Method for identifying whether a test c. moschata material is a honey-melon material and a special SNP primer set thereof

Abstract

The present application relates to a method for identifying whether a to-be-tested thin-skinned melon material is a honeydew melon material and a special SNP primer set thereof. The SNP primer set provided by the present application is used for identifying the genotype of the TGYJ_55 SNP site of the melon genome, preferably consisting of three primers, which are sequentially shown in SEQ ID NO: 1 to SEQ ID NO: 3 in the sequence table 2. Based on the genotype of the SNP site, whether the to-be-tested thin-skinned melon material is a honeydew melon can be identified, which has important application value for clarifying the population attribute of melon in the process of agricultural production and variety breeding. The SNP primer set in the present application not only greatly shortens the breeding period, but also provides a powerful tool for the improvement and identification of honeydew melon varieties for large, medium and small enterprises.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for identifying whether a thin-skinned melon material to be tested is a Yangjiao honey melon material and its dedicated SNP primer set. Background Art

[0002] Melon is one of the important economic crops in China. According to the pericarp thickness, it can be divided into thick-skinned melons and thin-skinned melons. China is the secondary origin center of thin-skinned melons, with a cultivation and breeding history of more than 2,000 years, among which Yangjiao honey is one of the most representative thin-skinned melon types. Yangjiao honey melon, also known as Yangjiao crisp, is a cultivated variety of melon in the genus Melon of the Cucurbitaceae family. The fruit is large at one end and slightly thin and pointed at the other end, and is named for its overall shape resembling a goat horn. The pericarp of Yangjiao honey is gray-green, the flesh is light green, with rich aroma and crisp texture, and is rich in carotene, inorganic salts, vitamin B, vitamin C, niacin, and various nutrients such as calcium, phosphorus, potassium and iron. At present, Yangjiao honey is cultivated in most areas of China, and plays an important role in promoting economic growth in rural areas and increasing the income level of farmers.

[0003] According to the statistics of the China Seed Industry Big Data website, more than 1060 thin-skinned melon varieties have been applied for registration in China, of which Yangjiao honey type varieties account for 1 / 5. Since the cultivation environment of Yangjiao honey melon is quite different from that of other thin-skinned melon types, it is necessary to confirm whether it is a Yangjiao honey melon type in the process of variety breeding and agricultural production. However, with the cross breeding and gene exchange between different types of thin-skinned melons, it is no longer possible to simply identify Yangjiao honey melon through seeds or seedlings. The traditional field phenotypic identification has a long cycle and high cost, and cannot meet the current identification demand brought by the sharp increase in the number of Yangjiao honey melon varieties.

[0004] With the continuous maturity and cost reduction of next-generation sequencing technology, it is possible to screen population-specific loci of Yangjiao honey melon from the whole genome and establish DNA-based molecular detection technology. SNP, namely single nucleotide polymorphism, has the advantages of abundant quantity, wide genomic distribution and stable heredity; KASP (Kompetitive Allele Specific PCR) is a high-throughput genotyping technology developed for SNP loci, which can realize accurate identification of SNP alleles. Therefore, mining SNP loci specific to Yangjiao honey melon varieties based on resequencing data of thin-skinned melons and developing specific KASP molecular markers therefrom can provide a powerful tool for the improvement and identification of Yangjiao honey melon varieties for large, medium and small enterprises. Contents of the Invention

[0005] In view of the deficiencies in the prior art, one of the objectives of the present invention is to provide a dedicated SNP primer set for identifying Yangjiao honey melon varieties. The primer set can be used for rapidly identifying whether a thin-skinned melon variety to be tested is a Yangjiao honey melon.

[0006] A second objective of this invention is to provide a kit containing the aforementioned primer set.

[0007] The third objective of this invention is to provide applications for the aforementioned primer sets or kits.

[0008] The fourth objective of this invention is to provide a method for identifying whether a thin-skinned melon material to be tested is a horned melon material.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a set of SNP primers for identifying the genotype of the TGYJ_55 SNP locus in the melon genome. The TGYJ_55 SNP site is located at nucleotide 22321018 on chromosome 5 of the melon genome, with bases A or G. The position of the SNP site on the genome was determined based on the alignment of the melon DHL92 reference genome sequence, version number V4.0.

[0010] The SNP primer set provided by this invention can be used to quickly identify whether a thin-skinned melon variety is a horned honeydew melon variety.

[0011] In the SNP primer set of the first aspect above, the primer set is designed for the above-mentioned SNP site based on competitive allele-specific PCR and fluorescence detection technology. Further, the primer set includes forward primer 1F, forward primer 2F and reverse primer 1R, wherein the nucleotide sequence of the forward primer 1F includes the nucleotide sequence shown in SEQ ID NO: 1 from position 22 to 48 from the 5' end, the nucleotide sequence of the forward primer 2F includes the nucleotide sequence shown in SEQ ID NO: 2 from position 22 to 47 from the 5' end, and the nucleotide sequence of the reverse primer R is shown in SEQ ID NO: 3. Furthermore, the 5' ends of the specific nucleotide sequences of forward primer 1F and forward primer 2F are respectively fitted with fluorescent tag sequences that emit different fluorescent signals. The fluorescent tags can be selected from commonly used tags in the field, such as FAM, HEX, etc.

[0012] Furthermore, in the SNP primer set, the nucleotide sequence of the forward primer 1F is shown in SEQ ID NO: 1, the nucleotide sequence of the forward primer 2F is shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer 1R is shown in SEQ ID NO: 3; Kits containing any of the SNP primer sets described above are also within the scope of protection of this invention. Therefore, a second aspect of this invention provides a kit comprising the SNP primer sets described in the first aspect. The kit may also contain other commonly used PCR reagents, such as DNA polymerase, dNTPs, buffer, and Mg²⁺. 2+ wait.

[0013] 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.

[0014] The third aspect of this invention provides the SNP primer set of the first aspect or the kit of the second aspect described above. Application in identifying or breeding thin-skinned melon materials that are either Goat's Horn Melon materials or non-Goat's Horn Melon materials; When identifying or breeding thin-skinned melon materials as Yangjiaomi melon or other types of melon varieties (i.e., non-Yangjiaomi melon), the following judgment is made based on the genotype of the SNP loci mentioned in the first aspect: If the genotype based on the TGYJ_55 SNP locus is homozygous AA, the thin-skinned melon material to be tested is identified as or suspected to be a Yangjiaomi melon material; if the genotype based on the TGYJ_55 SNP locus is homozygous GG or heterozygous GA, the thin-skinned melon material to be tested is identified as or suspected to be a non-Yangjiaomi melon material. Furthermore, the horn-shaped honeydew melon material is selected from any one of the following: Jinmi Cuibao, Huangjinmi No. 3, Tianyou Heipimian, Caihong No. 9, Xiaoheini, Shijixing Sumi, Jingyu Yangjiao Su, Bacui No. 1, Cuihua, Boyang No. 9, Pinfeng Huayang No. 9, Xiangli, Bainiu, Lvrou Sumi, Yinjiao Mi, Fengcui No. 1, Boyang No. 94, Fukangcuimi No. 61, Yinling, Huami No. 91, Xijing, Tianyou Yangjiao Su, Boyang No. 62, Jinke Yangjiao Cui, Keyuan Yangjiao Mi No. 8, Miyu, and Shenghe No. 156.

[0015] The non-Yangjiao Honey Melon materials are selected from Tongcui No. 1, Chucui No. 1, Jintian 117, Huayi Sweet and Crisp, Zhexiang Sweet No. 1, Ganlu No. 6, Tianyou Flower Bud, Ningbo White Skin Shoot Melon, Hangzhou Flower Skin Shoot Melon, Dongshui No. 1, Sanyou Jinxiang Ten-Vein Golden Melon, Zhenhai Ten-Ridge Golden Melon, Huxiang No. 1, Hangzhou Golden Melon, Nongyou Jinyu, Shenggong 108, Hami Xiang, Xuandian Bali Xiang, Huatianmi, Xingtian No. 28, Lintian 100, Zetian No. 4, Sheng Shijingshuang, Xiaoqingren, Xiaomatou, Xianyan, Longtian No. 6, Longtian No. 4, Lingyan Jinxin, Mengtian No. 1, Longtian No. 3, Baishami, Xiangfei No. 9, Shexiang, Xiangrui Liangchan, Jinglong Longfei, Xiangrui Jingqi, Xiangrui Qizhi, Xiangrui Jinwang, Xiangrui No. 18, Shengshi, Fengtian Jiamei, Tianjiao, Yingcai, Banyoupi, Xiangrui Zhanggui, Xiangrui Zhenqi Guomei, Zetian Chunyi, Herun Shangpin, Baichen Mixiang, Hatian No. 1, Youya, Ganlu No. 9, Haozai Laiyihao, Jinlong Lvmuggler, Baichen Jinxiangmi, Ganlu No. 4, Yucui Yutian Baiyu, Baimi 29, Fengtian V12, Jingyu No. 10, Bailu, Dingyan Baimeiren, Xiangrui Jinqianbao, Yucui Boyang 900, Liaotian 19, Qingya 22, Lvtianbao, Jingtian No. 7, Xuanshun Midian 11, Sweet No. 1, Golden Honey No. 2, Zetian Lvyuanshuai, Charlotte, Tianxian Lvcui, Qingmei Baobao, Lvbaoshi No. 2, Yingcui, Qingdie, Feicui No. 1 The following are some of the following: Lai Tian 100, Zhen Xiang Tian No. 7, Lv Bei Er, Jing Tian 208, Xue Tian No. 8, Yin Bao, Hangzhou Bai Li Gua, Mei Yu Bai Li, Qing Mi, Xiang Rui Bi Xi, Lv Mi, Bing Tang Lv Bao, Qiang Shi 268, Zhong Tian Xiang Fei, Yin QQ, Tai Di, Bai Yu, Pin Sheng, Linong, Lai Tian Tian Bao, Huai Tian No. 1, Tian You Cui Bao, Huang You Mei, Tian Xian 20, Shan Ke Mi No. 4, Bing Tang Gui Fei, Tian Mei 75, Ling Long Huang, Xiang Rui 15, Huang Fei, Huang Shuai.

[0016] As an alternative, the above-mentioned Yangjiaomi melon materials and non-Yangjiaomi melon materials constitute the thin-skinned melon materials to be tested in this invention. They are all commercially available varieties. The SNP primer set provided by this invention can be used to identify whether the above-mentioned thin-skinned melon varieties to be tested are Yangjiaomi melons.

[0017] A fourth aspect of this invention provides a method for identifying whether a thin-skinned melon material to be tested is a horned melon material, comprising: detecting the genotype of the TGYJ_55 SNP locus of the thin-skinned melon material to be tested, and then making the following judgment based on the genotype of the TGYJ_55 SNP locus: If the genotype of the TGYJ_55 SNP locus in the thin-skinned melon material to be tested is homozygous AA, then the thin-skinned melon material to be tested is or is suspected to be a horned melon material; if the genotype of the TGYJ_55 SNP locus in the thin-skinned melon material to be tested is homozygous GG or heterozygous GA, then the thin-skinned melon material to be tested is or is suspected to be a non-horned melon material.

[0018] Further, the steps for detecting the genotype of the TGYJ_55 SNP locus in the thin-skinned melon material to be tested are as follows: (1-1) Using the genomic DNA of the thin-skinned melon material to be tested as a template, PCR amplification was performed using the primer set described in the first aspect of the present invention to obtain PCR amplification products; (1-2) The fluorescence signal of the PCR amplification product described in step (1-1) is detected by an instrument, and the genotype of the thin-skinned melon material to be tested based on the TGYJ_55 SNP site is determined according to the color of the fluorescence signal. Preferably, the method for determining the genotype of the tested thin-skinned melon material based on the TGYJ_55 SNP site based on the color of the fluorescence signal is as follows: If the fluorescence signal color displayed by a tested thin-skinned melon material is the same as the color displayed by the fluorescent tag sequence connected to the 5' end of the forward primer F1, then the genotype of the tested thin-skinned melon material based on the SNP site is AA homozygous; if the fluorescence signal color displayed by a tested thin-skinned melon material is the same as the color displayed by the fluorescent tag sequence connected to the 5' end of the forward primer F2, then the genotype of the tested thin-skinned melon material based on the SNP site is GG homozygous; if the fluorescence signal color displayed by a tested thin-skinned melon material is a mixture of the colors displayed by the fluorescent tag sequence connected to the 5' end of the forward primer F1 and the fluorescent tag sequence connected to the 5' end of the forward primer F2, then the genotype of the tested thin-skinned melon material based on the SNP site is GA heterozygous.

[0019] Alternatively, the steps for detecting the genotype of the TGYJ_55 SNP locus in the thin-skinned melon material to be tested are as follows: (2-1) Using the genomic DNA of the thin-skinned melon material to be tested as a template, PCR amplification was performed using the primer set described in the first aspect of the present invention to obtain PCR amplification products; (2-2) Sequencing the PCR amplification product obtained in step (2-1); (2-3) Based on the sequencing results obtained in step (2-2), the genotype of the thin-skinned melon material to be tested based on the TGYJ_55 SNP site is obtained.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The SNP primer set provided by this invention can identify whether a tested thin-skinned melon variety is a Yangjiaomi melon or not, which has important application value for clarifying the population attributes of melons in agricultural production and variety breeding. This invention mines variety-specific SNP sites for Yangjiaomi melon based on resequencing data of thin-skinned melons, providing a powerful tool for variety improvement and identification of Yangjiaomi melons for enterprises of all sizes. Attached Figure Description

[0021] Figure 1PCA clustering results for resequencing data of 58 thin-skinned melon germplasm.

[0022] Figure 2 This study examines the distribution characteristics of Delta index along the physical location of chromosomes in the Yangjiao Honey Melon and other thin-skinned melons.

[0023] Figure 3 The allele frequency distribution of the Yangjiaomi type and other thin-skinned melons in the 20.0-25.0 Mb range on chromosome 5.

[0024] Figure 4 Six SNP markers were used to identify the similarity of the horned honeydew melon type.

[0025] Figure 5 The results of partial SNP typing of 126 tested melon varieties were obtained using the TGYJ_55 primer set.

[0026] Figure 6 The SNP typing results of 12 tested melon varieties were identified using the TGYJ_55 primer set. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] Example 1: The method for obtaining the SNP loci used to identify the muskmelon variety as Yangjiaomi muskmelon is as follows: I. Discovery of SNP sites used to identify the tested melon variety as Yangjiaomi melon. Based on the resequencing data of 149 representative melon germplasms included in the vegetable SNP variation database VegSNPDB (http: / / www.vegsnpdb.cn), this invention selected 58 thin-skinned melon germplasms for research. This thin-skinned melon germplasm is rich in types, including both horned and non-horned varieties, and basically encompasses the main ecological types and agronomic traits of melons, reflecting germplasm representativeness as much as possible and exhibiting high genetic diversity. Principal component analysis (PCA) clustering was performed using the resequencing data of the above 58 thin-skinned melons. The clustering results are as follows: Figure 1As shown; based on this, we developed the whole genome KASP molecular marker for melon to achieve accurate identification of whether the melon germplasm to be tested belongs to the Yangjiaomi type.

[0030] The screening criteria for SNP loci are as follows: First, SNP loci with MAF > 0.3, heterozygosity < 0.1, deletion rate < 0.1, and conserved 50bp sequences on both sides (without other variations) are selected throughout the genome; then, the SNP frequency difference between Yangjiaomi melon and other types of melon is calculated, and a significant peak is found on chromosome 5 in the Yangjiaomi melon type material. Figure 2 And within the 20.5-22.7 Mb range, six SNPs with a frequency difference of 1 between the two populations were obtained. Figure 3 The locations of the SNP sites in the genome were determined based on the alignment with the DHL92 melon reference genome sequence, version V4.0 (downloadable from: http: / / cucurbitgenomics.org / v2 / ftp / genome / melon / DHL92 / v4.0 / ). Basic information on the six SNP sites is detailed in Table 1.

[0031] Table 1. Basic information on SNP sites II. Obtaining the SNP primer set used to identify the melon variety as Yangjiaomi melon. Based on the six SNP loci discovered in step one, the inventors of this invention designed and synthesized six SNP primer combinations and performed SNP genotyping on the 58 tested melon varieties screened in the first part. According to the SNP genotype results ( Figure 4 ), and obtained a SNP locus with a high proportion of homozygous genotypes and 100% linkage to the horned melon, namely TGYJ_55.

[0032] The TGYJ_55 primer set consists of three primer sequences, the names of which are shown in column 2 of Table 2. These primers are used to amplify the TGYJ_55 locus to identify its genotype. The nucleotide sequences of each primer in the primer set are shown in column 4 of Table 2.

[0033] Table 2. SNP primer sets used to identify the melon variety as Yangjiaomi melon. Note: A single underscore indicates a FAM fluorescent tag sequence, and a double underscore indicates a HEX fluorescent tag sequence.

[0034] Example 2: Application of the developed SNP primer set in identifying whether the tested thin-skinned melon variety is the Yangjiaomi melon. The basic information of 126 tested thin-skinned melon varieties in this example is shown in Table 3, and all 126 tested melon varieties are common market varieties. According to the phenotype, 21 tested melon varieties are Yangjiaomi, and 105 tested melon varieties are non-Yangjiaomi, with details shown in Table 3.

[0035] Table 3. Basic information of 126 tested melon varieties 1. Obtaining genomic DNA of the tested melon varieties The SDS method is used to extract genomic DNA from the leaves of 126 tested melon varieties. The quality and concentration of the genomic DNA of the tested melon varieties must meet the PCR requirements, and the compliance criteria are as follows: agarose electrophoresis shows a single DNA band without obvious dispersion; the ultraviolet spectrophotometer Nanodrop2000 (Thermo) detects that the A260 / A280 ratio is between 1.7 and 2.1, and the A260 / A230 ratio is between 1.7 and 2.1; the concentration of the genomic DNA of the tested melon varieties is 50-80ng / μL.

[0036] 2. Using the genomic DNA of 126 tested melon varieties as templates respectively, the primer set in Table 2 is used for PCR amplification to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of the primer whose name contains "F1", the primer whose name contains "F2" and the primer whose name contains "R" is 2:2:5.

[0037] The reaction procedure is: pre-denaturation at 94°C for 15min; denaturation at 94°C for 20s, 61°C-55°C (touch down procedure is adopted, the temperature is reduced by 0.6°C per cycle) for 1min, for 10 cycles of amplification; denaturation at 94°C for 20s, annealing & extension at 55°C for 1min, and 26 more cycles of amplification are carried out.

[0038] 3. After completing step 2, once the temperature of each PCR amplification product has dropped below 40℃, the fluorescence values ​​are read using a microplate reader with FAM and HEX beams (FAM fluorescent tag sequences are read at excitation wavelengths of 485nm and emission wavelengths of 520nm, and HEX fluorescent tag sequences are read at excitation wavelengths of 528nm and emission wavelengths of 560nm). The genotypes of the 126 tested melon varieties based on each SNP locus are determined according to the fluorescence signal color. The specific determination principles are as follows: if a tested melon variety shows a blue fluorescence signal at a certain SNP locus, the genotype of that tested melon variety at that SNP locus is AA homozygous; if a tested melon variety shows a red fluorescence signal at a certain SNP locus, the genotype of that tested melon variety at that SNP locus is GG homozygous; if a tested melon variety shows a green fluorescence signal at a certain SNP locus, the genotype of that tested melon variety at that SNP locus is GA heterozygous.

[0039] 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.

[0040] Partial SNP genotyping results of the primer set are shown below Figure 5 The statistical results are shown in columns 4 and 8 of Table 3.

[0041] The results showed that the primer set could achieve good typing results in all 126 tested melon varieties.

[0042] 4. Efficiency evaluation of using primers targeting the TGYJ_55 locus to identify 126 tested melon varieties as Yangjiaomi melon. The genotypes of 126 tested melon varieties based on the TGYJ_55 locus were statistically analyzed, and the melon materials were used to determine whether they were Yangjiaomi melons. The method for determining melon materials based on the genotype of this SNP locus is as follows: if the genotype based on the TGYJ_55 SNP locus is homozygous AA, the tested thin-skinned melon material is identified as or suspected to be a Yangjiaomi melon material; if the genotype based on the TGYJ_55 SNP locus is homozygous GG or heterozygous GA, the tested thin-skinned melon material is identified as or suspected to be a non-Yangjiaomi melon material.

[0043] The results showed that 21 tested melon varieties with the homozygous AA genotype based on the TGYJ_55 locus were identified as Yangjiaomi melons, and all 21 melons exhibited the Yangjiaomi melon phenotype, with a consistency of 100%. 89 tested melon varieties with the homozygous GG genotype based on the TGYJ_55 locus were identified as non-Yangjiaomi melons, or 16 tested melon varieties with the heterozygous AG genotype were identified as non-Yangjiaomi melons, and all 105 melons exhibited the non-Yangjiaomi melon phenotype, with a consistency of 100%.

[0044] Therefore, the identification results of the SNP primer set developed in Example 1 are completely consistent with the material phenotype.

[0045] Example 3: Application of the SNP primer set developed in Example 1 to identify whether another 12 melon varieties to be tested are Yangjiaomi melons. The melon varieties to be tested included Miyu, Shenghe 156, Tianyou Yangjiaosu, Boyang 62, Jinke Yangjiaocui, Keyuan Yangjiaomi No. 8, Bingtang Guifei, Tianmei 75, Linglonghuang, Xiangrui 15, Huangfei, and Huangshuai. All 12 tested melon varieties were common market varieties.

[0046] 1. The primer set for the TGYJ_55 locus developed in Example 1 was used to identify whether the melon variety to be tested was the Yangjiaomi melon. (1) Obtaining genomic DNA from the melon variety to be tested Seeds of the melon variety to be tested were planted to obtain seedlings; leaves or roots of the melon variety to be tested were taken, and genomic DNA was extracted using the SDS method described in step 1 of Example 2 to obtain the genomic DNA of the melon variety to be tested.

[0047] (2) Using the genomic DNA of the melon variety to be tested as a template, PCR amplification was performed using the primer sets 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" in their names was 2:2:5.

[0048] The reaction program was 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, and continued amplification for 26 cycles.

[0049] 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 12 tested melon varieties based on each SNP site is determined according to the fluorescence signal color. The specific judgment principle is as follows: if a tested melon variety shows a blue fluorescence signal based on a certain SNP site, then the genotype of the tested melon variety based on that SNP site is AA homozygous; if a tested melon variety shows a red fluorescence signal based on a certain SNP site, then the genotype of the tested melon variety based on that SNP site is GG homozygous; if a tested melon variety shows a green fluorescence signal based on a certain SNP site, then the genotype of the tested melon variety based on that SNP site is GA heterozygous.

[0050] (3) It should be noted that if the fluorescence signal is weak after PCR amplification, which affects data analysis, additional cycles can be added (denaturation at 94℃ for 20s, annealing and extension at 55℃ for 1min, 5 cycles) until the results are satisfactory.

[0051] The SNP typing results of 12 melon varieties tested using the TGYJ_55 primer set are shown below. Figure 6 And Table 4.

[0052] (4) After completing step (3), make the following judgment: if the genotype based on the TGYJ_55 locus is AA homozygous, the melon variety to be tested is identified as or suspected to be Yangjiaomi melon; if the genotype based on the TGYJ_55 locus is GG homozygous or GA heterozygous, the melon variety to be tested is identified as or suspected to be non-Yangjiaomi melon. Table 4. Identification results of 12 tested melon varieties 2. Twelve different melon varieties were planted. Based on their phenotypes, the twelve varieties were determined to be either horned honey melons or non-horned honey melons.

[0053] The phenotypic statistics are shown in columns 3 and 6 of Table 4.

[0054] The results showed that the identification results of the SNP primer set developed in Example 1 were completely consistent with the phenotypic identification results.

[0055] Therefore, it can be seen that the SNP primer set developed in Example 1 can completely identify whether the thin-skinned melon variety is a Yangjiaomi melon or not.

[0056] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. SNP primer set, used to identify the genotype of the TGYJ_55 SNP locus in the melon genome, wherein the TGYJ_55 SNP locus is located at nucleotide 22321018 on chromosome 5 of the melon genome, and the base is A or G; The location of the TGYJ_55 SNP site on the genome was determined based on the alignment of the DHL92 melon reference genome sequence, version number V4.

0.

2. The SNP primer set as described in claim 1, characterized in that, The primer set includes forward primer 1F, forward primer 2F, and reverse primer 1R, wherein the nucleotide sequence of forward primer 1F includes the specific nucleotide sequence from position 22 to 48 from the 5' end as shown in SEQ ID NO: 1, the nucleotide sequence of forward primer 2F includes the specific nucleotide sequence from position 22 to 47 from the 5' end as shown in SEQ ID NO: 2, and the nucleotide sequence of reverse primer R is shown in SEQ ID NO:

3.

3. The SNP primer set according to claim 2, characterized in that, The 5' ends of the specific nucleotide sequences of forward primers 1F and 2F are respectively fitted with fluorescent tag sequences that emit different fluorescent signals.

4. The SNP primer set according to claim 3, characterized in that, The nucleotide sequence of the forward primer 1F is shown in SEQ ID NO: 1, and the nucleotide sequence of the forward primer 2F is shown in SEQ ID NO:

2.

5. A kit comprising the SNP primer set according to any one of claims 1-3.

6. The application of the SNP primer set according to any one of claims 1 to 4 or the kit according to claim 5 in identifying or selecting whether the thin-skinned melon material to be tested is a horned melon material or a non-horned melon material. When identifying or breeding a thin-skinned melon material to be tested as either Yangjiaomi melon or a non-Yangjiaomi melon, the following judgment is made based on the genotype of the TGYJ_55 SNP locus as described in claim 1: If the genotype of the thin-skinned melon material to be tested based on the TGYJ_55 SNP locus is homozygous AA, then the thin-skinned melon material to be tested is or is suspected to be Yangjiaomi melon material; if the genotype of the thin-skinned melon material to be tested based on the TGYJ_55 SNP locus is homozygous GG or heterozygous GA, then the thin-skinned melon material to be tested is or is suspected to be a non-Yangjiaomi melon material.

7. A method for identifying whether a thin-skinned melon sample is a horned honeydew melon sample, characterized in that: The method includes: detecting the genotype of the TGYJ_55 SNP locus as described in claim 1 of the thin-skinned melon material to be tested, and then making the following judgment based on the genotype of the TGYJ_55 SNP locus: If the genotype of the TGYJ_55 SNP locus in the thin-skinned melon material to be tested is homozygous AA, then the thin-skinned melon material to be tested is or is suspected to be a horned melon material; if the genotype of the TGYJ_55 SNP locus in the thin-skinned melon material to be tested is homozygous GG or heterozygous GA, then the thin-skinned melon material to be tested is or is suspected to be a non-horned melon material.

8. The method as described in claim 7, characterized in that, The steps for detecting the genotype of the TGYJ_55 SNP locus in the thin-skinned melon material to be tested are as follows: (1-1) Using the genomic DNA of the thin-skinned melon material to be tested as a template, PCR amplification was performed using the primer set described in claim 3 or 4 to obtain the PCR amplification product; (1-2) The fluorescence signal of the PCR amplification product described in step (1-1) is detected by an instrument, and the genotype of the thin-skinned melon material to be tested based on the TGYJ_55 SNP site is determined according to the color of the fluorescence signal.

9. The method as described in claim 8, characterized in that, The method for determining the genotype of the tested thin-skinned melon material based on the TGYJ_55 SNP site based on the color of the fluorescence signal is as follows: If the fluorescence signal color of a tested thin-skinned melon material is the same as the color of the fluorescent tag sequence connected to the 5' end of the forward primer F1, then the genotype of the tested thin-skinned melon material based on the SNP site is AA homozygous; if the fluorescence signal color of a tested thin-skinned melon material is the same as the color of the fluorescent tag sequence connected to the 5' end of the forward primer F2, then the genotype of the tested thin-skinned melon material based on the SNP site is GG homozygous; if the fluorescence signal color of a tested thin-skinned melon material is a mixture of the colors of the fluorescent tag sequence connected to the 5' end of the forward primer F1 and the fluorescent tag sequence connected to the 5' end of the forward primer F2, then the genotype of the tested thin-skinned melon material based on the SNP site is GA heterozygous.

10. The method according to claim 7, characterized in that: The steps for detecting the genotype of the TGYJ_55 SNP locus in the thin-skinned melon material to be tested are as follows: (2-1) Using the genomic DNA of the thin-skinned melon material to be tested as a template, PCR amplification was performed using the primer set described in claim 2 to obtain the PCR amplification product; (2-2) Sequencing the PCR amplification product obtained in step (2-1); (2-3) Based on the sequencing results obtained in step (2-2), the genotype of the thin-skinned melon material to be tested based on the TGYJ_55 SNP site is obtained.

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