Method for identifying purity of muskmelon hybrid and InDel primer combination used by method

Abstract

The invention discloses a method for identifying the purity of a muskmelon hybrid and an InDel primer combination used by the method. The InDel primer combination provided by the invention is composed of 12 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: 24. The InDel primer combination can be used for early identification in the seed or seedling stage of the muskmelon 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 muskmelon 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 seed purity identification, and particularly relates to a method for identifying the purity of melon hybrid seeds and an InDel primer combination used therein. Background Art

[0002] Muskmelon is an annual vine herb of the Cucurbitaceae family. Its fruit is rich in nutrients and is deeply loved by consumers. It is one of the best-selling fruits in the Chinese market. In recent years, with the development of the melon industry, more and more new varieties have entered the market. As of December 2024, there are 2,475 melon varieties registered nationwide, of which more than 95% are first-generation hybrids. Variety purity has become one of the important indicators affecting the quality of melon seeds. However, the traditional field planting method for melon seed purity identification has limitations such as complex operation, long time consumption, and susceptibility to environmental factors, which makes it difficult to meet the efficient and accurate management needs of the modern seed industry. Compared with traditional field identification methods, DNA-based molecular detection technology has the advantages of low cost, simple operation, and reliable results, which can significantly improve the efficiency and accuracy of melon seed purity identification.

[0003] InDel (Insertion-Deletion) markers are insertion-deletion markers, which refer to the presence of a certain number of nucleotide insertions or deletions in the genome of other samples relative to the reference genome. Compared with SNP markers, InDel markers do not require high DNA quality, have low detection costs, and can be detected on an ordinary electrophoresis platform. Compared with SSR markers, InDel markers have a high genomic distribution density, good variation stability, and are easy to type. At present, there are few reports on the development of InDel markers for purity identification of melon hybrids. Therefore, screening a set of InDel molecular marker combinations suitable for the purity of melon varieties is of great significance for improving seed quality, standardizing the melon seed market, and promoting the healthy development of the melon industry.

[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 for identifying the purity of melon 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 melon hybrid to be tested.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect of the present invention, an InDel primer combination is provided, including a first primer set to a twelfth primer set, corresponding to amplifying twelve InDel markers to respectively genotype each InDel marker or determine homozygosity or heterozygosity, wherein:

[0011] The twelve InDel markers include:

[0012] Marker TG_Ind01: Located at position 912,892 on chromosome 1, being nucleotide T or the nucleotide sequence shown in SEQ ID NO.25;

[0013] Marker TG_Ind02: Located at position 1,576,348 on chromosome 2, being nucleotide T or the nucleotide sequence shown in SEQ ID NO.26;

[0014] Marker TG_Ind03: Located at position 21,498,496 on chromosome 2, being nucleotide G or the nucleotide sequence shown in SEQ ID NO.27;

[0015] Marker TG_Ind04: Located at position 27,217,555 on chromosome 3, being nucleotide T or the nucleotide sequence shown in SEQ ID NO.28;

[0016] Marker TG_Ind05: Located at position 18,106,952 on chromosome 4, being nucleotide G or the nucleotide sequence shown in SEQ ID NO.29;

[0017] Marker TG_Ind06: Located at position 21,247,101 on chromosome 4, being nucleotide C or the nucleotide sequence shown in SEQ ID NO.30;

[0018] Marker TG_Ind07: Located at position 16,632,195 on chromosome 5, being nucleotide G or the nucleotide sequence shown in SEQ ID NO.31;

[0019] Marker TG_Ind08: Located at position 26,006,430 on chromosome 7, being nucleotide T or the nucleotide sequence shown in SEQ ID NO.32;

[0020] Marker TG_Ind09: Located at position 4,833,893 on chromosome 8, being nucleotide G or the nucleotide sequence shown in SEQ ID NO.33;

[0021] Marker TG_Ind10: Located at position 6770350 on chromosome 8, being nucleotide C or the nucleotide sequence shown in SEQ ID NO.34;

[0022] Marker TG_Ind11: Located at position 23860732 on chromosome 9, being nucleotide C or the nucleotide sequence shown in SEQ ID NO.35;

[0023] Marker TG_Ind12: Located at position 22505112 on chromosome 12, being nucleotide C or the nucleotide sequence shown in SEQ ID NO.36;

[0024] The positions of the twelve InDel markers on the chromosome were determined based on alignment with the melon DHL92 reference genome sequence, and the version number of the melon DHL92 reference genome sequence is V3.5.1;

[0025] The first primer set (primer set 1) is used to amplify molecular marker TG_Ind01; the second primer set (primer set 2) is used to amplify molecular marker TG_Ind02; the third primer set (primer set 3) is used to amplify molecular marker TG_Ind03; the fourth primer set (primer set 4) is used to amplify molecular marker TG_Ind04; the fifth primer set (primer set 5) is used to amplify molecular marker TG_Ind05; the sixth primer set (primer set 6) is used to amplify molecular marker TG_Ind06; the seventh primer set (primer set 7) is used to amplify molecular marker TG_Ind07; the eighth primer set (primer set 8) is used to amplify molecular marker TG_Ind08; the ninth primer set (primer set 9) is used to amplify molecular marker TG_Ind09; the tenth primer set (primer set 10) is used to amplify molecular marker TG_Ind10; the eleventh primer set (primer set 11) is used to amplify molecular marker TG_Ind11; the twelfth primer set (primer set 12) is used to amplify molecular marker TG_Ind12.

[0026] The InDel primer combination provided by the present invention can be used to identify the purity of melon hybrids. If the PCR product of the primer set that can be used to identify the purity of a certain melon hybrid is a heterozygous band, it represents that the melon variety to be tested is a hybrid. If it is a single band, it represents that the melon variety to be tested is not a hybrid and may be one of the parents, that is, the parent is mixed with the hybrid.

[0027] The hybrid mentioned in the present invention refers to the first-generation hybrid, and there may be parental mixing.

[0028] Furthermore, in the InDel primer combination,

[0029] The first primer set consists of the forward primer F1 shown in SEQ ID NO.1 and the reverse primer R1 shown in SEQ ID NO.2;

[0030] The second primer set consists of the forward primer F2 shown in SEQ ID NO.3 and the reverse primer R2 shown in SEQ ID NO.4.

[0031] 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.

[0032] 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.

[0033] The fifth primer set consists of the forward primer F5 shown in SEQ ID NO.9 and the reverse primer R5 shown in SEQ ID NO.10.

[0034] 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.

[0035] 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.

[0036] The eighth primer set consists of the forward primer F8 shown in SEQ ID NO.15 and the reverse primer R8 shown in SEQ ID NO.16.

[0037] The ninth primer set consists of the forward primer F9 shown in SEQ ID NO.17 and the reverse primer R9 shown in SEQ ID NO.18.

[0038] The tenth primer set consists of the forward primer F10 shown in SEQ ID NO.19 and the reverse primer R10 shown in SEQ ID NO.20.

[0039] The eleventh primer set consists of the forward primer F11 shown in SEQ ID NO.21 and the reverse primer R11 shown in SEQ ID NO.22.

[0040] The twelfth primer set consists of the forward primer F12 shown in SEQ ID NO.23 and the reverse primer R12 shown in SEQ ID NO.24.

[0041] The second aspect of the present invention provides a kit comprising the InDel primer combination of the first aspect.

[0042] 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, etc., and may also include conventional experimental equipment for PCR.

[0043] 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.

[0044] 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 melon hybrids.

[0045] Further, the melon hybrid is selected from Jinge Mi Nong No. 11, Jinmi No. 8, Nongjia No. 2, Fuqi Cuiyu, Cuidai M15, Xinli No. 6, Crown, Xiazhou Mi, White Pearl, Red Golden Crown, Guangmi No. 6, Baiyuhong, Network Times, Nongtian Baodian, Xiboluoto No. 3, Xinyuan Mi No. 12, Milv, Jingtian Jasper No. 2, Kaitian No. 9, Zhongtian 382, ​​Hongruihong, M1005, Hongxincui No. 2, Jinruibao, Lumi No. 1, Longxinmi No. 17, Shuangquanmi No. 13, Jingtian Xingkong No. 1, Arusxia, Bohongmi No. 5, Jingtianmi No. 3, Four Seasons Silver Red No. 2, Jiaohong No. 4, M3166, Smetuo, Jindian, Annas, Huozhoumi 125, Jincui, Qinghua Any of the following: Mi, Hongcuimi, Tiandu Yinmi, Youchuang Xiangxiu, Wanxiang, Bailing Beauty, Xinyuan Mi No. 6, Hengmi No. 2, Jinmiyue, Shengmi Qingmi, Longlin Twenty-Five, Meili, Mitian No. 17, Oriental Pride No. 25, Black Coral, Dumi No. 6, Tongcui No. 1, Qitian Taste, Tianyue Selection, Kentian No. 2, Shangyi Zaoxue, Baichen Xinya, Huatian No. 1, Bixiang, Wanjintian No. 12, Qingtian No. 2, Jinling, Xiangrui Jingrun, Jitian Melon No. 6, Snow Milk Fragrance No. 1, Shengyu No. 18, Pingya No. 1, Liaotian Cuiyu, Linglong Cui, Jingtian Cuibao, Yutian Jinyu, Xuandian Balixiang, Green Cuiyu, Jingtian Huayi, Huakui, Zhongtian 327, Qianyu No. 6, Ruitian No. 11, Xinmi No. 28.

[0046] The above-mentioned melon hybrids are all commercially available varieties. In the above-mentioned melon hybrids, at least one of the twelve InDel markers mentioned in the first aspect of the present invention is a heterozygous type, and the InDel primer combination provided by the present invention can be used for purity identification of the above-mentioned melon hybrids.

[0047] A fourth aspect of the present invention provides a method for identifying the purity of a melon hybrid to be tested, comprising the following steps:

[0048] (1) randomly selecting N melon hybrids to be tested and obtaining their genomic DNA;

[0049] (2) Using the genomic DNA of at least 8 (such as 10, 15 or 20) melon hybrid seeds to be tested selected from the N melon hybrid seeds to be tested obtained in step (1) as templates respectively, perform PCR amplification using the first to twelfth primer sets in the InDel primer combination provided in the first aspect above to obtain corresponding PCR amplification products;

[0050] (3) Perform agarose gel electrophoresis on the PCR amplification products obtained in step (2), and count the number of plants with heterozygous bands for the first to twelfth primer sets respectively according to the electrophoresis results; then select the primer set with a relatively large number of plants with heterozygous bands (such as more than 5 plants, the second most number of plants, the largest number of plants) and clear bands as the target primer set;

[0051] (4) Using the genomic DNA of the N melon hybrid seeds to be tested obtained in step (1) as templates respectively, perform PCR amplification using the target primer sets obtained in step (3) to obtain the PCR amplification products of each melon hybrid seed to be tested under the amplification of different target primer sets;

[0052] (5) Perform agarose gel electrophoresis on the PCR amplification products obtained in step (4), and count the number of plants with heterozygous bands in the amplification products of each target primer set respectively according to the electrophoresis results. Among them, the melon lines to be tested with heterozygous bands are hybrid seeds, and the purity of the melon hybrid seeds to be tested is obtained according to the number of plants with heterozygous bands under the amplification of each target primer set.

[0053] In the method of the fourth aspect of the present invention, the target primer set determined by step (3) can be 1, or 2 or more. When there are multiple target primer sets, the final purity of the melon hybrid seeds to be tested can be determined by the purity of the hybrid seeds obtained using multiple target primer sets. Specifically:

[0054] Count the number of plants with heterozygous bands and the number of plants without bands shown by each target primer set, calculate the purity of the melon hybrid seeds to be tested obtained by each target primer set respectively, and then calculate the average value;

[0055] The purity obtained by a certain target primer set = the number of plants with heterozygous bands shown by a certain target primer set / (N - the number of plants without bands of this target primer set) × 100%.

[0056] Further, the melon hybrid to be tested is selected from Jinge Mi Nong No. 11, Jinmi No. 8, Nongjia No. 2, Fuqi Cuiyu, Cuidai M15, Xinli No. 6, Crown, Xiazhou Mi, White Pearl, Red Golden Crown, Guangmi No. 6, Baiyuhong, Network Times, Nongtian Baodian, Xiboluoto No. 3, Xinyuan Mi No. 12, Milv, Jingtian Jasper No. 2, Kaitian No. 9, Zhongtian 382, ​​Hongruihong, M1005, Hongxincui No. 2, Jinruibao, Lumi No. 1, Longxinmi No. 17, Shuangquanmi No. 13, Jingtian Xingkong No. 1, Arusxia, Bohongmi No. 5, Jingtianmi No. 3, Four Seasons Silver Red No. 2, Jiaohong No. 4, M3166, Smetuo, Jindian, Annas, Huozhoumi 125, Jincui, Qing Any of the following: Huami, Hongcuimi, Tiandu Yinmi, Youchuang Xiangxiu, Wanxiang, White-collar Beauty, Xinyuan Mi No. 6, Hengmi No. 2, Jinmiyue, Shengmi Qingmi, Longlin Twenty-Five, Meili, Mitian No. 17, Oriental Pride No. 25, Black Coral, Dumi No. 6, Tongcui No. 1, Qitian Taste, Tianyue Selection, Kentian No. 2, Shangyi Zaoxue, Baichen Xinya, Huatian No. 1, Bixiang, Wanjintian No. 12, Qingtian No. 2, Jinling, Xiangrui Jingrun, Jitian Melon No. 6, Xuenaixiang No. 1, Shengyu No. 18, Pingya No. 1, Liaotian Cuiyu, Linglongcui, Jingtiancuibao, Yutian Jinyu, Xuandian Balixiang, Green Cuiyu, Jingtian Huayi, Huakui, Zhongtian 327, Qianyu No. 6, Ruitian No. 11, Xinmi No. 28.

[0057] 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.

[0058] In the above-described method, the larger the value of N is, the higher the accuracy of identifying the purity of the melon hybrid to be tested is, for example, N≥80, 100, 120, 150 or 200.

[0059] It should be noted that the present invention focuses on the purity assurance of melon 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 melon hybrids.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The InDel primer combination provided by the present invention enables the early identification of existing representative melon hybrids at the seed or seedling stage, thereby ensuring the purity of hybrids, effectively safeguarding the legitimate rights and interests of producers and breeders, and providing technical support for the seed quality management of melon 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. Description of the Drawings

[0062] Figure 1 shows the InDel genotyping effects of 12 primer sets in some tested melon hybrids.

[0063] Figure 2 shows the distribution of heterozygous sites of 12 primer sets in 83 melon species.

[0064] Figure 3 shows the band distribution effect of primer set 4 in 96 heterozygous seeds of Shengjingmi No. 8. Detailed Embodiments

[0065] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0066] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0067] Example 1. Obtaining of InDel primer combination for identifying the purity of melon hybrids.

[0068] I. Discovery of 12 InDel loci

[0069] Based on the resequencing data of 149 representative melon resources, the present invention developed whole-genome InDel markers for melons and used them for variety purity identification, obtaining 12 InDel loci. These 149 melon resources are rich in types, covering thin-skinned melons and thick-skinned melons, and basically include the main ecological types and agronomic traits of melons, reflecting the germplasm representativeness as much as possible and having high genetic diversity.

[0070] Specifically, the screening criteria for InDel loci are as follows: First, InDel loci of the dimorphic type are screened out on the whole-genome chromosomes, and InDel loci with 200 conserved flanks and >30 bp are selected. Then, InDel loci with Miss < 0.2, MAF > 0.2, and He < 0.05 are screened. Finally, blast specific analysis is performed, and 62 pairs of specific primers are designed. These primers were used to screen 83 tested melon hybrids in Table 3. During the screening process, through careful comparison and analysis, primers showing high heterozygosity and polymorphism were finally screened out. After strict screening and evaluation, 12 pairs of primers were finally determined.

[0071] The basic information of 12 InDel loci is shown in the first to fifth columns of Table 1. The positions of the InDel loci on the chromosome were determined based on the alignment with the melon DHL92 reference genome sequence, and the version number of the melon DHL92 reference genome sequence is V3.5.1 (download address: http: / / cucurbitgenomics.org / ftp / genome / melon / DHL92 / v3.5.1 / ).

[0072] Table 1. Basic information of 12 InDel loci

[0073]

[0074]

[0075]

[0076] II. Obtaining of InDel primer combinations for identifying the purity of melon hybrids

[0077] Based on the 12 InDel loci discovered in Step 1, the inventors of the present invention developed InDel primer combinations with high polymorphism and heterozygosity for identifying the purity of melon hybrids.

[0078] The InDel primer combination consists of 12 primer groups, and the name of each primer group is shown in the second column of Table 2. Each primer group consists of 2 primer sequences and is used to amplify one InDel locus. The nucleotide sequences of the respective primers in the 12 primer groups are shown in the fourth column of Table 2.

[0079] Table 2. 12 pairs of Indel primer sequences applicable to melon purity identification

[0080]

[0081]

[0082] Example 2: Validation of the effectiveness of the InDel primer combinations developed in Example 1

[0083] The basic information of the 83 tested melon hybrids in this example is shown in Table 3. The 83 tested melon hybrids are all common excellent hybrids or hybrids introduced from abroad.

[0084] Table 3. Basic information of 83 tested melon hybrids

[0085]

[0086]

[0087] 1. Obtaining genomic DNA of the tested melon hybrids (DNA extraction by magnetic bead method)

[0088] Take an appropriate amount of leaf samples of each melon material and collect them in a centrifuge tube. Add steel beads and place them in liquid nitrogen for freezing. Then use an ultra-high-throughput grinder for grinding. After grinding, add 800 μl of SDS extraction solution to each sample. Incubate in an oven at 60 °C for 30 - 40 min, and invert and mix evenly 3 times during this period. Add 240 μl (i.e., 0.3 times the volume of the SDS extraction solution) of 3M potassium acetate solution to each sample. After fully inverting and mixing evenly 5 times, place it in a refrigerator at 4 °C and let it stand for 20 min. Centrifuge at 4000 rpm for 10 minutes. Prepare a new centrifuge tube or deep well plate in advance, and add 600 μl of magnetic bead isopropanol to it (ensure that the volume is equal to the supernatant). Note that the operation sequence is to add magnetic bead isopropanol first, and then add the supernatant, and the volume of the supernatant aspirated should not exceed 2 / 3 of the total volume. Invert and mix evenly, let it stand at -20 °C until the magnetic beads precipitate for 30 min, invert and mix evenly again, adsorb with a magnetic rack, discard the supernatant, and invert and place it on absorbent paper to blot the residual liquid. Add 200 μl of 75% ethanol solution to the sample, fully invert and mix evenly, and let it stand until the magnetic beads are completely precipitated. Invert and mix evenly again, adsorb with a magnetic rack, discard the supernatant, and invert and place it on absorbent paper to blot the residual liquid. After the magnetic beads are completely air-dried, add 100 μl of pure water to it, fully invert and mix evenly to ensure that the DNA is completely dissolved. Use Nanodrop (micro-spectrophotometer) to take 2 μl of the DNA extraction solution for detection, measure its OD260 / 280 and OD260 / 230 ratios (the ideal range is 1.8 - 2.0), and record the DNA concentration. Subsequently, adjust the DNA concentration to 50 ng / μl for subsequent use.

[0089] 2. Using the genomic DNA of the 83 tested melon hybrids as templates respectively, perform PCR amplification with the 12 primer sets given in Table 2 to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer with "F" in its name and the primer with "R" in its name is 1:1.

[0090] The reaction system for PCR amplification was as follows: 5 μl of template DNA, 10 μl of 2× Mix, 1 μl of forward primer and 1 μl of reverse primer (20 μmol / L), and 3 μl of ultrapure water.

[0091] 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, for 35 cycles; extension at 72 °C for 10 min.

[0092] 3. After completing step 2, the PCR amplification products were subjected to agarose gel electrophoresis, and the results were photographed and read through a gel imaging system. The genotypes of 83 tested melon hybrids based on each InDel locus were judged by the banding pattern. The specific judgment principle was as follows: if a tested melon hybrid showed a heterozygous banding result based on a certain InDel locus, the genotype of the tested melon hybrid based on this InDel locus was heterozygous; if a tested melon hybrid showed a single banding result based on a certain InDel locus, the genotype of the tested melon hybrid based on this InDel locus was homozygous.

[0093] Partial results are shown in Figure 1 . The results showed that good genotyping effects could be obtained for each primer set in the tested melon hybrids.

[0094] 4. Distribution of the number of heterozygous loci and efficiency evaluation

[0095] Based on the genotypes of 83 tested melon hybrids at 12 InDel loci, the number of heterozygous loci for each tested melon hybrid was counted.

[0096] The distribution results of the number of heterozygous loci of 83 tested melon hybrids based on 12 primer sets are shown in Figure 2 . The results showed that each of the 12 primer sets could result in at least one heterozygous locus for each tested melon hybrid.

[0097] Thus, it can be seen that the InDel primer combination developed in Example 1 can be applied to the purity identification of melon hybrids.

[0098] Example 3: Detection of the purity of the hybrid of Shengjingmi No. 8 using the InDel primer combination developed in Example 1

[0099] 1. Obtaining the genomic DNA of the hybrid of Shengjingmi No. 8

[0100] (1) 200 seeds of the hybrid of Shengjingmi No. 8 were planted to obtain seedlings of the hybrid of Shengjingmi No. 8.

[0101] (2) Randomly select the leaves or roots of 96 seedlings of the Shengjingmi 8 hybrid variety, and use the magnetic bead method to extract genomic DNA respectively, obtaining 96 genomic DNAs of the Shengjingmi 8 hybrid variety in sequence.

[0102] 2. Screening of primer sets

[0103] (1) Select the genomic DNAs of 8 hybrid seedlings obtained in step (2), and use them as templates respectively. Perform PCR amplification using 12 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 with "F" in its name to the primer with "R" in its name is 1:1.

[0104] The specific PCR amplification reaction system can be: 5 μl of template DNA (50 ng / μl), 10 μl of 2×Mix, 1 μl of forward primer and 1 μl of reverse primer (20 μmol / L), 3 μl of ultrapure water.

[0105] The specific PCR amplification reaction program can be: 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, for 35 cycles; extension at 72°C for 10 min.

[0106] (2) After completing step (1), perform agarose gel electrophoresis, and use a gel imaging system to photograph and record the electrophoresis results to obtain the results. Compare the number of plants with heterozygous sites in 12 primer sets, and the primer set with the largest number of plants with heterozygous sites is the selected primer set.

[0107] The results show that the number of plants with heterozygous sites in primer set 4 is the largest, which is 8 plants. Therefore, primer set 4 is the selected primer set for subsequent experiments.

[0108] 3. Obtaining the purity of the Shengjingmi 8 hybrid variety

[0109] (1) Use the genomic DNAs of 96 Shengjingmi 8 hybrid varieties as templates respectively, and perform PCR amplification using the selected primer set 4 to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer with "F" in its name to the primer with "R" in its name is 1:1.

[0110] The specific PCR amplification reaction system can be: 5 μl of template DNA (50 ng / μl), 10 μl of 2×Mix, 1 μl of forward primer and 1 μl of reverse primer (20 μmol / L), 3 μl of ultrapure water.

[0111] The reaction procedure for PCR amplification can be specifically 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, for 35 cycles; extension at 72°C for 10 min.

[0112] (2) After completing step (1), perform agarose gel electrophoresis, and use a gel imaging system to photograph and record the electrophoresis results to obtain the results.

[0113] The InDel genotyping results are shown in Figure 3 .

[0114] (3) After completing step (2), count the number of plants with heterozygous sites and the number of plants without heterozygous sites shown by primer set 4; calculate the purity of the hybrid seeds of Shengjingmi 8 according to the following formula;.

[0115] Purity = the number of plants with heterozygous sites shown by the primer set / (96 - the number of plants without bands of the primer set) × 100%.

[0116] The results show that the number of plants with heterozygous bands shown by the primer set is 95, the number of plants with homozygous bands is 1, the number of plants without bands is 0, and the purity is 95 / 96 × 100% = 98.9%.

[0117] Among 200 hybrid seeds, 2 abnormal plants were identified through field identification, and the purity of the hybrid seeds of this variety is 99.0%. This shows that the identification method of the present invention has high accuracy.

[0118] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. An InDel primer combination, comprising a first primer set to a twelfth primer set, corresponding to amplifying twelve InDel markers to perform genotyping or homozygous or heterozygous determination on each InDel marker, wherein: The twelve InDel markers include: Marker TG_Ind01: located at position 912892 on chromosome 1, and is nucleotide T or the nucleotide sequence shown in SEQ ID NO.25; Marker TG_Ind02: located at position 1576348 on chromosome 2, and is nucleotide T or the nucleotide sequence shown in SEQ ID NO.26; Marker TG_Ind03: located at position 21498496 on chromosome 2, and is nucleotide G or the nucleotide sequence shown in SEQ ID NO.27; Marker TG_Ind04: located at position 27217555 on chromosome 3, and is nucleotide T or the nucleotide sequence shown in SEQ ID NO.28; Marker TG_Ind05: located at position 18106952 on chromosome 4, and is nucleotide G or the nucleotide sequence shown in SEQ ID NO.29; Marker TG_Ind06: located at position 21247101 on chromosome 4, nucleotide C or the nucleotide sequence shown in SEQ ID NO.30; Marker TG_Ind07: located at position 16632195 on chromosome 5, which is nucleotide G or the nucleotide sequence shown in SEQ ID NO.31; Marker TG_Ind08: located at position 26006430 on chromosome 7, and is nucleotide T or the nucleotide sequence shown in SEQ ID NO.32; Marker TG_Ind09: located at position 4833893 on chromosome 8, and is nucleotide G or the nucleotide sequence shown in SEQ ID NO.33; Marker TG_Ind10: located at position 6770350 on chromosome 8, nucleotide C or the nucleotide sequence shown in SEQ ID NO.34; Marker TG_Ind11: located at position 23860732 on chromosome 9, nucleotide C or the nucleotide sequence shown in SEQ ID NO.35; Marker TG_Ind12: located at position 22505112 on chromosome 12, which is nucleotide C or the nucleotide sequence shown in SEQ ID NO.36; The positions of the twelve InDel markers on the chromosome are determined based on the comparison of the melon DHL92 reference genome sequence, and the version number of the melon DHL92 reference genome sequence is V3.5.1; The first primer group is used to amplify the molecular marker TG_Ind01; the second primer group is used to amplify the molecular marker TG_Ind02; the third primer group is used to amplify the molecular marker TG_Ind03; the fourth primer group is used to amplify the molecular marker TG_Ind04; the fifth primer group is used to amplify the molecular marker TG_Ind05; the sixth primer group is used to amplify the molecular marker TG_Ind06; the seventh primer group is used to amplify the molecular marker TG_Ind07; the eighth primer group is used to amplify the molecular marker TG_Ind08; the ninth primer group is used to amplify the molecular marker TG_Ind09; the tenth primer group is used to amplify the molecular marker TG_Ind10; the eleventh primer group is used to amplify the molecular marker TG_Ind11; and the twelfth primer group is used to amplify the molecular marker TG_Ind12.

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; The ninth primer set consists of a forward primer F9 shown in SEQ ID NO.17 and a reverse primer R9 shown in SEQ ID NO.18; The tenth primer set consists of a forward primer F10 shown in SEQ ID NO.19 and a reverse primer R10 shown in SEQ ID NO.20; The eleventh primer set consists of a forward primer F11 shown in SEQ ID NO.21 and a reverse primer R11 shown in SEQ ID NO.22; The twelfth primer set consists of a forward primer F12 shown in SEQ ID NO.23 and a reverse primer R12 shown in SEQ ID NO.

24.

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 melon hybrids.

5. The use according to claim 4, characterized in that: The melon hybrids are selected from the group consisting of Jinge Mi Nong No. 11, Jinmi No. 8, Nongjia No. 2, Fuqi Cuiyu, Cuidai M15, Xinli No. 6, Crown, Xiazhou Mi, White Pearl, Red Golden Crown, Guangmi No. 6, Baiyuhong, Internet Times, Nongtian Baodian, Xiboluoto No. 3, Xinyuanmi No. 12, Milv, Jingtian Jasper No. 2, Kaitian No. 9, Zhongtian 382, ​​Hongruihong, M1005, Hongxincui No. 2, Jinruibao, Lumi No. 1, Longxinmi No. 17, Shuangquanmi No. 13, Jingtian Xingkong No. 1, Arusxia, Bohongmi No. 5, Jingtianmi No. 3, Four Seasons Silver Red No. 2, Jiaohong No. 4, M3166, Smetuo, Jindian, Annas, Huozhoumi 125, Jincui, Qinghua Mi, Hongcuimi, Tiandu Yinmi, Youchuang Xiangxiu, Wanxiang, White-collar Beauty, Xinyuan Mi No. 6, Hengmi No. 2, Jinmiyue, Shengmi Qingmi, Longlin Twenty-Five, Meili, Mitian No. 17, Oriental Pride No. 25, Black Coral, Dumi No. 6, Tongcui No. 1, Qitian Taste, Tianyue Selection, Kentian No. 2, Shangyi Zaoxue, Baichen Xinya, Huatian No. 1, Bixiang, Wanjintian No. 12, Qingtian No. 2, Jinling, Xiangrui Jingrun, Jitian Melon No. 6, Snow Milk Fragrance No. 1, Shengyu No. 18, Pingya No. 1, Liaotian Cuiyu, Linglong Cui, Jingtian Cuibao, Yutian Jinyu, Xuandian Balixiang, Green Cuiyu, Jingtian Huayi, Huakui, Zhongtian 327, Qianyu No. 6, Ruitian No. 11, Xinmi No.

28.

6. A method for identifying the purity of a melon hybrid to be tested, characterized in that: The steps include: (1) randomly selecting N melon hybrids to be tested and obtaining their genomic DNA; (2) using genomic DNA of at least eight of the N tested melon hybrids obtained in step (1) as templates, and using the first to twelfth 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 twelfth 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 strains of melon 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 strain of melon hybrids 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 melon strains to be tested with heterozygous bands are hybrids, and the purity of the melon hybrids 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 melon 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 melon 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 melon hybrids to be tested are selected from Jinge Mi Nong No. 11, Jinmi No. 8, Nongjia No. 2, Fuqi Cuiyu, Cuidai M15, Xinli No. 6, Crown, Xiazhou Mi, White Pearl, Red Golden Crown, Guangmi No. 6, Baiyuhong, Network Times, Nongtian Baodian, Xiboluoto No. 3, Xinyuan Mi No. 12, Milv, Jingtian Jasper No. 2, Kaitian No. 9, Zhongtian 382, ​​Hongruihong, M1005, Hongxincui No. 2, Jinruibao, Lumi No. 1, Longxinmi No. 17, Shuangquanmi No. 13, Jingtian Xingkong No. 1, Arusxia, Bohongmi No. 5, Jingtianmi No. 3, Four Seasons Silver Red No. 2, Jiaohong No. 4, M3166, Smeto, Jindian, Annas, Huozhoumi 125, Jincui, Qing Huami, Hongcuimi, Tiandu Yinmi, Youchuang Xiangxiu, Wanxiang, White-collar Beauty, Xinyuan Mi No. 6, Hengmi No. 2, Jinmiyue, Shengmi Qingmi, Longlin Twenty-Five, Meili, Honey Sweet No. 17, Oriental Pride No. 25, Black Coral, Dumi No. 6, Tongcui No. 1, Qitian Taste, Tianyue Selection, Kentian No. 2, Shangyi Zaoxue, Baichen Xinya, Huatian No. 1, Bixiang, Wanjintian No. 12, Qingtian No. 2, Jinling, Xiangrui Jingrun, Jitian Melon No. 6, Snow Milk Fragrance No. 1, Shengyu No. 18, Pingya No. 1, Liaotian Cuiyu, Linglong Cui, Jingtian Cuibao, Yutian Jinyu, Xuandian Balixiang, Green Cuiyu, Jingtian Huayi, Huakui, Zhongtian 327, Qianyu No. 6, Ruitian No. 11, Xinmi No. 28.

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