Method for identifying purity of melon hybrid and InDel primer combination used thereby

Abstract

The application discloses a method for identifying purity of melon hybrid and an InDel primer combination used in the method. The InDel primer combination provided by the application is composed of 12 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 sequentially shown in 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 melon hybrid, so as to ensure the purity of the hybrid, effectively protect the rights and interests of producers and breeders, and provide technical support for seed quality management of melon varieties. The method provided by the application has the advantages of accuracy, low cost, simple operation, clear results, saving of manpower and material resources, and has a very broad application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of hybrid purity identification, and particularly relates to a method for identifying the purity of a Cucumis melo hybrid and an InDel primer combination used in the method. BACKGROUND

[0002] Cucumis melo is an annual vine herb of the Cucurbitaceae family, and its fruit is rich in nutrients and deeply loved by consumers. It is one of the most popular fruits on the Chinese market. In recent years, with the development of the Cucumis melo industry, more and more new varieties have entered the market. As of December 2024, there were 2475 registered Cucumis melo varieties in China, of which more than 95% were first-generation hybrids. Variety purity has become one of the important indicators affecting the quality of Cucumis melo seeds. However, traditional Cucumis melo seed purity identification methods involve field planting, which is complex, time-consuming, and easily affected by environmental factors, and cannot meet the efficient and accurate management needs of 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, and can significantly improve the efficiency and accuracy of Cucumis melo seed purity identification.

[0003] InDel (Insertion-Deletion) markers are markers of insertion and deletion, which means that a certain number of nucleotide insertions or deletions exist in the genomes of other samples compared to the reference genome. Compared with SNP markers, InDel markers have lower requirements for DNA quality and lower detection costs, and can be detected by ordinary electrophoresis platforms. Compared with SSR markers, InDel markers have 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 Cucumis melo hybrid purity identification. Therefore, screening an InDel molecular marker combination suitable for Cucumis melo variety purity is of great significance for improving seed quality, regulating the Cucumis melo seed market, and promoting the healthy development of the Cucumis melo industry.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] In view of the deficiencies of the prior art, one of the purposes of the present application is to provide an InDel primer combination for identifying the purity of a Cucumis melo hybrid.

[0006] A second purpose of the present application is to provide a kit comprising the primer combination described above.

[0007] A third purpose of the present application is to provide the use of the primer combination or the kit described above.

[0008] A fourth purpose of the present application is to provide a method for identifying the purity of a Cucumis melo hybrid to be tested.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The first aspect of this invention provides an InDel primer set, comprising a first primer set to a twelfth primer set, corresponding to the amplification of twelve InDel markers to perform genotyping or homozygous / heterozygous determination for each InDel marker, wherein:

[0011] The twelve InDel markers include:

[0012] The marker TG_Ind01 is located at position 912892 on chromosome 1 and is either nucleotide T or the nucleotide sequence shown in SEQ ID NO.25.

[0013] The marker TG_Ind02 is located at position 1576348 on chromosome 2 and is either nucleotide T or the nucleotide sequence shown in SEQ ID NO.26.

[0014] The marker TG_Ind03 is located at position 21498496 on chromosome 2 and is either nucleotide G or the nucleotide sequence shown in SEQ ID NO.27.

[0015] The marker TG_Ind04 is located at position 27217555 on chromosome 3 and is either nucleotide T or the nucleotide sequence shown in SEQ ID NO.28.

[0016] The marker TG_Ind05 is located at position 18106952 on chromosome 4 and is either nucleotide G or the nucleotide sequence shown in SEQ ID NO.29.

[0017] The marker TG_Ind06 is located at position 21247101 on chromosome 4 and is either nucleotide C or the nucleotide sequence shown in SEQ ID NO.30.

[0018] The marker TG_Ind07 is located at position 16632195 on chromosome 5 and is either nucleotide G or the nucleotide sequence shown in SEQ ID NO.31.

[0019] The marker TG_Ind08 is located at position 26006430 on chromosome 7 and is either nucleotide T or the nucleotide sequence shown in SEQ ID NO.32.

[0020] The marker TG_Ind09 is located at position 4833893 on chromosome 8 and is either nucleotide G or the nucleotide sequence shown in SEQ ID NO.33.

[0021] The marker TG_Ind10 is located at position 6770350 on chromosome 8 and is either nucleotide C or the nucleotide sequence shown in SEQ ID NO.34.

[0022] The marker TG_Ind11 is located at chromosome 9, position 23860732, and is either nucleotide C or the nucleotide sequence shown in SEQ ID NO.35.

[0023] The marker TG_Ind12 is located at position 22505112 on chromosome 12 and is either 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 the alignment of the DHL92 melon reference genome sequence, version number V3.5.1.

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

[0026] The InDel primer set provided by this invention can be used to identify the purity of melon hybrids. If the PCR product of the primer set used to identify the purity of a melon hybrid is a heterozygous band, it means that the melon variety being tested is a hybrid. If it is a single band, it means that the melon variety being tested is not a hybrid, but may be one of the parents, that is, the parents and the hybrid are mixed.

[0027] The hybrid species mentioned in this invention refer to first-generation hybrids, which may contain a mixture of parental lines.

[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] A second aspect of the present invention provides a kit comprising the InDel primer combination described in the first aspect.

[0042] In addition to primer combinations, the kit may also include PCR auxiliary reagents such as ultrapure water, PCR buffer, dNTPs, and DNA polymerase, as well as standard experimental equipment for PCR.

[0043] In the primer set mentioned above, the molar ratio of primers containing "F" in their names to primers containing "R" in their names can be 1:1 during PCR.

[0044] The third aspect of this invention provides the application of the InDel primer combination of the first aspect or the kit of the second aspect in identifying the purity of melon hybrids.

[0045] Furthermore, the melon hybrids are selected from Jin Ge Mi Nong 11, Jin Mi 8, Nong Jia 2, Fu Qi Cui Yu, Cui Dai M15, Xin Li 6, Crown, Xia Zhou Mi, Bai Zhen Zhu, Hong Jin Guan, Guang Mi 6, Bai Yu Hong, Wanglu Shidai, Nong Tian Bao Dian, Xi Bo Luo Tuo 3, Xin Yuan Mi 12, Mi Lü, Jing Tian Bi Yu 2, Kai Tian 9, Zhong Tian 382, ​​Hong Rui Hong, M1005, Hong Xin Cui 2, Jin Rui Bao, Lu Mi 1, Long Xin Mi 17, Shuang Quan Mi 13, Jing Tian Xing Kong 1, A Lu Si Xia, Bo Hong Mi 5, Jing Tian Mi 3, Si Ji Yin Hong 2, Jiao Hong 4, M3166, S Mei Tuo, Jin Dian, Annas, Huo Zhou Mi 125, Jin Cui, and Qing Hua. The following are all of the following: Honey, Red Crisp Honey, Tiandu Silver Honey, Youchuang Xiangxiu, Wanxiang, White-collar Beauty, Xinyuan Honey No. 6, Hengmi No. 2, Jinmiyue, Shengmi Qingmi, Longlin Twenty-Five, Meili, Mitian No. 17, Dongfang Jiaozi No. 25, Black Coral, Dumi No. 6, Tongcui No. 1, Qitian Pinwei, Tianyue Zhenxuan, Kentian No. 2, Shangyi Zaoxue, Baichen Xinya, Huatian No. 1, Bixiang, Wanjintian No. 12, Qingtian No. 2, Jinling, Xiangrui Jingrun, Jitiangua No. 6, Xuenaixiang No. 1, Shengyu No. 18, Pingya No. 1, Liaotian Cuiyu, Linglong Cui, Jingtian Cuibao, Yutian Jinyu, Xuandian Balixiang, Lü Cuiyu, Jingtian Huayi, Huakui, Zhongtian 327, Qianyu No. 6, Ruitian No. 11, and Xinmi No. 28.

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

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

[0048] (1) Randomly select N hybrid melon plants to be tested and obtain their genomic DNA;

[0049] (2) Using the genomic DNA of at least 8 (e.g., 10, 15 or 20) melon hybrids selected from the N melon hybrids to be tested obtained in step (1) as templates, PCR amplification was performed using the first to twelfth primer sets in the InDel primer combination provided in the first aspect above, and the corresponding PCR amplification products were obtained.

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

[0051] (4) Using the genomic DNA of the N muskmelon hybrids to be tested obtained in step (1) as templates, PCR amplification was performed using the target primer sets obtained in step (3) to obtain the PCR amplification products of each muskmelon hybrid to be tested under different target primer sets.

[0052] (5) Perform agarose gel electrophoresis on the PCR amplification products obtained in step (4). According to the electrophoresis results, count the number of plants with heterozygous bands in the amplification products of each target primer group. The test melon lines with heterozygous bands are hybrids. The purity of the test melon hybrids is obtained according to the number of plants with heterozygous bands amplified by each target primer group.

[0053] In the fourth aspect of the method of the present invention, the target primer set determined by step (3) can be one, two or more. When there are multiple target primer sets, the purity of the hybrid obtained from multiple target primer sets can be used to determine the final purity of the melon hybrid to be tested, specifically:

[0054] The number of plants showing heterozygous bands and the number of plants without bands for each target primer set were counted. The purity of the melon hybrids obtained from each target primer set was calculated, and then the average value was calculated.

[0055] The purity of a target primer set = number of strains showing heterozygous bands with the target primer set / (N - number of strains without bands with the target primer set) × 100%.

[0056] Furthermore, the tested melon hybrids were selected from the following varieties: Jingeminong 11, Jinmi 8, Nongjia 2, Fuqi Cuiyu, Cuidai M15, Xinli 6, Crown, Xiazhoumi, Baizhenzhu, Hongjinguan, Guangmi 6, Baiyuhong, Wangluoshidai, Nongtianbaodian, Xiboluo 3, Xinyuanmi 12, Milu, Jingtian Biyu 2, Kaitian 9, Zhongtian 382, ​​Hongruihong, M1005, Hongxincui 2, Jinruibao, Lumi 1, Longxinmi 17, Shuangquanmi 13, Jingtian Xingkong 1, Arusxia, Bohongmi 5, Jingtianmi 3, Siji Yinhong 2, Jiaohong 4, M3166, Smeto, Jindian, Annas, Huozhoumi 125, Jincui, and Qing. Any one of the following: Hua Mi, Hong Cui Mi, Tian Du Yin Mi, You Chuang Xiang Xiu, Wan Xiang, Bai Ling Li Ren, Xin Yuan Mi No. 6, Heng Mi No. 2, Jin Mi Yue, Sheng Mi Qing Mi, Long Lin Twenty-Five, Mei Li, Mi Tian No. 17, Dong Fang Jiao Zi No. 25, Hei Shan Hu, Du Mi No. 6, Tong Cui No. 1, Qi Tian Pin Wei, Tian Yue Zhen Xuan, Ken Tian No. 2, Shang Yi Zao Xue, Bai Chen Xin Ya, Hua Tian No. 1, Bi Xiang, Wan Jin Tian No. 12, Qing Tian No. 2, Jin Ling, Xiang Rui Jing Run, Ji Tian Gua No. 6, Xue Nai Xiang No. 1, Sheng Yu No. 18, Ping Ya No. 1, Liao Tian Cui Yu, Ling Long Cui, Jing Tian Cui Bao, Yu Tian Jin Yu, Xuan Dian Ba ​​Li Xiang, Lü Cui Yu, Jing Tian Hua Yi, Hua Kui, Zhong Tian 327, Qian Yu No. 6, Rui Tian No. 11, Xin Mi No. 28.

[0057] As an optional implementation of the above method, the PCR amplification reaction system may specifically consist of: 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. The PCR amplification reaction program may specifically consist of: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 1 min, 35 cycles; and 72℃ extension for 10 min.

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

[0059] It should be noted that this invention focuses on ensuring the purity of melon hybrids, specifically referring to first-generation hybrids. The core challenge lies in avoiding parental mixing rather than mechanical mixing, i.e., not simply mixing multiple melon hybrids.

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

[0061] The InDel primer combination provided by this invention enables early identification of existing representative melon hybrids at the seed or seedling stage, thereby ensuring the purity of hybrids, effectively protecting the legitimate rights and interests of producers and breeders, and providing technical support for seed quality management of melon varieties. The method provided by this invention has advantages such as high throughput, accuracy, low cost, simple operation, and saving manpower and resources, and has a very broad application prospect. Attached Figure Description

[0062] Figure 1 The InDel typing effect of 12 primer sets in some tested melon hybrids was studied.

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

[0064] Figure 3 The banding effect of primer group 4 in 96 hybrid accessions of Shengjingmi 8 was shown. Detailed Implementation

[0065] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0066] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

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

[0068] I. Discovery of 12 InDel sites

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

[0070] Specifically, the screening criteria for InDel loci were as follows: First, InDel loci belonging to the dimorphic type were screened from the entire genome chromosome. InDel loci with a conserved 200 bp margin and >30 bp were selected. Then, InDel loci with Miss <0.2, MAF >0.2, and He <0.05 were screened. Finally, BLAST-specific analysis was performed, and 62 pairs of specific primers were designed. These primers were used to screen the 83 tested melon hybrids in Table 3. During the screening process, through careful comparison and analysis, primers exhibiting high heterozygosity and polymorphism were finally selected. After rigorous screening and evaluation, 12 pairs of primers were finally determined.

[0071] Basic information on the 12 InDel loci is detailed in columns 1 to 5 of Table 1. The positions of the InDel loci on the chromosome were determined based on alignment with the DHL92 melon reference genome sequence, version V3.5.1 (downloadable from: http: / / cucurbitgenomics.org / ftp / genome / melon / DHL92 / v3.5.1 / ).

[0072] Table 1.12 Basic Information of InDel Sites

[0073]

[0074]

[0075]

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

[0077] Based on the 12 InDel sites discovered in step one, the inventors of this invention developed an InDel primer combination with high polymorphism and heterozygosity for identifying the purity of melon hybrids.

[0078] The InDel primer set consists of 12 primer sets, the name of which is shown in column 2 of Table 2. Each primer set consists of 2 primer sequences and is used to amplify one InDel site. The nucleotide sequences of each primer in the 12 primer sets are shown in column 4 of Table 2.

[0079] Table 2. 12 pairs of Indel primer sequences suitable for melon purity identification.

[0080]

[0081]

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

[0083] The basic information of the 83 tested melon hybrids in this embodiment is shown in Table 3. All 83 tested melon hybrids are common superior hybrids or imported hybrids.

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

[0085]

[0086]

[0087] 1. Obtaining genomic DNA from the tested melon hybrids (DNA extraction using magnetic beads)

[0088] A suitable amount of leaf samples from each melon material were collected in centrifuge tubes, steel beads were added, and the samples were frozen in liquid nitrogen. Then, the samples were ground using an ultra-high throughput grinder. After grinding, 800 μl of SDS extraction buffer was added to each sample. The samples were dried in a 60℃ oven for 30-40 minutes, inverting and mixing three times during this period. 240 μl of 3M potassium acetate solution (0.3 times the volume of the SDS extraction buffer) was added to each sample, and the samples were thoroughly inverted and mixed five times. The samples were then placed in a 4℃ refrigerator and allowed to stand for 20 minutes. The samples were then centrifuged at 4000 rpm for 10 minutes. New centrifuge tubes or deep-well plates were prepared in advance, and 600 μl of magnetic bead isopropanol was added (ensuring the volume was equal to the supernatant). Note that the order of operations is to add the magnetic bead isopropanol first, followed by the supernatant, and the volume of the supernatant aspirated should not exceed [the specified volume]. Add 2 / 3 of the total volume; invert and mix well, let stand at -20℃ for 30 min until the magnetic beads precipitate, invert and mix again, adsorb using a magnetic rack, discard the supernatant, and invert onto absorbent paper to absorb residual liquid; add 200 μl 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 using a magnetic rack, discard the supernatant, and invert onto absorbent paper to absorb residual liquid; after the magnetic beads are completely air-dried, add 100 μl of purified water, invert and mix thoroughly to ensure complete DNA dissolution; use a Nanodrop (micro-spectrophotometer) to take 2 μl of the DNA extract for detection, measure its OD260 / 280 and OD260 / 230 ratios (ideal range for both 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 genomic DNA from 83 tested melon hybrids as templates, PCR amplification was performed using the 12 primer sets given 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.

[0090] The PCR amplification reaction system consisted of: 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.

[0091] The reaction program was as follows: 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.

[0092] 3. After completing step 2, the PCR amplification products were subjected to agarose gel electrophoresis, and the results were photographed and read using a gel imaging system. The genotypes of the 83 tested melon hybrids based on each InDel locus were determined by the banding pattern. The specific determination principles are as follows: if a tested melon hybrid shows a heterozygous band at a certain InDel locus, then the genotype of the tested melon hybrid based on that InDel locus is heterozygous; if a tested melon hybrid shows a single band at a certain InDel locus, then the genotype of the tested melon hybrid based on that InDel locus is homozygous.

[0093] Some results can be found in Figure 1 The results showed that each primer set could achieve good genotyping results in the tested melon hybrids.

[0094] 4. Distribution 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 of heterozygous loci in 83 tested melon hybrids established on 12 primer sets is shown in the figure. Figure 2 The results showed that the 12 primer sets enabled each tested melon hybrid to have at least one heterozygous site.

[0097] Therefore, 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 Shengjingmi No. 8 hybrid using the InDel primer combination developed in Example 1.

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

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

[0101] (2) Leaves or roots of 96 Shengjingmi No. 8 hybrid seedlings were randomly selected, and genomic DNA was extracted by magnetic bead method to obtain 96 Shengjingmi No. 8 hybrid genomic DNA samples.

[0102] 2. Primer set screening

[0103] (1) Genomic DNA from the 8 hybrid seedlings obtained in step (2) was selected and used as templates for PCR amplification using 12 primer sets from the InDel primer combination developed in Example 1, respectively, 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.

[0104] The specific reaction system for 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.

[0105] The specific reaction procedure for PCR amplification can be as follows: 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.

[0106] (2) After completing step (1), perform agarose gel electrophoresis and record the electrophoresis results by taking pictures using a gel imaging system. Compare the number of heterozygous sites in the 12 primer sets, and the primer set with the most heterozygous sites is the selected primer set.

[0107] The results showed that primer set 4 had the most heterozygous sites, with 8 plants. Therefore, primer set 4 was selected as the primer set for subsequent experiments.

[0108] 3. Obtain the purity of Shengjing Honey No. 8 hybrid.

[0109] (1) Using genomic DNA from 96 Shengjingmi No. 8 hybrids as templates, PCR amplification was performed using primer set 4 selected from the screening, and the corresponding PCR amplification products were obtained. 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.

[0110] The specific reaction system for 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.

[0111] The specific reaction procedure for PCR amplification can be as follows: 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.

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

[0113] InDel typing results are shown below 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 in primer set 4; calculate the purity of Shengjingmi No. 8 hybrid according to the following formula;

[0115] Purity = Number of strains with heterozygous sites in primer set / (96 - Number of strains with no band in primer set) × 100%.

[0116] The results showed that the primer set showed 95 heterozygous bands, 1 homozygous band, and 0 no bands, with a purity of 95 / 96 × 100% = 98.9%.

[0117] Two heteromorphic plants were obtained from 200 hybrid plants identified in the field. The purity of the hybrid variety was 99.0%, which 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 not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. InDel primer sets, including primer sets 1 through 12. 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; 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; 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 the forward primer F5 shown in SEQ ID NO.9 and the 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 the forward primer F8 shown in SEQ ID NO.15 and the reverse primer R8 shown in SEQ ID NO.16; 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; 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; 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; 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.

2. A kit comprising the InDel primer combination of claim 1.

3. The application of the InDel primer combination of claim 1 or the kit of claim 2 in identifying the purity of melon hybrids.

4. The application according to claim 3, characterized in that, The melon hybrids mentioned are selected from Jin Ge Minong 11, Jin Mi 8, Nong Jia 2, Fu Qi Cui Yu, Cui Dai M15, Xin Li 6, Crown, Xia Zhou Mi, Bai Zhen Zhu, Hong Jin Guan, Guang Mi 6, Bai Yu Hong, Wanglu Shidai, Nong Tian Bao Dian, Xi Bo Luo Tuo 3, Xin Yuan Mi 12, Mi Lü, Jing Tian Bi Yu 2, Kai Tian 9, Zhong Tian 382, ​​Hong Rui Hong, M1005, Hong Xin Cui 2, Jin Rui Bao, Lu Mi 1, Long Xin Mi 17, Shuang Quan Mi 13, Jing Tian Xing Kong 1, A Lu Si Xia, Bo Hong Mi 5, Jing Tian Mi 3, Si Ji Yin Hong 2, Jiao Hong 4, M3166, S Mei Tuo, Jin Dian, Annas, Huo Zhou Mi 125, Jin Cui, and Qing Hua. Mi, Hongcui Mi, Tiandu Yin Mi, Youchuang Xiangxiu, Wanxiang, Bailing Liren, Xinyuan Mi No. 6, Hengmi No. 2, Jinmiyue, Shengmi Qingmi, Longlin Twenty-Five, Meili, Mitian No. 17, Dongfang Jiaozi No. 25, Heishanhu, Dumi No. 6, Tongcui No. 1, Qitian Pinwei, Tianyue Zhenxuan, Kentian No. 2, Shangyi Zaoxue, Baichen Xinya, Huatian No. 1, Bixiang, Wanjintian No. 12, Qingtian No. 2, Jinling, Xiangrui Jingrun, Jitiangua No. 6, Xuenaixiang No. 1, Shengyu No. 18, Pingya No. 1, Liaotian Cuiyu, Linglong Cui, Jingtian Cuibao, Yutian Jinyu, Xuandian Balixiang, Lücuiyu, Jingtian Huayi, Huakui, Zhongtian 327, Qianyu No. 6, Ruitian No. 11, Xinmi No.

28.

5. A method for identifying the purity of a hybrid melon variety, characterized in that, Includes the following steps: (1) Randomly select N muskmelon hybrids to be tested and obtain their genomic DNA; (2) Using the genomic DNA of at least 8 muskmelon hybrids selected from the N muskmelon hybrids obtained in step (1) as templates, PCR amplification was performed using the first to twelfth primer sets in the InDel primer combination described in claim 1 to obtain the corresponding PCR amplification products. (3) Perform agarose gel electrophoresis on the PCR amplification products obtained in step (2), and count the number of heterozygous bands in the first to twelfth primer sets according to the electrophoresis results; then select the primer set with a relatively large number of heterozygous bands and clear bands as the target primer set. (4) Using the genomic DNA of the N muskmelon hybrids to be tested obtained in step (1) as templates, PCR amplification was performed using the target primer sets obtained in step (3) to obtain the PCR amplification products of each muskmelon hybrid to be tested under different target primer sets. (5) Perform agarose gel electrophoresis on the PCR amplification products obtained in step (4). According to the electrophoresis results, count the number of plants with heterozygous bands in the amplification products of each target primer group. The melon lines to be tested with heterozygous bands are hybrids. 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 group.

6. The method as described in claim 5, characterized in that, The method for "obtaining the purity of the melon hybrid to be tested based on the number of plants with heterozygous bands amplified by each target primer set" is as follows: count the number of plants that show heterozygous bands and the number of plants that do not show bands for each target primer set, calculate the purity of the melon hybrid to be tested obtained by each target primer set, and then calculate the average value. Purity = Number of strains showing heterozygous bands with a certain target primer set / (N - Number of strains without bands with that target primer set) × 100%.

7. The method as described in claim 5 or 6, characterized in that, The tested melon hybrids were selected from the following varieties: Jingeminong 11, Jinmi 8, Nongjia 2, Fuqi Cuiyu, Cuidai M15, Xinli 6, Crown, Xiazhoumi, Baizhenzhu, Hongjinguan, Guangmi 6, Baiyuhong, Wangluoshidai, Nongtianbaodian, Xiboluotuo 3, Xinyuanmi 12, Milu, Jingtian Biyu 2, Kaitian 9, Zhongtian 382, ​​Hongruihong, M1005, Hongxincui 2, Jinruibao, Lumi 1, Longxinmi 17, Shuangquanmi 13, Jingtian Xingkong 1, Arusxia, Bohongmi 5, Jingtianmi 3, Siji Yinhong 2, Jiaohong 4, M3166, Smeto, Jindian, Annas, Huozhoumi 125, Jincui, and Qing. Hua Mi, Hong Cui Mi, Tian Du Yin Mi, You Chuang Xiang Xiu, Wan Xiang, Bai Ling Li Ren, Xin Yuan Mi No. 6, Heng Mi No. 2, Jin Mi Yue, Sheng Mi Qing Mi, Long Lin Twenty-Five, Mei Li, Mi Tian No. 17, Dong Fang Jiao Zi No. 25, Hei Shan Hu, Du Mi No. 6, Tong Cui No. 1, Qi Tian Pin Wei, Tian Yue Zhen Xuan, Ken Tian No. 2, Shang Yi Zao Xue, Bai Chen Xin Ya, Hua Tian No. 1, Bi Xiang, Wan Jin Tian No. 12, Qing Tian No. 2, Jin Ling, Xiang Rui Jing Run, Ji Tian Gua No. 6, Xue Nai Xiang No. 1, Sheng Yu No. 18, Ping Ya No. 1, Liao Tian Cui Yu, Ling Long Cui, Jing Tian Cui Bao, Yu Tian Jin Yu, Xuan Dian Ba ​​Li Xiang, Lü Cui Yu, Jing Tian Hua Yi, Hua Kui, Zhong Tian 327, Qian Yu No. 6, Rui Tian No. 11, Xin Mi No. 28.

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