Method for identifying purity of tomato hybrid and InDel primer combination used by same

Abstract

The invention discloses a method for identifying the purity of tomato hybrids and an InDel primer combination used by the method. The InDel primer combination provided by the invention is composed of eight primer groups; each primer group consists of two primer sequences and is used for amplifying an InDel site; the nucleotide sequences of the primers are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 16. The InDel primer combination can be used for early identification in the seed or seedling stage of the tomato hybrid, so that the purity of the hybrid is ensured, the rights and interests of producers and breeders are practically protected, and technical support is provided for seed quality management of tomato varieties. The method provided by the invention has the advantages of being accurate, low in cost, simple to operate, clear in result, capable of saving 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 purity identification, and particularly relates to a method for identifying the purity of tomato hybrids and an InDel primer combination used therein. Background Art

[0002] As one of the main vegetables in my country, tomato is one of the most important vegetables in the world. According to statistics, the planting area of ​​tomato in my country is about 95.14hm2. 2 , the annual output can reach 48.749 million tons. At present, 3,305 tomato varieties have been registered nationwide. In tomato production, hybrids have become the main force due to their excellent genetic characteristics and production performance. However, before hybrids enter the market, they must pass strict purity identification. According to the "Regulations for the Inspection of Crop Seeds" (GB / T3543.1-3543.7-1995), the purity of tomato hybrids is required to exceed 98%. Since my country's tomato breeding enterprises are generally small in scale and not concentrated in distribution, seed quality management has become an urgent problem to be solved. The frequent appearance of counterfeit and inferior tomato seeds with insufficient purity in the market not only infringes on the interests of farmers, but also has a negative impact on the sustainable development of the tomato industry. At present, traditional field purity detection methods are costly and time-consuming, and cannot meet the rapid development of the modern seed industry. Therefore, DNA-based molecular detection technology came into being, and with its low cost and high efficiency, it quickly became the preferred method for identifying the purity of tomato hybrids. By selecting the right combination of molecular markers for DNA testing technology, we can complete purity identification more accurately and quickly, effectively prevent the circulation of counterfeit and inferior seeds, protect farmers' rights and interests, and promote the sustainable development of the tomato industry. Therefore, it is particularly important to select a molecular marker combination for identifying the purity of tomato hybrids based on DNA testing technology.

[0003] InDel (Insertion-Deletion) markers are insertion-deletion markers, that is, relative to the reference genome, there are a certain number of nucleotide insertions or deletions in the genomes of other samples. InDel mutations are widely distributed in the genome, second only to SNP mutations in number, and specific InDel marker primers can be designed based on the sequences on both sides of the insertion-deletion site. Compared with SNP and SSR markers, Indel markers have a wider range of applications, can be detected on a common agarose electrophoresis platform, and have lower requirements for sample DNA quality. Their banding stability and interpretation effect are also better than SSR; they have the characteristics of good stability, wide distribution, high polymorphism, strong versatility, simple typing system and low cost.

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

[0005] In view of the defects of the prior art, one of the objectives of the present invention is to provide an InDel primer combination, which 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] The third object of the present invention is to provide the 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] The first aspect of the present invention provides an InDel primer combination, including a first primer set to an eighth primer set, corresponding to amplifying eight InDel markers to perform genotyping (or homozygous or heterozygous determination) on 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, 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 set (primer set 1) is used to amplify the molecular marker FQ_Ind18; the second primer set (primer set 2) is used to amplify the molecular marker FQ_Ind24; the third primer set (primer set 3) is used to amplify the molecular marker FQ_Ind26; the fourth primer set (primer set 4) is used to amplify the molecular marker FQ_Ind27; the fifth primer set (primer set 5) is used to amplify the molecular marker FQ_Ind29; the sixth primer set (primer set 6) is used to amplify the molecular marker FQ_Ind33; the seventh primer set (primer set 7) is used to amplify the molecular marker FQ_Ind34; the eighth primer set (primer set 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 group that can be used to identify the purity of a certain tomato hybrid is a heterozygous band, it means that the tomato variety to be tested is a hybrid. If it is a single band, it means that the tomato variety to be 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 hybrids of the first generation, 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, dNTP 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 is selected from Angelia, Weigens Xiachuan No. 7, Weigens Hanxiang, Weigens Xiayu No. 10, Meishuai, Xia Rihong, 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, IVF617 2. 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, Findlay 897, Lee Garden Sky Red, Luoxi, Oriental Beauty, Fushan 88, Rila, Baofa 666, Feiteng 158, Xiwo, Unidya Fenli, 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, Ainila, CapitaLand Shuangli 87170, CapitaLand Yucheng 8722, N6426, Duoxi 8, Guanshu Mingzhu, CapitaLand 87553, SVTG6210, and Nongge 521.

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

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

[0040] (1) randomly selecting N tomato hybrids to be tested and obtaining their genomic DNA;

[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 using the first to eighth primer groups in the InDel primer combination provided in the first aspect to perform PCR amplification to obtain corresponding PCR amplification products;

[0042] (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 a primer set with a relatively large number of 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, respectively, and using the target primer sets obtained in step (3) to perform PCR amplification to obtain PCR amplification products of each tomato hybrid to be tested under amplification with different target primer sets;

[0044] (5) performing agarose gel electrophoresis on the PCR amplification products obtained in step (4), and counting the number of plants with heterozygous bands in the amplification products of each target primer set according to the electrophoresis results, wherein the tomato strains to be tested with heterozygous bands are hybrids, and the purity of the tomato hybrid to be tested is obtained according to 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 may 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 hybrids 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 by a certain target primer set / (N-the number of strains without bands by the target primer set)×100%.

[0048] Further, the tomato hybrid to be tested is selected from Angelia, Weigens Xiachuan No. 7, Weigens Hanxiang, Weigens Xiayu No. 10, Meishuai, Xia Rihong, 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, Gutian, Boya, IVF3116, IVF6201, IVF1106, Hong Xiaobei, Emily, Bertis, Sakura 101, Nicola, Jinxiu 202, He Ruisi No. 1, SV7846TH, Jingfan Black Arhat, Jidali 5605, Findlay 897, Lee Garden Sky Red, Luoxi, Oriental Beauty, Fushan 88, Rila, Baofa 666, Feiteng 158, Xiwo, Unidya Fenli, 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, Ainila, 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 reaction system of the PCR amplification can be specifically: 5μl template DNA (50ng / μl), 10μl 2×Mix, 1μl forward primer and 1μl reverse primer (20μmol / L), 3μl ultrapure water. The reaction procedure of the PCR amplification can be specifically: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s; 58℃ annealing for 30s; 72℃ extension for 1min, 35 cycles; 72℃ extension for 10min.

[0050] In the above 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, and the core challenge is to avoid parental mixing rather than mechanical mixing, that is, not a simple mixture of 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 present invention realizes the early identification of existing representative tomato hybrids at the seed or seedling stage, thereby ensuring the purity of the hybrids, effectively safeguarding the legitimate rights and interests of producers and breeders, and providing technical support for seed quality management of tomato varieties. The method provided by the present invention has the advantages of high throughput, accuracy, low cost, simple operation, saving manpower and material resources, etc., and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The InDel typing effects of eight primer sets in some tested tomato hybrids.

[0055] Figure 2 The figure shows the distribution of heterozygous loci of 8 primer sets in 96 tomato varieties.

[0056] Figure 3 The purity identification result of primer set 7 in TM2402 hybrid. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0058] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0059] Example 1: Obtaining an InDel primer combination for identifying the purity of tomato hybrids.

[0060] 1. Discovery of 8 InDel sites

[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 is used for variety purity identification, and obtains 8 InDel sites. These 96 tomato resources are rich in types, covering types such as pink fruit type, red fruit type, cherry type, etc., basically including the main ecological types and agronomic traits of tomatoes, reflecting germplasm representativeness as much as possible, and having high genetic diversity. Specifically, the screening criteria of InDel sites are as follows: first, InDel sites belonging to dimorphic types are screened out on the whole genome chromosome, InDel sites with 200 conservative and >30bp on both wings are selected, and then InDel sites with Miss<0.2, MAF>0.2, and He<0.05 are screened, and finally blast specific analysis is performed, and 48 pairs of specific primers are designed. These primers are used to screen 96 test tomato hybrids. In the screening process, by careful comparison and analysis, primers showing higher 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 8 InDel sites is shown in Table 1. The positions of the InDel sites on the chromosome were determined based on the sequence alignment of the tomato reference genome SL3.0 (URL: 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 8 InDel sites found 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 combination consists of 8 primer sets, and the name of each primer set is shown in the second column of Table 2. Each primer set consists of 2 primer sequences and is used to amplify one InDel site. The nucleotide sequence of each primer in the 8 primer sets is shown in the fourth column 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 tested tomato hybrids in this example is shown in Table 3. The 96 tested tomato hybrids 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 of test tomato hybrids (DNA extraction by magnetic beads)

[0076] Take an appropriate amount of leaf samples from each tomato material and collect them in a centrifuge tube, add steel beads, and freeze them in liquid nitrogen, then use an ultra-high throughput grinder for grinding; add 800μl of SDS extract to each sample after grinding; place in a 60℃ oven for 30-40min, invert and mix 3 times during the period; add 240μl (i.e. 0.3 times the volume of SDS extract) of 3M potassium acetate solution to each sample, invert and mix thoroughly 5 times, and place in a 4℃ refrigerator for 20min; centrifuge at 4000 rpm for 10 minutes; prepare a new centrifuge tube or deep-well plate in advance, add 600μl of magnetic bead isopropanol to it (make sure the volume is equal to the supernatant), note that the order of operation is to add magnetic bead isopropanol first, then add supernatant, and the volume of supernatant absorbed should not exceed 2 / 3 of the total volume; invert and mix, let stand at -20℃ until the magnetic beads are precipitated for 30 minutes, invert and mix again, adsorb with a magnetic stand, discard the supernatant, and invert on absorbent paper to pick up the residual liquid; add 200μl of 75% ethanol solution to the sample, invert and mix thoroughly, and let stand until the magnetic beads are completely precipitated; invert and mix again, adsorb with a magnetic stand, discard the supernatant, and invert on absorbent paper to pick up the residual liquid; after the magnetic beads are completely air-dried, add 100μl of pure water to it, invert and mix thoroughly to ensure that the DNA is completely dissolved; use Nanodrop (micro spectrophotometer) to take 2μl of DNA extract for detection, determine its OD260 / 280 and OD260 / 230 ratios (the ideal range is 1.8-2.0), and record the DNA concentration. Subsequently, the DNA concentration was adjusted to 50ng / μl for subsequent use.

[0077] 2. Using the genomic DNA of the 96 tested tomato hybrids as templates, PCR amplification was performed using the 8 primer sets given in Table 2 to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F" in the name to the primer containing "R" in the name was 1:1.

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

[0079] The reaction program was as follows: 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.

[0080] 3. After completing step 2, the PCR amplification product was subjected to agarose gel electrophoresis, and the gel imaging system was used to take pictures and read the results. The genotype of each InDel site of the 96 tested tomato hybrids was determined by the band situation. The specific judgment principle is as follows: if a tested tomato hybrid shows a heterozygous band based on a certain InDel site, the genotype of the tested tomato hybrid based on the InDel site is heterozygous; if a tested tomato hybrid shows a single band based on a certain InDel site, the genotype of the tested tomato hybrid based on the InDel site is homozygous.

[0081] Some results can be found in Figure 1 The results showed that each primer set could achieve good typing results in the tested tomato hybrids.

[0082] 4. Distribution of heterozygous sites and efficiency evaluation

[0083] (1) Based on the genotypes of 96 tested tomato hybrids at 8 InDel loci, the number of heterozygous loci in each tested tomato hybrid was counted.

[0084] The distribution results of the number of heterozygous loci of 96 tested tomato hybrids based on 8 primer sets are shown in Figure 2 The results showed that the eight primer sets could make each tested tomato hybrid have at least one heterozygous site.

[0085] (2) Hybrid purity identification can be carried out by using multiplex PCR to reduce the workload.

[0086] The results showed that the coverage rate of heterozygous sites in 96 tested tomato hybrids by multiplex PCR (8 primer sets) reached 100%.

[0087] It can be seen that the InDel primer combination developed in Example 1 can be applied to the purity identification of tomato hybrids.

[0088] Example 3: Using the InDel primer combination developed in Example 1 to detect the purity of TM2402 hybrids

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

[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) Selecting genomic DNA of 10 hybrid seedlings obtained in step (2), using them as templates, respectively, and performing PCR amplification using 8 primer sets in 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 the name to the primer containing "R" in the name is 1:1.

[0095] The specific reaction system of PCR amplification can be: 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 procedure of PCR amplification can be: 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; extension at 72°C for 10 minutes.

[0097] (2) After completing step (1), perform agarose gel electrophoresis, and use a gel imaging system to take pictures of the electrophoresis results to obtain the results. Compare the number of strains at the heterozygous sites of the 8 primer sets, and 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 at the heterozygous site of primer set 7 was the largest. Therefore, primer set 7 was selected as the primer set for subsequent experiments.

[0099] 3. Obtaining the purity of TM2402 hybrids

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

[0101] The specific reaction system of PCR amplification can be: 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.

[0102] The specific reaction procedure of PCR amplification can be: 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; extension at 72°C for 10 minutes.

[0103] (2) After completing step (1), perform agarose gel electrophoresis and use a gel imaging system to photograph the electrophoresis results to obtain the results.

[0104] InDel typing results are shown in Figure 3 .

[0105] (3) After completing step (2), the number of strains showing heterozygous sites and the number of strains without heterozygous sites in primer set 7 were counted; the purity of the TM2402 hybrid was calculated according to the following formula;.

[0106] Purity = number of strains showing heterozygous sites with the primer set / (96 - number of strains with no bands with the primer set) x 100%.

[0107] The results showed that primer set 7 showed heterozygous bands in 95 strains and homozygous bands in 1 strain, with a purity of 95 / 96×100%=98.9%.

[0108] The purity of the hybrids obtained by field identification of 200 hybrids was 98.5%, which shows that the identification method of the present invention has high accuracy.

[0109] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. InDel primer combinations, including the first primer set to the eighth primer set, corresponding to amplifying eight InDel markers to perform genotyping on 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, 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, characterized in that: 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 a forward primer F3 shown in SEQ ID NO.5 and a reverse primer R3 shown in SEQ ID NO.6; 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; 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 a forward primer F6 shown in SEQ ID NO.11 and a reverse primer R6 shown in SEQ ID NO.12; 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; 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 hybrids are selected from Angelia, Weigens Xiachuan No. 7, Weigens Hanxiang, Weigens Xiayu No. 10, Meishuai, Xia Rihong, 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, Hong Yali, Grammy, Baoliyuan No. 5, Gutian, Boya, IVF3116, IVF6201, IVF1106, Red Bei, Emily, Bertis, Sakura 101, Nicola, Jinxiu 202, Horis No. 1, SV7846TH, Jingfan Black Arhat, Jidali 5605, Findlay 897, Lee Garden Sky Red, Luoxi, Oriental Beauty, Fushan 88, Rila, Baofa 666, Feiteng 1 58. Xiwo, Unidya Fenli, 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, Ainila, 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 to be tested, characterized in that: The steps include: (1) randomly selecting N tomato hybrids to be tested and obtaining 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 using the first to eighth primer groups in the InDel primer combination of claim 1 or 2 to perform PCR amplification 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; and then selecting a primer set with a relatively large number of clear 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, respectively, and using the target primer sets obtained in step (3) to perform PCR amplification to obtain PCR amplification products of each tomato hybrid to be tested under amplification with different target primer sets; (5) performing agarose gel electrophoresis on the PCR amplification products obtained in step (4), and counting the number of plants with heterozygous bands in the amplification products of each target primer set according to the electrophoresis results, wherein the tomato strains to be tested with heterozygous bands are hybrids, and the purity of the tomato hybrid to be tested is obtained according to the number of plants with heterozygous bands amplified by each target primer set.

7. The method according to claim 6, characterized in that The method of "obtaining the purity of the tomato hybrid to be tested according to the number of plants with heterozygous bands amplified by each target primer set" is: counting the number of plants showing heterozygous bands and the number of plants without bands by each target primer set, respectively calculating the purity of the tomato hybrid to be tested obtained by each target primer set, and then calculating the average value; Purity = 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%.

8. The method according to claim 6 or 7, characterized in that The tomato hybrids to be tested are selected from Angelia, Weigens Xiachuan No. 7, Weigens Hanxiang, Weigens Xiayu No. 10, Meishuai, Xia Rihong, 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 4, Jiafan 1, Yifan 1, Jingfan 309, Makuta Powder 80, Jingfan 203, Haoying 220, Jingfan 701, Furuite 1681, Weigens Big Brother, Jingfan 401, Jingfan Powder Star 1, Jingfan Yellow Star 1, Jingfan Purple Star 2, Yingguan 218, Hangfen 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, Findlay 897, Lee Garden Sky Red, Luoxi, Oriental Beauty, Fushan 88, Rila, Baofa 666, Feiteng 1 58. Xiwo, Unidya Fenli, 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, Ainila, CapitaLand Shuangli 87170, CapitaLand Yucheng 8722, N6426, Duoxi 8, Guanshu Mingzhu, CapitaLand 87553, SVTG6210, and Nongge 521.

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