Methods for identifying the authenticity of tomato varieties and their specific SSR primer combinations

Abstract

This invention belongs to the field of SSR molecular markers and their detection, specifically relating to a method for identifying the authenticity of tomato varieties and a dedicated SSR primer combination. The SSR loci used to identify the authenticity of tomato varieties are selected from any 1 to 28 of the following SSR loci, from the first to the twenty-eighth. The aforementioned SSR primer combination is selected from the first to the twenty-eighth SSR primer pairs, respectively used for PCR amplification of the first to the twenty-eighth SSR loci. This invention can be used for early identification of tomato varieties at the seed or seedling stage, ensuring the authenticity of varieties, effectively protecting the rights and interests of producers and breeders, and providing technical support for the protection of tomato germplasm resources and new varieties.

Description

Technical Field

[0001] This invention belongs to the field of SSR molecular markers and their detection, specifically relating to a method for identifying the authenticity of tomato varieties and a dedicated SSR primer combination. Background Technology

[0002] Tomatoes (Solanum lycopersicum L.), originating in South America, are also known as tomatoes, foreign persimmons, etc. They are annual or perennial herbaceous plants belonging to the Solanaceae family and the Solanum genus, and are one of the main cultivated vegetables in my country. Tomatoes are rich in nutrients. Studies have shown that consuming 50-100g of fresh tomatoes daily can meet the body's needs for various vitamins and minerals. Tomatoes also contain abundant antioxidants, offering certain beauty and anti-wrinkle benefits. Because they serve as both a vegetable and a fruit, they are widely loved by consumers, and China ranks first globally in annual production among vegetable crops.

[0003] The tomato seed industry is a crucial component of the tomato industry; for the stable development of the tomato industry, the seed industry must take the lead. my country's tomato industry has developed steadily, with domestic breeding units undertaking extensive work in germplasm innovation, resulting in new varieties with significant improvements in combined disease resistance and quality. However, my country's tomato germplasm resources are relatively scarce. Through long-term artificial selection, the genetic background has gradually narrowed, leading to small genetic differences among bred varieties. Currently, 3215 tomato varieties have been registered, necessitating effective regulation of tomato varieties in the market. Because closely related varieties are difficult to distinguish using appearance alone, illegal activities such as using the same name for the same species, or passing off inferior varieties as superior ones are common. Therefore, establishing an accurate and efficient method for identifying the authenticity of tomato varieties to serve the regulatory monitoring of seed enforcement agencies and the internal seed quality control of breeding units has become one of the most pressing issues to be addressed in tomato production.

[0004] With the rapid development of molecular biology, it has become possible to analyze and identify the authenticity of seed varieties at the DNA molecular level, offering advantages such as rapid identification, high accuracy, and immunity to environmental factors. Simple sequence repeat (SSR) molecular markers are widely present in the genomes of eukaryotes and prokaryotes. They exhibit good repeatability, site specificity, and co-dominance, making them one of the commonly used markers in crop variety identification research.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a method for identifying the authenticity of tomato varieties and a dedicated SSR primer combination. The method and primer combination of this invention can produce stable, efficient and reliable identification results, and can distinguish whether the tomato variety to be tested belongs to a certain type of standard tomato variety, and which type it is, and can also distinguish whether two unknown tomato varieties are the same tomato variety.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] The first aspect of this invention provides an SSR primer set for identifying the authenticity of tomato varieties. The SSR primer set includes a first SSR primer pair to a twenty-eighth SSR primer pair, each used to amplify the following SSR loci:

[0009] The first SSR locus is located at position 24204297-24204403 on chromosome 11 of the tomato reference genome; the second SSR locus is located at position 63878565-63878713 on chromosome 9 of the tomato reference genome; the third SSR locus is located at position 55664286-55664424 on chromosome 11 of the tomato reference genome; the fourth SSR locus is located at position 47297032-47297290 on chromosome 5 of the tomato reference genome; the fifth SSR locus is located at position 60943735-60943925 on chromosome 3 of the tomato reference genome; and the sixth SSR locus is located at position 4827710 on chromosome 11 of the tomato reference genome. -4827882; the seventh SSR locus, located at positions 4611948-4612079 on chromosome 3 of the tomato reference genome; the eighth SSR locus, located at positions 7102250-7102471 on chromosome 10 of the tomato reference genome; the ninth SSR locus, located at positions 62287690-62287862 on chromosome 10 of the tomato reference genome; the tenth SSR locus, located at positions 86705211-86705441 on chromosome 1 of the tomato reference genome; the eleventh SSR locus, located at positions 43539414-43539519 on chromosome 1 of the tomato reference genome; the twelfth SSR locus, located at position -4827882 on chromosome 4 of the tomato reference genome. SSR loci 47994650-47994908; the thirteenth SSR locus, located on chromosome 7 of the tomato reference genome at positions 66874993-66875136; the fourteenth SSR locus, located on chromosome 12 of the tomato reference genome at positions 67862946-67863137; the fifteenth SSR locus, located on chromosome 6 of the tomato reference genome at positions 42014670-42014771; the sixteenth SSR locus, located on chromosome 2 of the tomato reference genome at positions 38481245-38481428; the seventeenth SSR locus, located on chromosome 7 of the tomato reference genome at positions 9960607-9960757; the eighteenth SSR locus, located at... The tomato reference genome has the following SSR loci: loci 96843682-96843974 on chromosome 1; loci 19 on chromosome ...The 24th SSR locus is located on chromosome 6 of the tomato reference genome, at positions 19271156-19271289; the 25th SSR locus is located on chromosome 2 of the tomato reference genome, at positions 46769738-46769920; the 26th SSR locus is located on chromosome 8 of the tomato reference genome, at positions 55139338-55139629; the 27th SSR locus is located on chromosome 12 of the tomato reference genome, at positions 6759631-6759785; and the 28th SSR locus is located on chromosome 4 of the tomato reference genome, at positions 6220284-6220510. The tomato reference genome is the Heinz 1706v3.0 reference genome sequence.

[0010] In the above-mentioned SSR primer combinations for identifying the authenticity of tomato varieties, as an optional implementation, the nucleotide sequences of the first SSR primer pair are as shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing; the nucleotide sequences of the second SSR primer pair are as shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing; the nucleotide sequences of the third SSR primer pair are as shown in SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing; the nucleotide sequences of the fourth SSR primer pair are as shown in SEQ ID NO: 7 and SEQ ID NO: 8 in the sequence listing; the nucleotide sequences of the fifth SSR primer pair are as shown in SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing; the nucleotide sequences of the sixth SSR primer pair are as shown in SEQ ID NO: 11 and SEQ ID NO: 12 in the sequence listing; the nucleotide sequences of the seventh SSR primer pair are as shown in SEQ ID NO: 13 and SEQ ID NO: 14 in the sequence listing; and the nucleotide sequences of the eighth SSR primer pair are as shown in SEQ ID NO: 15 and SEQ ID NO: 16 in the sequence listing. The 9th SSR primer pair has nucleotide sequences shown in SEQ ID NO: 16; the 10th SSR primer pair has nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20; the 11th SSR primer pair has nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22; the 12th SSR primer pair has nucleotide sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24; the 13th SSR primer pair has nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26; the 14th SSR primer pair has nucleotide sequences shown in SEQ ID NO: 27 and SEQ ID NO: 28; the 15th SSR primer pair has nucleotide sequences shown in SEQ ID NO: 29 and SEQ ID NO: 30; the 16th SSR primer pair has nucleotide sequences shown in SEQ ID NO: 31 and SEQ ID NO: 28. The 17th SSR primer pair is shown in SEQ ID NO: 32; the nucleotide sequences of the 17th SSR primer pair are shown in SEQ ID NO: 33 and SEQ ID NO: 34 in the sequence listing; the 18th SSR primer pair is shown in SEQ ID NO: 35 and SEQ ID NO: 36 in the sequence listing; the 19th SSR primer pair is shown in SEQ ID NO: 37 and SEQ ID NO: 38 in the sequence listing.The nucleotide sequences of the 20th SSR primer pair are shown in SEQ ID NO: 39 and SEQ ID NO: 40 in the sequence listing; the nucleotide sequences of the 21st SSR primer pair are shown in SEQ ID NO: 41 and SEQ ID NO: 42 in the sequence listing; the nucleotide sequences of the 22nd SSR primer pair are shown in SEQ ID NO: 43 and SEQ ID NO: 44 in the sequence listing; the nucleotide sequences of the 23rd SSR primer pair are shown in SEQ ID NO: 45 and SEQ ID NO: 46 in the sequence listing; the nucleotide sequences of the 24th SSR primer pair are shown in SEQ ID NO: 47 and SEQ ID NO: 48 in the sequence listing; the nucleotide sequences of the 25th SSR primer pair are shown in SEQ ID NO: 49 and SEQ ID NO: 50 in the sequence listing; the nucleotide sequences of the 26th SSR primer pair are shown in SEQ ID NO: 51 and SEQ ID NO: 52 in the sequence listing; and the nucleotide sequences of the 27th SSR primer pair are shown in SEQ ID NO: 53 and SEQ ID NO: 44 in the sequence listing. The 28th SSR primer pair is shown in SEQ ID NO: 54; its nucleotide sequences are shown in SEQ ID NO: 55 and SEQ ID NO: 56 in the sequence listing.

[0011] Furthermore, one primer in each primer pair is linked to a fluorescent molecule. The fluorescent molecule can be selected from ROX, TAMRA, FAM, and HEX.

[0012] In the primer pairs mentioned above, the 5′ end of the upstream primer may carry a fluorescent tag sequence for fluorescent PCR detection. For example, the fluorescence signal of the FAM fluorescent tag sequence is blue, and the fluorescence signal of the HEX fluorescent tag sequence is green.

[0013] The second aspect of the present invention provides a kit for identifying the authenticity of tomato varieties, the kit being prepared as a PCR reaction system; the PCR reaction system comprising: the SSR primer combination described in the first aspect of the present invention.

[0014] Furthermore, the concentration ratio of the upstream primer to the downstream primer in each pair of the SSR primer set is 1:1 in the system;

[0015] The final concentration of both the upstream and downstream primers in the system is preferably 0.25 μmol / L;

[0016] Preferably, the system further includes: dNTPs: a final concentration of 0.15 mmol / L for each; magnesium chloride: a final concentration of 2.5 mmol / L; DNA polymerase: a final concentration of 0.05 U / μL; and PCR buffer: prepared by mixing potassium chloride (final concentration of 10-50 mmol / L) and Tris-HCl (pH 7.5-9.0) (final concentration of 1-10 mmol / L).

[0017] The third aspect of this invention provides the application of the SSR primer combination described in the first aspect or the kit of the second aspect in the following X1 or X2:

[0018] X1: To determine whether the tomato variety to be tested belongs to one of the standard tomato varieties;

[0019] X2: Identify which specific standard tomato variety the tomato being tested belongs to;

[0020] X3: Determine whether the tomato varieties being tested belong to the same variety.

[0021] The fourth aspect of this invention provides a method for establishing a DNA fingerprint of a standard tomato variety, comprising: using the genomic DNA of the standard tomato variety as a template, performing PCR amplification using 28 primer pairs from the SSR primer combination provided in the first aspect, and then detecting the size of the PCR amplification product to obtain the genotype (i.e., the length of the allelic variant amplification fragment) of each SSR locus, thereby constituting the DNA fingerprint of the standard tomato variety.

[0022] The fifth aspect of this invention provides a detection method for identifying the authenticity of a tomato variety, comprising the following steps:

[0023] Step 1: Detect the genotypes of the tomato sample and standard tomato varieties based on the first to twenty-eighth SSR loci mentioned above;

[0024] Step 2: Determining the variety of the tomato to be tested:

[0025] If the number of genotypes of the tomato under test based on the first SSR locus to the twenty-eighth SSR locus is 0, and the number of genotypes of a specified variety of standard tomato varieties based on the first SSR locus to the twenty-eighth SSR locus is 0, then the tomato under test and the specified variety of standard tomato varieties belong to the same variety.

[0026] If the number of genotypes of the tomato under test based on the first SSR locus to the twenty-eighth SSR locus is 1, and the number of genotypes of a specified variety of standard tomato varieties based on the first SSR locus to the twenty-eighth SSR locus is 1, then the tomato under test and the specified variety of standard tomato varieties are similar varieties.

[0027] If the number of genotypes of the tomato under test based on the first SSR locus to the twenty-eighth SSR locus is greater than or equal to 2 compared with the number of genotypes of each of the standard tomato varieties based on the first SSR locus to the twenty-eighth SSR locus, then the tomato under test is different from each of the standard tomato varieties.

[0028] The genotypic differences from the first to the twenty-eighth SSR loci specifically refer to the differences in the number and / or length of DNA fragments in the PCR products of each primer pair of each tomato sample among different tomato samples. These differences can be determined using a DNA analyzer and / or DNA analysis software and / or modules. Specifically, the DNA analyzer can be an ABI 3730xl DNA analyzer. The DNA analysis software can be GENEMARKER.

[0029] In the above detection method, as an optional implementation, the step of detecting the genotype of each SSR locus in the tomato and standard tomato varieties includes the following sub-steps:

[0030] Step 1: Using the genomic DNA of the tomato to be tested and the genomic DNA of the standard tomato variety as templates, PCR amplification is performed using the primer pairs in the SSR primer combination described in the first aspect, respectively, to obtain PCR amplification products;

[0031] Step 2: Detect the PCR amplification products to obtain the genotypes of the tomato to be tested and the standard tomato variety based on the first SSR locus to the twenty-eighth SSR locus.

[0032] Further, the detection method in step two is as follows: amplification product fragment detection method: detecting the fragment size of the PCR amplification product to obtain the genotypes of the tomato to be tested and the standard tomato variety based on the first SSR site to the twenty-eighth SSR site.

[0033] Furthermore, the size of the PCR amplification product can be detected by fluorescence signal to obtain the genotypes of the tomato to be tested and the standard tomato variety based on the first SSR locus to the twenty-eighth SSR locus.

[0034] Furthermore, in step two, the determination result is obtained based on cluster analysis.

[0035] The standard tomato varieties described in this invention are selected from the following 84 tomato varieties: Jingfan 101, Jingfan 102, Jingfan 401, Jingfan 404, Jingfan 303, Jingfan 309, JFSY1603, JFSY1608, Jingfan 403, Jingfan 302, Jingfan 308, ND2301, ND2302, ND2303, ND2304, ND2305, TM-12, TM-13, TM -14, TM-15, 230029, 230030, 230034, 230049, 230061, 230062, 230063, 230083, 230085 , 230089, 230091, 230096, 238222, 238234, 233057, TM-1, TM-2, TM-3, TM-4, TM-5, TM-6, TM-7, TM-8, TM-9, TM-10, TM-11, Kyoto Animation 502, Kyoto Animation 603, Kyoto Animation 310, Kyoto Animation 501, Kyoto Animation 604, Kyoto Animation 701, Kyoto Animation Green Angel, Kyoto Animation Yellow Luo No. 1, Kyoto Animation Red Luo No. 2, 22S-457, 22S-467, 22S-478, 22S-484, 22S-555, 22S-575, 22-C24, 22-C38 22-C42, 22-C45, 22-C72, 22-C101, 22-C103, 22-C136, M601, ND2306, ND2307, ND2308, ND2309, Jingfan Pink Star No. 3, Jingfan Red Star No. 2, Jingfan Yellow Star No. 7, Jingfan Colorful Star No. 1, Jingfan Purple Star No. 2, Jingfan Green Star No. 3, Jingfan Blue Elf, Jingfan Pink Star No. 1, Jingfan Yellow Star No. 1, Jingfan Green Elf.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] 1. The SSR primer combination provided by this invention can be used for early identification of tomato varieties in the seed or seedling stage, ensuring the authenticity of varieties, effectively protecting the rights and interests of producers and breeders, and providing technical support for the protection of tomato germplasm resources and new varieties.

[0038] 2. The method provided by this invention can identify whether a tomato variety belongs to a specific standard tomato variety, and if so, which one, or whether two or more unknown tomato varieties belong to the same variety. Therefore, this method can identify both unknown tomato varieties and verify the authenticity of known varieties.

[0039] 3. The method provided by this invention has the advantages of high throughput, accuracy, low cost, simple operation, and saving manpower and material resources, and has a very broad application prospect. Attached Figure Description

[0040] Figure 1This is a cluster diagram of 84 tested tomato varieties established on 28 primer pairs in Example 1.

[0041] Figure 2 This is a graph showing the relationship between the number of SSR markers (i.e., the number of SSR loci) and the differentiation of 84 tested tomato varieties in Example 2.

[0042] Figure 3 The SSR typing effect of the 28 primer pairs in Example 2 on some of the tested tomato varieties is shown. Detailed Implementation

[0043] 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.

[0044] 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.

[0045] In this invention, the authenticity of tomato varieties refers to the true correspondence between a tomato variety and its genetic background.

[0046] Example 1: Obtaining SSR primer combinations for identifying the authenticity of tomato varieties

[0047] I. Discovery of 28 SSR loci

[0048] This invention, based on resequencing data from 157 representative tomato resources, has for the first time discovered and obtained 28 SSR loci. These 157 tomato resources are diverse in type, covering fresh tomatoes, processing tomatoes, and cherry tomatoes, and basically include the main ecological types and agronomic traits of tomatoes currently on the market, reflecting germplasm representativeness as much as possible and possessing high genetic diversity.

[0049] Specifically, the selection criteria for SSR loci are as follows: SSR loci with uniform location across the entire genome, good polymorphism, low heterozygosity, MAF > 0.3, good PCA clustering effect, high discriminative power, and conserved 50bp sequences on both sides (no InDel, no SNP, no other SSRs). Basic information on the 28 SSR loci is detailed in columns 2 to 4 of Table 1. The chromosomal positions of the SSR loci were determined based on the Heinz 1706v3.0 reference genome sequence alignment (downloadable from: https: / / plants.ensembl.org / Solanum_lycopersicum / Info / Index?db=core).

[0050] Table 1. Basic information of 28 SSR loci

[0051]

[0052]

[0053]

[0054] The "major allelic variation" was obtained from the statistics of the 84 tested tomato varieties in Table 3. For example, if a sample has only one allelic variation at a certain locus with a size of 99 bp, then the genotype of the major allelic variation at that locus is written as 99 / 99; if a sample has two allelic variations at a certain locus with sizes of 147 bp and 152 bp respectively, then the genotype of the major allelic variation at that locus is written as 147 / 152.

[0055] II. Obtaining SSR primer combinations for identifying the authenticity of tomato varieties

[0056] Based on the 28 SSR loci discovered in step one, the inventors of this invention developed an SSR primer set with high polymorphism information content (i.e., PIC value, which refers to the value of a marker used to detect polymorphism in a population; the PIC value depends on the number of alleles detected and their frequency distribution; the PIC value equals 1 minus the sum of the squares of all allele frequencies, see column 4 in Table 1) for identifying the authenticity of tomato varieties. Primers were designed based on the upstream and downstream sequences of the aforementioned SSR loci in the tomato Heinz 1706v3.0 reference genome. The SSR primer set consists of 28 primer pairs, each numbered as shown in column 2 of Table 2, corresponding to the primer numbers in Table 1. Each primer pair consists of two primer sequences used to amplify one SSR locus. The nucleotide sequences of each primer in the 28 primer pairs are shown in column 3 of Table 2. Primers within each group can be combined for electrophoresis.

[0057] Table 2

[0058]

[0059]

[0060] Example 2: Validation of the SSR primer combination developed in Example 1.

[0061] The basic information of the 84 tomato varieties tested in this embodiment is shown in Table 3. All 84 tomato varieties tested are common superior varieties or some are introduced varieties from abroad.

[0062] Table 3: Basic Information of 84 Tested Tomato Varieties

[0063]

[0064]

[0065] 1. Obtaining genomic DNA from the tested tomato varieties

[0066] Genomic DNA was extracted from the leaves (with true leaves from 30 seeds mixed in) of 84 tested tomato varieties using the CTAB method to obtain the genomic DNA of the tested tomato varieties.

[0067] The specific operation of the CTAB method described above is as follows:

[0068] Leaves from the above 84 varieties at the seedling stage were collected and dehydrated in a freeze dryer (CoolSafe 55-4). The leaves were then crushed using a high-throughput grinder (Geno / Grind6875). 20 μg of the dried leaf powder was taken and 800 μL of CTAB extraction solution (2% CTAB, 1.4 mM NaCl, 100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1% PVP-40, 0.2% β-mercaptoethanol) was added. The mixture was incubated in a 65°C water bath for 30 min. An equal volume of chloroform / isoamyl alcohol (24:1) was added, and the mixture was centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a new centrifuge tube, and 0.8 times the volume of pre-cooled isopropanol was added. The mixture was gently inverted and mixed. After incubation at -20°C for 30 min, the mixture was centrifuged at 4°C and 12,000 rpm for 10 min. Discard the supernatant, wash twice with 70% ethanol solution, dry under natural conditions, dissolve the DNA in 100 μL ddH2O to obtain the genomic DNA of the tested tomato variety, and store at 4℃ after determining the concentration.

[0069] The quality and concentration of genomic DNA from the tested tomato varieties must meet the requirements of PCR. The standards are as follows: the A260 / A280 ratio detected by the Nanodrop2000 (Thermo) UV spectrophotometer should be around 1.8, and the A260 / A230 ratio should be greater than 1.8; the concentration of genomic DNA from the tested tomato varieties should be 50-100 ng / μL.

[0070] 2. Using genomic DNA from 84 tested tomato varieties as templates, PCR amplification was performed using 28 primer pairs to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F" in their names to primers containing "R" in their names was 1:1.

[0071] The reaction system includes:

[0072] The concentration ratio of the upstream primer (with "F" in its name) to the downstream primer (with "R" in its name) in the system is 1:1.

[0073]

[0074] The reaction procedure was as follows: pre-denaturation: 95℃ for 5 min; amplification: 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 34 cycles; final extension: 72℃ for 10 min. The amplified products were stored at 4℃ before electrophoresis.

[0075] 3. Fluorescent capillary electrophoresis

[0076] After completing step 2, depending on the size of the SSR molecular marker amplified fragment, multiple primer pairs can be selected for electrophoresis depending on the instrument. According to the pre-determined primer combinations (i.e., the groups in Table 2), take equal volumes of amplified products from different fluorescently labeled primer combinations, dilute 200-fold, and mix thoroughly. Pipette 1.5 μL from the mixture and add it to the wells of the DNA analyzer's sample plate. Add 0.1 μL of molecular weight internal standard and 8.9 μL of deionized formamide to each well. Denature at 95°C for 5 min on a PCR instrument, then immediately place in a -20°C freezer or on ice for 5 min. Centrifuge briefly for 10 s and place on the DNA analyzer. Turn on the DNA analyzer and check the instrument's operating status and reagent status. Place the sample plate containing the sample on the sample holder base, and place the buffer plate containing electrode buffer on the buffer plate holder base. Open the data collection software and operate according to the DNA analyzer's user manual. The DNA analyzer will automatically run the parameters and save the raw electrophoresis data. The excitation wavelength and color used for detecting fluorescent primers should be referenced to the instrument's default values ​​(maximum excitation wavelength of FAM: 494nm, maximum excitation wavelength of HEX: 535nm, maximum excitation wavelength of TAMRA: 560nm, maximum excitation wavelength of ROX: 587nm). The capillary electrophoresis equipment needs to be spectrally calibrated periodically.

[0077] Some results can be found Figure 3 The results showed that each primer set could achieve good typing results in the tested tomato varieties.

[0078] 4. Cluster analysis

[0079] Based on the genotypes of 84 tested tomato varieties at 28 SSR loci, cluster analysis was performed on the 84 tested tomato varieties using MEGA7 software.

[0080] Cluster diagram of 84 tested tomato varieties based on 28 primer pairs is shown below. Figure 1 As shown in the figure. The results showed that the 28 primer pairs could completely distinguish the 84 tested tomato varieties in Table 3. Therefore, the SSR primer combinations developed in Example 1 can be applied to the construction of a tomato variety DNA fingerprint database and the identification of variety authenticity.

[0081] 5. Efficiency Evaluation

[0082] Species authenticity identification can be reduced by using sequential analysis. The inventors of this invention compared the relationship between the number of SSR markers (i.e., the number of primer pairs) and the discrimination rate of 84 tested tomato varieties.

[0083] like Figure 2 The number of difference markers between each of the 84 varieties shown is counted and compared pairwise. The number of pairwise comparisons is C.84 2 =84 × 83 ÷ 2 = 3486 results; among these 3486 results, approximately 0.06% had 1 differentially expressed locus, approximately 0.11% had 2 differentially expressed loci, and approximately 99.83% had more than 2 differentially expressed loci. This result indicates that these markers showed good polymorphism in the 84 varieties; the 28 primer pairs (i.e., 28 SSR markers) achieved a 100% discrimination rate among the 84 tested tomato varieties.

[0084] Example 3: Method for detecting whether a tomato variety belongs to the standard tomato varieties

[0085] 1. Obtaining genomic DNA from the tomato variety to be tested

[0086] Leaves of the tomato variety being tested were taken from the experimental base of the Vegetable Research Center of the Beijing Academy of Agricultural and Forestry Sciences.

[0087] Following the method in step 1 of Example 2, replace "the leaf of the tomato variety being tested" with "the leaf of the tomato variety to be tested", and keep all other steps unchanged to obtain the genomic DNA of the tomato variety to be tested.

[0088] 2. Preparation of SSR primers and PCR reaction system

[0089] Following the method in step 2 of Example 2, replace "genomic DNA of the tested tomato variety" with "genomic DNA of the tomato variety to be tested", and keep all other steps unchanged to obtain the PCR product of the tomato variety to be tested.

[0090] 3. Fluorescent capillary electrophoresis detection

[0091] Collect the PCR product of the tomato variety to be tested.

[0092] The fragment sizes of the 28 SSR amplification products from the tomato varieties to be tested were compared with the 28 SSR loci from 84 tested tomato varieties (as shown in Table 3). The number of differentially expressed SSR loci between the tomato varieties to be tested and the 28 standard tomato varieties was counted, and then the following judgments were made:

[0093] If the number of different loci between the tomato variety being tested and a standard tomato variety is two or more, then the tomato variety being tested and the standard tomato variety are different tomato varieties; the more different loci, the more distant the genetic relationship.

[0094] If the number of different sites between the tomato variety being tested and a standard tomato variety is 1 or 0, then the tomato variety being tested and the standard tomato variety are suspected to be the same tomato variety.

[0095] The results showed that the tested tomato variety had more than one different site at each of the 28 SSR loci compared to the 84 tested tomato varieties. Therefore, the tested tomato variety did not belong to any of the 84 tested tomato varieties, meaning that the tested tomato variety was not the same as any of the 84 tested tomato varieties.

[0096] Example 4

[0097] This embodiment uses capillary electrophoresis to compare fragment size to determine the tomato variety, rather than using fluorescence signal determination.

[0098] This case study uses the ABI 3730 fluorescence capillary detection platform as a reference. If other platforms are used, adjustments should be made according to the equipment's operating requirements.

[0099] Depending on the size of the SSR molecular marker amplified fragment, multiple primer combinations can be selected for electrophoresis according to different instruments.

[0100] S1: Using a pre-determined primer combination, dilute equal volumes of amplification products from different fluorescently labeled primer combinations 200-fold and mix thoroughly. Pipette 1.5 μL of the mixture into the wells of a sample plate for the DNA analyzer. Add 0.1 μL of internal standard and 8.9 μL of deionized formamide to each well. Denature at 95°C for 5 min on a PCR instrument, then immediately place on ice and cool for 5 min. Centrifuge briefly for 10 s and then transfer to the DNA analyzer.

[0101] S2: Turn on the ABI 3730 DNA Analyzer and check the instrument's operating status and reagent status. Place the sample plate containing the sample on the sample holder base, and place the buffer plate containing the electrode buffer on the buffer plate holder base. Open the data collection software and operate according to the DNA analyzer's user manual. The DNA analyzer will automatically run the parameters and save the raw electrophoresis data. Refer to the instrument's default values ​​for the excitation wavelength and color used for detecting fluorescent primers.

[0102] S3: Export the raw electrophoresis data file and use data analysis software to perform data screening according to the following steps: In the data analysis software, pre-set the SSR primer names and fluorescence categories, molecular weight internal standards, and amplification fragment sizes of the corresponding primers; import the raw electrophoresis data file into the analysis software, and select panel, molecular weight internal standards, Bin, quality control parameters, etc. for analysis; the analysis software will assign color indicators to score the detection quality, with green indicating reliable quality that requires no intervention, red indicating substandard quality or that the fragment size does not fall within the specified range, and yellow indicating doubt that the original image needs to be checked for confirmation.

[0103] S4: Using standard samples and reference samples (with a small number of controls selected based on primers) that were tested simultaneously, calibrate the data deviation between different electrophoresis plates before reading the amplified fragment size. If the identified specific peaks fall within the specified fragment size range, the amplified fragment size is read directly; if most of the peaks are outside the specified range, the entire fragment can be shifted to bring them within the set range before reading the data.

[0104] S5: The fragment sizes of the 28 SSR amplification products of the tomato varieties to be tested were compared with the 28 SSR loci of 84 tomato varieties tested (as shown in Table 3). The number of differential loci between the tomato varieties to be tested and the 84 standard tomato varieties was counted, and then the following judgments were made:

[0105] If the number of different loci between the tomato variety being tested and a standard tomato variety is two or more, then the tomato variety being tested and the standard tomato variety are different tomato varieties; the more different loci, the more distant the genetic relationship.

[0106] If the number of different sites between the tomato variety being tested and a standard tomato variety is 1 or 0, then the tomato variety being tested and the standard tomato variety are suspected to be the same tomato variety.

[0107] Example 5: A method for detecting whether two unknown tomato varieties belong to the same variety

[0108] 1. Obtaining genomic DNA from the tomato variety to be tested

[0109] Leaves of the two tomato varieties to be tested were taken from the experimental base of the Vegetable Research Center of the Beijing Academy of Agricultural and Forestry Sciences.

[0110] Following the method in step 1 of Example 2, the "leaf of the tested tomato variety" was replaced with the "leaf of the tomato variety to be tested", and all other steps remained unchanged, and the genomic DNA of the two tomato varieties to be tested was obtained respectively.

[0111] 2. Preparation of SSR primers and PCR reaction system

[0112] Following the method in step 2 of Example 2, replace "genomic DNA of the tested tomato variety" with "genomic DNA of the tomato variety to be tested", with all other steps remaining unchanged, and obtain PCR products of the two tomato varieties to be tested.

[0113] 3. Fluorescent capillary electrophoresis detection

[0114] The detection method is the same as in Example 2.

[0115] The results showed that the two tomato varieties tested had 4 different loci at 28 SSR sites, therefore, the two tomato varieties tested were not the same variety.

[0116] Field phenotypes can confirm that the two tested tomato varieties are different. Therefore, the SSR primer combination of this invention can be used to determine whether two or more tested tomato varieties belong to the same variety.

[0117] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An SSR primer combination for identifying the authenticity of tomato varieties, characterized in that, The SSR primer combination includes the first SSR primer pair to the twenty-eighth SSR primer pair, which are used to amplify the following first SSR sites to the twenty-eighth SSR sites respectively: The first SSR locus is located at position 24204297-24204403 on chromosome 11 of the tomato reference genome; The second SSR locus is located at position 63878565-63878713 on chromosome 9 of the tomato reference genome; The third SSR locus is located at position 55664286-55664424 on chromosome 11 of the tomato reference genome; The fourth SSR locus is located on chromosome 5 of the tomato reference genome at positions 47297032-47297290; The fifth SSR locus is located at position 60943735-60943925 on chromosome 3 of the tomato reference genome; The sixth SSR locus is located on chromosome 11 of the tomato reference genome at positions 4827710-4827882. The seventh SSR locus is located on chromosome 3 of the tomato reference genome at positions 4611948-4612079. The eighth SSR locus is located on chromosome 10 of the tomato reference genome at positions 7102250-7102471. The ninth SSR locus is located on chromosome 10 of the tomato reference genome at positions 62287690-62287862. The tenth SSR locus is located at position 86705211-86705441 on chromosome 1 of the tomato reference genome; The eleventh SSR locus is located at positions 43539414-43539519 on chromosome 1 of the tomato reference genome; The twelfth SSR locus is located on chromosome 4 of the tomato reference genome at positions 47994650-47994908. The thirteenth SSR locus is located on chromosome 7 of the tomato reference genome at positions 66874993-66875136. The fourteenth SSR locus is located on chromosome 12 of the tomato reference genome at positions 67862946-67863137. The fifteenth SSR locus is located on chromosome 6 of the tomato reference genome at positions 42014670-42014771. The sixteenth SSR locus is located at position 38481245-38481428 on chromosome 2 of the tomato reference genome; The seventeenth SSR locus is located at position 9960607-9960757 on chromosome 7 of the tomato reference genome; The eighteenth SSR locus is located at position 96843682-96843974 on chromosome 1 of the tomato reference genome; The nineteenth SSR locus is located on chromosome 9 of the tomato reference genome, at positions 9632069-9632270. The twentieth SSR locus is located on chromosome 7 of the tomato reference genome, at positions 7450891-7451019. The 21st SSR locus is located on chromosome 8 of the tomato reference genome at positions 65372995-65373163. The 22nd SSR locus is located at positions 61099092-61099200 on chromosome 5 of the tomato reference genome; The 23rd SSR locus is located on chromosome 5 of the tomato reference genome at positions 9256610-9256821. The 24th SSR locus is located on chromosome 6 of the tomato reference genome at positions 19271156-19271289. The 25th SSR locus is located at position 46769738-46769920 on chromosome 2 of the tomato reference genome; The 26th SSR locus is located on chromosome 8 of the tomato reference genome at positions 55139338-55139629. The 27th SSR locus is located on chromosome 12 of the tomato reference genome at positions 6759631-6759785. The 28th SSR locus is located on chromosome 4 of the tomato reference genome at positions 6220284-6220510. The tomato reference genome is the Heinz 1706 v3.0 reference genome sequence; The first SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing; The second SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing; The nucleotide sequences of the third SSR primer pair are shown in SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing; The fourth SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8 in the sequence listing; The fifth SSR primer pair has the nucleotide sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing; The sixth SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 11 and SEQ ID NO: 12 in the sequence listing; The nucleotide sequences of the seventh SSR primer pair are shown in SEQ ID NO: 13 and SEQ ID NO: 14 in the sequence listing; The eighth SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16 in the sequence listing; The nucleotide sequences of the ninth SSR primer pair are shown in SEQ ID NO: 17 and SEQ ID NO: 18 in the sequence listing; The 10th SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20 in the sequence listing; The eleventh SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22 in the sequence listing; The 12th SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 23 and SEQ ID NO: 24 in the sequence listing; The 13th SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 25 and SEQ ID NO: 26 in the sequence listing; The nucleotide sequences of the fourteenth SSR primer pair are shown in SEQ ID NO: 27 and SEQ ID NO: 28 in the sequence listing; The 15th SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 29 and SEQ ID NO: 30 in the sequence listing; The sixteenth SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 31 and SEQ ID NO: 32 in the sequence listing; The 17th SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 33 and SEQ ID NO: 34 in the sequence listing; The 18th SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 35 and SEQ ID NO: 36 in the sequence listing; The nucleotide sequences of the nineteenth SSR primer pair are shown in SEQ ID NO: 37 and SEQ ID NO: 38 in the sequence listing. The 20th SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 39 and SEQ ID NO: 40 in the sequence listing; The nucleotide sequences of the twenty-first SSR primer pair are shown in SEQ ID NO: 41 and SEQ ID NO: 42 in the sequence listing. The nucleotide sequences of the twenty-second SSR primer pair are shown in SEQ ID NO: 43 and SEQ ID NO: 44 in the sequence listing. The nucleotide sequences of the 23rd SSR primer pair are shown in SEQ ID NO: 45 and SEQ ID NO: 46 in the sequence listing. The nucleotide sequences of the 24th SSR primer pair are shown in SEQ ID NO: 47 and SEQ ID NO: 48 in the sequence listing. The nucleotide sequences of the 25th SSR primer pair are shown in SEQ ID NO: 49 and SEQ ID NO: 50 in the sequence listing. The nucleotide sequences of the twenty-sixth SSR primer pair are shown in SEQ ID NO: 51 and SEQ ID NO: 52 in the sequence listing. The nucleotide sequences of the 27th SSR primer pair are shown in SEQ ID NO: 53 and SEQ ID NO: 54 in the sequence listing. The 28th SSR primer pair has nucleotide sequences as shown in SEQ ID NO: 55 and SEQ ID NO: 56 in the sequence listing.

2. The SSR primer combination for identifying the authenticity of tomato varieties according to claim 1, characterized in that, One primer in each primer pair is linked to a fluorescent molecule.

3. The SSR primer combination for identifying the authenticity of tomato varieties according to claim 2, characterized in that, The fluorescent molecules are selected from ROX, TAMRA, FAM, and HEX.

4. A kit for identifying the authenticity of tomato varieties, characterized in that: The kit is configured as a PCR reaction system; the PCR reaction system includes: The SSR primer combination according to any one of claims 1-3.

5. The use of the SSR primer combination according to any one of claims 1-3 or the kit according to claim 4 in the following X 1 or X 2: X1: To determine whether the tomato variety to be tested belongs to one of the standard tomato varieties; X2: Identify which specific standard tomato variety the tomato being tested belongs to; The standard tomato varieties are selected from the following 22 tomato varieties: Jingfan 101, Jingfan 102, Jingfan 401, Jingfan 404, Jingfan 303, Jingfan 309, Jingfan 403, Jingfan 302, Jingfan 308, Jingfan 502, Jingfan 603, Jingfan 310, Jingfan 501, Jingfan 701, Jingfan Huangluo No. 1, Jingfan Hongluo No. 2, Jingfan Huangxing No. 7, Jingfan Caixing No. 1, Jingfan Zixing No. 2, Jingfan Lvxing No. 3, Jingfan Fenxing No. 1, and Jingfan Huangxing No.

1.

6. A method for identifying the authenticity of a tomato variety, characterized in that: The detection method includes the following steps: Step 1: Detect the genotypes of the tomato sample and the standard tomato variety at the first to twenty-eighth SSR loci as described in claim 1; Step 2: Determining the variety of the tomato to be tested: If the number of genotypes of the tomato under test based on the first SSR locus to the twenty-eighth SSR locus is 0, and the number of genotypes of a specified variety of standard tomato varieties based on the first SSR locus to the twenty-eighth SSR locus is 0, then the tomato under test and the specified variety of standard tomato varieties belong to the same variety. If the number of genotypes of the tomato under test based on the first SSR locus to the twenty-eighth SSR locus is 1, and the number of genotypes of a specified variety of standard tomato varieties based on the first SSR locus to the twenty-eighth SSR locus is 1, then the tomato under test and the specified variety of standard tomato varieties are similar varieties. If the number of genotypes of the tomato under test based on the first SSR locus to the twenty-eighth SSR locus is greater than or equal to 2 compared with the number of genotypes of each of the standard tomato varieties based on the first SSR locus to the twenty-eighth SSR locus, then the tomato under test is different from each of the standard tomato varieties. The steps for detecting the genotypes of various SSR loci in the test tomato and standard tomato varieties include the following sub-steps: Step 1: Using the genomic DNA of the tomato to be tested and the genomic DNA of the standard tomato variety as templates, PCR amplification is performed using the primer pairs in the SSR primer combination described in any one of claims 1-3 to obtain PCR amplification products; Step 2: Detect the PCR amplification products to obtain the genotypes of the tomato to be tested and the standard tomato varieties based on the first SSR locus to the twenty-eighth SSR locus; The standard tomato varieties are selected from the following 22 tomato varieties: Jingfan 101, Jingfan 102, Jingfan 401, Jingfan 404, Jingfan 303, Jingfan 309, Jingfan 403, Jingfan 302, Jingfan 308, Jingfan 502, Jingfan 603, Jingfan 310, Jingfan 501, Jingfan 701, Jingfan Huangluo No. 1, Jingfan Hongluo No. 2, Jingfan Huangxing No. 7, Jingfan Caixing No. 1, Jingfan Zixing No. 2, Jingfan Lvxing No. 3, Jingfan Fenxing No. 1, and Jingfan Huangxing No.

1.

7. The detection method according to claim 6, characterized in that: The detection method for step two is as follows: Method for detecting fragments of amplified products: Detect the fragment size of the PCR amplified products to obtain the genotypes of the tomato to be tested and the standard tomato variety based on the first SSR locus to the twenty-eighth SSR locus.

8. The detection method according to claim 6, characterized in that: In step two, the result of the determination is obtained based on cluster analysis.

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