A method for identifying the purity of melon hybrids and the SNP primer combination used therein.

Abstract

This invention relates to a method for identifying the purity of melon hybrids and the SNP primer combination used therein. The SNP primer combination provided by this invention consists of 12 primer sets; each primer set consists of 3 primer sequences used to amplify one SNP site; the nucleotide sequences of each primer are shown sequentially as SEQ ID NO:1 to SEQ ID NO:36. This SNP primer combination can be used for early identification of melon hybrids in the seed or seedling stage, ensuring the purity of the hybrids, effectively protecting the rights and interests of producers and breeders, and providing technical support for the 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.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for identifying the purity of melon hybrids and the SNP primer combination used therein; hybrid refers to a first-generation hybrid, which may contain parental contamination. Background Technology

[0002] Muskmelon, an annual vine belonging to the Cucurbitaceae family and the Cucurbita genus, is a popular fruit among consumers and one of the best-selling fruits in the Chinese market. In recent years, with the development of the muskmelon industry, more and more new varieties have entered the market, with over 2,000 muskmelon varieties registered nationwide. This has made seed quality increasingly important. As is well known, the vast majority of muskmelon varieties on the market are hybrids. During the production process, incomplete or untimely emasculation or human error leading to the introduction of hybrid seeds can reduce their purity. The purity assessment of hybrid seeds is a crucial indicator for determining the quality of commercially available seeds, an important means of preventing counterfeit and substandard seeds from entering the market, and a vital channel for seed companies to control product quality.

[0003] There are two methods for determining the purity of melon seeds: field identification and molecular identification. Traditional field identification, while providing direct statistical results, is time-consuming, energy-intensive, and requires breeders with extensive experience to determine difficult-to-determine phenotypes. Furthermore, close parental relationships and low hybridization rates can introduce errors in field morphological observations, affecting purity test results. With the development of molecular biology techniques, molecular markers based on DNA polymorphism have been widely applied in commercial breeding and purity testing. Therefore, screening for molecular marker combinations based on DNA detection technology to determine the purity of melon hybrids is particularly important.

[0004] Currently, the SSR marker method is mainly used for DNA molecular detection to identify the purity of melon hybrids in my country. However, the number of varieties used for SSR primer screening is limited, and the detection method is prone to inaccurate, false positive, and false negative results, making it unsuitable for the requirements of automation, high throughput, and large-scale testing. SNPs, as third-generation molecular markers, are widely distributed in the genome, with an average of one SNP per 1000 bp. They are genetically stable and easy to automate. SNP genotyping does not require control varieties, presents results with accurate base pairs, has high reliability and accuracy, and is simple to statistically analyze. KASP (Kompetitive Allele Specific PCR) is a commonly used method for SNP genotyping, characterized by high stability, accuracy, and low cost, and has been widely applied in high-throughput molecular-assisted breeding and variety identification. Therefore, identifying the purity of melon hybrids through SNP molecular markers is the most practical and feasible method.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, one of the objectives of this invention is to provide a method for identifying the purity of melon hybrids and the SNP primer combination used therein.

[0007] A second objective of this invention is to provide the use of the above-mentioned SNP primer combinations.

[0008] The third objective of this invention is to provide a combination of SNP sites.

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

[0010] The first aspect of this invention provides a combination of SNP sites, including 12 SNP sites from the melon genome, wherein the 12 SNP sites are as follows: TGSNP01 is located at nucleotide 8779544 on chromosome 1 of the melon DHL92 reference genome, and the nucleotide base of this site is A or G; TGSNP02 is located at nucleotide 1937599 on chromosome 2 of the melon DHL92 reference genome, and the nucleotide base of this site is G or C; TGSNP03 is located at... The TGSNP04 site is located at nucleotide 28641302 on chromosome 3 of the DHL92 reference genome, with a nucleotide base of G or A; the TGSNP05 site is located at nucleotide 16951407 on chromosome 5 of the DHL92 reference genome, with a nucleotide base of A or T; the TGSNP06 site is located at nucleotide 35 on chromosome 6 of the DHL92 reference genome. Nucleotide 52531, the nucleotide base at this site is T or A; TGSNP07 is located at nucleotide 21326763 on chromosome 7 of the DHL92 reference genome of melon, the nucleotide base at this site is G or A; TGSNP08 is located at nucleotide 176762 on chromosome 8 of the DHL92 reference genome of melon, the nucleotide base at this site is G or A; TGSNP09 is located at nucleotide 21606061 on chromosome 9 of the DHL92 reference genome of melon, this site... The nucleotide bases at this site are G or A; the TGSNP10 site is located at nucleotide 8407131 on chromosome 10 of the DHL92 reference genome of melon, and the nucleotide base at this site is C or A; the TGSNP11 site is located at nucleotide 20770253 on chromosome 11 of the DHL92 reference genome of melon, and the nucleotide base at this site is T or C; the TGSNP12 site is located at nucleotide 1189283 on chromosome 12 of the DHL92 reference genome of melon, and the nucleotide base at this site is T or C.

[0011] A second aspect of the present invention provides an SNP primer set comprising a first primer set to a twelfth primer set, wherein,

[0012] The first primer set was used to amplify the SNP site TGSNP01 in the melon genome to determine the genotype corresponding to the SNP site TGSNP01. The SNP site TGSNP01 is located at nucleotide 8779544 on chromosome 1 of the melon DHL92 reference genome, and the nucleotide base of this site is A or G.

[0013] The second primer set was used to amplify the SNP site TGSNP02 in the melon genome to determine the genotype corresponding to the SNP site TGSNP02. The SNP site TGSNP02 is located at nucleotide 1937599 on chromosome 2 of the melon DHL92 reference genome, and the nucleotide base of this site is G or C.

[0014] The third primer set was used to amplify the SNP site TGSNP03 in the melon genome to determine the genotype corresponding to the SNP site TGSNP03. The SNP site TGSNP03 is located at nucleotide 28641302 on chromosome 3 of the melon DHL92 reference genome, and the nucleotide base of this site is G or A.

[0015] The fourth primer set was used to amplify the SNP site TGSNP04 in the melon genome to determine the genotype corresponding to the SNP site TGSNP04. The SNP site TGSNP04 is located at nucleotide 12956460 on chromosome 4 of the melon DHL92 reference genome, and the nucleotide base of this site is A or G.

[0016] The fifth primer set was used to amplify the SNP site TGSNP05 in the melon genome to determine the genotype corresponding to the SNP site TGSNP05. The SNP site TGSNP05 is located at nucleotide 16951407 on chromosome 5 of the melon DHL92 reference genome, and the nucleotide base of this site is A or T.

[0017] The sixth primer set was used to amplify the SNP site TGSNP06 in the melon genome to determine the genotype corresponding to the SNP site TGSNP06. The SNP site TGSNP06 is located at nucleotide 3552531 on chromosome 6 of the melon DHL92 reference genome, and the nucleotide base of this site is T or A.

[0018] The seventh primer set was used to amplify the SNP site TGSNP07 in the melon genome to determine the genotype corresponding to the SNP site TGSNP07. The SNP site TGSNP07 is located at nucleotide 21326763 on chromosome 7 of the melon DHL92 reference genome, and the nucleotide base of this site is G or A.

[0019] The eighth primer set was used to amplify the SNP site TGSNP08 in the melon genome to determine the genotype corresponding to the SNP site TGSNP08. The SNP site TGSNP08 is located at nucleotide 176762 on chromosome 8 of the melon DHL92 reference genome, and the nucleotide base of this site is G or A.

[0020] The ninth primer set was used to amplify the SNP site TGSNP09 in the melon genome to determine the genotype corresponding to the SNP site TGSNP09. The SNP site TGSNP09 is located at nucleotide 21606061 on chromosome 9 of the melon DHL92 reference genome, and the nucleotide base of this site is G or A.

[0021] The tenth primer set was used to amplify the SNP site TGSNP10 in the melon genome to determine the genotype corresponding to the SNP site TGSNP10. The SNP site TGSNP10 is located at nucleotide 8407131 on chromosome 10 of the melon DHL92 reference genome, and the nucleotide base of this site is C or A.

[0022] The eleventh primer set was used to amplify the SNP site TGSNP11 in the melon genome to determine the genotype corresponding to the SNP site TGSNP11. The SNP site TGSNP11 is located at nucleotide 20770253 on chromosome 11 of the melon DHL92 reference genome, and the nucleotide base of this site is T or C.

[0023] The twelfth primer set was used to amplify the SNP site TGSNP12 in the melon genome to determine the genotype corresponding to the SNP site TGSNP12. The SNP site TGSNP12 is located at nucleotide 1189283 on chromosome 12 of the melon DHL92 reference genome, and the nucleotide base of this site is T or C.

[0024] In the above SNP primer combination, as a preferred embodiment, the first primer set consists of forward primer 1F1 shown in SEQ ID NO: 1, forward primer 1F2 shown in SEQ ID NO: 2, and reverse primer 1R shown in SEQ ID NO: 3;

[0025] The second primer set consists of forward primer 2F1 shown in SEQ ID NO: 4, forward primer 2F2 shown in SEQ ID NO: 5, and reverse primer 2R shown in SEQ ID NO: 6.

[0026] The third primer set consists of forward primer 3F1 shown in SEQ ID NO: 7, forward primer 3F2 shown in SEQ ID NO: 8, and reverse primer 3R shown in SEQ ID NO: 9.

[0027] The fourth primer set consists of forward primer 4F1 shown in SEQ ID NO: 10, forward primer 4F2 shown in SEQ ID NO: 11, and reverse primer 4R shown in SEQ ID NO: 12, as shown in the sequence listing;

[0028] The fifth primer set consists of forward primer 5F1 shown in SEQ ID NO: 13, forward primer 5F2 shown in SEQ ID NO: 14, and reverse primer 5R shown in SEQ ID NO: 15, as shown in the sequence listing;

[0029] The sixth primer set consists of forward primer 6F1 shown in SEQ ID NO: 16, forward primer 6F2 shown in SEQ ID NO: 17, and reverse primer 6R shown in SEQ ID NO: 18, as shown in the sequence listing.

[0030] The seventh primer set consists of forward primer 7F1 shown in SEQ ID NO: 19, forward primer 7F2 shown in SEQ ID NO: 20, and reverse primer 7R shown in SEQ ID NO: 21, as listed in the sequence listing;

[0031] The eighth primer set consists of forward primer 8F1 shown in SEQ ID NO: 22, forward primer 8F2 shown in SEQ ID NO: 23, and reverse primer 8R shown in SEQ ID NO: 24;

[0032] The ninth primer set consists of forward primer 9F1 shown in SEQ ID NO: 25, forward primer 9F2 shown in SEQ ID NO: 26, and reverse primer 9R shown in SEQ ID NO: 27, as shown in the sequence listing;

[0033] The tenth primer set consists of forward primer 10F1 shown in SEQ ID NO: 28, forward primer 10F2 shown in SEQ ID NO: 29, and reverse primer 10R shown in SEQ ID NO: 30.

[0034] The eleventh primer set consists of the forward primer 11F1 shown in SEQ ID NO: 31, the forward primer 11F2 shown in SEQ ID NO: 32, and the reverse primer 11R shown in SEQ ID NO: 33;

[0035] The twelfth primer set consists of the forward primer 12F1 shown in SEQ ID NO: 34, the forward primer 12F2 shown in SEQ ID NO: 35, and the reverse primer 12R shown in SEQ ID NO: 36.

[0036] In sequences (SEQ ID NO:) 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, and 34, the nucleotide sequences represented from position 1 to 21 starting from the 5' end are fluorescent tag sequences (i.e., FAM fluorescent tag sequences), with a specific blue fluorescence signal. In sequences (SEQ ID NO:) 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, and 35, the nucleotide sequences represented from position 1 to 21 starting from the 5' end are also fluorescent tag sequences (i.e., HEX fluorescent tag sequences), with a specific red fluorescence signal.

[0037] In the above SNP primer combination, as a preferred embodiment, the first primer set consists of forward primer 1F1 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 1, forward primer 1F2 shown in positions 22 to 47 from the 5' end of SEQ ID NO: 2, and reverse primer 1R shown in SEQ ID NO: 3;

[0038] The second primer set consists of forward primer 2F1 shown at positions 22 to 44 from the 5' end of SEQ ID NO: 4, forward primer 2F2 shown at positions 22 to 44 from the 5' end of SEQ ID NO: 5, and reverse primer 2R shown at SEQ ID NO: 6.

[0039] The third primer set consists of forward primer 3F1 shown in positions 22 to 50 from the 5' end of SEQ ID NO: 7, forward primer 3F2 shown in positions 22 to 52 from the 5' end of SEQ ID NO: 8, and reverse primer 3R shown in SEQ ID NO: 9;

[0040] The fourth primer set consists of forward primer 4F1 shown in positions 22 to 49 from the 5' end of SEQ ID NO: 10, forward primer 4F2 shown in positions 22 to 47 from the 5' end of SEQ ID NO: 11, and reverse primer 4R shown in SEQ ID NO: 12;

[0041] The fifth primer set consists of forward primer 5F1 shown in positions 22 to 46 from the 5' end of SEQ ID NO: 13, forward primer 5F2 shown in positions 22 to 46 from the 5' end of SEQ ID NO: 14, and reverse primer 5R shown in SEQ ID NO: 15;

[0042] The sixth primer set consists of forward primer 6F1 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 16, forward primer 6F2 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 17, and reverse primer 6R shown in SEQ ID NO: 18;

[0043] The seventh primer set consists of forward primer 7F1 shown in positions 22 to 46 from the 5' end of SEQ ID NO: 19, forward primer 7F2 shown in positions 22 to 47 from the 5' end of SEQ ID NO: 20, and reverse primer 7R shown in SEQ ID NO: 21;

[0044] The eighth primer set consists of forward primer 8F1 shown in positions 22 to 52 from the 5' end of SEQ ID NO: 22, forward primer 8F2 shown in positions 22 to 55 from the 5' end of SEQ ID NO: 23, and reverse primer 8R shown in SEQ ID NO: 24;

[0045] The ninth primer set consists of forward primer 9F1 shown in positions 22 to 46 from the 5' end of SEQ ID NO: 25, forward primer 9F2 shown in positions 22 to 47 from the 5' end of SEQ ID NO: 26, and reverse primer 9R shown in SEQ ID NO: 27;

[0046] The tenth primer set consists of forward primer 10F1 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 28, forward primer 10F2 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 29, and reverse primer 10R shown in SEQ ID NO: 30;

[0047] The eleventh primer set consists of forward primer 11F1 shown in positions 22 to 46 from the 5' end of SEQ ID NO: 31, forward primer 11F2 shown in positions 22 to 45 from the 5' end of SEQ ID NO: 32, and reverse primer 11R shown in SEQ ID NO: 33;

[0048] The twelfth primer set consists of forward primer 12F1 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 34, forward primer 12F2 shown in positions 22 to 47 from the 5' end of SEQ ID NO: 35, and reverse primer 12R shown in SEQ ID NO: 36;

[0049] Since the nucleotide sequences shown from position 1 to position 21 from the 5′ end in sequences (SEQ ID NO:) 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, and 35 are fluorescent tag sequences, and the primer-specific fragments only begin from position 22, the two forward primers in each of the above-mentioned preferred primer sets are both specific sequences in sequences (SEQ ID NO:) 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, and 35.

[0050] When identifying the purity of a melon hybrid, if sequencing is used to determine the genotype of the SNP site, the twelve preferred primer sets mentioned above can be used directly. Each primer set consists of a specific fragment in the forward primer and does not contain a fluorescent tag sequence. Of course, sequences containing fluorescent tags can also be used. If fluorescence signals are used to determine the genotype of the SNP site, fluorescent tag sequences emitting different colors can be added to the 5' ends of the two forward primers in each of the twelve preferred primer sets mentioned above. The fluorescent tag sequences can be selected from FAM, TET, VIC, and HEX.

[0051] When performing PCR using any of the primer sets described above, the molar ratio of primers containing "F1" in their names, primers containing "F2" in their names, and primers containing "R" in their names can specifically be 2:2:5.

[0052] A third aspect of this invention provides a kit containing the aforementioned SNP primer combinations. Kits containing any of the aforementioned SNP primer combinations also fall within the scope of this invention. The kit is used to identify the purity of melon hybrids.

[0053] The kit may also include reagents for other competitive allele-specific PCR.

[0054] The preparation method of the kit is also within the scope of protection of this invention. The preparation method of the kit includes the step of individually packaging each primer from any of the primer sets described above.

[0055] The fourth aspect of the present invention provides the application of any of the above-described SNP site combinations or any of the above-described SNP primer combinations, which can be x1) or x2): x1) preparing a kit for identifying the purity of melon hybrids; x2) identifying the purity of melon hybrids.

[0056] The fifth aspect of this invention also provides a method for identifying the purity of a melon hybrid to be tested.

[0057] The method for identifying the purity of a melon hybrid provided by this invention includes two methods, specifically method one, which may include the following steps:

[0058] (a1) Obtain the genomic DNA of N muskmelon hybrids to be tested; N is a natural number greater than 95;

[0059] (a2) Select 8-12 (e.g., 8-10, 10-12, 8, 10 or 12) muskmelon hybrids to be tested from step (a1). For each selected muskmelon hybrid to be tested, use the genome of the muskmelon hybrid to be tested as a template, and perform PCR amplification using the 12 primer sets in the SNP primer combination (each primer contains a fluorescent tag sequence) to obtain the corresponding PCR amplification products.

[0060] (a3) After completing step (a2), use an instrument to detect the fluorescence signal of each PCR amplification product, and count the number of strains that represent heterozygotes, such as green fluorescence signals, in each of the 12 primer sets; the primer set that shows the most strains that represent heterozygotes, such as green fluorescence signals, is the target primer set.

[0061] (a4) Using the genomic DNA of the N muskmelon hybrids to be tested obtained in step (a1) as templates, PCR amplification was performed using the target primer set to obtain the corresponding PCR amplification products.

[0062] (a5) After completing step (a4), the fluorescence signal of each PCR amplification product is detected by an instrument, and the purity of the melon hybrid to be tested is obtained according to the color of the fluorescence signal.

[0063] In Method 1 above, the method of "obtaining the purity of the melon hybrid to be tested based on the color of the fluorescence signal" can be as follows: count the number of plants that show fluorescence signals representing heterozygosity (e.g., green fluorescence), the number of plants that show fluorescence signals representing homozygosity, and the number of plants that do not show fluorescence signals. Calculate the purity according to the following formula. When there are two or more target primer sets, calculate the purity of each target primer set separately, and then take the average value as the final purity of the melon hybrid to be tested.

[0064] Number of plants without fluorescent signal = N - Number of plants displaying fluorescence representing heterozygote, such as green fluorescence - Number of plants displaying fluorescence representing homozygote (e.g., subtract the sum of the number of plants displaying red fluorescence and the number of plants displaying blue fluorescence).

[0065] Purity = Number of strains showing a heterozygous green fluorescent signal (e.g., N) / (Number of strains without fluorescent signal in the target primer set) × 100%.

[0066] In Method 1 above, the specific reaction program for PCR amplification can be as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touch-down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, continued amplification for 26 cycles. If the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20 s, 55℃ annealing and extension for 1 min, 5 cycles) until satisfactory results are obtained.

[0067] The second method for identifying the purity of a melon hybrid provided by this invention may include the following steps:

[0068] (b1) Obtain the genomic DNA of N melon hybrids to be tested; N is a natural number greater than 95;

[0069] (b2) Select genomic DNA from 8-12 (e.g., 8-10, 10-12, 8, 10 or 12) muskmelon hybrids to be tested from step (b1). For each selected muskmelon hybrid to be tested, use the genome of the muskmelon hybrid to be tested as a template and perform PCR amplification using the 12 primer sets in the above SNP primer combination to obtain the corresponding PCR amplification products.

[0070] (b3) Take each PCR amplification product obtained in step (b2) and sequence it; based on the sequencing results, obtain the number of heterozygous strains based on 12 SNP sites, and the primer set with the largest number of strains is the target primer set.

[0071] (b4) Using the genomic DNA of the N muskmelon hybrids to be tested obtained in step (b1) as templates, PCR amplification was performed using the target primer set to obtain the corresponding PCR amplification products.

[0072] (b5) Take each PCR amplification product obtained in step (b4) and sequence it; obtain the purity of the melon hybrid to be tested based on the sequencing results.

[0073] In Method 2 above, the primers in the 12 primer sets of the SNP primer combination may or may not contain fluorescent tag sequences.

[0074] In Method 2 above, the method for "obtaining the purity of the melon hybrid to be tested based on the sequencing results" can be as follows: count the number of plants with heterozygous genotypes based on SNP sites of the target primer set and the number of plants that did not obtain PCR amplification products, and calculate the purity according to the following formula. When there are two or more target primer sets, calculate the purity of each target primer set separately, and then take the average value as the final purity of the melon hybrid to be tested.

[0075] Purity = Number of heterozygous strains with the target primer set based on the SNP site / (N - Number of strains with the target primer set that did not yield PCR amplification products) × 100%.

[0076] In any of the methods described above, the muskmelon hybrid to be tested may be: Xin Hongxin Cui, Jinlong, Xue Li Hong, Huangpi 9818, RX-99, Xiangrui No. 1, Jinyu, Elizabeth, Honglv Zaocui, E'mian Gua No. 5, Shiji Mi, Huangguan, Zhuguan, Xuetian No. 1, Fengtian No. 1, Xiboluo, Jinlu No. 3, Jiu Hongrui, Jiu Qing Mi, RX-14-3, Yulu No. 1, Xin Chengxiang, Xin Shiji, Yinlu No. 1, Fengtian No. 7, Huang Jingyu, Hongjia, Elizabeth, Basu 317, Jingtian 208, Xianghua, Jinli, Jinmilong, Yongtian No. 5, Dongfangmi No. 1, Cuitian No. 1, Xinhui, Jianghuaimi No. 3, Hongyou, Zhongtian No. 5, Baiyun, Gansu Bailangua, Hongsushou 1401, Hongsushou 1402, Yanyang, Cicui No. 2, Jianghuaimi No. 6, Yongtian No. 7, Yongyue No. 1, Cixi Caigua, Yongtian No. 8, Xiaobaigua, Zhongtian No. 7, Zhongtian No. 2, Zhongtian No. 8, Jianghuaimi No. 2, Jianghuaimi No. 1, Mifeng No. 1, Xiangrui 301, Jinfei, Zhongyun 20, Ruixue No. 8 Fengtian No. 7, Fengwei No. 4, Regua Danzi, Fengwei No. 5, Moxiangyu, Baiyu Mantang, Tianxiangyu, Jincheng, Yuruyi, Qingsu, Jianghuai Mi No. 7, Xizhou Mi No. 25, Zhongtian Cuixue, Zhongtian Cuibao, Dongfang Huacui, Huacui, Chuangyan Cuicui Tian, ​​Riben Tianbao, M16002, Dongzhixing, Hongyu Cui, Yuhong, Dongfang Cuimi, Fengmi 29, Xizhou Mi No. 25, Yinmi 58, Cuixue No. 5, Xiangshan Xuemi, Jingyu 357, Jingyu 280, Jingyu 30, Jiaoao, Jingyu 200 8. Jingyu Yangjiao Su, Jingyu 10, Jingyu No. 3, Jingyu Moon, Jingyu Green Treasure, Jingyu Xiangshuai, Jingyu Mobao, Jingyu White Meteor, Jingyu Yellow Meteor, Jingyu No. 5, Jingyu No. 4, Jingyu No. 2, Yu Meiren, Tianguan No. 3, Jingyu Mi 28, Tianguan No. 1, Tianmi Mi, Jingmi No. 8, Jingmi No. 10, Zaoshu Yichun, Jiujiu Hong, Jingmi No. 7, Jingmi No. 15, Yite Jin, Yite Bai, Yangjiao Cui, Mi Cui Xiangyuan, Jingmi No. 11, Chunpeng No. 5, Zhongmi 198, Chizi Niu (any one).

[0077] In any of the methods described above, the larger the value of N, the higher the accuracy of identifying the purity of the melon hybrid to be tested.

[0078] It should be noted that the melon hybrids mentioned in this invention refer to first-generation hybrids, which can only be a mixture of parent varieties; they are not mechanically mixed, that is, a mixture of several melon hybrids.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] The SNP primer combinations provided by this invention can be used for early identification of melon hybrids in seeds or seedlings, ensuring the purity of hybrids, effectively protecting the 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

[0081] Figure 1 The SNP genotyping effects of 12 primer sets in some tested melon hybrids were studied.

[0082] Figure 2 The distribution of heterozygous loci in 126 tested melon hybrids using 12 primer sets.

[0083] Figure 3 The SNP genotyping effects of primer set 3 and primer set 5 in 96 Jingyu No. 1 hybrids were determined. Detailed Implementation

[0084] The following examples are provided to help to better understand the present invention, but are not intended to limit the invention.

[0085] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0086] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0087] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0088] Example 1: Obtaining SNP primer combinations for identifying the purity of melon hybrids

[0089] I. Discovery of 12 SNP sites

[0090] This invention, based on resequencing data from 49 representative melon resources, obtained 12 SNP loci. These 49 melon resources are diverse, covering non-netted thick-skinned melons (15), netted thick-skinned melons (18), thin-skinned melons (7), and thin-skinned vegetable melons (9), basically encompassing the main ecological types and agronomic traits of melons, reflecting germplasm representativeness as much as possible, and possessing high genetic diversity.

[0091] Specifically, the screening criteria for SNP sites are as follows: SNP sites with uniform location, good polymorphism, low heterozygosity, MAF>0.3, good PCA clustering effect, high discrimination, and conserved 50bp sequences on both sides (no InDel, no SSR, no other SNPs) are selected throughout the genome.

[0092] Basic information on the 12 SNP loci is detailed in columns 1 to 5 of Table 1. The positions of the SNP 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 / ).

[0093] Table 1.12 Basic Information of SNP Loci

[0094] Name of SNP locus Chromosome Location on chromosomes Base type (ref) Base type (alt) TGSNP01 Chr1 8779544 A G TGSNP02 Chr2 1937599 G C TGSNP03 Chr3 28641302 G A TGSNP04 Chr4 12956460 A G TGSNP05 Chr5 16951407 A T TGSNP06 Chr6 3552531 T A TGSNP07 Chr7 21326763 G A TGSNP08 Chr8 176762 G A TGSNP09 Chr9 21606061 G A TGSNP10 Chr10 8407131 C A TGSNP11 Chr11 20770253 T C TGSNP12 Chr12 1189283 T C

[0095] II. Obtaining SNP primer combinations for identifying the purity of melon hybrids

[0096] Based on the 12 SNP sites discovered in step one, the inventors of this invention developed a combination of SNP primers with high polymorphism for identifying the purity of melon hybrids.

[0097] The SNP primer sets consist of 12 primer groups, the names of which are shown in column 2 of Table 2. Each primer group consists of 3 primer sequences and is used to amplify one SNP site. The nucleotide sequences of each primer in the 12 primer groups are shown in column 4 of Table 2.

[0098] Table 2

[0099]

[0100]

[0101]

[0102] Note: A single underscore indicates a FAM fluorescent tag sequence, and a double underscore indicates a HEX fluorescent tag sequence.

[0103] Example 2: Validation of the SNP primer combinations developed in Example 1

[0104] The basic information of the 126 tested melon hybrids in this embodiment is shown in Table 3. All 126 tested melon hybrids are common superior hybrids or imported hybrids.

[0105] Table 3. Basic information of 126 tested melon hybrids

[0106]

[0107]

[0108] 1. Obtaining genomic DNA from the tested melon hybrids

[0109] Genomic DNA was extracted from the leaves (with true leaves from 30 seeds mixed in) of 126 tested melon hybrids using the CTAB method to obtain the genomic DNA of the tested melon hybrids.

[0110] The quality and concentration of genomic DNA from the tested melon hybrids must meet the requirements for PCR. The standards are as follows: agarose gel electrophoresis shows a single DNA band without obvious diffusion; the A260 / A280 ratio detected by a Nanodrop2000 (Thermo) UV spectrophotometer is around 1.8, and the A260 / A230 ratio is greater than 1.8; the concentration of genomic DNA from the tested melon hybrids is 10-30 ng / μL.

[0111] 2. Using genomic DNA from 126 tested melon hybrids as templates, PCR amplification was performed using the 12 primer sets listed in Table 2 to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2", and primers containing "R" was 2:2:5.

[0112] The reagents, consumables, and instruments used in this reaction system were all provided by LGC. The dosage, usage, and experimental procedures were all carried out in accordance with LGC's KASP user guide and manual (www.lgcgenomics.com). The KASPar reaction was carried out in 384-well plates (Part No. KBS-0750-001) or 96-well plates (Part No. KBS-0751-001), with a reaction volume of 3 μl or 10 μl, as shown in Table 4 below.

[0113] Table 4: KASP reaction systems in 384-well or 96-well plates

[0114]

[0115] The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touchdown program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, and continued amplification for 26 cycles.

[0116] 3. After completing step 2, when the temperature of each PCR amplification product drops below 40℃, the fluorescence value is read by scanning with the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is read at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is read at an excitation wavelength of 528nm and an emission wavelength of 560nm). The genotype of the 126 tested melon hybrids based on each SNP locus is determined according to the fluorescence signal color. The specific judgment principles are as follows: If a tested melon hybrid shows a blue or red fluorescent signal based on a certain SNP locus, it means that the genotype of the tested melon hybrid based on that SNP locus is homozygous, meaning that the SNP locus and the corresponding primer set cannot be used to identify the purity of the tested melon hybrid; if a tested melon hybrid shows a green fluorescent signal based on a certain SNP locus, it means that the genotype of the tested melon hybrid based on that SNP locus is heterozygous, meaning that the SNP locus and the corresponding primer set can be used to identify the purity of the tested melon hybrid.

[0117] It should be noted that if the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20s, 55℃ annealing and extension for 1min, 5 cycles) until the results are satisfactory.

[0118] 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.

[0119] 4. Distribution of heterozygous loci and efficiency evaluation

[0120] (1) Based on the genotypes of 126 tested melon hybrids at 12 SNP loci, the number of heterozygous loci for each tested melon hybrid was counted.

[0121] The distribution of heterozygous loci in 126 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.

[0122] (2) Sequential analysis can be used to reduce the workload of hybrid purity identification.

[0123] The results showed that the 12 primer sets achieved 100% coverage of heterozygous sites in 126 tested melon hybrids.

[0124] Therefore, the SNP primer combination developed in Example 1 can be applied to the purity identification of melon hybrids.

[0125] Example 3: Detection of the purity of Jingyu No. 1 hybrid using the SNP primer combination developed in Example 1.

[0126] I. Detection of the purity of Jingyu No. 1 hybrid using the SNP primer combination developed in Example 1

[0127] 1. Obtaining genomic DNA from the Jingyu No. 1 hybrid

[0128] (1) Plant 200 commercially available Jingyu No. 1 hybrid seeds to obtain Jingyu No. 1 hybrid seedlings.

[0129] (2) Leaves or roots of 96 Jingyu No.1 hybrid seedlings were randomly selected and genomic DNA was extracted by CTAB method to obtain 96 genomic DNA samples of Jingyu No.1 hybrid.

[0130] 2. Primer set screening

[0131] (1) Twelve genomic DNA samples were randomly selected from 96 samples of Jingyu No. 1 hybrid. Using these 12 samples as templates, PCR amplification was performed using the 12 primer sets from the SNP primer combinations developed in Example 1, and the corresponding PCR amplification products were obtained. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2", and primers containing "R" in their names was 2:2:5. The reaction systems are shown in Table 4.

[0132] The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touchdown program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, and continued amplification for 26 cycles.

[0133] (2) After completing step (1), when the temperature of each PCR amplification product drops below 40℃, the fluorescence value is read by scanning with the FAM and HEX beams of an enzyme-linked immunosorbent assay (ELISA) reader (the FAM fluorescent tag sequence is read at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is read at an excitation wavelength of 528nm and an emission wavelength of 560nm) to obtain the fluorescence signal color. Compare the number of plants showing green fluorescence signals among the 12 primer sets, and the primer set with the most green fluorescence signals is the selected primer set.

[0134] The rules for determining the genotype of the corresponding SNP loci when testing the genome of each individual melon plant with each primer set are as follows:

[0135] Primer set 1: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP01, then the genotype of the test strain based on the SNP locus TGSNP01 is A:A homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP01, then the genotype of the test strain based on the SNP locus TGSNP01 is G:G homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP01, then the genotype of the test strain based on the SNP locus TGSNP01 is A:G heterozygous.

[0136] Primer set 2: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP02, then the genotype of the test strain based on the SNP locus TGSNP02 is G:G homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP02, then the genotype of the test strain based on the SNP locus TGSNP02 is C:C homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP02, then the genotype of the test strain based on the SNP locus TGSNP02 is G:C heterozygous. Primer set 3: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP03, then the genotype of the test strain based on the SNP locus TGSNP03 is G:G homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP03, then the genotype of the test strain based on the SNP locus TGSNP03 is A:A homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP03, then the genotype of the test strain based on the SNP locus TGSNP03 is G:A heterozygous.

[0137] Primer set 4: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP04, then the genotype of the test strain based on the SNP locus TGSNP04 is A:A homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP04, then the genotype of the test strain based on the SNP locus TGSNP04 is G:G homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP04, then the genotype of the test strain based on the SNP locus TGSNP04 is A:G heterozygous.

[0138] Primer set 5: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP05, then the genotype of the test strain based on the SNP locus TGSNP05 is A:A homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP05, then the genotype of the test strain based on the SNP locus TGSNP05 is T:T homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP05, then the genotype of the test strain based on the SNP locus TGSNP05 is A:T heterozygous.

[0139] Primer set 6: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP06, then the genotype of the test strain based on the SNP locus TGSNP06 is T:T homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP06, then the genotype of the test strain based on the SNP locus TGSNP06 is A:A homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP06, then the genotype of the test strain based on the SNP locus TGSNP06 is T:A heterozygous.

[0140] Primer set 7: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP07, then the genotype of the test strain based on the SNP locus TGSNP07 is G:G homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP07, then the genotype of the test strain based on the SNP locus TGSNP07 is A:A homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP07, then the genotype of the test strain based on the SNP locus TGSNP07 is G:A heterozygous.

[0141] Primer set 8: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP08, then the genotype of the test strain based on the SNP locus TGSNP08 is G:G homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP08, then the genotype of the test strain based on the SNP locus TGSNP08 is A:A homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP08, then the genotype of the test strain based on the SNP locus TGSNP08 is G:A heterozygous.

[0142] Primer set 9: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP09, then the genotype of the test strain based on the SNP locus TGSNP09 is G:G homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP09, then the genotype of the test strain based on the SNP locus TGSNP09 is A:A homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP09, then the genotype of the test strain based on the SNP locus TGSNP09 is G:A heterozygous.

[0143] Primer set 10: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP10, then the genotype of the test strain based on the SNP locus TGSNP10 is C:C homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP10, then the genotype of the test strain based on the SNP locus TGSNP10 is A:A homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP10, then the genotype of the test strain based on the SNP locus TGSNP10 is C:A heterozygous.

[0144] Primer set 11: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP11, then the genotype of the test strain based on the SNP locus TGSNP11 is T:T homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP11, then the genotype of the test strain based on the SNP locus TGSNP11 is C:C homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP11, then the genotype of the test strain based on the SNP locus TGSNP11 is T:C heterozygous.

[0145] Primer set 12: If a test strain shows a blue fluorescent signal based on the SNP locus TGSNP12, then the genotype of the test strain based on the SNP locus TGSNP12 is T:T homozygous; if a test strain shows a red fluorescent signal based on the SNP locus TGSNP12, then the genotype of the test strain based on the SNP locus TGSNP12 is C:C homozygous; if a test strain shows a green fluorescent signal based on the SNP locus TGSNP12, then the genotype of the test strain based on the SNP locus TGSNP12 is T:C heterozygous.

[0146] The results showed that primer sets 3 and 5 produced the most green fluorescent signals, with 8 plants exhibiting each. Therefore, primer sets 3 and 5 were selected for subsequent experiments.

[0147] 3. Obtain the purity of the Jingyu No. 1 hybrid.

[0148] (1) Using genomic DNA from 96 Jingyu No. 1 hybrids as templates, PCR amplification was performed using primer sets 3 and 5, respectively, to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2", and primers containing "R" was 2:2:5. See Table 4 for the reaction system.

[0149] The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touchdown program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, and continued amplification for 26 cycles.

[0150] (2) After completing step (1), when the temperature of each PCR amplification product drops below 40°C, the fluorescence value is read by scanning the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is observed at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is observed at an excitation wavelength of 528nm and an emission wavelength of 560nm) to obtain the fluorescence signal color.

[0151] SNP typing results are shown in Figure 3 (The left image shows primer set 3, and the right image shows primer set 5).

[0152] (3) After completing step (2), count the number of plants that showed green fluorescence and the number of plants that did not show fluorescence for primer set 3 and primer set 5 respectively (number of plants without fluorescence = 96 - number of plants showing green fluorescence - number of plants showing red fluorescence - number of plants showing blue fluorescence); calculate the purity of Jingyu No. 1 hybrid according to the following formula; further calculate the average value to obtain the average purity.

[0153] Purity = Number of plants with green fluorescence in primer set / (96 - Number of plants with no fluorescence in primer set) × 100%.

[0154] The results showed that 93 plants exhibited green fluorescence with primer set 3, and 1 plant did not, with a purity of 93 / (96-1) = 97.89%; 93 plants exhibited green fluorescence with primer set 5, and 1 plant did not, with a purity of 93 / (96-1) = 97.89%; the average purity of the Jingyu No. 1 hybrid was (97.89% + 97.89%) / 2 = 97.89%. 200 Jingyu No. 1 hybrids were planted at the experimental base, with 3 heteromorphic plants and 185 normal plants. The purity identified in the field was 98.40%, consistent with the results of the two pairs of SNP primer markers mentioned above, indicating that the SNP marker identification results are accurate and reliable.

Claims

1. A KASP primer set, comprising primer sets 1 through 12, wherein, The first primer set was used to amplify the SNP site TGSNP01 in the melon genome to determine the genotype corresponding to the SNP site TGSNP01. The SNP site TGSNP01 is located at nucleotide 8779544 on chromosome 1 of the melon DHL92 reference genome, and the nucleotide base of this site is A or G. The second primer set was used to amplify the SNP site TGSNP02 in the melon genome to determine the genotype corresponding to the SNP site TGSNP02. The SNP site TGSNP02 is located at nucleotide 1937599 on chromosome 2 of the melon DHL92 reference genome, and the nucleotide base of this site is G or C. The third primer set was used to amplify the SNP site TGSNP03 in the melon genome to determine the genotype corresponding to the SNP site TGSNP03. The SNP site TGSNP03 is located at nucleotide 28641302 on chromosome 3 of the melon DHL92 reference genome, and the nucleotide base of this site is G or A. The fourth primer set was used to amplify the SNP site TGSNP04 in the melon genome to determine the genotype corresponding to the SNP site TGSNP04. The SNP site TGSNP04 is located at nucleotide 12956460 on chromosome 4 of the melon DHL92 reference genome, and the nucleotide base of this site is A or G. The fifth primer set was used to amplify the SNP site TGSNP05 in the melon genome to determine the genotype corresponding to the SNP site TGSNP05. The SNP site TGSNP05 is located at nucleotide 16951407 on chromosome 5 of the melon DHL92 reference genome, and the nucleotide base of this site is A or T. The sixth primer set was used to amplify the SNP site TGSNP06 in the melon genome to determine the genotype corresponding to the SNP site TGSNP06. The SNP site TGSNP06 is located at nucleotide 3552531 on chromosome 6 of the melon DHL92 reference genome, and the nucleotide base of this site is T or A. The seventh primer set was used to amplify the SNP site TGSNP07 in the melon genome to determine the genotype corresponding to the SNP site TGSNP07. The SNP site TGSNP07 is located at nucleotide 21326763 on chromosome 7 of the melon DHL92 reference genome, and the nucleotide base of this site is G or A. The eighth primer set was used to amplify the SNP site TGSNP08 in the melon genome to determine the genotype corresponding to the SNP site TGSNP08. The SNP site TGSNP08 is located at nucleotide 176762 on chromosome 8 of the melon DHL92 reference genome, and the nucleotide base of this site is G or A. The ninth primer set was used to amplify the SNP site TGSNP09 in the melon genome to determine the genotype corresponding to the SNP site TGSNP09. The SNP site TGSNP09 is located at nucleotide 21606061 on chromosome 9 of the melon DHL92 reference genome, and the nucleotide base of this site is G or A. The tenth primer set was used to amplify the SNP site TGSNP10 in the melon genome to determine the genotype corresponding to the SNP site TGSNP10. The SNP site TGSNP10 is located at nucleotide 8407131 on chromosome 10 of the melon DHL92 reference genome, and the nucleotide base of this site is C or A. The eleventh primer set was used to amplify the SNP site TGSNP11 in the melon genome to determine the genotype corresponding to the SNP site TGSNP11. The SNP site TGSNP11 is located at nucleotide 20770253 on chromosome 11 of the melon DHL92 reference genome, and the nucleotide base of this site is T or C. The twelfth primer set was used to amplify the SNP site TGSNP12 in the melon genome to determine the genotype corresponding to the SNP site TGSNP12. The SNP site TGSNP12 is located at nucleotide 1189283 on chromosome 12 of the melon DHL92 reference genome, and the nucleotide base of this site is T or C. The first primer set consists of forward primer 1F1 shown at positions 22 to 48 from the 5' end of SEQ ID NO: 1, forward primer 1F2 shown at positions 22 to 47 from the 5' end of SEQ ID NO: 2, and reverse primer 1R shown at SEQ ID NO: 3; The second primer set consists of forward primer 2F1 shown at positions 22 to 44 from the 5' end of SEQ ID NO: 4, forward primer 2F2 shown at positions 22 to 44 from the 5' end of SEQ ID NO: 5, and reverse primer 2R shown at SEQ ID NO:

6. The third primer set consists of forward primer 3F1 shown in positions 22 to 50 from the 5' end of SEQ ID NO: 7, forward primer 3F2 shown in positions 22 to 52 from the 5' end of SEQ ID NO: 8, and reverse primer 3R shown in SEQ ID NO: 9; The fourth primer set consists of forward primer 4F1 shown in positions 22 to 49 from the 5' end of SEQ ID NO: 10, forward primer 4F2 shown in positions 22 to 47 from the 5' end of SEQ ID NO: 11, and reverse primer 4R shown in SEQ ID NO: 12; The fifth primer set consists of forward primer 5F1 shown in positions 22 to 46 from the 5' end of SEQ ID NO: 13, forward primer 5F2 shown in positions 22 to 46 from the 5' end of SEQ ID NO: 14, and reverse primer 5R shown in SEQ ID NO: 15; The sixth primer set consists of forward primer 6F1 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 16, forward primer 6F2 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 17, and reverse primer 6R shown in SEQ ID NO: 18; The seventh primer set consists of forward primer 7F1 shown in positions 22 to 46 from the 5' end of SEQ ID NO: 19, forward primer 7F2 shown in positions 22 to 47 from the 5' end of SEQ ID NO: 20, and reverse primer 7R shown in SEQ ID NO: 21; The eighth primer set consists of forward primer 8F1 shown in positions 22 to 52 from the 5' end of SEQ ID NO: 22, forward primer 8F2 shown in positions 22 to 55 from the 5' end of SEQ ID NO: 23, and reverse primer 8R shown in SEQ ID NO: 24; The ninth primer set consists of forward primer 9F1 shown in positions 22 to 46 from the 5' end of SEQ ID NO: 25, forward primer 9F2 shown in positions 22 to 47 from the 5' end of SEQ ID NO: 26, and reverse primer 9R shown in SEQ ID NO: 27; The tenth primer set consists of forward primer 10F1 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 28, forward primer 10F2 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 29, and reverse primer 10R shown in SEQ ID NO: 30; The eleventh primer set consists of forward primer 11F1 shown in positions 22 to 46 from the 5' end of SEQ ID NO: 31, forward primer 11F2 shown in positions 22 to 45 from the 5' end of SEQ ID NO: 32, and reverse primer 11R shown in SEQ ID NO: 33; The twelfth primer set consists of forward primer 12F1 shown in positions 22 to 48 from the 5' end of SEQ ID NO: 34, forward primer 12F2 shown in positions 22 to 47 from the 5' end of SEQ ID NO: 35, and reverse primer 12R shown in SEQ ID NO:

36.

2. The KASP primer combination as described in claim 1, characterized in that: The 5' ends of the two forward primers in any of the first to twelfth primer sets are added with fluorescent tag sequences that emit different colors.

3. The KASP primer combination as described in claim 2, characterized in that: The fluorescent tag sequence is selected from one of FAM, TET, VIC, and HEX.

4. The KASP primer combination as described in claim 2, characterized in that: The first primer set consists of forward primer 1F1 shown in SEQ ID NO: 1, forward primer 1F2 shown in SEQ ID NO: 2, and reverse primer 1R shown in SEQ ID NO:

3. The second primer set consists of forward primer 2F1 shown in SEQ ID NO: 4, forward primer 2F2 shown in SEQ ID NO: 5, and reverse primer 2R shown in SEQ ID NO:

6. The third primer set consists of forward primer 3F1 shown in SEQ ID NO: 7, forward primer 3F2 shown in SEQ ID NO: 8, and reverse primer 3R shown in SEQ ID NO:

9. The fourth primer set consists of forward primer 4F1 shown in SEQ ID NO: 10, forward primer 4F2 shown in SEQ ID NO: 11, and reverse primer 4R shown in SEQ ID NO: 12, as shown in the sequence listing. The fifth primer set consists of forward primer 5F1 shown in SEQ ID NO: 13, forward primer 5F2 shown in SEQ ID NO: 14, and reverse primer 5R shown in SEQ ID NO: 15, as shown in the sequence listing; The sixth primer set consists of forward primer 6F1 shown in SEQ ID NO: 16, forward primer 6F2 shown in SEQ ID NO: 17, and reverse primer 6R shown in SEQ ID NO: 18, as shown in the sequence listing. The seventh primer set consists of forward primer 7F1 shown in SEQ ID NO: 19, forward primer 7F2 shown in SEQ ID NO: 20, and reverse primer 7R shown in SEQ ID NO: 21, as shown in the sequence listing; The eighth primer set consists of forward primer 8F1 shown in SEQ ID NO: 22, forward primer 8F2 shown in SEQ ID NO: 23, and reverse primer 8R shown in SEQ ID NO: 24; The ninth primer set consists of forward primer 9F1 shown in SEQ ID NO: 25, forward primer 9F2 shown in SEQ ID NO: 26, and reverse primer 9R shown in SEQ ID NO: 27, as shown in the sequence listing; The tenth primer set consists of forward primer 10F1 shown in SEQ ID NO: 28, forward primer 10F2 shown in SEQ ID NO: 29, and reverse primer 10R shown in SEQ ID NO:

30. The eleventh primer set consists of the forward primer 11F1 shown in SEQ ID NO: 31, the forward primer 11F2 shown in SEQ ID NO: 32, and the reverse primer 11R shown in SEQ ID NO: 33; The twelfth primer set consists of the forward primer 12F1 shown in SEQ ID NO: 34, the forward primer 12F2 shown in SEQ ID NO: 35, and the reverse primer 12R shown in SEQ ID NO:

36.

5. A kit comprising any one of the KASP primer combinations of claims 1-4.

6. The application of any of the KASP primer combinations according to claims 1 to 4, for example, x1) or x2). x1) Prepare a kit for identifying the purity of melon hybrids; x2) Identify the purity of melon hybrids.

7. A method for identifying the purity of a hybrid melon variety, characterized in that, The method includes the following steps: (a1) Obtain the genomic DNA of N muskmelon hybrids to be tested; N is a natural number greater than 95; (a2) Select genomic DNA from 8-12 muskmelon hybrids to be tested in step (a1). For each selected muskmelon hybrid to be tested, use the genome of the muskmelon hybrid to be tested as a template and perform PCR amplification using the twelve primer sets in the KASP primer combination described in any one of claims 2-4 to obtain the corresponding PCR amplification products. (a3) After completing step (a2), use an instrument to detect the fluorescence signal of each PCR amplification product, and count the number of strains that show heterozygous fluorescence signals in each of the twelve primer sets; the primer set that shows the most heterozygous fluorescent strains is the target primer set. (a4) Using the genomic DNA of the N muskmelon hybrids to be tested obtained in step (a1) as templates, PCR amplification was performed using the target primer set to obtain the corresponding PCR amplification products. (a5) After completing step (a4), use an instrument to detect the fluorescence signal of each PCR amplification product, and obtain the purity of the melon hybrid to be tested based on the color of the fluorescence signal. The method for obtaining the purity of the melon hybrid to be tested based on the color of the fluorescence signal is as follows: count the number of plants that display fluorescence signals representing heterozygous type, the number of plants that display fluorescence signals representing homozygous type, and the number of plants that do not display fluorescence signals using the target primer set; calculate the purity according to the following formula; when there are two or more target primer sets, calculate the purity of each target primer set separately, and then take the average value as the final purity of the melon hybrid to be tested. Number of plants without fluorescent signal = N - Number of plants displaying fluorescent signals representing heterozygous type - Number of plants displaying fluorescent signals representing homozygous type; Purity = Number of strains showing fluorescent signals representing heterozygotes using the target primer set / (N - Number of strains without fluorescent signals using the target primer set) × 100%.

8. The method as described in claim 7, characterized in that: The tested melon hybrids are: Xin Hongxin Cui, Jinlong, Xue Li Hong, Huangpi 9818, RX-99, Xiangrui No. 1, Jinyu, Elizabeth, Honglv Zaocui, E'miangua No. 5, Shiji Mi, Huangguan, Zhuguan, Xuetian No. 1, Fengtian No. 1, Xiboluo, Jinlu No. 3, Jiu Hongrui, Jiu Qingmi, RX-14-3, Yulu No. 1, Xin Chengxiang, Xin Shiji, Yinlu No. 1, Fengtian No. 7, Huang Jingyu, Hongjia, Elizabeth, Basu 317, Jingtian 208, Xianghua, and Jinli. Jinmilong, Yongtian No. 5, Dongfangmi No. 1, Cuitian No. 1, Xinhui, Jianghuaimi No. 3, Hongyou, Zhongtian No. 5, Baiyun, Gansu Bailangua, Hongsushou 1401, Hongsushou 1402, Yanyang, Cicui No. 2, Jianghuaimi No. 6, Yongtian No. 7, Yongyue No. 1, Cixi Caigua, Yongtian No. 8, Xiaobaigua, Zhongtian No. 7, Zhongtian No. 2, Zhongtian No. 8, Jianghuaimi No. 2, Jianghuaimi No. 1, Mifeng No. 1, Xiangrui 301, Jinfei, Zhongyun 20, Ruixue No. 8, Fengtian No. 7, Fengwei No. 4, Hot Melon Egg, Flavor No. 5, Ink Fragrance Jade, White Jade Abundance, Sweet Fragrance Jade, Golden Orange, Jade Ruyi, Green Crisp, Jianghuai Honey No. 7, Xizhou Honey No. 25, Zhongtian Cuixue, Zhongtian Cuibao, Oriental Flower Crisp, Flower Crisp, Creative Crispy Sweet, Japanese Sweet Treasure, M16002, Dongzhixing, Red Jade Crisp, Jade Red, Oriental Crispy Honey, Fengmi 29, Xizhou Honey No. 25, Silver Honey 58, Cuixue No. 5, Xiangshan Snow Honey, Jingyu 357, Jingyu 280, Jingyu 30, Pride, Jingyu 2008, Jingyu Sheep The following are listed: Jiao Su, Jingyu 10, Jingyu No. 3, Jingyu Moon, Jingyu Green Treasure, Jingyu Fragrant Handsome, Jingyu Ink Treasure, Jingyu White Meteor, Jingyu Yellow Meteor, Jingyu No. 5, Jingyu No. 4, Jingyu No. 2, Jade Beauty, Sweet Crown No. 3, Jingyu Honey 28, Sweet Crown No. 1, Sweet Honey, Jingmi No. 8, Jingmi No. 10, Early Spring, Long-lasting Red, Jingmi No. 7, Jingmi No. 15, Special Gold, Special White, Yangjiao Crisp, Honey Crisp Fragrant Garden, Jingmi No. 11, Spring Shed No. 5, Zhongmi 198, and Chizi Niu.

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