Watermelon snp marker combination based on kasp technology and application thereof in germplasm identification

Abstract

The application discloses a watermelon SNP marker combination based on KASP technology and application thereof in germplasm identification, and belongs to the technical field of molecular markers.The application discloses a watermelon SNP marker combination based on KASP technology, which comprises 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 16 KASP-SNP markers provided by the application are simple to operate, good in stability and low in cost when used 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.

Description

TECHNICAL FIELD

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

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

[0003] Single nucleotide polymorphism (SNP) markers, known as the third generation of molecular markers, have been widely used as an important tool in molecular breeding due to their high genetic stability, wide distribution, and simple 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 simple operation. It has great application potential in crop genotyping, fingerprinting and germplasm identification. In rice and other crops, KASP technology has been successfully applied to the development and application of molecular markers, showing its important value in crop genetic research.

[0004] Xu et al. developed 18 high-quality KASP markers for barley variety identification, genetic relationship analysis and population division using KASP technology, which verified the accuracy and efficiency of the technology in barley genetic diversity research, and contributed to the scientific and standardized management of barley germplasm resources and genetic diversity research. Cao et al. detected the important trait functional genes of four national wheat varieties by KASP markers, and clarified the key trait genes contained in these varieties, which provided important reference data for the effective use and breeding of wheat varieties. Shikari et al. analyzed 470 temperate rice germplasm resources at the molecular level by using KASP genotyping technology, revealed three different population substructures, and pointed out that KASP markers are a cost-effective tool for effective maintenance and use of rice genetic resources. Lu et al. developed 700 KASP molecular markers based on high-throughput sequencing data for corn germplasm resource analysis, genetic map construction and heterosis group division, which showed the efficiency and practicability of KASP technology in corn genetic research. Li et al. developed Brassica SNP markers using KASP technology and constructed a DNA fingerprint database of 59 Brassica varieties, which provided a basis for the specificity and authenticity identification of Brassica varieties. The application of KASP molecular marker technology in tomato resistance breeding shows that this technology can effectively assist in screening tomato materials with multiple resistance genes, significantly improving the efficiency and accuracy of disease resistance breeding, and providing a powerful molecular assisted tool for rapid breeding of tomato varieties with multiple resistance.

[0005] Although KASP technology has achieved remarkable results in other crops, 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. Therefore, developing KASP markers suitable for watermelon and constructing watermelon fingerprint have important scientific significance and application value for the protection, variety identification and genetic improvement of watermelon germplasm resources.

[0006] Therefore, it is an urgent problem for those skilled in the art to provide a combination of watermelon SNP markers based on KASP technology and its application in germplasm identification. SUMMARY

[0007] Therefore, the present application provides a combination of watermelon SNP markers based on KASP technology and its application in germplasm identification, the main purpose of which is to provide a set of markers (a total of 16, 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) that can be used for watermelon germplasm relationship identification and its application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A watermelon SNP tagging combination based on KASP technology, wherein the SNP tags 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;

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

[0011] The WM1_4 is located at position 13529716 on chromosome 1, and its nucleotide is either T or A.

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

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

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

[0015] The WM4_1 is located at position 2546332 on chromosome 4, and its nucleotide is either T or A.

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

[0017] The WM6_2 is located at position 1313817 on chromosome 6, and its nucleotide is either T or C.

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

[0019] The WM7_1 is located at position 27571332 on chromosome 7, and its nucleotide is either T or C.

[0020] The WM7_2 is located at position 2357842 on chromosome 7, and its nucleotide is either T or C.

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

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

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

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

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

[0026] Furthermore, a set of KASP primers for detecting SNP markers in watermelon germplasm was provided, and the nucleotide sequences of the SNP-marked KASP primers were amplified as shown in SEQ ID NO.17-64.

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

[0028] SEQ ID NO.20~22 amplify WM1_4;

[0029] SEQ ID NO.23~25 amplify WM1_5;

[0030] SEQ ID NO.26~28 amplify WM3_2;

[0031] SEQ ID NO.29~31 amplify WM3_4;

[0032] SEQ ID NO.32~34 amplify WM4_1;

[0033] SEQ ID NO.35~37 amplify WM5_4;

[0034] SEQ ID NO.38~40 amplify WM6_2;

[0035] SEQ ID NO.41~43 amplify WM6_5;

[0036] SEQ ID NO.44~46 amplify WM7_1;

[0037] SEQ ID NO.47~49 amplify WM7_2;

[0038] SEQ ID NO.50~52 amplify WM7_3;

[0039] SEQ ID NO.53~55 amplify WM8_1;

[0040] SEQ ID NO.56~58 amplify WM9_2;

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

[0042] SEQ ID NO.62~64 Amplification of WM10_2.

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

[0044] Furthermore, the application of the SNP marker combination or the KASP primer set in the identification of germplasm relationships in watermelon.

[0045] Furthermore, the application of the SNP marker combination or the KASP primer set in watermelon population segmentation.

[0046] Furthermore, the application of the SNP marker combination or the KASP primer set in watermelon fingerprinting.

[0047] Furthermore, the application of the SNP marker combination or the KASP primer set in watermelon progeny-assisted breeding.

[0048] As can be seen from the above technical solution, 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, have good stability and low cost when used for germplasm or variety identification, and can effectively improve the accuracy and timeliness of watermelon germplasm or variety identification, authenticity detection and DNA fingerprinting. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0050] Figure 1 The attached figure shows the SNP clustering analysis diagram of 43 watermelon materials from this invention;

[0051] Figure 2 The attached figure shows the QR codes of 43 germplasm fingerprints of watermelon according to this invention. Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0053] Example 1

[0054] 1) Design of KASP tags

[0055] Based on unpublished watermelon germplasm resequencing data, combined with published watermelon genome data (Guo et al., 2013, doi:10.1038 / ng.2470), sequence alignment was performed using BWA software to locate the watermelon reference genome (genotype: 97103, version 2; Guo et al., 2019, doi:10.1038 / s41588-019-0518-4). High-quality SNP sites were identified using GATK analysis.

[0056] Detailed information on a set of 16 KASP-SNP markers:

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

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

[0059] WM1_4: Located at position 13529716 on chromosome 1, its nucleotides are T or A. The sequence extending 50 bp before and after this site is as follows:

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

[0061] WM1_5: Located at position 17173816 on chromosome 1, its nucleotide is G or A. The sequence extending 50 bp before and after this site is as follows:

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

[0063] WM3_2: Located at position 3674510 on chromosome 3, its nucleotides are C or A. The sequence extending 50 bp before and after this site is as follows:

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

[0065] WM3_4: Located at position 15053314 on chromosome 3, its nucleotides are G or A. The sequence extending 50 bp before and after this site is as follows:

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

[0067] WM4_1: Located at position 2546332 on chromosome 4, its nucleotides are T or A. The sequence extending 50 bp before and after this site is as follows:

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

[0069] WM5_4: Located at position 16987238 on chromosome 5, its nucleotides are G or A. The sequence extending 50 bp before and after this site is as follows:

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

[0071] WM6_2: Located at position 1313817 on chromosome 6, its nucleotides are T or C. The sequence extending 50 bp before and after this site is as follows:

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

[0073] WM6_5: Located at position 13216125 on chromosome 6, its nucleotides are G or C. The sequence extending 50 bp before and after this site is as follows:

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

[0075] WM7_1: Located at position 27571332 on chromosome 7, its nucleotides are T or C. The sequence extending 50 bp before and after this site is as follows:

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

[0077] WM7_2: Located at position 2357842 on chromosome 7, its nucleotides are T or C. The sequence extending 50 bp before and after this site is as follows:

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

[0079] WM7_3: Located at position 7097337 on chromosome 7, its nucleotide is A or G. The sequence extending 50 bp before and after this site is as follows:

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

[0081] WM8_1: Located at position 16329485 on chromosome 8, its nucleotides are G or A. The sequence extending 50 bp before and after this site is as follows:

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

[0083] WM9_2: Located at position 2196636 on chromosome 9, its nucleotides are G or C. The sequence extending 50 bp before and after this site is as follows:

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

[0085] WM9_4: Located at position 11424607 on chromosome 9, its nucleotides are T or G, and the sequence extending 50 bp before and after this site is as follows:

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

[0087] WM10_2: Located at position 2426164 on chromosome 10, its nucleotides are T or A. The sequence extending 50 bp before and after this site is as follows:

[0088] ATCATATGTTGTCAAGAAAAAGACACAAGAA TGGAGTAATCCCCGGTGTCWACTAT TGAGTTCTCTTTTGTTCATGTAGCAATTTGTTTAAAATTTAAGGA; SEQ ID NO.16. W is either A or T.

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

[0090] Information on a set of 16 KASP-SNP marker primers:

[0091] Table 1. Numbers and sequences of 116 successfully genotyping 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 came from different countries including South Africa, Zaire, Namibia, Zimbabwe, Spain, Egypt, Swaziland, Ethiopia, Iran, Chad, Cyprus, and Nigeria, as well as the Watermelon and Melon Germplasm Resources and Genetic Breeding Team of Northwest A&F University. These included 17 feed watermelon materials (CA), 12 cultivated watermelon materials (CL), 10 sticky-seeded 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 showed signs of sprouting, they were sown in the research greenhouse of Northwest A&F University in October 2023. Seedlings were raised in 50-cell trays, with 3 seeds sown per germplasm. After sowing, good substrate temperature and humidity conditions were maintained to ensure normal seedling growth.

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

[0097] Table 2 Watermelon germplasm resources tested

[0098]

[0099] Note: CM: Mucosospermus (sticky-seeded watermelon); CA: Amarus (feed watermelon); CC: Colocynthis (medicinal watermelon); CL: Lanatus L. (cultivated watermelon).

[0100] 3) DNA extraction:

[0101] Select watermelon germplasm to be tested, and extract total DNA from its leaves using the CTAB method: Take young upper leaves, rapidly grind them into powder in liquid nitrogen, and place them in a 1.5 ml centrifuge tube; add 800 μl of preheated... CTAB extraction buffer was used, and the mixture was incubated at 65°C for 30 min. An equal volume of chloroform-isoamyl alcohol (chloroform to isoamyl alcohol volume ratio 24:1) was added, and the mixture was centrifuged at 12000 rpm for 15 min. The supernatant was transferred to a new centrifuge tube, and an equal volume of isopropanol was added. The mixture was gently mixed and incubated on ice for at least 1 hour. The mixture was then centrifuged at 12000 rpm for 15 min. The supernatant was discarded, and the precipitate was washed twice with 75% ethanol. After drying, 200 μl of TE buffer was added to dissolve the precipitate, followed by the addition of 10 μg / ml RNase to remove RNA. The mixture was incubated at 37°C for 30 min. Electrophoresis was performed on a 0.8% agarose gel, and the concentration of the obtained DNA was estimated using 50 ng / μl of λDNA as a standard. The final concentration was then diluted with TE buffer to 100 ng / μl and stored at -20°C for later use.

[0102] 4) PCR reaction:

[0103] First, the KASP primers (Table 1) were diluted to 100 μmol·L⁻¹. -1 Prepare the primer mixture at the working concentration as follows: 12 μL each of forward primers Forward1 and Forward2, 30 μL of reverse primer, and distilled water to a final volume of 100 μL. After preparation, store the primer mixture at 4°C for later use; for long-term storage, store at -20°C. PCR reactions were performed using 96-well plates. 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, using ddH2O as a template) were included for each DNA sample. The reaction plates were sealed after preparation.

[0104] PCR reactions were performed using a quantitative real-time fluorescence instrument. The specific procedure included: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, annealing and extension at 61–55℃ for 1 min, for a total of 10 cycles, with the temperature decreasing by 0.6℃ per cycle; followed by 95℃ denaturation for 15 s and 55℃ annealing for 40 s, for a total of 35 cycles. After the PCR reaction, the data were read. If the genotyping results were insufficient, amplification was continued with the following program: 95℃ denaturation for 20 s and 55℃ annealing for 40 s, checking the genotyping results every 3 cycles until a clear genotyping result was obtained.

[0105] 5) Genotyping:

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

[0107] 6) Cluster analysis of watermelon materials

[0108] Based on the KASP genotyping results, fluorescence signals were converted into genotypes at different loci, and phylogenetic analysis was performed on 43 watermelon accessions using MEGA X software. In the dendrogram, each node represents a watermelon sample, and the lines connecting nodes represent the genetic distance between samples. Node merging indicates clustering among samples; that is, genetically similar samples will first cluster together to form branches. The results of the clustering analysis are as follows: Figure 1 As shown, red dots represent feed watermelons (CA), yellow dots represent medicinal watermelons (CC), blue dots represent sticky-seed watermelons (CM), and green dots represent cultivated watermelons (CL). The results indicate that watermelon materials can be divided into different groups based on their genetic background. For example, the feed watermelons represented by red dots, except for W019 and W023, all 15 materials cluster together. At the same cluster of feed watermelons, sticky-seed watermelons W044, W046, and W054 are also clustered together, indicating that these three types of watermelon materials are closely related to feed watermelons and may share a common ancestor. Among the cultivated watermelons, 9 materials are in the same branch, and another 3 materials are clustered in the same branch as medicinal watermelon W031 and sticky-seed watermelon W045. The remaining 3 medicinal watermelon materials and 5 sticky-seed watermelon materials are clustered relatively tightly in different branches, indicating that these two types of watermelon materials are closely related in terms of genetic evolution.

[0109] 7) Construction of fingerprint map

[0110] The KASP genotyping results at 16 SNP loci for 43 watermelon germplasm materials 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 at each locus is represented by two letters, corresponding to the two alleles. For example, "TT" indicates homozygote and "TA" indicates heterozygote. Undetected genotypes are represented by "NN".

[0155] Analysis of these loci reveals genetic diversity among different materials. For example, excluding undetected gene loci, material W003 exhibited homozygosity at 12 detected loci, while material W005 showed homozygosity at 10 detected loci. This genetic diversity is of significant value for watermelon breeding and genetic improvement.

[0156] To facilitate rapid identification and retrieval of genetic information from watermelon materials, a fingerprint map can be constructed using KASP genotyping results and converted into a QR code. Figure 2 Each QR code corresponds to the genotypic information of 16 SNP loci for a single watermelon material. By scanning the QR code, the genetic characteristics of the material can be quickly obtained, enabling efficient management and utilization of watermelon germplasm resources.

[0157] The watermelon fingerprint map constructed using KASP technology provides an important molecular tool for the identification and breeding of watermelon germplasm resources. This map not only reveals the genetic diversity of watermelon materials but also helps identify superior germplasm with specific traits. Furthermore, the application of QR codes on the fingerprint map facilitates rapid identification and information sharing of watermelon germplasm resources.

[0158] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A set of KASP primers for detecting SNP markers in watermelon germplasm, characterized in that, The nucleotide sequences of the SNP-tagged KASP primers are shown in SEQ ID NO.17-64; SEQ ID NO.17~19 amplify WM1_2; SEQ ID NO.20~22 amplify WM1_4; SEQ ID NO.23~25 amplify WM1_5; SEQ ID NO.26~28 amplify WM3_2; SEQ ID NO.29~31 amplify WM3_4; SEQ ID NO.32~34 amplify WM4_1; SEQ ID NO.35~37 amplify WM5_4; SEQ ID NO.38~40 amplify WM6_2; SEQ ID NO.41~43 amplify WM6_5; SEQ ID NO.44~46 amplify WM7_1; SEQ ID NO.47~49 amplify WM7_2; SEQ ID NO.50~52 amplify WM7_3; SEQ ID NO.53~55 amplify WM8_1; SEQ ID NO.56~58 amplify WM9_2; SEQ ID NO.59~61 amplifies WM9_4; SEQ ID NO.62~64 amplify WM10_2; The SNP tags 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 on chromosome 1, and its nucleotide is either T or A. The WM1_4 is located at position 13529716 on chromosome 1, and its nucleotide is either T or A. The WM1_5 is located at position 17173816 on chromosome 1, and its nucleotide is G or A; The WM3_2 is located at position 3674510 on chromosome 3, and its nucleotide is C or A; The WM3_4 is located at position 15053314 on chromosome 3, and its nucleotide is G or A; The WM4_1 is located at position 2546332 on chromosome 4, and its nucleotide is either T or A. The WM5_4 is located at position 16987238 on chromosome 5, and its nucleotide is G or A; The WM6_2 is located at position 1313817 on chromosome 6, and its nucleotide is either T or C. The WM6_5 is located at position 13216125 on chromosome 6, and its nucleotide is G or C; The WM7_1 is located at position 27571332 on chromosome 7, and its nucleotide is either T or C. The WM7_2 is located at position 2357842 on chromosome 7, and its nucleotide is either T or C. The WM7_3 is located at position 7097337 on chromosome 7, and its nucleotide is A or G; The WM8_1 is located at position 16329485 on chromosome 8, and its nucleotide is G or A; The WM9_2 is located at position 2196636 on chromosome 9, and its nucleotide is G or C; The WM9_4 is located at position 11424607 on chromosome 9, and its nucleotide is T or G; The WM10_2 is located at position 2426164 on chromosome 10, and its nucleotide is either T or A. The reference genome for the watermelon is: genotype 97103, version 2.

2. The application of the KASP primer set described in claim 1 in watermelon germplasm identification.

3. The application of the KASP primer set as described in claim 1 in the identification of germplasm relationships in watermelon.

4. The application of the KASP primer set as described in claim 1 in watermelon population segmentation.

5. The application of the KASP primer set according to claim 1 in watermelon fingerprinting.

6. The application of the KASP primer set as described in claim 1 in watermelon progeny-assisted breeding.

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