SNP site related to sugar content of watermelon fruit, molecular marker based on the site and application

Abstract

The application discloses a SNP site related to sugar content of watermelon fruits, a molecular marker based on the site and application, and belongs to the technical field of genetic engineering. The application discloses for the first time that the application of detecting the polymorphism or genotype of the SNP site Chr1-33089964 in identifying or assisting in identifying the sugar content of watermelon fruits; the SNP site Chr1-33089964 is located at the 33089964th nucleotide of the 1st chromosome of the V2 genome of watermelon '97103', and the polymorphism is A or T. The application can be used for molecular assisted breeding of watermelon fruit high-sugar content materials, improving the selection efficiency of breeding and accelerating the breeding process; meanwhile, the application can lay a foundation for ultimately realizing cloning of a watermelon fruit sugar content gene and analysis of a regulation network.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to SNP sites related to the sugar content of watermelon fruits, molecular markers based on these sites, and their applications. Background Technology

[0002] Genotype selection is one of the most crucial steps in breeding, referring to the selection of genotypes that meet specific requirements within a population for subsequent cultivation. In traditional breeding processes, selection is often based on plant morphology, which is time-consuming and prone to errors and inefficiency due to ambiguity in the genotype and potential deviations from the desired traits. However, marker-assisted breeding allows for rapid detection of co-segregating loci with the target gene or trait at the seedling stage, achieving highly efficient and precise selection of the target trait. It offers advantages such as speed, accuracy, and independence from environmental conditions.

[0003] Watermelon (Citrullus lanatus), belonging to the genus Citrus in the family Cucurbitaceae, is an annual vine-like herbaceous plant, hailed as the king of summer fruits, and holds a very important position among horticultural crops worldwide. As a fruit primarily consumed fresh, the sugar content of watermelon is a core indicator for evaluating its commercial quality and has always been a crucial target trait in variety improvement and cultivation. Soluble sugars are important nutrients and flavor compounds in watermelon fruits, and their content and proportion directly affect the fruit's flavor, texture, quality, and commercial value. Research on the composition and content of soluble sugars on fruit flavor and quality is of great significance for watermelon quality evaluation and variety improvement. However, current research on watermelon fruit sugar content is in its early stages, with limited markers available for watermelon breeding. In 2014, Ren et al. used an integrated watermelon genetic linkage map to locate QTLs related to soluble sugar content in the fruit, detecting 5 QTLs related to fructose, 1 QTL related to glucose, and 3 QTLs related to sucrose. In 2018, further research confirmed that the expression of the gene ClTST2 at the sugar-related QBRX2-1 locus on chromosome 2 is related to the uptake and accumulation of sugar by vacuolar plasmids in watermelon fruit pulp cells. In 2019, Guo et al. used resequencing data from 414 watermelon materials for GWAS analysis and discovered two other regions on chromosome 10 closely related to fruit sweetness, containing the sucrose synthase gene Cla97C10G194010 and the raffinose synthase gene Cla97C10G196740, which are involved in the synthesis of sucrose and raffinose, respectively. Currently, research on sugar accumulation in watermelons is still very weak, making it difficult to meet the needs of high-quality watermelon molecular breeding. Therefore, further in-depth research into the molecular mechanisms of sugar accumulation in watermelons and the identification of more key genes or loci controlling sugar content in watermelon fruits are crucial for establishing an efficient molecular system for watermelon quality breeding.

[0004] In recent years, research on watermelon molecular breeding has developed rapidly. Many superior traits have been gene-mapped, and some molecular markers co-segregating with important agronomic traits have been applied to marker-assisted breeding. In-depth research on the sugar content trait of watermelon fruits, and the development of molecular markers closely linked to or co-segregating with this trait, can not only effectively assist in the marker-assisted selection of new watermelon varieties with high sugar content, but also significantly shorten the breeding process and improve the accuracy of selection. Summary of the Invention

[0005] One of the objectives of this invention is to provide an SNP site related to the sugar content of watermelon fruit and an SNP molecular marker developed based on this site.

[0006] The second objective of this invention is to provide the aforementioned SNP sites and the application of SNP molecular markers based on these sites in watermelon molecular breeding.

[0007] The third objective of this invention is to provide a method for identifying watermelon varieties / genotypes with high or low sugar content.

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

[0009] The SNP locus associated with the sugar content of watermelon fruit is located at nucleotide 33089964 on chromosome 1 of the “97103”V2 version genome, named Chr1-33089964. This SNP locus associated with the sugar content of watermelon fruit is developed based on the QTL locus ssc1.2 for the sugar content of watermelon fruit.

[0010] The SNP site is polymorphic either A or T. Materials with the A genotype exhibit high fruit sugar content, while materials with the T genotype exhibit low fruit sugar content. In this invention, the preferred fruit sugar content range for the low fruit sugar content trait is 1.3-7.25°Bx, and the preferred fruit sugar content range for the high fruit sugar content trait is 7.91-10.82°Bx.

[0011] The SNP molecular marker based on this site has a nucleotide sequence as shown in SEQ ID NO.3 or SEQ ID NO.4, where the SNP site is located at position 139 of the sequence in SEQ ID NO.3 or SEQ ID NO.4. The upstream primer sequence for amplifying the SNP molecular marker is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2.

[0012] This invention also discloses the application of SNP molecular markers related to watermelon fruit sugar content in watermelon molecular breeding. These SNP molecular markers are associated with the fruit sugar content trait and can assist in identifying watermelon plants with high fruit sugar content at the molecular level. Furthermore, the sugar content trait can be determined at the seed or seedling stage, thereby improving selection efficiency and accelerating the breeding process. Those skilled in the art will understand that screening for materials or varieties with high fruit sugar content can be achieved by detecting the genotype of SNP loci. The detection is performed using Sanger sequencing.

[0013] This invention also discloses a method for determining watermelon varieties / genotypes with high or low sugar content, which uses PCR amplification followed by sequencing for detection. The method includes the following steps:

[0014] (1) Extracting DNA from watermelon tissue;

[0015] (2) PCR amplification: The sample extracted in step (1) was amplified by using the primer pair with the upstream primer sequence of the SNP molecular marker as shown in SEQ ID NO.1 and the downstream primer sequence as shown in SEQ ID NO.2.

[0016] (3) Perform Sanger sequencing on the amplification products;

[0017] (4) Make a judgment based on the results of step (3), and the specific criteria are as follows:

[0018] If the 139th base (Chr1-33089964) of the cloned product is A, and its sequence is shown in SEQ ID NO.3 in the sequence listing, then the watermelon plant to be tested is a homozygous material with high sugar content in watermelon fruit; if the 139th base (Chr1-33089964) of the cloned product is T, and its sequence is shown in SEQ ID NO.4 in the sequence listing, then the watermelon plant to be tested is a material with low sugar content in watermelon fruit; if the 139th base (Chr1-33089964) of the cloned product has two forms, A and T, then the watermelon plant to be tested is a heterozygous material with high sugar content in watermelon fruit;

[0019] Specifically, the PCR amplification reaction system consisted of: 1 μL DNA, 5 μL 2×PCR Mix, 0.5 μL upstream primer, 0.5 μL downstream primer, and 3 μL sterile distilled water, for a total volume of 10 μL. The PCR amplification conditions were: 95℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, and 72℃ for 50 s, for a total of 35 cycles; 72℃ for 10 min; and storage at 4℃.

[0020] This invention also provides a kit for detecting SNP molecular markers related to sugar content in watermelon fruit, comprising the primer pairs described above for detecting SNP molecular markers related to fruit sugar content. The kit can be used to identify the sugar content trait in watermelon fruit. Specifically, the kit can be made using reagents containing the primer pairs described above, wherein each component of the reagent in the kit can be packaged independently, or two or more components can be packaged together.

[0021] This invention also protects vectors containing the aforementioned molecular markers. The recombinant vector may be an expression vector or a cloning vector containing the molecular markers of this invention. After obtaining the aforementioned recombinant vector, those skilled in the art can transform the recombinant vector into suitable cells according to different needs to obtain recombinant cells containing the recombinant vector. Therefore, this invention also protects recombinant cells containing the aforementioned recombinant vector.

[0022] Advantages of this invention:

[0023] This invention uses genome-wide association analysis to screen for a single SNP locus on chromosome 1 of watermelon that is associated with the sugar content of watermelon fruit. The SNP locus is named Chrl-33089964. Genotype A of this SNP locus indicates high sugar content in the fruit, and genotype T indicates low sugar content in the fruit. This SNP locus is used as an auxiliary selection marker for the sugar content trait in watermelon breeding.

[0024] SNP molecular markers based on this SNP site, which are associated with the sugar content of watermelon fruits, can be directly used for marker-assisted breeding of watermelons with high sugar content. In watermelon selection breeding, a large number of segregating populations are often generated. By utilizing the polymorphism of this SNP site, desired plants can be identified during the seedling stage. This not only reduces the land area required for breeding but also reduces the manpower and resources needed for identification after the plants have grown, greatly improving breeding efficiency and shortening the selection period. Therefore, the SNP molecular markers provided by this invention have significant application value in the breeding of new varieties. Attached Figure Description

[0025] Figure 1 The Manhattan plot is a genome-wide association analysis (GWAS) plot of the sugar content trait in watermelon fruit, where: the horizontal axis represents the genomic location of the SNP site on the 5K liquid microarray; the vertical axis represents the negative logarithm of the P-value of each marker site under the MLM model, base 10.

[0026] Figure 2 The QQ plot is a genome-wide association analysis of sugar content in watermelon fruit, where: the horizontal axis represents the expected negative logarithm of the observed P-values, which are assumed to follow a uniform distribution [0, 1]; the vertical axis represents the observed negative logarithm of the observed P-values, which are base 10.

[0027] Figure 3 Linkage disequilibrium analysis of SNP sites on 5K liquid phase chip in watermelon in 89 watermelon samples;

[0028] Figure 4 The primer pair used to amplify the SNP locus Chr1-33089964, which is associated with sugar content in watermelon fruit, was compared with the amplified product sequence and fruit characteristics in different watermelon materials. In the figure, Figure A shows the phenotypic map of the 10 selected watermelon materials; Figure B shows the box plot of the sugar content trait distribution of the samples of different genotypes corresponding to the significantly associated locus Chr1-33089964; where: *** represents the extremely significant difference in fruit sugar content among different genotypes at the 0.001 level; Figure C shows the Sanger sequencing results of the 10 selected watermelon materials. Detailed Implementation

[0029] The following is a further explanation based on the application. Before introducing the specific embodiments, the biological materials, experimental reagents and related experimental background of the following embodiments are briefly introduced as follows.

[0030] Biomaterials:

[0031] The 89 watermelon materials used for SNP locus development were obtained by our laboratory, the Cucurbit Germplasm Genetic Improvement and Molecular Breeding Laboratory of Henan Agricultural University, through multiple generations of self-pollination, separation, and purification.

[0032] The aforementioned 89 watermelon materials are consistent with the 89 watermelon materials used in the published article "Phenological Identification and Genetic Diversity Analysis of 89 Watermelon Germplasm Resources".

[0033] All of the above materials can be obtained through commercial channels or from the Cucurbit Germplasm Genetic Improvement and Molecular Breeding Laboratory of Henan Agricultural University.

[0034] It should be explained that the use of this material as the basis for research is solely due to the ease of obtaining experimental materials, and should not be construed as implying that the implementation of the relevant technical solutions in this application must depend on this experimental material.

[0035] This invention primarily utilizes a population constructed for GWAS analysis using 89 watermelon accessions. The watermelons were grown in a greenhouse at the Maozhuang Science and Education Park of Henan Agricultural University. During cultivation, seedlings were raised in plug trays after germination, following standard watermelon cultivation management methods. Fifteen days after transplanting, the terminal young leaves of each accession were collected as samples for genomic DNA extraction. Fruit sugar content was assessed 35 days after pollination as phenotypic data for GWAS analysis. Genome-wide association sites (SNPs) used in the 89 watermelon accessions were obtained using a watermelon 5K liquid chromatography-mass spectrometry chip developed in our laboratory. Through genome-wide association analysis of sugar content-related traits, SNPs associated with watermelon sugar content were identified.

[0036] Association analysis between the results of the 5K liquid phase microarray of watermelon and the sugar content trait of watermelon fruit showed that a SNP site associated with the sugar content trait of watermelon fruit was screened on chromosome 1 of the watermelon genome. The SNP site is located at position 33089964 on chromosome 1 of the watermelon genome and is named Chr1-33089964.

[0037] The 5K liquid chromatography-mass spectrometry (LC-MS) analysis of 89 watermelon samples was completed by Borui Biotechnology Co., Ltd.

[0038] The primers used for PCR amplification and gene sequencing were provided by Beijing Qingke Biotechnology Co., Ltd.

[0039] Experimental reagents:

[0040] PCR amplification was performed using 2x 3G Taq Master Mix, purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0041] Experimental equipment:

[0042] PCR instrument, Zhuhai Heima Medical Instrument Co., Ltd. Hema9600 gene amplification instrument;

[0043] Sugar analyzer, ATAGO China Branch PAL-1 model;

[0044] Example 1: Obtaining SNP molecular markers related to sugar content in watermelon fruit

[0045] 1.1 Construction of the Genome-Wide Association Analysis Population

[0046] Using 89 watermelon materials purified through multiple generations of self-pollination by the Cucurbit Germplasm Genetic Improvement and Molecular Breeding Laboratory of Henan Agricultural University, a watermelon genome-wide association analysis population was constructed. In 2022, all watermelon materials were planted in plastic greenhouses at the Maozhuang Science and Education Park of Henan Agricultural University in Zhengzhou, with 10 plants per material. Production management included soaking seeds at room temperature for 4 hours, germinating them in 28℃ dark light, sowing them in 50-cell trays after the seeds showed signs of germination, and then raising them in a greenhouse. Seedlings were transplanted into plastic greenhouses when they reached the three-leaf stage. Transplanting methods included: raised ridges, single-row double-row planting, triangular planting, and wide and narrow row spacing (35cm plant spacing, 60cm narrow row spacing, and 80cm wide row spacing). Pruning methods included: single-vine pruning, artificial pollination before 10:00 AM during peak flowering, single fruit set per plant, and normal fertilization, irrigation, and pest and disease management.

[0047] 1.2 Sample collection and phenotypic data investigation

[0048] Thirty-five days after pollination, five well-developed and uniformly grown fruits from each sample were selected for sugar content (measured using a sugar analyzer). The average value of the five measurements was taken as the final sugar content of each sample.

[0049] 1.3 Extraction of watermelon genomic DNA

[0050] Fifteen days after the watermelon plants were transplanted, on a sunny morning at 8:00 AM, tender leaves from the tips of lateral branches were taken and quickly frozen in 2ml centrifuge tubes. After being brought back to the laboratory, they were stored in a -20℃ freezer.

[0051] Total DNA was extracted using a modified CTAB method. The experimental procedure included the following steps:

[0052] (1) Add mercaptoethanol solution to CTAB solution (2 ml mercaptoethanol per 100 ml CTAB solution) and preheat in a 65°C water bath. Precool isopropanol solution in a -20°C refrigerator.

[0053] (2) Take out the 2ml centrifuge tube sample stored in the -20℃ refrigerator and grind it. Then add 800ul of preheated CTAB solution to the 2ml centrifuge tube containing the ground sample, mix well and place it in a 65℃ water bath for 90min. Invert the sample once every 15min to ensure that the leaf tissue and solution are thoroughly mixed and heated.

[0054] (3) Remove the centrifuge tubes from the water bath and place them at room temperature for 2 minutes. Add 800 μL of chloroform / isoamyl alcohol (24:1) mixture. Invert the tubes 8-10 times and place them in a centrifuge. Centrifuge at 10,000 rpm for 10 minutes.

[0055] (4) Remove the centrifuge tube and transfer the supernatant to a new 1.5ml centrifuge tube (do not aspirate the lower impurities). Quickly add an equal volume of pre-cooled isopropanol solution to the centrifuge tube. Invert the tube to mix well and place it in a -20℃ refrigerator for 1 hour.

[0056] (5) Take out the centrifuge tube stored at -20℃, centrifuge at 10000rpm for 10min, and discard the liquid. The DNA should precipitate at the bottom of the centrifuge tube.

[0057] (6) Add 500 μL of 70% ethanol solution to the centrifuge tube and gently tap the bottom of the centrifuge tube to suspend the precipitate. Centrifuge at 10,000 rpm for 5 minutes and discard the liquid.

[0058] (7) Repeat step (6) above and place the DNA precipitate in a vent to dry.

[0059] (8) Dissolve the DNA in sterilized 1×TE solution and store at -20℃ for later use.

[0060] 1.4 Watermelon 5K Liquid Chromatography Chip Detection

[0061] Genotyping of 89 DNA samples was performed using a watermelon 5K liquid phase chip developed in the laboratory.

[0062] The specific steps are as follows:

[0063] (1) Fragment the DNA, attach fixed adapters, and amplify the DNA fragments to construct a library.

[0064] (2) Mix the SNP probe, DNA library and buffer, and amplify the detection fragment by PCR.

[0065] (3) The amplified fragments were purified using Beckman AMPure Beads, the library was detected using qubit and qPCR, and the amplified fragments were sequenced using Illumina HiSeq X.

[0066] (4) The obtained sequencing data is filtered and mapped onto the reference genome of watermelon “97103”V2 version. GATK is used to detect SNPs in different samples to obtain SNP locus genotype data for all samples.

[0067] 1.5 Genome-wide association analysis and acquisition of associated SNP loci

[0068] The SNP genotype data from section 1.4 were used to perform association analysis using a mixed linear model (MLM) in TASSEL 5.0 software to identify SNPs associated with sugar content in watermelon fruit. When the p-value of the detected variant was less than 2 × 10⁻⁶, the association was considered complete. -9 At that time, it was considered a significant association site. In this experiment, the sugar content trait in watermelon fruit was associated with one significant SNP site on chromosome 1: Chr1-33089964, as shown in Table 1. Manhattan plots and QQplots of the p-values ​​of the whole genome locus were plotted using Rpackages, as shown in Table 1. Figure 1 and Figure 2 As shown, the GWAS results have significant peak values ​​and the QQ plot shows high consistency, proving that the correlation analysis localization results are accurate.

[0069] Table 1. SNP loci significantly associated with sugar content in watermelon fruit.

[0070] name chromosome position / bp refer to Mutations Chr1-33089964 Chr1 33089964 A T

[0071] Further linkage disequilibrium (LD) analysis was performed using PopLDdecay software, and the results showed that 100 kb LD was correlated with soluble sugar content. Figure 3Therefore, the 100kb region flanking the significant site Chr1-33089964 was designated as a candidate QTL region and named ssc1.2. In ssc1.2, 35 annotated genes (Cla97C01G020260 to Cla97C01G020600) were identified, among which the Cla97C01G020520 gene encodes monosaccharide-sensing protein 2, which shares 66.13% amino acid similarity with the Arabidopsis thaliana vacuolar membrane monosaccharide transporter gene AtTST2. Its homologous gene ClTST2 in watermelon has been identified as the pathogenic gene for the watermelon sugar content QTL site QBRX2-1, associated with sugar uptake and accumulation by vacuolar plastids in watermelon fruit pulp cells, and the two share 69.47% amino acid similarity. Therefore, we speculate that Cla97C01G020520 is a candidate gene for ssc1.2, and at the same time, we demonstrate that the polymorphism of AT at the Chr1-33089964 site is related to the sugar content of watermelon fruit.

[0072] As shown in Table 2, when the genotype at Chr1-33089964 is A, the fruit exhibits a high sugar content trait, with the preferred sugar content range being 7.91-10.82 (°Bx); when the genotype at Chr1-33089964 mutates from A to T, the fruit exhibits a low sugar content trait, with the preferred sugar content range being 1.3-7.25 (°Bx).

[0073] Table 2. Phenotypic values ​​of sugar content in watermelon fruits and genotypic data corresponding to the Chr1-33089964 locus in 77 melon-related populations.

[0074]

[0075]

[0076] Note: Since this site was not detected in 12 of the materials in the Watermelon 5K liquid phase chip, this table only shows the data for 77 of the materials.

[0077] Example 2: Primer development for detecting the Chr1-33089964 locus genotype

[0078] 2.1 Primer development for amplifying the Chr1-33089964 locus genotype

[0079] For the SNP site Chr1-33089964, which is significantly associated with the sugar content of watermelon fruit, the nucleotide sequences of the first and last 300 bp of Chr1-33089964 site were retrieved from the watermelon “97103” V2 version genome. Using Primer3, a pair of primers with a length not exceeding 300 bp were designed to amplify the molecular marker. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2.

[0080] SEQ ID NO.1: 5'-AAAAAGAACATCAAAGAATGGAA-3';

[0081] SEQ ID NO. 2: 5'-CTGAATCGAAAAACCCTCTCTG-3'.

[0082] Example 3: Application of the Chr1-33089964 locus genotype in detecting the sugar content trait in watermelon fruit.

[0083] 3.1 Selection of materials to be verified

[0084] Five samples each of high and low fruit sugar content were randomly selected from 89 samples. The samples with high sugar content were WM104 (7.91), WM106 (9.23), WM108 (10.82), WM225 (8.93), and WM259 (8.00), while the samples with low sugar content were WM232 (3.90), WM257 (3.00), WM274 (3.35), WM275 (3.55), and WM289 (3.28). Phenotypic results are shown below. Figure 4 See Figure A in the diagram. Germinate the above materials and sow them in seedling trays (see 1.1 for specific methods). Take samples after the seedlings have fully expanded their true leaves.

[0085] 3.2 DNA extraction, PCR amplification, and Sanger sequencing of the watermelon variety to be validated

[0086] DNA extraction was performed on the collected samples (see 1.2 for specific methods).

[0087] The PCR reaction system was performed according to the standard procedure, and the PCR reaction system is shown in Table 3.

[0088] Table 3 PCR reaction system

[0089]

[0090] The PCR amplification program was as follows: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 40 s, 35 cycles; 72℃ for 5 min.

[0091] The PCR products were sequenced using the Sanger sequencing method.

[0092] 3.3 Genotype and Phenotype Verification

[0093] The results of Sanger sequencing of the amplified products are as follows: Figure 4 As shown, the amplification products of the five materials with low sugar content all had a base of T at position 139, while the amplification products of the five materials with high sugar content all had a base of A at position 139. Figure 4 (See Figure C in the diagram). It can be seen that the genotype detection results and the actual sugar content measurement results (phenotype) are consistent. Box plots showing the sugar content trait distribution of different genotypes corresponding to the Chr1-33089964 locus are shown in Figure C. Figure 4 Figure B in the diagram demonstrates that the primers used to detect the Chr1-33089964 locus genotype are effective in distinguishing between watermelon materials with high and low sugar content.

[0094] Therefore, Sanger sequencing can be used to verify the sugar content of watermelon samples by detecting the Chr1-33089964 locus genotype. The specific method is as follows:

[0095] The amplified products were subjected to Sanger sequencing. If the 139th base of the cloned product was A, its sequence is shown in SEQ ID NO.3 in the sequence listing. This indicates that the genotype at Chr1-33089964 is A, and the tested watermelon plant is a homozygous material with high sugar content in the watermelon fruit. If the 139th base (Chr1-33089964) of the cloned product was T, its sequence is shown in SEQ ID NO.4 in the sequence listing. This indicates that the genotype at Chr1-33089964 is T, and the tested watermelon plant is a material with low sugar content in the watermelon fruit. If the 139th base of the cloned product has two bases, A and T, it indicates that the genotype at Chr1-33089964 is A and T, and the tested watermelon plant is a heterozygous material with high sugar content in the watermelon fruit. The sequences of SEQ ID NO.3 and SEQ ID NO.4 are as follows:

[0096] SEQ ID NO.3:

[0097]

[0098] SEQ ID NO.4:

[0099]

[0100]

[0101] In summary, the polymorphism of the Chr1-33089964 site A / T disclosed in this invention can be used for large-scale identification of sugar content traits in watermelon fruits. It is not only fast and effective, but can also be identified at the seedling stage, greatly shortening the breeding cycle and enabling large-scale application in production.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of SNP molecular markers related to sugar content in watermelon fruit in identifying the sugar content trait / genotype of watermelon fruit, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.

3. The SNP molecular marker has an A / T mutation at position 139. If position 139 is A, the watermelon plant is a homozygous material / genotype with high sugar content in the watermelon fruit; if position 139 is T, the watermelon plant is a material / genotype with low sugar content in the watermelon fruit; if position 139 has two bases, A and T, the watermelon plant is a heterozygous material / genotype with high sugar content in the watermelon fruit. The upstream primer sequence for amplifying the SNP molecular marker is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.

2.

2. A method for determining the sugar content / genotype of watermelon fruit, characterized in that, The method includes the following steps: (1) Extract genomic DNA from the watermelon sample to be tested; (2) Using the genomic DNA extracted in step (1) as a template, the sample extracted in step (1) is subjected to PCR amplification using the primer pair of the SNP molecular marker described in claim 1 to obtain the amplification product; (3) Perform Sanger sequencing on the amplified products; (4) The determination is based on the sequencing results of step (3), and the specific criteria are as follows: If the cloning product has an A base at position 139, and its sequence is as shown in SEQ ID NO.3 in the sequence listing, then the watermelon plant being tested is a homozygous material / genotype with high sugar content in its watermelon fruit; if the cloning product has a T base at position 139, and its sequence is as shown in SEQ ID NO.4 in the sequence listing, then the watermelon plant being tested is a material / genotype with low sugar content in its watermelon fruit; if the cloning product has two bases at position 139, namely A and T, then the watermelon plant being tested is a heterozygous material / genotype with high sugar content in its watermelon fruit.

3. The application of a reagent kit in identifying the sugar content trait / genotype of watermelon fruit, characterized in that, The kit contains primer pairs of the SNP molecular markers described in claim 1. Using the primer pairs of the SNP molecular markers, PCR amplification is performed with the genomic DNA of the watermelon sample to be tested as a template. If the cloning product has an A base at position 139, and its sequence is shown in SEQ ID NO.3 in the sequence listing, then the watermelon plant to be tested is a homozygous material / genotype with high sugar content in the watermelon fruit. If the cloning product has a T base at position 139, and its sequence is shown in SEQ ID NO.4 in the sequence listing, then the watermelon plant to be tested is a material / genotype with low sugar content in the watermelon fruit. If the cloning product has two bases at position 139, namely A and T, then the watermelon plant to be tested is a heterozygous material / genotype with high sugar content in the watermelon fruit.

Images