Watermelon SNP marker combination based on KASP technology and application of watermelon SNP marker combination in germplasm identification

Abstract

The invention discloses a watermelon SNP marker combination based on a KASP technology and application of the watermelon SNP marker combination in germplasm identification, and belongs to the technical field of molecular markers. The invention discloses a watermelon SNP (Single Nucleotide Polymorphism) marker combination based on a KASP technology. The watermelon SNP marker combination comprises WM11, WM14, WM15, WM32, WM34, WM41, WM54, WM62, WM65, WM71, WM72, WM73, WM81, WM92, WM94 and WM102. When the 16 KASP-SNP markers provided by the invention are used for germplasm or variety identification, the operation is simple, the stability is good, the cost is low, and the accuracy and timeliness of watermelon germplasm or variety identification, authenticity detection and DNA fingerprint drawing can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, and more particularly to a watermelon SNP marker combination based on KASP technology and an application thereof in germplasm identification. Background Art

[0002] Watermelon (Citrullus lanatus L.) is an annual vine plant of the genus Citrullus and the family Cucurbitaceae. It is one of the most widely cultivated fruit melon vegetables in the world. With the development of molecular biology techniques, molecular marker technology has become an important tool for crop genetic research and variety identification. These technologies provide strong support for the study of crop genetic diversity, germplasm resource evaluation, variety identification and genetic improvement by detecting specific sequence variations in crop genomes. In the genetic research of crops such as watermelon, the application of molecular marker technology has greatly promoted the progress of related fields.

[0003] Single nucleotide polymorphism (SNP) markers, known as the third generation of molecular markers, have become an important tool in molecular breeding and are widely used due to their high genetic stability, wide distribution, and easy detection. KASP (Kompetitive Allele Specific PCR) technology is a SNP-based molecular marker technology with the advantages of low cost, high throughput, high accuracy, and easy operation. It has shown great application potential in crop genotyping, fingerprint map construction, and germplasm resource identification. In other crops such as rice, KASP technology has been successfully used in the development and application of molecular markers, demonstrating its important value in crop genetic research.

[0004] Xu Tingting et al. used KASP technology to develop 18 high-quality KASP markers for barley variety identification, genetic relationship analysis and population division, verifying the accuracy and efficiency of this technology in barley genetic diversity research, which is helpful for the scientific and standardized management of barley germplasm resources and genetic diversity research. Cao Yanyan et al. used KASP markers to detect the important trait functional genes of four national wheat varieties, clarified the key trait genes contained in these varieties, and provided important reference data for the effective utilization and breeding of wheat varieties. Shikari et al. used KASP genotyping technology to analyze 470 temperate rice germplasm resources at the molecular level, revealing three different population substructures, and pointed out that KASP markers are a high-efficiency and low-cost tool that can be used to effectively maintain and utilize rice genetic resources. Lu Haiyan et al. developed 700 KASP molecular markers based on high-throughput sequencing data for maize germplasm resource analysis, genetic map construction and heterosis group division, showing the efficiency and practicality of KASP technology in maize genetic research. Li Zhiyuan and others used KASP technology to develop cabbage SNP markers and constructed a DNA fingerprint database of 59 cabbage varieties, providing a basis for the specificity and authenticity of cabbage varieties. The application of KASP molecular marker technology in tomato resistance breeding showed that the technology can effectively assist in the screening of tomato materials with multiple resistance genes, significantly improving the efficiency and accuracy of disease resistance breeding, and providing a powerful molecular auxiliary tool for the rapid breeding of new tomato varieties with aggregated multi-resistance.

[0005] Although the application of KASP technology in other crops has achieved remarkable results, its application in watermelon is still in its infancy. The diversity and complexity of watermelon varieties require more accurate and efficient molecular marker technology for variety identification and genetic research. Based on this, the development of KASP markers suitable for watermelon and the construction of watermelon fingerprints have important scientific significance and application value for the protection of watermelon germplasm resources, variety identification and genetic improvement.

[0006] Therefore, providing a watermelon SNP marker combination based on KASP technology and its application in germplasm identification is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a watermelon SNP marker combination based on KASP technology and its application in germplasm identification. The main purpose is to provide a set of markers that can be used for watermelon germplasm kinship identification (a total of 16 markers, namely WM1_2, WM1_4, WM1_5, WM3_2, WM3_4, WM4_1, WM5_4, WM6_2, WM6_5, WM7_1, WM7_2, WM7_3, WM8_1, WM9_2, WM9_4 and WM10_2) and their applications.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A watermelon SNP marker combination based on KASP technology, the SNP markers comprising: WM1_2, WM1_4, WM1_5, WM3_2, WM3_4, WM4_1, WM5_4, WM6_2, WM6_5, WM7_1, WM7_2, WM7_3, WM8_1, WM9_2, WM9_4 and WM10_2;

[0010] The WM1_2 is located at position 1003654 of chromosome 1, and its nucleotide is T or A (i.e., the variant site is T or A);

[0011] The WM1_4 is located at position 13529716 of chromosome 1, and its nucleotide is T or A;

[0012] The WM1_5 is located at position 17173816 of chromosome 1, and its nucleotide is G or A;

[0013] The WM3_2 is located at position 3674510 of chromosome 3, and its nucleotide is C or A;

[0014] The WM3_4 is located at position 15053314 of chromosome 3, and its nucleotide is G or A;

[0015] The WM4_1 is located at position 2546332 of chromosome 4, and its nucleotide is T or A;

[0016] The WM5_4 is located at position 16987238 of chromosome 5, and its nucleotide is G or A;

[0017] The WM6_2 is located at position 1313817 of chromosome 6, and its nucleotide is T or C;

[0018] The WM6_5 is located at position 13216125 of chromosome 6, and its nucleotide is G or C;

[0019] The WM7_1 is located at position 27571332 of chromosome 7, and its nucleotide is T or C;

[0020] The WM7_2 is located at position 2357842 of chromosome 7, and its nucleotide is T or C;

[0021] The WM7_3 is located at position 7097337 of chromosome 7, and its nucleotide is A or G;

[0022] The WM8_1 is located at position 16329485 of chromosome 8, and its nucleotide is G or A;

[0023] The WM9_2 is located at position 2196636 of chromosome 9, and its nucleotide is G or C;

[0024] The WM9_4 is located at position 11424607 of chromosome 9, and its nucleotide is T or G;

[0025] The WM10_2 is located at position 2426164 of chromosome 10, and its nucleotide is T or A.

[0026] Further, a set of KASP primers for detecting SNP markers of watermelon germplasm, the nucleotide sequences of KASP primers for amplifying the SNP markers are shown in SEQ ID NOs. 17 to 64;

[0027] SEQ ID NO. 17-19 amplify WM1_2;

[0028] SEQ ID NO. 20-22 amplifies WM1_4;

[0029] SEQ ID NO. 23-25 ​​amplifies WM1_5;

[0030] SEQ ID NO. 26-28 amplifies WM3_2;

[0031] SEQ ID NO. 29-31 amplifies WM3_4;

[0032] SEQ ID NO. 32-34 amplifies WM4_1;

[0033] SEQ ID NO. 35-37 amplifies WM5_4;

[0034] SEQ ID NO. 38-40 amplifies WM6_2;

[0035] SEQ ID NO. 41-43 amplifies WM6_5;

[0036] SEQ ID NO. 44-46 amplifies WM7_1;

[0037] SEQ ID NO. 47-49 amplifies WM7_2;

[0038] SEQ ID NO. 50-52 amplifies WM7_3;

[0039] SEQ ID NO. 53-55 amplifies WM8_1;

[0040] SEQ ID NO. 56-58 amplifies WM9_2;

[0041] SEQ ID NO. 59-61 amplifies WM9_4;

[0042] SEQ ID NOs. 62 to 64 amplify WM10_2.

[0043] Furthermore, the SNP marker combination or the KASP primer set is used in watermelon germplasm identification.

[0044] Furthermore, the SNP marker combination or the KASP primer set is used in identifying the genetic relationship of watermelon germplasm.

[0045] Furthermore, the SNP marker combination or the KASP primer set is used in watermelon population division.

[0046] Furthermore, the SNP marker combination or the KASP primer set is used in watermelon fingerprint.

[0047] Furthermore, the SNP marker combination or the KASP primer set is used in assisted breeding of watermelon offspring.

[0048] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a watermelon SNP marker combination based on KASP technology and its application in germplasm identification. The 16 KASP-SNP markers provided are simple to operate, stable and low in cost for germplasm or variety identification, and can effectively improve the accuracy and timeliness of watermelon germplasm or variety identification, authenticity detection, and DNA fingerprint mapping. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0050] Figure 1 The accompanying drawing is a SNP cluster analysis diagram of 43 watermelon materials of the present invention;

[0051] Figure 2 The attached drawing is the QR code of 43 watermelon germplasm fingerprints of the present invention. DETAILED DESCRIPTION

[0052] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] Example 1

[0054] 1) Design of KASP Mark

[0055] Based on the unpublished watermelon germplasm resequencing data and the published watermelon genome data (Guo et al., 2013, doi:10.1038 / ng.2470), the sequence was aligned using BWA software and located to the watermelon reference genome (genotype: 97103, version 2; Guo et al., 2019, doi:10.1038 / s41588-019-0518-4). The sequence data was analyzed using the GATK tool to identify high-quality SNP sites.

[0056] A set of 16 KASP-SNP markers details:

[0057] WM1_2: Located at position 1003654 of chromosome 1, its nucleotide is T or A, and the sequence extending 50 bp before and after the site is:

[0058] TATGTAATAGATTGATTATATCGAGT CAAAGCTTACACCAGCAAATACTT WAACAT GTTTGAAATCAATTCCACATATAATTCTTATATAATATGGTCAAT; SEQ ID NO.1. W is A or T.

[0059] WM1_4: Located at position 13529716 of chromosome 1, its nucleotide is T or A, and the sequence extending 50 bp before and after the site is:

[0060] GTCAATTACAAAGGTGAAAGTAGTAGAG CCAATGAGATGATGCCAGAATG WAGTA TTATCCCAAAATATTTCATAGATTTTCAAGAACTAATCTTGAGTAA; SEQ ID NO. 2. W is A or T.

[0061] WM1_5: Located at position 17173816 of chromosome 1, its nucleotide is G or A, and the sequence extending 50 bp before and after the site is:

[0062] AAATTGCAGAAAAATACTTTCTGCCGGTTC TCCCTGCAAGTTTCGCTAGA RAAGCAG CGAAATCAGTCTTGCTAAGTTTCGCCAACCACTTTCTGTCTCAC; SEQ ID NO. 3. R is A or G.

[0063] WM3_2: Located at position 3674510 on chromosome 3, its nucleotide is C or A, and the sequence extending 50 bp before and after the site is:

[0064] ATCTACCTGTGATGCATAATCACAAAG CCGTTGTAGGGATGGCAATATAA MAGCTG CATTCATGTTAAAGAGAATTAATATCATCTTAAACTGTGAAGTAC; SEQ ID NO. 4. M is A or C.

[0065] WM3_4: Located at position 15053314 of chromosome 3, its nucleotide is G or A, and the sequence extending 50 bp before and after the site is:

[0066] TTCGTGATCAAAAGCTTGAAGGATTTCAGATCAATTTTGAGGTACAACAAR TAAGGC ACCATGCATT CAACC TTCCCCTCTATGACTTGGGATTCCCCAAA; SEQ ID NO.5. R is A or G.

[0067] WM4_1: Located at position 2546332 of chromosome 4, its nucleotide is T or A, and the sequence extending 50 bp before and after the site is:

[0068] ATGAGTTTTACGCAAGAAACTTGGAAGGATTTGGTTGGGAATTGATGATTW TGGAG CTAAAAGATGG GTGTGA AATTTTGTCAAACACAGGGTAGCAGTAG; SEQ ID NO. 6. W is A or T.

[0069] WM5_4: Located at position 16987238 of chromosome 5, its nucleotide is G or A, and the sequence extending 50 bp before and after the site is:

[0070] AGTAGCACAAGCGGCAAGTTCGGGAT CGAACCACATGGAGTCTAACATAC RATCTC TCTTAAGTTAAATATGTCTATGGAAATTAGTAAAAATAGGAGTTC;SEQ ID NO. 7. R is A or G.

[0071] WM6_2: Located at position 1313817 on chromosome 6, its nucleotide is T or C, and the sequence extending 50 bp before and after the site is:

[0072] GTTACTAAAATAATAAGTAGTTTTGCA CGCAATGTGTTCAACTATCATGA YTTTCACTTAAATTAAAAATTCAAGAGTTTTTTTGACATCAAAATATAGGA; SEQ ID NO.8. Y is C or T.

[0073] WM6_5: Located at position 13216125 of chromosome 6, its nucleotide is G or C, and the sequence extending 50 bp before and after the site is:

[0074] GCTACTAATCTAGCCTTAAAGGTATGTACCTTTCCATCTACACCTCTTTTTS CTCTTTTA GATCCACT TGGAACC TATAAATTTTACCGTATTAAGTTGATC;SEQ ID NO. 9. S is C or G.

[0075] WM7_1: Located at position 27571332 of chromosome 7, its nucleotide is T or C, and the sequence extending 50 bp before and after the site is:

[0076] CTCGCTTGTTGATTTCCATTTTCATTCTAGTAAGCTCCCTTTGCGAGCTCY GGTAAGG AGAGGTTGA ATGGG ACGATTATGGGCGAAGGCTACGATGATGA; SEQ ID NO. 10. Y is C or T.

[0077] WM7_2: Located at position 2357842 of chromosome 7, its nucleotide is T or C, and the sequence extending 50 bp before and after the site is:

[0078] TCATCTTGGAATCATCGTGTTGGTGTG GGATGAGATTAGTTGGATTGAGC YTGACTA CGTTAGGTCATTCAGAATTAAGGTAAAGAACGATAAATATGAAA; SEQ ID NO. 11. Y is C or T.

[0079] WM7_3: Located at position 7097337 of chromosome 7, its nucleotide is A or G, and the sequence extending 50 bp before and after the site is:

[0080] TGTATAGAGTTTGTTGTAAGATGAAGTTCCACAATAAATGTGCAAAATAGTR AAAGA GCTGAAGATGA AGTTTCTC GATAAATGTGCAAACTTCAATAGTGT; SEQ ID NO. 12. R is A or G.

[0081] WM8_1: Located at position 16329485 of chromosome 8, its nucleotide is G or A, and the sequence extending 50 bp before and after the site is:

[0082] GTGCCCCTTTAATTTACCCATGATGAATGGATCGTAGGTTCAAGTTTTAGR GGGTCA TTATCTTGCT ATTCAGA GGATTGAGGTGGTTTCGACAAGATTTT; SEQ ID NO. 13. R is A or G.

[0083] WM9_2: Located at position 2196636 of chromosome 9, its nucleotide is G or C, and the sequence extending 50 bp before and after the site is:

[0084] GGAATCCTATGTGGGAGACCACATGGTAACCAATATAGGACGTATTTTTTS TTT TCAAACGTCATGT CGTCT ACAAATACTATCTTTTTATTTTTTTAGTC; SEQ ID NO. 14. S is C or G.

[0085] WM9_4: Located at position 11424607 of chromosome 9, its nucleotide is T or G, and the sequence extending 50 bp before and after the site is:

[0086] TGCCATCCGTTCTATTCTGTGAATGT CAATAAACTTATCCAGCCACTTGT KTTTGCCG TCGGCCATTCATTCTATTCTGAAAAGGTGGCATCAACAAGCTT; SEQ ID NO. 15. K is G or T.

[0087] WM10_2: Located at position 2426164 of chromosome 10, its nucleotide is T or A, and the sequence extending 50 bp before and after the site is:

[0088] ATCATATGTTGTCAAGAAAAAGACACAAGAA TGGAGTAATCCCCGGTGTCWACTATTGAGTTCTCTTTTGTTCATGTAGCAATTTGTTTAAAATTTAAGGA; SEQ ID NO. 16. W is A or T.

[0089] Subsequently, KASP primers for the selected SNP sites were designed according to the SNP_Primer_Pipeline2-master software package developed by Dr. Zhang Junli (Table 1). Each set of KASP primers includes two forward competitive primers (F1 / F2) and one reverse universal primer, in which the tail of the F1 primer is designed with a specific sequence that binds to the FAM fluorescent marker, while the tail of the F2 primer is designed with a specific sequence that binds to the HEX fluorescent marker.

[0090] A set of 16 KASP-SNP marker primers information:

[0091] Table 1 Numbers and sequences of 16 successful typing primers

[0092]

[0093]

[0094] 2) Material planting

[0095] The names of the tested watermelon germplasms are shown in Table 2. The 43 tested watermelon germplasms come from different countries such as South Africa, Zaire, Namibia, Zimbabwe, Spain, Egypt, Swaziland, Ethiopia, Iran, Chad, Cyprus, Nigeria, and the West Melon Germplasm Resources and Genetic Breeding Team of Northwest Agriculture and Forestry University, including 17 forage watermelon materials (CA), 12 cultivated watermelon materials (CL), 10 sticky seed watermelon materials (CM) and 4 medicinal watermelon materials (CC). All watermelon materials were soaked in 55℃ warm water, then germinated in a 25℃ incubator. After the watermelon seeds turned white, they were sown in the research greenhouse of Northwest Agriculture and Forestry University in October 2023. 50-hole plug trays were used for seedling cultivation, and 3 seeds were sown for each germplasm. After sowing, good substrate temperature and humidity conditions were maintained to ensure normal growth of seedlings.

[0096] Information on the 43 watermelon materials to be tested:

[0097] Table 2 Watermelon germplasm resources tested

[0098]

[0099] Note: CM: C. mucosospermus; CA: C. amarus; CC: C. colocynthis; CL: C. lanatus L.

[0100] 3) DNA extraction:

[0101] Select the watermelon germplasm to be tested and extract the total DNA from its leaves using the CTAB method: take the young leaves from the upper part and quickly grind them into powder in liquid nitrogen, then put them in a 1.5 ml centrifuge tube; add 800 μl of preheated CTAB extraction buffer, 65℃ water bath for 30min; add equal volume of chloroform and isopropanol, where the volume ratio of chloroform to isopropanol is 24:1, mix well and centrifuge at 12000r / min for 15min; transfer the supernatant to a new centrifuge tube, add equal volume of isopropanol, mix gently, and ice bath for more than 1h; centrifuge at 12000r / min for 15min; pour out the supernatant, rinse the precipitate twice with 75% volume fraction ethanol, dry, add TE buffer 200μl to dissolve, add 10μg / ml RNase to remove RNA, 37℃ water bath for 30min; electrophoresis on 0.8% agarose gel, use 50ng / μl λDNA as the standard to estimate the concentration of the obtained DNA; dilute with TE to a final concentration of 100ng / μl, and store at -20℃ for use.

[0102] 4) PCR reaction:

[0103] First, dilute the KASP primers (Table 1) to 100 μmol·L -1 The working concentration was set at 1 μL, and the primer mixture was prepared in the following proportions: 12 μL each of the forward primers Forward1 and Forward2, 30 μL of the reverse primer, and distilled water was added to make up to 100 μL. After the primer mixture was configured, it was stored at 4°C for use. If it needs to be stored for a long time, it was placed at -20°C. The experiment used a 96-well plate for PCR reaction. Each reaction system contained 1 μL DNA template, 0.14 μL primer mixture, 5 μL 2×KASP Mix (provided by Beijing Jiacheng Biotechnology Co., Ltd.), and 4 μL distilled water. Two negative controls (NTC, with ddH2O as the template) were set for each group of DNA samples, and the reaction plate was sealed after configuration.

[0104] The PCR reaction was carried out in a fluorescence quantitative detection instrument. The specific procedure included: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 15 seconds, annealing and extension at 61-55°C for 1 minute, for a total of 10 cycles, with a decrease of 0.6°C in each cycle; followed by denaturation at 95°C for 15 seconds, annealing at 55°C for 40 seconds, for a total of 35 cycles. After the PCR reaction was completed, the data was read. If the typing result was not sufficient, amplification was continued. The amplification procedure was denaturation at 95°C for 20 seconds, annealing at 55°C for 40 seconds, and the typing was checked every 3 cycles until a clear typing result was obtained.

[0105] 5) Genotyping:

[0106] After the PCR reaction was completed, a real-time fluorescence quantitative PCR instrument (QuantStudio 3, Thermo Fisher Scientific) was used for genotyping and data analysis. The genotype of each sample was determined by detecting the fluorescence signal.

[0107] 6) Cluster analysis of watermelon materials

[0108] Based on the KASP typing results, the fluorescence signals were converted into genotypes at different sites, and the 43 watermelon materials were analyzed by phylogenetic tree analysis using MEGA X software. In the tree diagram, each node represents a watermelon sample, and the lines between the nodes represent the genetic distance between the samples. The merging of nodes represents the clustering between samples, that is, genetically similar samples will first cluster together to form branches. The results of the cluster analysis are shown in Figure 2. Figure 1 As shown in the figure, the red dots represent fodder watermelon (CA), the yellow dots represent medicinal watermelon (CC), the blue dots represent sticky-seed watermelon (CM), and the green dots represent cultivated watermelon (CL). The results show that watermelon materials can be divided into different groups according to their genetic background. For example, except for W019 and W023, the other 15 materials represented by red dots for fodder watermelon are clustered together, and where the fodder watermelon is clustered, sticky-seed watermelons W044, W046, and W054 are also clustered, indicating that these three watermelon materials are closely related to fodder watermelon and may have the same ancestor; 9 cultivated watermelon materials are in the same branch, and the other 3 materials are clustered in the same branch with medicinal watermelon W031 and sticky-seed watermelon W045; the remaining 3 medicinal watermelon materials and 5 sticky-seed watermelon materials are clustered together more compactly on different branches, indicating that the two watermelon materials are relatively close in genetic evolution.

[0109] 7) Construction of fingerprint

[0110] The KASP genotyping results of 43 watermelon germplasm materials at 16 SNP loci are as follows:

[0111] W003:AAAANNCCGGAANNTCCCCCCCGGGGGCGGTT;

[0112] W005:AAAANNCCNNNNAGTCCCCCCCGGGGGCGGTT;

[0113] W006:AAAAAGCCGGAAAGTCCCCCCCGGGGGCGGTT;

[0114] W007:AAAAAGNNGGAANNTTCCCCCCGGAGGCGGTT;

[0115] W009:AAAANNCCGGAANNNNCCCCCCGGGGGCGGTT;

[0116] W011:AAAAAGCCGGATAGNNCCCCCCGGNNNNGGTT;

[0117] W012:AAAAAGCCGGAAAGNNCCCCCCGGGGGCGGTT;

[0118] W015:AAAAAGNNGGAAAGTCCCTCCCGGGGGCGGTT;

[0119] W016:AAAAAGCCGGTTAGTCCCCCCCGGGGGCGGTT;

[0120] W017:AAAAGGCCGGNNAGNNCCCCCCGGGGGCGGTT;

[0121] W019:NNNNGGCCAGATAATTCCNNTTNNNNGCGGAT;

[0122] W021:AAAAAGCCGGAAAGTCCCCCCCGGGGGCGGTT;

[0123] W022:AAAANNCCGGAAAGTTCCCCNNGGGGGCGGTT;

[0124] W023:AAAAAACCAAAAAGTTGGTTTTNNNNGGTTTT;

[0125] W025:AAAANNCCGGAAAGTTCCCCCCGGGGGCGGNN;

[0126] W026:AAAANNCCGGAANNNNCCCCCCGGGGGCGGTT;

[0127] W027:AAAAAGCCGGNNAGTCCCCCCCGGGGNNGGTT;

[0128] W031:AAAAGGACNNAANNCCGGTTCCGGGGCCTTTT;

[0129] W033:ATATAGCCGGAAAACCGGNNCCGGGGGCTGTT;

[0130] W036:NNNNNNCCGGNNGGCCGGNNNNGGGGGCTGTT;

[0131] W039:AAAAGGCCGGAAAGTTCCCCCCAGGGGGGGTT;

[0132] W041:AAAAGGCCGGAAGGTTGGCCCCAANNGGTTTT;

[0133] W044:AAAAAGCCGGAAAGTCCCCCCCGGGGGCGGTT;

[0134] W045:AAAAGGCCNNTTGGTTGGNNCCAAAGGGTTTT;

[0135] W046:AAAANNCCGGATAGTCCCCCCCGGGGGCGGTT;

[0136] W048:AAAAGGCCGGTTNNTTGGCCCCAAGGGGTTTT;

[0137] W053:TTTTGGCCGGTTAATTGGCCCCAAAAGGTTTT;

[0138] W054:AAAAAGCCGGTTNNTCCCCCCCGGGGNNGGNN;

[0139] W056:AAAAGGCCGGAAAATTGGCCCCAAGGGGTTTT;

[0140] W057:AAAAGGCCGGNNAATTGCCCCCNNGGGGTGTT;

[0141] W058:AAAAGGCCNNAAAATTGGCCNNAAGGGGTTTT;

[0142] W062:TTTTGGAAAATTGGCCGGCCTTAAAAGCTTAA;

[0143] C002:TTTTGGAAAAAAGGTTCCCCTTAAAACCGGAA;

[0144] C003:AAAAGGAAAAAAGGTTCCCCTTGGGGGGGGAA;

[0145] C004:TTTTAACCGGTTAATTCCTTTTGGAACCGGAA;

[0146] C005:AAAAGGAAAAAANNCCGGTTCCAAGGCCTTAA;

[0147] C006:TTTTAACCGGAAAATTCCCCTTGGAACCGGAA;

[0148] C007:TTTTAACCAGAAAGTTCCTTTTGGAANNGGAA;

[0149] C008:TTTTAANNGGAAAATTGCTTTTGGAACCGGAA;

[0150] M08M:AAAANNCCGGTTAATTCCTTCCGGAACCGGAA;

[0151] Middle 10:AAAAAAACCAATTGGTTGGTTTCCAAGGGGTTTT;

[0152] M08:TTTTAACCGGTTAATTNNTTTTTGGAANNGGAA;

[0153] BLQ:AAAAGGAAAAAAAACCGGTTCCGGGGCCTTTT.

[0154] The genotype of each locus is represented by two letters, corresponding to the two alleles. For example, "TT" represents homozygous and "TA" represents heterozygous. Undetected genotypes are represented by "NN".

[0155] By analyzing these loci, we can observe the genetic diversity among different materials. For example, excluding the undetected gene loci, material W003 showed homozygosity at 12 detected loci, while material W005 showed homozygosity at 10 detected loci. This genetic diversity is of great value for watermelon breeding and genetic improvement.

[0156] In order to quickly identify and query the genetic information of watermelon materials, the KASP genotyping results can be used to construct a fingerprint map and convert it into a QR code ( Figure 2 ). Each QR code corresponds to the genotype information of 16 SNP sites of a watermelon material. By scanning the QR code, the genetic characteristics of the material can be quickly obtained, realizing the efficient management and utilization of watermelon germplasm resources.

[0157] The watermelon fingerprint constructed by KASP technology provides an important molecular tool for the identification and breeding of watermelon germplasm resources. The map not only reveals the genetic diversity of watermelon materials, but also helps to identify excellent germplasm with specific traits. In addition, the application of fingerprint QR code facilitates the rapid identification and information sharing of watermelon germplasm resources.

[0158] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A watermelon SNP marker combination based on KASP technology, characterized in that: The SNP markers include: WM1_2, WM1_4, WM1_5, WM3_2, WM3_4, WM4_1, WM5_4, WM6_2, WM6_5, WM7_1, WM7_2, WM7_3, WM8_1, WM9_2, WM9_4 and WM10_2; The WM1_2 is located at position 1003654 of chromosome 1, and its nucleotide is T or A; The WM1_4 is located at position 13529716 of chromosome 1, and its nucleotide is T or A; The WM1_5 is located at position 17173816 of chromosome 1, and its nucleotide is G or A; The WM3_2 is located at position 3674510 of chromosome 3, and its nucleotide is C or A; The WM3_4 is located at position 15053314 of chromosome 3, and its nucleotide is G or A; The WM4_1 is located at position 2546332 of chromosome 4, and its nucleotide is T or A; The WM5_4 is located at position 16987238 of chromosome 5, and its nucleotide is G or A; The WM6_2 is located at position 1313817 of chromosome 6, and its nucleotide is T or C; The WM6_5 is located at position 13216125 of chromosome 6, and its nucleotide is G or C; The WM7_1 is located at position 27571332 of chromosome 7, and its nucleotide is T or C; The WM7_2 is located at position 2357842 of chromosome 7, and its nucleotide is T or C; The WM7_3 is located at position 7097337 of chromosome 7, and its nucleotide is A or G; The WM8_1 is located at position 16329485 of chromosome 8, and its nucleotide is G or A; The WM9_2 is located at position 2196636 of chromosome 9, and its nucleotide is G or C; The WM9_4 is located at position 11424607 of chromosome 9, and its nucleotide is T or G; The WM10_2 is located at position 2426164 of chromosome 10, and its nucleotide is T or A.

2. A KASP primer set for detecting SNP markers in watermelon germplasm, characterized in that: The nucleotide sequences of the KASP primers for amplifying the SNP markers are shown in SEQ ID NOs. 17 to 64; SEQ ID NO. 17-19 amplify WM1_2; SEQ ID NO. 20-22 amplifies WM1_4; SEQ ID NO. 23-25 ​​amplifies WM1_5; SEQ ID NO. 26-28 amplifies WM3_2; SEQ ID NO. 29-31 amplifies WM3_4; SEQ ID NO. 32-34 amplifies WM4_1; SEQ ID NO. 35-37 amplifies WM5_4; SEQ ID NO. 38-40 amplifies WM6_2; SEQ ID NO. 41-43 amplifies WM6_5; SEQ ID NO. 44-46 amplifies WM7_1; SEQ ID NO. 47-49 amplifies WM7_2; SEQ ID NO. 50-52 amplifies WM7_3; SEQ ID NO. 53-55 amplifies WM8_1; SEQ ID NO. 56-58 amplifies WM9_2; SEQ ID NO. 59-61 amplifies WM9_4; SEQ ID NOs. 62 to 64 amplify WM10_2.

3. Use of the SNP marker combination according to claim 1 or the KASP primer set according to claim 2 in watermelon germplasm identification.

4. Use of the SNP marker combination according to claim 1 or the KASP primer set according to claim 2 in identifying the genetic relationship of watermelon germplasm.

5. Use of the SNP marker combination according to claim 1 or the KASP primer set according to claim 2 in watermelon population division.

6. Use of the SNP marker combination described in claim 1 or the KASP primer set described in claim 2 in watermelon fingerprint.

7. Use of the SNP marker combination of claim 1 or the KASP primer set of claim 2 in assisted breeding of watermelon progeny.

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