Molecular marker related to germination capability of tomato seeds and application of molecular marker
By using genome-wide association analysis and KASP-SNP molecular marker technology, the germination ability of tomato seeds was identified, solving the problem of screening tomato seed germination ability and enabling rapid and efficient variety screening and breeding.
Patent Information
- Application Number
- CN202511465663.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-14
- Publication Date
- 2025-12-09
AI Technical Summary
In the current technology, the genetic variation patterns and regulatory mechanisms of tomato seed germination ability have not been fully studied, making it difficult to quickly screen out tomato varieties with strong germination ability.
Single nucleotide polymorphism sites associated with tomato seed germination were identified through genome-wide association analysis. KASP-SNP molecular markers were designed, and PCR reactions were performed using specific primer sets to determine the germination ability of tomato seeds.
It achieves highly consistent and clearly defined population typing results in tomato populations, enabling rapid and efficient screening of superior tomato varieties and shortening the breeding cycle.
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Figure CN121087218A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of nucleic acid molecular marker detection technology, specifically relating to a molecular marker related to the germination ability of tomato seeds and its application. Background Technology
[0002] Seed germination marks the beginning of a plant's life cycle and is a prerequisite for plant growth, development, and reproduction. For annual crops, successful seed germination leads to rapid and uniform emergence, which is essential for improving crop yield and quality. Research indicates that the seed germination stage is finely regulated by multiple factors, including plant hormones, genetic factors, and environmental factors. Among these, the regulatory effects of plant hormones gibberellin and abscisic acid on seed germination are relatively well-studied (Reed, et al . Seedgermination and vigor: ensuring crop sustainability in a changing climate. Hereditas , 2022, 128(6), 450-459.). In addition, DOG1 , DOG18 These genetic loci participate in the regulation of plant seed germination through multiple pathways, including hormones (Krüger, et al . DOG1 controls dormancyindependently of ABA core signaling kinases regulation by preventing AFPdephosphorylation through AHG1. Science Advances, 2025, 11(9).; Xiang, et al .Sequence polymorphisms at the REDUCED DORMANCY5 pseudophosphatase underlienatural variation in Arabidopsis dormancy. Plant Physiology, 2016, 171(4), 2659-2670.). Therefore, exploring the genetic variation patterns and regulatory mechanisms of seed germination is of great significance for creating crops with strong germination capabilities.
[0003] Genome-wide association study (GWAS) is an important genetic analysis technique that identifies phenotypic genetic variants and discovers key regulatory genes by analyzing associations between phenotypes and variations in large populations. Key variants located by GWAS can be precisely distinguished from different genotypes using single nucleotide polymorphism (SNP)-based molecular markers, helping researchers quickly screen genetic material. Kompetitive Allele Specific PCR (KASP) is a commonly used SNP molecular marker technique that can meet genotyping requirements of varying throughputs, offering high accuracy, ease of operation, and low detection costs. It is currently widely used in marker-assisted breeding of animals and plants.
[0004] tomato( Solanum lycopersicum Tomato (L.) is an annual herbaceous plant belonging to the genus *Solanum* of the Solanaceae family. Its fruit is widely loved by consumers for its sweet and sour taste, rich nutrition, and diverse varieties, making it one of the world's most important vegetable crops. my country is a major tomato producer, and good seed quality is an important prerequisite for the healthy and orderly development of the tomato seed market. Previous research has identified genetic loci affecting tomato seed germination, including... LeMAN2 , SpMPK3 , LeXET4 However, its variation patterns and distribution in natural tomato populations still need further study (Belotserkovsky, etc.). et al . Specific role ofLeMAN2 in the control of seed germination exposed by overexpression of theLeMAN3 gene in tomato plants. Planta , 2007, 227(1), 199-209.; Li, et al .Silencing the SpMPK1, SpMPK2, and SpMPK3 genes in tomato reduces abscisic acid-mediated drought tolerance. International Journal of Molecular Sciences ,2013, 14(11), 21983-21996.; Chen, et alA gibberellin-regulated xyloglucan endotransglycosylase gene is expressed in the endosperm cap during tomato seed germination. Journal of Experimental Botany , 2002, 53(367), 215-223.
[0005] Therefore, identifying SNP sites associated with tomato seed germination by GWAS analysis method, and developing KASP-SNP molecular markers have important significance for creating tomato germplasm and new varieties with strong germination ability. SUMMARY
[0006] The purpose of the present application is to provide a KASP-SNP marker related to tomato seed germination, which is used for comparing and identifying the seed germination ability of different varieties of tomatoes, and assisting in breeding tomato varieties with rapid germination and uniform emergence.
[0007] To achieve the above purpose, the present application adopts the following technical solutions: The present application obtains a plurality of single nucleotide polymorphism sites related to tomato seed germination by whole genome association analysis of large-scale samples, further designs KASP primers, and performs genotyping identification in a tomato population to develop a molecular marker with good genotyping effect.
[0008] The present application uses the screened single nucleotide polymorphism site as a detection target for identifying the seed germination ability of tomatoes, the single nucleotide polymorphism site is the 59512287th site of the full-length sequence of chromosome 3 of the tomato genome, there is a G>A polymorphism, and the version of the tomato genome is ITAG4.0.
[0009] Specifically, the single nucleotide polymorphism site is the SL3_59512287 site, which is located at the 59512287th site of the full-length sequence of chromosome 3 of the tomato genome ITAG4.0 version, and the base is G or A. When the genotype is GG, the germination rate of tomato seeds is low and the germination potential is weak; when the genotype is AA, the germination rate of tomato seeds is high and the germination potential is strong.
[0010] The present application research shows that using the above-mentioned single nucleotide polymorphism site as a detection target in a tomato population verifies a highly consistent and clear population genotyping effect, which can clearly distinguish tomato materials of different genotypes, and the seed germination index of different genotypic materials reaches a highly significant level.
[0011] Further, the application comprises: using the genomic DNA of the tomato plant to be tested as a template, and using a primer set for specifically detecting the single nucleotide polymorphism site to perform a PCR reaction, so as to determine the genotyping of the tomato to be tested, and to predict the germination ability of the tomato seeds.
[0012] Another object of the application is to provide a method for detecting the germination ability of tomato seeds, which comprises the following steps: (1) extracting the genomic DNA of the tomato plant to be tested; (2) using the genomic DNA of the tomato plant as a template, and using a primer set for amplifying the molecular marker related to the germination of tomato seeds to perform PCR amplification, wherein the primer set is used for specifically detecting the G>A single nucleotide polymorphism existing at position 59512287 of the full-length sequence of chromosome 3 of the tomato genome, and the version of the tomato genome is ITAG4.0; (3) detecting the PCR product, and genotyping the tomato to be tested; when the genotyping is identified as GG, it is determined that the germination rate of the tomato seeds is low and the germination potential is weak; when the genotyping is identified as AA, it is determined that the germination rate of the tomato seeds is high and the germination potential is strong.
[0013] The application develops a KASP-SNP molecular marker related to the germination of tomato seeds according to the single nucleotide polymorphism site, and specifically, a nucleotide fragment upstream and downstream of the single nucleotide polymorphism site is intercepted as the KASP-SNP molecular marker.
[0014] Preferably, the molecular marker is a nucleotide sequence of 40-110 bp upstream and downstream of the single nucleotide polymorphism site.
[0015] More preferably, the nucleotide sequence of the molecular marker is shown in SEQ ID NO. 1.
[0016] The application designs a primer set according to the single nucleotide polymorphism site in the molecular marker, and the primer set comprises a specific forward primer 1, a specific forward primer 2 and a universal reverse primer.
[0017] The research of the application shows that KASP amplification is performed using the primer set, and a highly consistent and clear population genotyping effect is obtained in the verification of the tomato population.
[0018] As preferred, two different fluorescent label sequences are connected to the 5' end of the specific forward primer 1 and the specific forward primer 2 respectively. The identification of different genotypes can be achieved by identifying the fluorescent labels.
[0019] More preferably, the 5' end of the two specific forward primers is labeled with a FAM group and a HEX group respectively.
[0020] As preferred, in step (2), the PCR reaction system is: 20 ng / μL of template DNA 0.8 μL, 2 × KASP premix 0.75 μL, primer mixture 0.05 μL, wherein the preparation of the primer mixture is to mix the forward primer 1, the forward primer 2, the reverse primer and ddH2O with a volume ratio of 12:12:30:46.
[0021] As preferred, in step (2), the PCR reaction program is: first step: 94℃ pre-denaturation for 15 minutes; second step: 94℃ for 20 seconds, 61~55℃ annealing for 60 seconds, a total of 10 cycles, and the temperature decreases by 0.6℃ for each cycle; third step: 94℃ for denaturation for 20 seconds, 55℃ for annealing for 60 seconds, a total of 26 cycles.
[0022] Another object of the present application is to provide the application of the above-mentioned primer set in the preparation of a kit for detecting the germination ability of tomato seeds. The single nucleotide polymorphism site with G>A polymorphism at position 59512287 of the full-length sequence of chromosome 3 of the tomato genome is used as a detection target to develop a corresponding detection kit, and the detection kit comprises a primer set for specifically detecting the single nucleotide polymorphism site in the tomato genome, and the primer set comprises two specific forward primers and one universal reverse primer.
[0023] The application of the kit includes: a. identifying or assisting in identifying the germination ability of tomato seeds; b. assisting in breeding tomato single plants or varieties; c. genetic analysis of tomato materials.
[0024] The present application has the following beneficial effects: (1) The present application identifies a representative and universal single nucleotide polymorphism site related to the germination of tomato seeds through whole genome association analysis, further develops a primer set, detects by KASP method, and obtains a highly consistent and clear population genotyping effect in different materials, thereby providing a rapid and reliable detection method for genetic improvement of tomatoes.
[0025] (2) The seed germination ability assisted screening is carried out by using the molecular marker, high-throughput detection can be realized, excellent tomato varieties are screened quickly and efficiently, and the breeding period is greatly shortened. BRIEF DESCRIPTION OF DRAWINGS
[0026] Figure 1 The Manhattan plot for whole genome association analysis related to seed germination ability, and the dotted line is the threshold level of -log 10 P value =8.39 significantly related threshold level line.
[0027] Figure 2 The allelic typing diagram of SL3_59512287 KASP-SNP marker in 22 materials.
[0028] Figure 3 The allelic typing diagram of SL3_59496397 KASP-SNP marker in 22 materials.
[0029] Figure 4 The allelic typing diagram of SL3_59498965 KASP-SNP marker in 22 materials.
[0030] Figure 5 The allelic typing diagram of SL3_59500049 KASP-SNP marker in 22 materials.
[0031] Figure 6 The allelic typing diagram of SL3_59504907 KASP-SNP marker in 22 materials.
[0032] Figure 7 The allelic typing diagram of SL3_59506701 KASP-SNP marker in 22 materials.
[0033] Figure 8 The allelic typing diagram of SL3_59508380 KASP-SNP marker in 22 materials.
[0034] Figure 9 The allelic typing diagram of SL3_59508846 KASP-SNP marker in 22 materials.
[0035] Figure 10 The allelic typing diagram of SL3_59510138 KASP-SNP marker in 22 materials.
[0036] Figure 11 The allelic typing diagram of SL3_59512398 KASP-SNP marker in 22 materials.
[0037] Figure 12 Figure 2. Allele genotyping plot for SL2_59512287 KASP-SNP marker in 319 materials.
[0038] Figure 13 Figure 3. Phenotypic difference plot of seed germination ability allelic variation for SL2_59512287 KASP-SNP marker in 319 materials. DETAILED DESCRIPTION
[0039] The preferred embodiments of the present application will be described in detail below with reference to the attached drawings. It should be understood that the following embodiments are given by way of example only and are not intended to limit the scope of the present application. Various modifications and alterations of the present application will become apparent to those skilled in the art from this disclosure, without departing from the spirit and scope of the present application.
[0040] The experimental methods used in the following examples are generally in accordance with conventional conditions or as suggested by the manufacturer, unless otherwise specified.
[0041] The materials, reagents, etc. used in the following examples can be purchased commercially, unless otherwise specified.
[0042] Example 1: Mining of SNP sites related to tomato germination ability 1. Materials: A tomato core population was constructed using 319 tomato natural populations. The materials were derived from the germplasm resources preserved by the Tomato and Pepper Group of the Zhejiang Academy of Agricultural Sciences, and the specific information is shown in Table 1.
[0043] Table 1. 319 tomato materials
[0044] 2. The method for investigating seed germination ability is as follows: 50-100 full and glossy seeds without defects were selected from each material and placed in a culture dish lined with two layers of wet filter paper for 7 days, and constant light and humidity were maintained. The emergence of radicles was used as the germination standard, and the number of germinated seeds was recorded every day for 7 days.
[0045] Statistical analysis of germination rate was achieved using the germination index, which was calculated as follows: Germination index = (n3x7 + n4x6 + n5x5 + n6x4 + n7x3) ni represents the germination rate on the i-th day (3≤i≤7) (germination rate = number of germinated seeds / total number of total seeds).
[0046] 3. The SNP site results obtained by resequencing 319 tomato materials were screened, and the screening criteria were minimum allele frequency (MAF) > 0.05 and deletion rate < 20%, only sites with double alleles were reserved, and finally 12392992 SNP sites were obtained. GEMMA was used to perform association analysis on the correlation traits of different populations, and the -log 10 P value = 8.39 as the threshold level of significant correlation to screen out potential candidate SNPs, such as Figure 1 .
[0047] 4. Screening results: two segments significantly related to germination index were identified in the whole genome, and the segment on chromosome 3 of the tomato genome had the largest genetic contribution to seed germination. Ten SNP sites were selected for preliminary screening of the sites. The above tomato genome version is ITAG4.0. The 10 SNP sites are: 1. SL3_59512287 site, located at position 59512287 of the full-length sequence of chromosome 3 of the tomato genome ITAG4.0 version, base G or A; 2. SL3_59496397 site, located at position 59496397 of the full-length sequence of chromosome 3 of the tomato genome ITAG4.0 version, base G or T; 3. SL3_59498965 site, located at position 59498965 of the full-length sequence of chromosome 3 of the tomato genome ITAG4.0 version, base C or T; 4. SL3_59500049 site, located at position 59500049 of the full-length sequence of chromosome 3 of the tomato genome ITAG4.0 version, base G or C; 5. SL3_59504907 site, located at position 59504907 of the full-length sequence of chromosome 3 of the tomato genome ITAG4.0 version, base C or T; 6. SL3_59506701 site, located at position 59506701 of the full-length sequence of chromosome 3 of the tomato genome ITAG4.0 version, base G or deletion; 7. SL3_59508380 site, located at position 59508380 of the full-length sequence of chromosome 3 of the tomato genome ITAG4.0 version, base C or T; 8. SL3_59508846 locus, located at the 59508846th base of the full-length sequence of chromosome 3 in the ITAG4.0 version of the tomato genome, and the base is G or deletion; 9. SL3_59510138 locus, located at the 59510138th base of the full-length sequence of chromosome 3 in the ITAG4.0 version of the tomato genome, and the base is C or T; 10. SL3_59512398 locus, located at the 59512398th base of the full-length sequence of chromosome 3 in the ITAG4.0 version of the tomato genome, and the base is T or G.
[0048] Example 2: Development of KASP-SNP molecular markers related to the germination ability of tomato 1. According to the SNP locus provided in Example 1, download the flanking sequence containing the above-mentioned SNP locus from Sol Genomics Network, design KASP primers, design two specific forward primers and one universal reverse primer for each KASP-SNP molecular marker, and the 5' ends of the two specific forward primers are respectively connected with a fluorescent tag sequence carrying a FAM group and a fluorescent tag sequence carrying a HEX group. The specific sequences are shown in Table 2.
[0049] Table 2. 10 pairs of KASP primer groups
[0050] 2. Use the KASP primer group of the above-mentioned SNP locus to amplify and fluorescently detect 22 tomato materials (see Table 3, the materials are derived from the germplasm resources preserved by the Tomato and Pepper Group of the Vegetable Institute of Zhejiang Academy of Agricultural Sciences), and further screen KASP-SNP molecular markers with good population typing effect.
[0051] Table 3. 22 tomato materials
[0052] Fresh leaves of 22 tomato materials were collected, and tomato genomic DNA was extracted by magnetic bead method. Using the IntelliQube genotyping platform, the genomic DNA samples of the above-mentioned 22 tomato materials were amplified and fluorescently detected using 10 pairs of KASP primer groups with the template of the tomato genomic DNA to be tested.
[0053] PCR system: 20 ng / μL template DNA 0.8 μL, 2 × KASP premix 0.75 μL, primer mixture 0.05 μL, primer mixture contains 100 μM specific primer Primer1: 100 μM specific forward primer Primer2: 100 μM universal primer: ddH2O (volume ratio) = 12: 12: 30: 46; PCR reaction program: step 1: 94℃, 15min; step 2: 94℃, 20s, 61~55℃, 60s, each cycle of annealing temperature decreases by 0.6℃, 55℃, 60s, a total of 10 cycles; step 3: 94℃, 20s, 55℃, 60s, a total of 26 cycles.
[0054] IntelliQube full-automatic PCR instrument is used for fluorescence data reading and analysis, and the typing chart presented after the human color setting of the detection result is as follows Figures 2-11 The red dot close to the abscissa represents the sample (homozygous genotype 1) which can combine with the forward primer F1 carrying FAM fluorescent linker to produce PCR amplification signal, the blue dot arranged along the Y axis direction close to the ordinate represents the sample (homozygous genotype 2) which can combine with the forward primer F2 carrying HEX fluorescent linker to produce PCR amplification signal, and the purple dot gathered in the middle represents the sample (heterozygous) which can simultaneously combine with the primer carrying FAM and HEX two kinds of fluorescent signals to produce PCR amplification, the black hollow dot represents the amplification signal of negative control (NTC), and the gray hollow dot represents that the sample does not detect amplification signal or the typing result is not clear and cannot be accurately assigned.
[0055] Finally, the primer group SL3_59512287 with significant fluorescence signal clustering typing trend and sample signal amplification data is selected, and the site and upstream and downstream sequences and primer group sequences are shown in SEQ ID NO. 1~4, see Table 2.
[0056] Example 3: Application of KASP-SNP molecular marker in selection of tomato germination ability In spring 2025, 319 population materials (see Table 1) were planted in Sangyuan, Xinqü, Zhejiang Academy of Agricultural Sciences, 6 holes of each tomato material were planted in the seedling tray, and after germination, the most robust 4 plants were selected and transplanted in the water culture greenhouse in Sangyuan, and managed regularly.
[0057] The seed germination ability investigation method is referred to example 1.
[0058] Fresh leaves of the tomato materials to be tested were collected, genomic DNA was extracted by magnetic bead method, and the primer group of SL3_59512287 was used to amplify and fluorescently detect the genomic DNA samples of the above population by using IntelliQube genotyping platform.
[0059] The PCR system was as follows: 0.8 μL of 20 ng / μL template DNA, 0.75 μL of 2 × KASP premix, and 0.05 μL of primer mixture. The primer mixture consisted of 100 μM specific primer Primer1: 100 μM specific forward primer Primer2: 100 μM universal primer: ddH2O (volume ratio) = 12:12:30:46. PCR reaction procedure: Step 1: 94℃, 15 minutes; Step 2: 94℃, 20 seconds, 61~55℃, 60 seconds, decreasing the annealing temperature by 0.6℃ for each cycle, 55℃, 60 seconds, for a total of 10 cycles; Step 3: 94℃, 20 seconds, 55℃, 60 seconds, for a total of 26 cycles.
[0060] like Figure 12 As shown, the 319 samples were clearly distinguished and clustered into three categories. The red signal points indicate that the sample carries the FAM fluorescence signal, and the corresponding tomato genotype is GG; the blue signal points indicate that the sample carries the HEX signal, and the corresponding tomato genotype is AA; the purple signal points indicate that the sample carries both FAM and HEX signals, and the corresponding tomato genotype is GA; the black dots near the origin represent the amplification signal of the negative control (sample without added DNA).
[0061] like Figure 13 As shown in Table 4, combining the germination index data and genotyping results, the average germination index of materials with genotype GG was 13.43, and the average germination index of materials with genotype AA was 17.14. The difference in germination index between the two genotypes reached a highly significant level. p (≤0.0001), AA genotype materials have a higher germination index, higher germination rate, and faster germination speed than GG genotype materials.
[0062] Table 4. Genotypes and germination indices of 319 materials
[0063] In summary, this invention provides an SNP locus SL3_59512287, which is closely linked to the tomato germination ability gene and exhibits G / A polymorphism at 59512287 bp on chromosome 3 of the tomato genome. Further, related KASP molecular markers and primers were developed for verification. These primers were then used to amplify the material to be identified, and favorable allelic variations in germination ability were determined based on the G / A genotype. This invention can be applied to marker-assisted breeding.
[0064] Finally, it is to be understood that the application is not limited to the particulars of the foregoing description and / or the specific embodiments described above, as modifications and / or improvements thereto will readily occur to those skilled in the art. Consequently, the present application is to cover any and all modifications and / or improvements which fall within the scope of the application as claimed.
Claims
1. Use of a single nucleotide polymorphism site as a detection target in identifying the germination ability of tomato seeds, characterized in that, The single nucleotide polymorphism site is the 59512287th site of the full-length sequence of chromosome 3 of a tomato genome, and a G>A polymorphism exists, and the tomato genome version is ITAG4.
0.
2. Use according to claim 1, wherein The application comprises: using a genome DNA of a tomato plant to be tested as a template, and performing a PCR reaction by using a primer group for specifically detecting the single nucleotide polymorphism site, so as to determine the genotyping of the tomato to be tested, and to predict the seed germination ability of the tomato.
3. A method for detecting the germination capacity of tomato seeds, characterized in that, The application comprises the following steps: (1) extracting the genome DNA of the tomato plant to be tested; (2) using the genome DNA of the tomato plant as a template, and performing PCR amplification by using a primer group for amplifying a tomato seed germination-related molecular marker, wherein the primer group is used for specifically detecting the G>A single nucleotide polymorphism existing at the 59512287th site of the full-length sequence of chromosome 3 of a tomato genome, and the tomato genome version is ITAG4.0; (3) detecting the PCR product, and genotyping the tomato to be tested, wherein when the genotyping is identified as GG, it is determined that the seed germination rate of the tomato is low, and the germination potential is weak; and when the genotyping is identified as AA, it is determined that the seed germination rate of the tomato is high, and the germination potential is strong.
4. The method for testing the germination capacity of tomato seeds according to claim 3, characterized in that, The tomato seed germination-related molecular marker is a nucleotide sequence of 40-110 bp obtained from the upstream and downstream of the single nucleotide polymorphism site.
5. The method for testing the germination capacity of tomato seeds according to claim 4, characterized in that, The nucleotide sequence of the tomato seed germination-related molecular marker is shown in SEQ ID NO.
1.
6. The method for testing the germination capacity of tomato seeds according to any one of claims 3 to 5, characterized in that, The primer group comprises a specific forward primer 1, a specific forward primer 2 and a universal reverse primer, and the nucleotide sequences are shown in SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
7. The method for testing the germination capacity of tomato seeds according to claim 6, characterized in that, The 5' ends of the specific forward primer 1 and the specific forward primer 2 are respectively connected with two different fluorescent label sequences.
8. The method for testing the germination capacity of tomato seeds according to claim 7, characterized in that, The 5' ends of the two specific forward primers are respectively labeled with FAM groups and HEX groups.
9. The method for testing the germination capacity of tomato seeds according to claim 3, characterized in that In step (2), the PCR reaction system is: 20 ng / μL of template DNA 0.8 μL, 2 × KASP premix 0.75 μL, and primer mixture 0.05 μL, wherein the primer mixture is prepared by mixing the forward primer 1, the forward primer 2 and the reverse primer with a concentration of 100 μM with ddH2O in a volume ratio of 12:12:30:
46. The PCR reaction program is: the first step: 94℃ pre-denaturation for 15 minutes; the second step: 94℃ for 20 seconds, 61-55℃ for 60 seconds, a total of 10 cycles, and the temperature decreases by 0.6℃ for each cycle; the third step: 94℃ for 20 seconds, 55℃ for 60 seconds, a total of 26 cycles.
10. Use of a primer set for the preparation of a kit for detecting the germination capacity of tomato seeds, characterized in that, The primer group is used for specifically detecting the G>A single nucleotide polymorphism existing at the 59512287th site of the full-length sequence of chromosome 3 of a tomato genome, and the tomato genome version is ITAG4.0; and the application of the kit comprises: identifying or assisting in identifying the seed germination ability of the tomato; assisting in breeding a single tomato plant or variety; and gene analysis of tomato materials.