozygous or CT heterozygous, then the tomato variety to be tested is identified or suspected to be identified as cherry tomato; if the genotype based on ToSNP01 locus is AA homozygous and / or the genotype based on ToSNP07 locus is CC homozygous, then the tomato variety to be tested is identified or suspected to be identified as non-cherry tomato.

[0024] ToSNP01 locus is the 44955024th nucleotide on chromosome 2;

[0025] ToSNP07 locus is the 46612819th nucleotide on chromosome 2;

[0026] The positions of ToSNP01 locus and ToSNP07 locus on the chromosome are determined based on the alignment of tomato HEINZ 1706 reference genome sequence, and the version number of the tomato HEINZ 1706 reference genome sequence is V3.0.

[0027] In the above method, the step of detecting the genotype of the tomato variety to be tested based on ToSNP01 locus and / or ToSNP07 locus can be as follows:

[0028] (1) Taking the genomic DNA of the tomato variety to be tested as a template, using the primer set 1 of the first aspect and / or the primer set 2 of the first aspect to perform PCR amplification, to obtain a PCR amplification product;

[0029] (2) After completing step (1), detecting the fluorescence signal of the PCR amplification product using an instrument, and obtaining the genotype of the tomato variety to be tested based on ToSNP01 locus and / or ToSNP07 locus 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 ToSNP01 locus and / or ToSNP07 locus is as follows:

[0031] (1) Taking the genomic DNA of the tomato variety to be tested as a template, using the primer set 1 of the first aspect and / or the primer set 2 of the first aspect to perform PCR amplification, to obtain a PCR amplification product;

[0032] (2) taking the PCR amplification product obtained in step (1) for sequencing;

[0033] (3) obtaining the genotype of the tomato variety to be tested based on ToSNP01 and / or ToSNP07 according to the sequencing result obtained in step (2).

[0034] In any of the above-mentioned methods, the reaction procedure of "PCR amplification using the primer set 1 and / or the primer set 2 of the first aspect" can be specifically as follows: pre-denaturation at 94℃ for 15 min; denaturation at 94℃ for 20 s, annealing at 61℃-55℃ (touch down program is selected, and the temperature is reduced by 0.6℃ per cycle), 1 min, 10 cycles of amplification; denaturation at 94℃ for 20 s, annealing and extension at 55℃ for 1 min, and 26 cycles of continuous amplification. If the fluorescence signal is weak after the PCR amplification, which affects data analysis, additional cycles (denaturation at 94℃ for 20 s, annealing and extension at 55℃ for 1 min, 5 cycles) can be added until the results are satisfactory.

[0035] Advantages of the present application:

[0036] With the development of genomics technology, the tomato genome has been published and more than 1000 tomato resources have been resequenced, and the rich variation group big data provides marker resources for screening population-specific genetic loci of tomatoes. Using the resequencing data of cherry tomato and non-cherry tomato germplasm resources, population-specific genetic loci are screened from the whole genome, and then SNP primer combinations for identifying cherry tomatoes and non-cherry tomatoes are developed. The SNP primer combinations provided by the present application can be used to identify whether the tomato variety to be tested is a cherry tomato or a non-cherry tomato, which has important application value for clarifying the population properties of tomatoes in agricultural production and variety breeding process. The method provided by the present application has the advantages of high throughput, accuracy, low cost, simple operation, saving of manpower and material resources, etc., and has very broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 PCA clustering results of resequencing data of 96 tomato representative resources.

[0038] Figure 2 Whole genome SNP frequency difference between cherry tomatoes and other types of tomatoes.

[0039] Figure 3 Distribution of 8 cherry tomato-specific SNP loci located on chromosome 2.

[0040] Figure 4 Part of the SNP genotyping results of the test tomato varieties identified by primer set 1.

[0041] Figure 5Part of SNP typing results of the tested tomato varieties identified by primer set 2.

[0042] Figure 6 Part of SNP typing results of the tested tomato varieties identified by primer set 1.

[0043] Figure 7 Part of SNP typing results of the tested tomato varieties identified by primer set 2. DETAILED DESCRIPTION

[0044] The application will be further described in conjunction with the specific embodiments. The examples given are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0045] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0046] Example 1, obtaining of SNP primer combination for identifying the tested tomato variety as cherry tomato

[0047] I. Discovery of 8 SNP loci

[0048] The application is based on the resequencing data of 96 representative tomato resources in the vegetable SNP variation database (http: / / www.vegsnpdb.cn), and 31 perfect SNP loci without other variations in the 50bp flanking sequences and specific to the genome are screened. The 96 representative tomato resources represent a wide genetic diversity of tomatoes, including 68 non-cherry tomatoes and 28 cherry tomatoes. The PCA clustering results of the resequencing data of the 96 representative tomato resources are shown in Figure 1 .

[0049] Specifically, the screening criteria for the SNP loci are as follows: first, select SNP loci with MAF>0.3, heterozygosity less than 0.1, deletion rate less than 0.1, and 50bp sequence conservative (no other variations) in the whole genome, a total of 33656 tomato perfect SNPs are obtained; then, calculate the SNP frequency difference between cherry tomatoes and other types of tomatoes, find that cherry tomato type materials have a significant peak on chromosome 2 ( Figure 2 ), and a total of 8 loci with SNP frequency difference of 1 between the two populations are obtained in this interval ( Figure 3The basic information of the 8 SNP sites is shown in Table 1. The position of the SNP site on the chromosome is determined based on the alignment of the tomato HEINZ 1706 reference genome sequence, the version number of which is V3.0 (download address: https: / / plants.ensembl.org / Solanum_ lycopersicum).

[0050] Table 1. Basic information of 8 SNP sites

[0051]

[0052] II. Obtaining of SNP primer combination for identifying the tested tomato variety as cherry tomato

[0053] Based on the 8 SNP sites discovered in step one, the inventors of the present application designed and synthesized 8 SNP primer combinations and performed SNP typing on 24 cherry tomato varieties. According to the SNP genotype results, 2 SNP sites with higher proportion of homozygous genotypes and 100% linkage with cherry tomato, i.e. ToSNP01 and ToSNP07, were obtained.

[0054] The SNP primer combination consists of primer group 1 and / or primer group 2. The primer group for amplifying ToSNP01 is primer group 1. The primer group for amplifying ToSNP07 is primer group 2. Each primer group consists of 3 primer sequences for amplifying one SNP site. The nucleotide sequences of the primers in primer group 1 and primer group 2 are shown in Table 2, column 4.

[0055] Table 2. SNP primer combination for identifying the tested tomato variety as cherry tomato

[0056]

[0057] Note: single underline is FAM fluorescent tag sequence, double underline is HEX fluorescent tag sequence.

[0058] Example 2, verification of the effectiveness of the SNP primer combination developed in Example 1

[0059] The basic information of the 159 tested tomato varieties in this example is shown in Table 3, columns 1-4. The 159 tested tomato varieties are all common market varieties. According to the phenotype, 46 of the tested tomato varieties are cherry tomatoes, and 113 of the tested tomato varieties are non-cherry tomatoes.

[0060] Table 3. Basic information of 159 tested tomato varieties

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] 1. Obtaining genomic DNA of the tested tomato varieties

[0067] Genomic DNA of 159 tested tomato varieties was extracted by SDS method. The quality and concentration of the genomic DNA of the tested tomato varieties must meet the PCR requirements, and the standard for meeting the requirements is: agarose electrophoresis shows that the DNA band is single and has no obvious dispersion; the A260 / A280 ratio detected by ultraviolet spectrophotometer Nanodrop 2000 (Thermo) is about 1.8, and the A260 / A230 ratio is greater than 1.8; the concentration of the genomic DNA of the tested tomato varieties is 10-30 ng / μL.

[0068] 2. PCR amplification was performed by using primer group 1 or primer group 2 with the genomic DNA of 159 tested tomato varieties as templates, respectively, to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing “F1” in the name, the primer containing “F2” in the name and the primer containing “R” in the name is 2:2:5.

[0069] The reaction procedure is: 94℃ pre-denaturation, 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (touch down program is selected, and the temperature is reduced by 0.6℃ per cycle), 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ recombination & extension for 1 min, and continue to amplify for 26 cycles.

[0070] 3. After completing step 2, when the temperature of each PCR amplification product drops below 40°C, the fluorescence value is read by scanning the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is observed and read at an excitation light wavelength of 485 nm and an emission light wavelength of 520 nm, and the HEX fluorescent tag sequence is observed and read at an excitation light wavelength of 528 nm and an emission light wavelength of 560 nm). The genotype of the 159 test tomato varieties based on each SNP site is determined according to the color of the fluorescent signal. The specific judgment principles are as follows: if a test tomato variety shows a blue fluorescent signal based on a certain SNP site, the genotype of the test tomato variety based on the SNP site is the homozygous type of "the first base at the 3' end of the primer that amplifies the SNP site and contains "F1" in its name"; if a test tomato variety shows a red fluorescent signal based on a certain SNP site, the genotype of the test tomato variety based on the SNP site is the homozygous type of "the first base at the 3' end of the primer that amplifies the SNP site and contains "F2" in its name"; if a test tomato variety shows a green fluorescent signal based on a certain SNP site, the genotype of the test tomato variety based on the SNP site is heterozygous, one base is "the first base at the 3' end of the primer that amplifies the SNP site and contains "F1" in its name", and the other base is "the first base at the 3' end of the primer that amplifies the SNP site and contains "F2" in its name".

[0071] It should be noted that if the fluorescence signal is weak after PCR amplification, which affects data analysis, additional cycles can be added (denaturation at 94°C for 20 seconds, annealing and extension at 55°C for 1 minute, 5 cycles) until the results are satisfactory.

[0072] Some SNP typing results of primer set 1 are shown in Figure 4 The statistical results are shown in the fifth column of Table 3.

[0073] Some SNP typing results of primer set 2 are shown in 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 obtain good typing results in 159 tested tomato varieties.

[0075] 4. Evaluation of the efficiency of identifying 159 test tomato varieties as cherry tomatoes using primer set 1 or primer set 2

[0076] (1) The genotypes of 159 tested tomato varieties based on the ToSNP01 locus were counted.

[0077] The results show that the genotype of the 27 tomato varieties based on ToSNP01 site is GG homozygous, the genotype of the 19 tomato varieties based on ToSNP01 site is AG heterozygous, and the 46 tomatoes are determined as cherry tomatoes, and the consistency is 100%; the genotype of the 113 tomato varieties based on ToSNP01 site is AA homozygous, and the 113 tomatoes are determined as large fruit tomatoes, and the consistency is 100%.

[0078] (2) The genotypes of 159 test tomato varieties based on ToSNP07 site were counted.

[0079] The results show that the genotype of the 37 tomato varieties based on ToSNP07 site is TT homozygous, the genotype of the 9 tomato varieties based on ToSNP07 site is CT heterozygous, and the 46 tomatoes are determined as cherry tomatoes, and the consistency is 100%; the genotype of the 113 tomato varieties based on ToSNP07 site is CC homozygous, and the 113 tomatoes are determined as non-cherry tomatoes, and the consistency is 100%.

[0080] It can be seen that the SNP primer combination developed in Example 1 can identify the test tomato varieties as cherry tomatoes.

[0081] Example 3, the accuracy of using the SNP primer combination developed in Example 1 to identify the test tomato varieties as cherry tomatoes

[0082] The test tomato varieties are test tomato variety 1 to test tomato variety 12, a total of 12.

[0083] 1. Use the SNP primer combination developed in Example 1 to identify each test tomato variety as cherry tomato. The specific steps are as follows:

[0084] (1) Plant the seeds of the test tomato varieties to obtain test tomato seedlings; take the leaves or roots of the test tomato seedlings, and use the SDS method to extract the genomic DNA to obtain the genomic DNA of the test tomato varieties.

[0085] (2) Using the genomic DNA of the test tomato varieties as the template, PCR amplification is carried out using primer set 1 or primer set 2 to obtain the PCR amplification product. In each PCR reaction system, the concentration ratio of the primer containing "F1" in the name, the primer containing "F2" in the name and the primer containing "R" in the name is 2:2:5.

[0086] The reaction program is the same as part 2 of Example 2.

[0087] (3) After step (2) is completed, each PCR product is subjected to fluorescence analysis by using the same method as that in part 3 of Example 2, and the genotype of each SNP site is determined based on the same judgment principle as that in part 3 of Example 2.

[0088] It should be noted that if the fluorescence signal is weak after PCR amplification, which affects data analysis, the cycle (94℃ denaturation for 20s, 55℃ annealing and extension for 1min, 5 cycles) can be added until the result is satisfactory.

[0089] Part of the SNP typing results of primer group 1 are shown in Table 3 and the 2nd column of Table 4. Figure 6

[0090] Part of the SNP typing results of primer group 2 are shown in Table 3 and the 3rd column of Table 4. Figure 7

[0091] (4) After step (3) is completed, the following judgment is made: if the genotype based on ToSNP01 site is GG homozygous or AG heterozygous, and / or the genotype based on ToSNP07 site is TT homozygous or CT heterozygous, it is identified as cherry tomato; if the genotype based on ToSNP01 site is AA homozygous and / or the genotype based on ToSNP07 site is CC homozygous, it is identified as non-cherry tomato.

[0092] Table 4 Identification results of 12 tomato varieties to be tested

[0093] Tomato variety to be tested Genotype based on ToSNP01 Genotype based on ToSNP07 Type Tomato variety to be tested 1 GG TT Cherry tomato Tomato variety to be tested 2 AG TT Cherry tomato Tomato variety to be tested 3 GG TT Cherry tomato Tomato variety to be tested 4 GG TT Cherry tomato Tomato variety to be tested 5 AG CT Cherry tomato Tomato variety to be tested 6 AA CC Non-cherry tomato Tomato variety to be tested 7 AA CC Non-cherry tomato Tomato variety to be tested 8 AA CC Non-cherry tomato Tomato variety to be tested 9 AA CC Non-cherry tomato Tomato variety to be tested 10 AA CC Non-cherry tomato Tomato variety to be tested 11 AA CC Non-cherry tomato Tomato variety to be tested 12 AA CC Non-cherry tomato

[0094] 2, 12 tomato varieties to be tested are planted respectively. According to the phenotype, it is determined whether the 12 tomato varieties to be tested are cherry tomato or non-cherry tomato.

[0095] The phenotype statistical results are shown in the 4th column of Table 4.

[0096] The results show that the identification results of the SNP primer combination developed in Example 1 are completely consistent with the phenotype identification results.

[0097] It can be seen that the SNP primer combination developed in Example 1 can completely identify whether the tomato is cherry tomato or non-cherry tomato.

[0098] ​​The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. A SNP primer combination comprising primer set 1 for amplifying the ToSNP01 locus of the tomato genome and / or primer set 2 for amplifying the ToSNP07 locus of the tomato genome; The ToSNP01 site is nucleotide 44955024 on chromosome 2; The ToSNP07 locus is nucleotide position 46612819 on chromosome 2; The positions of the ToSNP01 and ToSNP07 loci on the chromosomes were determined based on the alignment of the tomato HEINZ 1706 reference genome sequence, and the version number of the tomato HEINZ 1706 reference genome sequence is V3.

0.

2. The SNP primer combination according to claim 1, wherein: The primer set 1 comprises a first forward primer, a second forward primer and a first reverse primer, wherein the nucleotide sequence of the first forward primer is as shown in SEQ ID NO: 1 at positions 22 to 48 from the 5' end, the nucleotide sequence of the second forward primer is as shown in SEQ ID NO: 2 at positions 22 to 47 from the 5' end, and the nucleotide sequence of the first reverse primer is as shown in 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 in SEQ ID NO: 4 at positions 22 to 46 from the 5' end, the nucleotide sequence of the fourth forward primer is shown in SEQ ID NO: 5 at positions 22 to 47 from the 5' end, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO:

6.

3. The SNP primer combination according to claim 1, wherein: The primer set 1 consists of a forward primer 1F1 shown in SEQ ID NO: 1, a forward primer 1F2 shown in SEQ ID NO: 2, and a reverse primer 1R shown in SEQ ID NO: 3; The primer set 2 consists of a forward primer 2F1 shown in SEQ ID NO: 4, a forward primer 2F2 shown in SEQ ID NO: 5, and a reverse primer 2R shown in SEQ ID NO:

6.

4. A kit, characterized in that: The kit comprises the SNP primer combination according to any one of claims 1 to 3.

5. Use of the SNP primer combination according to any one of claims 1 to 3 in preparing a kit for identifying whether a tomato variety to be tested is a cherry tomato or a non-cherry tomato.

6. Use of the SNP primer combination according to any one of claims 1 to 3 in identifying whether a tomato variety to be tested is a cherry tomato or a non-cherry tomato.

7. 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 performing the following judgment: 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 locus is nucleotide position 46612819 on chromosome 2; The positions of the ToSNP01 and ToSNP07 loci on the chromosomes were determined based on the alignment of the tomato HEINZ 1706 reference genome sequence, and the version number of the tomato HEINZ 1706 reference genome sequence is V3.

0.

8. The method according to claim 7, wherein: The steps of detecting the genotype of the tomato variety to be tested based on the ToSNP01 site and / or the ToSNP07 site are as follows: (1) Using the genomic DNA of the tomato variety to be tested as a template, PCR amplification is performed using the primer set 1 and / or primer set 2 described in claim 3 to obtain a PCR amplification product; (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 the ToSNP07 site is obtained according to the color of the fluorescence signal.

9. The method according to claim 7, wherein: The steps of detecting the genotype of the tomato variety to be tested based on the ToSNP01 site and / or the ToSNP07 site are as follows: (1) Using the genomic DNA of the tomato variety to be tested as a template, PCR amplification is performed using the primer set 1 and / or primer set 2 described in claim 2 to obtain a PCR amplification product; (2) taking the PCR amplification product obtained in step (1) and sequencing it; (3) According to the sequencing results obtained in step (2), the genotype of the tomato variety to be tested based on the ToSNP01 site and / or the ToSNP07 site is obtained.

Images