A method for identifying the purity of a tomato hybrid and an InDel primer combination used thereby

Abstract

The application discloses a method for identifying purity of a tomato hybrid and an InDel primer combination used in the method. The InDel primer combination provided by the application is composed of eight primer groups; each primer group is composed of two primer sequences, and is used for amplifying an InDel site; and the nucleotide sequences of the primers are shown in SEQ ID NO:1 to SEQ ID NO:16 in sequence. The InDel primer combination can be used for early identification in the seed or seedling stage of the tomato hybrid, so as to guarantee the purity of the hybrid, protect the rights and interests of producers and breeders, and provide technical support for seed quality management of the tomato variety. The method provided by the application has the advantages of accuracy, low cost, simple operation, clear results, saving of manpower and material resources, and the like, and has a very wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of hybrid seed purity identification, and particularly relates to a method for identifying the purity of tomato hybrid seeds and an InDel primer combination used therein. Background Art

[0002] As one of the main vegetables in my country, tomato is one of the largest vegetables in the world in terms of planting scale and yield. According to statistics, the planting area of ​​tomatoes in my country is about 95.14hm2. 2 , with an annual output of 48.749 million tons. Currently, 3,305 tomato varieties have been registered nationwide. Hybrids have become a major force in tomato production due to their excellent genetic characteristics and production performance. However, before hybrids can enter the market, they must undergo rigorous purity testing. According to the "Regulations for the Inspection of Crop Seeds" (GB / T3543.1-3543.7-1995), tomato hybrids must have a purity level exceeding 98%. Currently, traditional field purity testing methods are costly and time-consuming, failing to meet the rapid development of the modern seed industry. Therefore, DNA-based molecular detection technology has emerged. With its low cost and high efficiency, it has quickly become the preferred method for determining the purity of tomato hybrids. By selecting the appropriate molecular marker combination for DNA detection technology, we can more accurately and rapidly complete purity testing, effectively preventing the circulation of counterfeit and substandard seeds, protecting farmers' rights, and promoting the sustainable development of the tomato industry. Therefore, selecting the right molecular marker combination for DNA-based tomato hybrid purity testing is crucial.

[0003] InDel (Insertion-Deletion) markers are insertion-deletion markers, meaning that a certain number of nucleotides are inserted or deleted in the genome of other samples relative to the reference genome. InDel variations are widely distributed throughout the genome, second only to SNP variations in number. Specific InDel marker primers can be designed based on the sequences flanking the insertion-deletion site. Compared to SNP and SSR markers, Indel markers have a wider range of applicability and can be detected using a standard agarose electrophoresis platform. They also have lower requirements for sample DNA quality, and their banding stability and interpretation are superior to SSR markers. They offer advantages such as good stability, wide distribution, high polymorphism, strong versatility, a simple typing system, and low cost.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, one of the objectives of the present invention is to provide an InDel primer combination that can be used to identify the purity of tomato hybrids.

[0006] A second object of the present invention is to provide a kit comprising the above primer combination.

[0007] A third object of the present invention is to provide an application of the above primer combination or kit.

[0008] A fourth object of the present invention is to provide a method for identifying the purity of a tomato hybrid to be tested.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides an InDel primer set comprising a first primer set to an eighth primer set, which amplify eight InDel markers to perform genotyping (or determining homozygosity or heterozygosity) for each InDel marker, wherein:

[0011] The eight InDel markers include:

[0012] Marker FQ_Ind18: located at position 71483275 on chromosome 9, and is nucleotide G or the nucleotide sequence shown in SEQ ID NO. 17;

[0013] Marker FQ_Ind24: located at position 58381796 on chromosome 12, and is nucleotide A or the nucleotide sequence shown in SEQ ID NO. 18;

[0014] Marker FQ_Ind26: located at position 67342246 on chromosome 3, and is nucleotide A or the nucleotide sequence shown in SEQ ID NO.19;

[0015] Marker FQ_Ind27: located at position 35307300 on chromosome 4, and is nucleotide A or the nucleotide sequence shown in SEQ ID NO. 20;

[0016] Marker FQ_Ind29: located at position 46541839 on chromosome 5, and is nucleotide A or the nucleotide sequence shown in SEQ ID NO. 21;

[0017] Marker FQ_Ind33: located at position 35630796 on chromosome 11, and is nucleotide T or the nucleotide sequence shown in SEQ ID NO. 22;

[0018] Marker FQ_Ind34: located at position 17984202 on chromosome 12, and is nucleotide A or the nucleotide sequence shown in SEQ ID NO. 23;

[0019] Marker FQ_Ind47: located at position 48618696 on chromosome 11, and is nucleotide T or the nucleotide sequence shown in SEQ ID NO. 24;

[0020] The positions of the eight InDel markers on the chromosome were determined based on the sequence alignment of the tomato reference genome SL3.0;

[0021] The first primer group (primer group 1) is used to amplify the molecular marker FQ_Ind18; the second primer group (primer group 2) is used to amplify the molecular marker FQ_Ind24; the third primer group (primer group 3) is used to amplify the molecular marker FQ_Ind26; the fourth primer group (primer group 4) is used to amplify the molecular marker FQ_Ind27; the fifth primer group (primer group 5) is used to amplify the molecular marker FQ_Ind29; the sixth primer group (primer group 6) is used to amplify the molecular marker FQ_Ind33; the seventh primer group (primer group 7) is used to amplify the molecular marker FQ_Ind34; and the eighth primer group (primer group 8) is used to amplify the molecular marker FQ_Ind47.

[0022] The InDel primer combination provided by the present invention can be used to identify the purity of tomato hybrids. If the PCR product of the primer set used to identify the purity of a certain tomato hybrid is a heterozygous band, it means that the tomato variety being tested is a hybrid. If it is a single band, it means that the tomato variety being tested is not a hybrid, but may be one of the parents, that is, the parent is mixed with the hybrid.

[0023] The hybrids described in the present invention refer to first-generation hybrids, in which the parents may be mixed.

[0024] Furthermore, in the InDel primer combination,

[0025] The first primer set consists of a forward primer F1 shown in SEQ ID NO.1 and a reverse primer R1 shown in SEQ ID NO.2;

[0026] The second primer set consists of a forward primer F2 shown in SEQ ID NO.3 and a reverse primer R2 shown in SEQ ID NO.4.

[0027] The third primer set consists of a forward primer F3 shown in SEQ ID NO.5 and a reverse primer R3 shown in SEQ ID NO.6.

[0028] The fourth primer set consists of a forward primer F4 shown in SEQ ID NO.7 and a reverse primer R4 shown in SEQ ID NO.8.

[0029] The fifth primer set consists of a forward primer F5 shown in SEQ ID NO.9 and a reverse primer R5 shown in SEQ ID NO.10.

[0030] The sixth primer set consists of a forward primer F6 shown in SEQ ID NO.11 and a reverse primer R6 shown in SEQ ID NO.12.

[0031] The seventh primer set consists of a forward primer F7 shown in SEQ ID NO.13 and a reverse primer R7 shown in SEQ ID NO.14.

[0032] The eighth primer set consists of a forward primer F8 shown in SEQ ID NO.15 and a reverse primer R8 shown in SEQ ID NO.16.

[0033] The second aspect of the present invention provides a kit comprising the InDel primer combination of the first aspect.

[0034] In addition to the primer combination, the kit may also include auxiliary reagents for PCR, such as ultrapure water, PCR buffer, dNTPs, and DNA polymerase.

[0035] In the above primer set, when performing PCR, the molar ratio of the primer containing "F" in its name to the primer containing "R" in its name can be specifically 1:1.

[0036] The third aspect of the present invention provides the use of the InDel primer combination of the first aspect or the kit of the second aspect in identifying the purity of tomato hybrids.

[0037] Further, the tomato hybrid varieties are selected from the group consisting of Angelia, Weigens Xiachuan No. 7, Weigens Hanxiang, Weigens Xiayu No. 10, Meishuai, Xiarihong, Shengtao T610, Jing T308, Baowadi, Thomas, Buffett, Daxi, Senna, Zhongyan TV1, Hui 1617, Xiariyangguang, Tianbei, Hongzha 33, IVF6260, IVF6172, IVF3311, Meishengda, Newnham 1718, Huanxi, Dedesi, Jingfen 801, Xindandan No. 4, Jiaban No. 1, Yiban No. 1, Jingban 309, Mucida Fen 80, Jingban 203, Haoyin 220, Jingban 701, Fureite 1681, Weigens Dageda, Jingban Fenxing No. 1, Jingban Huangxing No. 1, Jingban Zixing No. 2, Yingguan 218, Hangfen No. 2, Chuntao 70, Hongyali, Grammy, Baoliangyuan No. 5, Gutan, Boya, IVF3116, IVF6201, IVF1106, Hongxiaobei, Emily, Baidisi, Yinghua 101, Nicola, Jinxiu 202, Heweisi No. 1, SV7846TH, Jingban Heiluohan, Jidali 5605, Fenderli 897, Liyuantianhong, Luoshi, Dongfangmei, Fushan 88, Ruila, Baofa 666, Fiten 158, Xiwu, Younidya Fenli, Beli 1319, Xishido, Baoliangyuan No. 1, Pengyou No. 1, Hejunsheng No. 1, Temete 56, Yinhui 99, Huachen 70, Kevin, Sibaidi, Hanyu 522, Ainila, Kede Shuangli 87170, Kede Yucheng 8722, N6426, Duo xi 8, Guan Shu Mingzhu, Kede 87553, SVTG6210, Nongge 521.

[0038] The above-mentioned tomato hybrid varieties are all commercially available. Among the above-mentioned tomato hybrid varieties, at least one of the eight InDel markers mentioned in the first aspect of the present application is heterozygous, and the tomato hybrid varieties with one heterozygous site are 19, the tomato hybrid varieties with two heterozygous sites are 14, the tomato hybrid varieties with three heterozygous sites are 24, the tomato hybrid varieties with four heterozygous sites are 24, the tomato hybrid varieties with five heterozygous sites are 9, and the tomato hybrid varieties with six heterozygous sites are 6. Therefore, the InDel primer combination provided by the present application can be used for purity identification of the above-mentioned 96 tomato hybrid varieties.

[0039] The fourth aspect of the present application provides a method for identifying the purity of a tomato hybrid variety to be tested, comprising the following steps:

[0040] (1) Randomly select N strains of tomato hybrid varieties to be tested and obtain the genomic DNA thereof;

[0041] (2) using genomic DNA of at least 10 (e.g., 10, 15, or 20) tomato hybrids to be tested selected from the N tomato hybrids to be tested obtained in step (1) as templates, and performing PCR amplification using the first to eighth primer groups in the InDel primer combination provided in the first aspect to obtain corresponding PCR amplification products;

[0042] (3) performing agarose gel electrophoresis on the PCR amplification products obtained in step (2), and counting the number of strains with heterozygous bands of the first to eighth primer sets according to the electrophoresis results; then selecting the primer set with relatively more strains with heterozygous bands (e.g., more than 5 strains, the most strains, the most strains) and clear as the target primer set;

[0043] (4) using the genomic DNA of the N tomato hybrids to be tested obtained in step (1) as templates, and performing PCR amplification using the target primer sets obtained in step (3) to obtain PCR amplification products of each tomato hybrid to be tested under different target primer sets;

[0044] (5) The PCR amplification products obtained in step (4) are subjected to agarose gel electrophoresis. The number of plants with heterozygous bands in the amplification products of each target primer set is counted according to the electrophoresis results. The tomato strains to be tested with heterozygous bands are hybrids. The purity of the tomato hybrid to be tested is obtained based on the number of plants with heterozygous bands amplified by each target primer set.

[0045] In the method of the fourth aspect of the present invention, the target primer set determined in step (3) may be one or two or more. When there are multiple target primer sets, the purity of the hybrid obtained by the multiple target primer sets can be used to determine the final purity of the tomato hybrid to be tested, specifically:

[0046] Count the number of plants showing heterozygous bands and the number of plants without bands for each target primer set, calculate the purity of the tomato hybrid obtained by each target primer set, and then calculate the average value;

[0047] The purity obtained by a certain target primer set = the number of strains showing heterozygous bands for a certain target primer set / (N-the number of strains without bands for the target primer set)×100%.

[0048] Furthermore, the tomato hybrid to be tested is selected from Angelia, Weigens Xiachuan No. 7, Weigens Hanxiang, Weigens Xiayu No. 10, Meishuai, Xiarihong, Shengtao T610, Jing T308, Baowadi, Thomas, Buffett, Daxi, Senna, Zhongyan TV1, Hui 1617, Summer Sunshine, Tianbei, Europe and America, Huajin No. 1, Silver Moon 619, Hongsui, Hongza 33, IVF6260, IVF61 72, IVF3311, Meishengda, Nunhems 1718, Huanxi, Deruisi, Jingfen 801, Xindandan No. 4, Jiafan No. 1, Yifan No. 1, Jingfan 309, Makuta Powder 80, Jingfan 203, Haoying 220, Jingfan 701, Furuite 1681, Weigens Big Brother, Jingfan 401, Jingfan Pink Star No. 1, Jingfan Yellow Star No. 1, Jingfan Purple Star No. 2, Yingguan 218, Hangfen No. 2, Chuntao 7 0, Hong Yali, Grammy, Baoliyuan No. 5, Gu Tian, ​​Boya, IVF3116, IVF6201, IVF1106, Hong Xiaobei, Emily, Bertis, Sakura 101, Nicola, Jinxiu 202, He Ruisi No. 1, SV7846TH, Jingfan Black Arhat, Jidali 5605, Fendley 897, Lee Garden Sky Red, Luo Xi, Oriental Beauty, Fushan 88, Ella, Baofa 666, Feiteng 158, Xiwo, Unidya Pink, Belle 1319, Xishiduo, Zhongqinghong No. 1, Baoliyuan No. 1, Pengyou No. 1, Herunsheng No. 1, Temeite 56, Yinhe 99, Huachen 70, Kevin, Sibed, Hanyu 522, Aini, CapitaLand Shuangli 87170, CapitaLand Yucheng 8722, N6426, Duoxi 8, Guanshu Mingzhu, CapitaLand 87553, SVTG6210, and Nongge 521.

[0049] As an optional embodiment of the above method, the PCR amplification reaction system can specifically include: 5 μl template DNA (50 ng / μl), 10 μl 2× Mix, 1 μl forward primer, 1 μl reverse primer (20 μmol / L), and 3 μl ultrapure water. The PCR amplification reaction procedure can specifically include: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 1 min, 35 cycles; and extension at 72°C for 10 min.

[0050] In the above-described method, the larger the value of N is, the higher the accuracy of identifying the purity of the tomato hybrid to be tested is.

[0051] It should be noted that the present invention focuses on the purity assurance of tomato hybrids, specifically first-generation hybrids. The core challenge lies in avoiding parental mixing rather than mechanical mixing, that is, not simply mixing multiple tomato hybrids.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The InDel primer combination provided by the application realizes early identification of existing representative tomato hybrid seeds or seedlings, thereby ensuring the purity of the hybrid seeds, effectively maintaining the legal rights and interests of producers and breeders, and providing technical support for seed quality management of tomato varieties. The method provided by the application has the advantages of high throughput, accuracy, low cost, simple operation, saving of manpower and material resources, and has a very broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 InDel typing effect of 8 primer groups in part of the tested tomato hybrid seeds.

[0055] Figure 2 Distribution of 8 primer groups in 96 tomato hybrid loci.

[0056] Figure 3 Purity identification result of primer group 7 in TM2402 hybrid seeds. DETAILED DESCRIPTION

[0057] The application will be further described in detail below in conjunction with 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 skilled in the art, and do not in any way constitute a limitation on the application.

[0058] The experimental methods in the following examples are all conventional methods, and are carried out 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.

[0059] Example 1: Obtaining of InDel primer combination for identifying purity of tomato hybrid seeds.

[0060] I. Discovery of 8 InDel loci

[0061] The present invention is based on the resequencing data of 96 representative tomato resources recorded in Table 3, develops tomato whole-genome InDel markers, and uses them for variety purity identification, obtaining 8 InDel sites. These 96 tomato resources are rich in types, covering types such as pink fruit type, red fruit type, and cherry type, basically covering the main ecological types and agronomic traits of tomatoes, embodying as much germplasm representativeness as possible, and having high genetic diversity. Specifically, the screening criteria for InDel sites are as follows: first, InDel sites belonging to dimorphic types are screened on the whole genome chromosomes, InDel sites with 200 conserved flanks and >30bp are selected, and then InDel sites with Miss <0.2, MAF>0.2, and He <0.05 are screened. Finally, blast specific analysis is performed and 48 pairs of specific primers are designed. These primers are used to screen 96 test tomato hybrids. During the screening process, through careful comparison and analysis, primers that show high heterozygosity and polymorphism are finally screened out. After strict screening and evaluation, 8 pairs of primers are finally retained.

[0062] The basic information of the eight InDel sites is detailed in Table 1. The positions of the InDel sites on the chromosomes were determined based on the sequence alignment of the tomato reference genome SL3.0 (https: / / solgenomics.net / organism / Solanum_lycopersicum / genome).

[0063] Table 1. Basic information of 8 InDel sites

[0064]

[0065] 2. Obtaining InDel Primer Combinations for Identifying the Purity of Tomato Hybrids

[0066] Based on the eight InDel sites discovered in step 1, the inventors of the present invention developed an InDel primer combination with high polymorphism and heterozygosity for identifying the purity of tomato hybrids.

[0067] The InDel primer set consists of eight primer sets. The names of each primer set are shown in column 2 of Table 2. Each primer set consists of two primer sequences and is used to amplify a single InDel site. The nucleotide sequences of each primer in the eight primer sets are shown in column 4 of Table 2.

[0068] Table 2 8 pairs of Indel primer sequences suitable for tomato purity identification

[0069]

[0070] Example 2: Validation of the InDel primer combination developed in Example 1

[0071] The basic information of the 96 tomato hybrids tested in this example is shown in Table 3. The 96 tomato hybrids tested are all common high-quality hybrids or hybrids introduced from abroad.

[0072] Table 3. Basic information of 96 tested tomato hybrids

[0073]

[0074]

[0075] 1. Obtaining genomic DNA from test tomato hybrids (DNA extraction using magnetic beads)

[0076] For each tomato material, an appropriate amount of leaf samples were collected in a centrifuge tube, steel balls were added, and the samples were frozen in liquid nitrogen, and then ground using an ultra-high throughput grinder; after grinding, 800 μl of SDS extract was added to each sample; the samples were dried in a 60°C oven for 30-40 minutes, and the mixture was mixed by inversion 3 times during the process; 240 μl (0.3 times the volume of the SDS extract) of 3M potassium acetate solution was added to each sample, the mixture was mixed by inversion 5 times, and the sample was placed in a 4°C refrigerator for 20 minutes; the sample was centrifuged at 4000 rpm for 10 minutes; a new centrifuge tube or deep-well plate was prepared in advance, and 600 μl of magnetic bead isopropanol was added to the sample (ensuring that the volume was equal to that of the supernatant). Note that the order of operation is to add magnetic bead isopropanol first, and then add supernatant, and the volume of supernatant should not exceed Fill the sample with 2 / 3 of the total volume; invert to mix, let it stand at -20°C until the magnetic beads are precipitated for 30 minutes, invert again to mix, adsorb with a magnetic rack, discard the supernatant, and invert onto absorbent paper to remove the remaining liquid; add 200 μl of 75% ethanol solution to the sample, invert thoroughly to mix, and let it stand until the magnetic beads are completely precipitated; invert again to mix, adsorb with a magnetic rack, discard the supernatant, and invert onto absorbent paper to remove the remaining liquid; after the magnetic beads are completely air-dried, add 100 μl of purified water to it and invert thoroughly to mix to ensure that the DNA is completely dissolved; use Nanodrop (microspectrophotometer) to take 2 μl of DNA extract for detection, determine its OD260 / 280 and OD260 / 230 ratios (ideal range is 1.8-2.0), and record the DNA concentration. Subsequently, the DNA concentration is adjusted to 50 ng / μl for subsequent use.

[0077] 2. PCR amplification was performed using genomic DNA from each of the 96 test tomato hybrids as templates using the eight primer sets listed in Table 2 to obtain the corresponding 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.

[0078] The PCR amplification reaction system is: 5 μl template DNA, 10 μl 2 x Mix, 1 μl forward primer and 1 μl reverse primer (20 μmol / L), 3 μl ultrapure water.

[0079] The reaction procedure is: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; 72℃ extension for 10 min.

[0080] 3、After completing step 2, the PCR amplification product is subjected to agarose gel electrophoresis, and the result is photographed and read by a gel imaging system. The genotype of each InDel site of the 96 test tomato hybrid seeds is determined by the band condition. The specific determination principle is as follows: if a test tomato hybrid seed shows a heterozygous band result based on an InDel site, the genotype of the test tomato hybrid seed based on the InDel site is heterozygous; if a test tomato hybrid seed shows a single band result based on an InDel site, the genotype of the test tomato hybrid seed based on the InDel site is homozygous.

[0081] Some results are shown in Figure 1 . The results show that each primer group can obtain good typing effect in the test tomato hybrid seeds.

[0082] 4、Heterozygous site number distribution and efficiency evaluation

[0083] (1) According to the genotype of 96 test tomato hybrid seeds based on 8 InDel sites, the number of heterozygous sites of each test tomato hybrid seed is counted.

[0084] The distribution results of the number of heterozygous sites of 96 test tomato hybrid seeds based on 8 primer groups are shown in Figure 2 . The results show that 8 primer groups can make each test tomato hybrid seed have at least one heterozygous site.

[0085] (2) Hybrid seed purity identification can use multiplex PCR to reduce workload.

[0086] The results show that the coverage rate of heterozygous sites in 96 test tomato hybrid seeds by multiplex PCR (8 primer groups) reaches 100%.

[0087] As can be seen, the InDel primer combination developed in Example 1 can be applied to tomato hybrid seed purity identification.

[0088] Example 3, using the InDel primer combination developed in Example 1 to detect the purity of TM2402 hybrid seed

[0089] I. Using the InDel primer combination developed in Example 1 to detect the purity of TM2402 hybrid seed

[0090] 1. Obtaining genomic DNA of TM2402 hybrid

[0091] (1) Plant 200 TM2402 hybrid seeds to obtain TM2402 hybrid seedlings.

[0092] (2) Leaves or roots of 96 TM2402 hybrid seedlings were randomly selected and genomic DNA was extracted using the magnetic bead method to obtain 96 genomic DNAs of TM2402 hybrids in turn.

[0093] 2. Screening of primer sets

[0094] (1) Genomic DNA of the ten hybrid seedlings obtained in step (2) was selected and used as a template, and PCR amplification was performed using eight primer sets from the InDel primer combination developed in Example 1 to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F" in its name to the primer containing "R" in its name was 1:1.

[0095] The reaction system for PCR amplification can be specifically: 5 μl template DNA (50 ng / μl), 10 μl 2×Mix, 1 μl forward primer and 1 μl reverse primer (20 μmol / L), and 3 μl ultrapure water.

[0096] The specific reaction program of PCR amplification can be as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds; annealing at 58°C for 30 seconds; extension at 72°C for 1 minute, 35 cycles; and extension at 72°C for 10 minutes.

[0097] (2) After completing step (1), perform agarose gel electrophoresis and take photos of the electrophoresis results using a gel imaging system to obtain the results. Compare the number of strains at the heterozygous sites of the eight primer sets. The primer set with the largest number of strains at the heterozygous sites is the selected primer set.

[0098] The results showed that the number of strains with heterozygous sites was the largest for primer set 7. Therefore, primer set 7 was selected as the primer set for subsequent experiments.

[0099] 3. Obtaining the purity of TM2402 hybrids

[0100] (1) PCR amplification was performed using 96 genomic DNA samples of the TM2402 hybrid as templates using the screened primer set 7 to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F" in its name to the primer containing "R" in its name was 1:1.

[0101] The reaction system of PCR amplification can be specifically 5ul of template DNA (50ng / ul), 10ul of 2x Mix, 1ul of forward primer and 1ul of reverse primer (20umol / L), and 3ul of ultrapure water.

[0102] The reaction procedure of PCR amplification can be specifically pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 58℃ for 30s, extension at 72℃ for 1min, 35 cycles, and extension at 72℃ for 10min.

[0103] (2) After step (1) is completed, agarose gel electrophoresis is carried out, and the electrophoresis result is recorded by taking a picture using a gel imaging system to obtain the result.

[0104] The InDel typing result is shown in Table 2. Figure 3 .

[0105] (3) After step (2) is completed, the number of strains showing heterozygous sites and the number of strains without heterozygous sites of the primer group 7 are counted, and the purity of the TM2402 hybrid is calculated according to the following formula.

[0106] Purity = number of strains showing heterozygous sites of the primer group / (96-number of strains without bands of the primer group) x 100%.

[0107] The results show that the number of strains showing heterozygous bands of the primer group 7 is 95, the number of strains showing homozygous bands is 1, and the purity is 95 / 96x100%=98.9%.

[0108] The purity of the hybrid obtained by field identification of 200 strains of hybrid is 98.5%, which shows that the identification method has high accuracy.

[0109] Finally, it should be explained that the above specific embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. InDel primer combinations, including the first to eighth primer sets, corresponding to amplifying eight InDel markers to perform genotyping for each InDel marker, wherein: The eight InDel markers include: Marker FQ_Ind18: located at position 71483275 on chromosome 9, and is nucleotide G or the nucleotide sequence shown in SEQ ID NO. 17; Marker FQ_Ind24: located at position 58381796 on chromosome 12, and is nucleotide A or the nucleotide sequence shown in SEQ ID NO. 18; Marker FQ_Ind26: located at position 67342246 on chromosome 3, and is nucleotide A or the nucleotide sequence shown in SEQ ID NO.19; Marker FQ_Ind27: located at position 35307300 on chromosome 4, and is nucleotide A or the nucleotide sequence shown in SEQ ID NO. 20; Marker FQ_Ind29: located at position 46541839 on chromosome 5, and is nucleotide A or the nucleotide sequence shown in SEQ ID NO. 21; Marker FQ_Ind33: located at position 35630796 on chromosome 11, and is nucleotide T or the nucleotide sequence shown in SEQ ID NO. 22; Marker FQ_Ind34: located at position 17984202 on chromosome 12, and is nucleotide A or the nucleotide sequence shown in SEQ ID NO. 23; Marker FQ_Ind47: located at position 48618696 on chromosome 11, and is nucleotide T or the nucleotide sequence shown in SEQ ID NO. 24; The positions of the eight InDel markers on the chromosome were determined based on the sequence alignment of the tomato reference genome SL3.0; The first primer set is used to amplify the molecular marker FQ_Ind18; the second primer set is used to amplify the molecular marker FQ_Ind24; the third primer set is used to amplify the molecular marker FQ_Ind26; the fourth primer set is used to amplify the molecular marker FQ_Ind27; the fifth primer set is used to amplify the molecular marker FQ_Ind29; the sixth primer set is used to amplify the molecular marker FQ_Ind33; the seventh primer set is used to amplify the molecular marker FQ_Ind34; and the eighth primer set is used to amplify the molecular marker FQ_Ind47.

2. The InDel primer combination according to claim 1, wherein: The first primer set consists of a forward primer F1 shown in SEQ ID NO.1 and a reverse primer R1 shown in SEQ ID NO.2; The second primer set consists of a forward primer F2 shown in SEQ ID NO.3 and a reverse primer R2 shown in SEQ ID NO.4; The third primer set consists of the forward primer F3 shown in SEQ ID NO.5 and the reverse primer R3 shown in SEQ ID NO.6; The fourth primer set consists of the forward primer F4 shown in SEQ ID NO.7 and the reverse primer R4 shown in SEQ ID NO.8; The fifth primer set consists of a forward primer F5 shown in SEQ ID NO.9 and a reverse primer R5 shown in SEQ ID NO.10; The sixth primer set consists of the forward primer F6 shown in SEQ ID NO.11 and the reverse primer R6 shown in SEQ ID NO.12; The seventh primer set consists of the forward primer F7 shown in SEQ ID NO.13 and the reverse primer R7 shown in SEQ ID NO.14; The eighth primer set consists of a forward primer F8 shown in SEQ ID NO.15 and a reverse primer R8 shown in SEQ ID NO.

16.

3. A kit comprising the InDel primer combination according to claim 1 or 2.

4. Use of the InDel primer combination according to claim 1 or 2, or the kit according to claim 3, in identifying the purity of tomato hybrids.

5. The use according to claim 4, characterized in that The tomato hybrid is selected from Angelia, Weigens Xiachuan No. 7, Weigens Hanxiang, Weigens Xiayu No. 10, Meishuai, Xiarihong, Shengtao T610, Jing T308, Baowadi, Thomas, Buffett, Daxi, Senna, Zhongyan TV1, Hui 1617, Summer Sunshine, Tianbei, Europe and America, Huajin No. 1, Silver Moon 619, Hongsui, Hongza 33, IVF6260, IVF6172, IV F3311, Meishengda, Nunhems 1718, Huanxi, Deruisi, Jingfen 801, Xindandan No. 4, Jiafan No. 1, Yifan No. 1, Jingfan 309, Makuta Powder 80, Jingfan 203, Haoying 220, Jingfan 701, Furuite 1681, Weigens Big Brother, Jingfan 401, Jingfan Pink Star No. 1, Jingfan Yellow Star No. 1, Jingfan Purple Star No. 2, Yingguan 218, Hangfen No. 2, Chuntao 70, Red Yali, Grammy, Baoliyuan No. 5, Gutian, Boya, IVF3116, IVF6201, IVF1106, Hong Xiaobei, Emily, Bertis, Sakura 101, Nicola, Jinxiu 202, Heruisi No. 1, SV7846TH, Jingfan Black Arhat, Jidali 5605, Fendley 897, Lee Garden Sky Red, Luo Xi, Oriental Beauty, Fushan 88, Rila, Baofa 666, Feiteng 1 58. Xiwo, Unidya Pink, Belle 1319, Xishiduo, Zhongqinghong No. 1, Baoliyuan No. 1, Pengyou No. 1, Herunsheng No. 1, Temeite 56, Yinhe 99, Huachen 70, Kevin, Sibed, Hanyu 522, Aini, CapitaLand Shuangli 87170, CapitaLand Yucheng 8722, N6426, Duoxi 8, Guanshu Mingzhu, CapitaLand 87553, SVTG6210, and Nongge 521.

6. A method for identifying the purity of a tomato hybrid, characterized in that: The steps include: (1) Randomly select N tomato hybrids to be tested and obtain their genomic DNA; (2) using genomic DNA of at least 10 tomato hybrids to be tested selected from the N tomato hybrids to be tested obtained in step (1) as templates, and performing PCR amplification using the first to eighth primer groups in the InDel primer combination of claim 1 or 2 to obtain corresponding PCR amplification products; (3) performing agarose gel electrophoresis on the PCR amplification product obtained in step (2), and counting the number of strains with heterozygous bands of the first to eighth primer sets according to the electrophoresis results; then selecting the primer set with relatively more and clearer heterozygous bands as the target primer set; (4) using the genomic DNA of the N tomato hybrids to be tested obtained in step (1) as templates, and performing PCR amplification using the target primer sets obtained in step (3) to obtain PCR amplification products of each tomato hybrid to be tested under different target primer sets; (5) The PCR amplification products obtained in step (4) are subjected to agarose gel electrophoresis. The number of plants with heterozygous bands in the amplification products of each target primer set is counted according to the electrophoresis results. The tomato strains to be tested with heterozygous bands are hybrids. The purity of the tomato hybrid to be tested is obtained based on the number of plants with heterozygous bands amplified by each target primer set.

7. The method according to claim 6, wherein The method of "obtaining the purity of the tomato hybrid to be tested based on the number of plants showing heterozygous bands under amplification by each target primer set" is as follows: counting the number of plants showing heterozygous bands and the number of plants without bands under each target primer set, calculating the purity of the tomato hybrid to be tested obtained by each target primer set respectively, and then calculating the average value; Purity = number of strains showing heterozygous bands for a certain target primer set / (N - number of strains showing no bands for the target primer set) × 100%.

8. The method according to claim 6 or 7, wherein: The tomato hybrids to be tested are selected from Angelia, Weigens Xiachuan No. 7, Weigens Hanxiang, Weigens Xiayu No. 10, Meishuai, Xiarihong, Shengtao T610, Jing T308, Baowadi, Thomas, Buffett, Daxi, Senna, Zhongyan TV1, Hui 1617, Summer Sunshine, Tianbei, Europe and America, Huajin No. 1, Silver Moon 619, Hongsui, Hongza 33, IVF6260, IVF6172, IVF3311, Meishengda, Nunhems 1718, Huanxi, Deruisi, Jingfen 801, Xindandan No. 4, Jiafan No. 1, Yifan No. 1, Jingfan 309, Makuta Powder 80, Jingfan 203, Haoying 220, Jingfan 701, Furuite 1681, Weigens Big Brother, Jingfan 401, Jingfan Pink Star No. 1, Jingfan Yellow Star No. 1, Jingfan Purple Star No. 2, Yingguan 218, Hangfen No. 2, Chuntao 70, Hong Yali, Grammy, Baoliyuan No. 5, Gutian, Boya, IVF3116, IVF6201, IVF1106, Hong Xiaobei, Emily, Bertis, Sakura 101, Nicola, Jinxiu 202, He Ruisi No. 1, SV7846TH, Jingfan Black Arhat, Jidali 5605, Fendley 897, Lee Garden Sky Red, Luo Xi, Oriental Beauty, Fushan 88, Eila, Baofa 666, Feiteng 1 58. Xiwo, Unidya Pink, Belle 1319, Xishiduo, Zhongqinghong No. 1, Baoliyuan No. 1, Pengyou No. 1, Herunsheng No. 1, Temeite 56, Yinhe 99, Huachen 70, Kevin, Sibed, Hanyu 522, Aini, CapitaLand Shuangli 87170, CapitaLand Yucheng 8722, N6426, Duoxi 8, Guanshu Mingzhu, CapitaLand 87553, SVTG6210, and Nongge 521.

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