SNP (Single Nucleotide Polymorphism) molecular marker for detecting watermelon pulp color and application

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker for detecting the color of watermelon pulp and application of the SNP molecular marker. The SNP molecular marker corresponds to a Chr06: 24308944 site on a No.6 chromosome of a watermelon Citrullus lanatus 97103 v2.5 genome, the polymorphism of the SNP molecular marker is G / A, and the SNP molecular marker is closely linked with the thick yellow / light yellow color character of watermelon pulp. When the basic group of the SNP site is G, the watermelon shows a thick yellow pulp character; and when the basic group is A, the watermelon shows a light yellow pulp character. The invention also provides a KASP primer group for detecting the SNP molecular marker, and a detection method based on the primer group, and KASP-PCR amplification and genetic typing are performed on a sample to be detected, so that the color character of watermelon pulp is judged. The invention also provides a kit containing the primer group and application of the kit in watermelon molecular marker-assisted breeding. The method has the advantages of being rapid in detection, high in accuracy, free of environmental influence, suitable for high-throughput application and the like, and the breeding efficiency of the yellow pulp watermelon variety is improved.

Description

Technical Field

[0001] This invention relates to the field of biological breeding technology, and more specifically, to an SNP molecular marker for detecting the color of watermelon flesh and its application. Background Technology

[0002] Watermelon (Citrullus lanatus) is an annual vine belonging to the genus Citrullus in the family Cucurbitaceae. It is one of the most widely cultivated economic crops globally. Watermelon fruit is characterized by its sweet taste, high water content, and rich content of vitamins and minerals, making it a popular choice among consumers. Among the commercial traits of watermelons, flesh color is a crucial factor influencing consumer purchasing intentions and market value, and it is also a key trait focused on during watermelon variety breeding. Based on the differences in the composition and accumulation of carotenoids in the flesh, watermelon flesh colors can be mainly classified into red (including scarlet and coral), yellow (including deep yellow and light yellow), orange, and white. The formation of different flesh colors is closely related to the accumulation of specific carotenoid components in the chromoplasms of the flesh cells. For example, the accumulation of all-trans lycopene and lutein-like substances (such as zeaxanthin, neoxanthin, and azathoicin) usually results in red or bright yellow flesh; the accumulation of ξ-carotene and pro-lycopene can result in orange-yellow flesh; the large accumulation of β-carotene results in orange flesh; and the formation of white flesh is related to the accumulation of phytolycopene and pale yellow ξ-carotene.

[0003] Existing research has shown that the color trait of watermelon flesh is jointly regulated by multiple gene loci in the carotenoid biosynthesis pathway. Among them, the Y gene locus has multiple alleles, and different allele combinations are associated with the formation of coral-red, orange, or orange-yellow flesh, exhibiting a certain dominance-recession relationship among different flesh colors. Furthermore, other chromosomes in the watermelon genome contain major quantitative trait loci controlling the content of specific carotenoids, which have a significant impact on flesh color formation. For yellow-fleshed watermelons, studies have indicated that the C gene locus is closely related to the yellow flesh trait; the lycopene β-cyclase encoded by this locus plays a crucial role in carotenoid metabolism. Yellow-fleshed watermelons can generally be further distinguished into light yellow and dark yellow flesh types based on the depth of flesh color. Compared to light yellow flesh, dark yellow flesh watermelons have a more vibrant color and higher content of carotenoids and other nutrients, resulting in higher market acceptance and economic value. Therefore, the distinction between light yellow and dark yellow flesh is not only of significant commercial importance, but also an important target trait in the breeding of yellow-fleshed watermelons.

[0004] In actual breeding processes, the identification of watermelon flesh color traits mainly relies on field phenotypic observation at maturity, that is, judging the flesh color type by visual inspection after the fruit matures. However, this method requires waiting for a long growth cycle to complete the trait identification, resulting in low breeding efficiency. At the same time, the phenotypic expression of flesh color is also easily affected by factors such as cultivation environment, growth conditions, and differences in human judgment, leading to insufficient stability and accuracy of the identification results, making it difficult to meet the needs of modern breeding for high-efficiency and high-precision screening.

[0005] With the development of molecular biology techniques, marker-assisted selection (MAG) technology has been increasingly applied to crop genetic improvement and breeding practices. This technology enables early prediction and screening of target traits by detecting molecular markers closely linked to them, thereby effectively shortening the breeding cycle and improving breeding efficiency. In recent years, studies have reported several molecular markers related to watermelon flesh color, such as markers based on the lycopene β-cyclase gene to distinguish between red and bright yellow flesh, markers based on carotenoid isomerization-related genes or precursor synthesis-related genes to screen for orange flesh types, and markers to distinguish between scarlet and coral-red flesh.

[0006] However, current research and applications of molecular markers primarily focus on distinguishing between different major categories of fruit flesh color. For the sub-trait of light yellow and dark yellow flesh within yellow-fleshed watermelons, the availability of specific molecular markers remains limited. In practical breeding, the lack of a stable and accurate molecular detection method to differentiate between light and dark yellow flesh means that this trait still relies mainly on phenotypic identification at maturity, hindering the efficient breeding of yellow-fleshed watermelons, especially dark yellow-fleshed varieties. Therefore, under current technological conditions, there is an urgent need for a detection method that can accurately distinguish between light and dark yellow flesh within yellow-fleshed watermelons to meet the practical needs of early, rapid, and stable identification of this trait in watermelon molecular breeding. Summary of the Invention

[0007] One of the technical problems to be solved by the present invention is to provide an SNP molecular marker for detecting the color of watermelon flesh, so as to solve the problem that the prior art lacks specific molecular markers for the light yellow / dark yellow trait inside yellow flesh, making it difficult to achieve early and accurate identification of this trait.

[0008] To overcome the shortcomings of the prior art, the present invention provides an SNP molecular marker for detecting the color of watermelon flesh. The molecular marker corresponds to the SNP site Chr06:24308944 on chromosome 6 of the watermelon Citrullus lanatus 97103 v2.5 genome. The polymorphism of the SNP site is closely linked to the dark yellow / light yellow color trait of watermelon flesh, and its polymorphism is G / A.

[0009] Preferably, the SNP site Chr06:24308944 is located in a watermelon nucleotide sequence, and is numbered starting from the 5′ end of the nucleotide sequence: When the nucleotide sequence is as shown in SEQ ID NO:4, and the base at the corresponding SNP site is G, the watermelon exhibits a deep yellow flesh texture. When the nucleotide sequence is as shown in SEQ ID NO:5, and the base corresponding to the SNP site is A, the watermelon exhibits a light yellow flesh appearance.

[0010] Compared with existing technologies, the SNP molecular markers provided by this invention target the subtle differences between light and dark yellow flesh within watermelon's yellow flesh, rather than merely distinguishing between broad categories like red and yellow flesh colors. The molecular markers provided by this invention are based on specific SNP loci obtained through genetic mapping and validation in segregating populations. These alleles exhibit a stable and highly consistent correspondence with the flesh color phenotype, allowing the markers to directly reflect the genetic differences controlling the light / dark yellow trait. This invention transforms the traditional identification method, which relies on phenotypic observation at maturity, into a molecular detection method based on SNP polymorphism at the genome level. This not only avoids interference from environmental factors in phenotypic judgment but also enables prediction and screening of target traits at the seedling stage. It solves the problems of long identification cycles, low efficiency, and difficulty in accurately distinguishing subtle traits in the prior art, providing a reliable molecular basis for the precise breeding of yellow-fleshed watermelons, especially dark yellow-fleshed varieties.

[0011] Another technical problem to be solved by the present invention is to provide a KASP primer set for detecting the SNP molecular marker, so as to solve the problem in the prior art of lacking a stable, high-throughput detection tool suitable for practical breeding applications for the light yellow / dark yellow flesh trait.

[0012] To overcome the shortcomings of the prior art, the present invention provides a KASP primer set for detecting the SNP molecular marker, the primer set comprising: The first allele-specific forward primer named ClCyf-FAM has the nucleotide sequence shown in SEQ ID NO:1; The second allele-specific forward primer named ClCyf-HEX has the nucleotide sequence shown in SEQ ID NO:2; The reverse primer named ClCyf-COMMON has the nucleotide sequence shown in SEQ ID NO:3; Among them, SEQ ID NO:1 is: 5′-GAAGGTGACCAAGTTCATGCTAAACAGAGTCGTCTCACGAAG-3′; SEQ ID NO:2 is: 5′-GAAGGTCGGAGTCAACGGATTAAACAGAGTCGTCTTCACGAAA-3′; SEQ ID NO:3 is: 5′-TGTCGAATATGAACTCACAAACCCA-3′.

[0013] This invention provides a method for detecting the color traits of watermelon flesh using the aforementioned primer set, comprising the following steps: (a) Extracting genomic DNA from the watermelon sample to be tested; (b) Using the DNA extracted in step (a) as a template, perform KASP-PCR amplification using the primer set described above; (c) Detect the fluorescence signal of the PCR product and perform genotyping; (d) Determine the flesh color trait of the watermelon to be tested based on the genotyping results: If the genotype is GG, it is determined to be a dark yellow flesh type; If the genotype is AA, it is determined to be a light yellow flesh type; If the genotype is AG, it is determined to be a light yellow flesh type.

[0014] Preferably, the DNA extraction in step (a) is performed using the TPS method, which includes: grinding young watermelon leaves, adding TPS solution, incubating in a 65°C water bath, centrifuging, collecting the supernatant, precipitating the DNA with isopropanol, and dissolving it with an aqueous solution.

[0015] Preferably, the KASP reaction system in step (b) comprises: 2.5 μL of 2× KASP MasterMix, 2.5 μL of template DNA, and 0.07 μL of KASP primers, wherein the KASP primers are prepared from the primer set, and the remaining volume is made up by ddH2O.

[0016] Preferably, the PCR amplification procedure in step (b) includes: Step 1: Pre-denaturation at 94 ℃ for 15 minutes; Step 2: 10 cycles of landing PCR, each cycle consisting of denaturation at 94 °C for 20 seconds, and annealing / extension at 61 °C starting at 0.6 °C and holding for 60 seconds each cycle. Step 3: 29 cycles of standard PCR, each cycle consisting of denaturation at 94 °C for 20 seconds and annealing / extension at 55 °C for 60 seconds.

[0017] Compared with existing technologies, the KASP primer set and detection method of this invention are based on the principle of competitive allele-specific amplification. By distinguishing different alleles in the same reaction system, it achieves rapid and accurate genotyping of target SNP sites. The method provided by this invention does not require electrophoretic separation, and the detection results are presented intuitively in the form of fluorescence signals. It has the advantages of simple operation, high throughput, good repeatability, and low detection cost. In this invention, the primer sequences are highly matched with the target SNP sites, resulting in good consistency between the genotyping results and the flesh color phenotype, which can meet the needs of rapid screening of large-scale segregating populations or breeding materials. By applying this detection method to watermelon molecular marker-assisted breeding, the identification of light yellow or dark yellow flesh traits can be completed at the seedling stage, significantly shortening the breeding cycle, improving the breeding efficiency of dark yellow flesh watermelon varieties, and solving the problem of difficult efficient and accurate breeding of yellow flesh sub-traits in the background technology.

[0018] The present invention provides a kit for detecting the color of watermelon flesh, the kit comprising a primer set of the KASP molecular marker, and at least one of a DNA extraction reagent, a PCR reaction buffer, or a fluorescent detection reagent.

[0019] Preferably, the primer set is provided in the kit as a premixed KASP primer mixture and is prepared by dilution with ddH2O.

[0020] This invention provides an application of the aforementioned SNP molecular markers in watermelon breeding, used to select watermelon flesh with the aid of molecular markers during the seedling stage for the trait of dark yellow or light yellow color. This includes using KASP technology to genotype watermelon F2 or F3 segregating populations and comparing the results with phenotypic data to screen individual plants with dark yellow or light yellow flesh.

[0021] The kit of this invention integrates a set of KASP primers closely linked to the target trait with relevant detection components, making the detection process more standardized and convenient, reducing human error, and improving the consistency and stability of detection results. Simultaneously, by applying the kit and detection method to molecular marker-assisted selection in watermelon breeding, rapid genotyping of F2 or F3 segregating populations can be performed at the seedling stage, and compared with phenotypic data, thereby accurately screening individual plants with the target trait at an early stage. The above-mentioned application of this invention effectively solves the problem of long breeding cycles and high resource input caused by relying on phenotypic identification at maturity in traditional breeding, improving the breeding efficiency of dark yellow and light yellow flesh watermelon varieties, and meeting the actual needs of modern watermelon molecular breeding for high efficiency and precision. Attached Figure Description

[0022] Figure 1 This diagram illustrates the flesh color of the parent watermelons and the F1 generation. The maternal parent "Tianyi 2054" has dark yellow flesh, the paternal parent "Huangti 1804" has light yellow flesh, and the F1 generation has light yellow flesh. Figure 2 This is a schematic diagram of the gene mapping results for the dark yellow / light yellow color trait of watermelon flesh based on BSA mixed pool analysis, including the SNP-index distribution and ΔSNP-index analysis results for dark yellow flesh mixed pool and light yellow flesh mixed pool. Figure 3 A schematic diagram showing the fine localization results of the deep yellow / light yellow color trait of watermelon flesh based on the F3 family; Figure 4 This is a schematic diagram showing the results of SNP typing in the watermelon F2 segregating population using ClCyf KASP primers. Detailed Implementation

[0023] First, those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make adjustments as needed to adapt to specific application scenarios.

[0024] This invention provides an SNP molecular marker for detecting the color of watermelon flesh. The molecular marker corresponds to the SNP site Chr06:24308944 on chromosome 6 of the watermelon Citrullus lanatus 97103 v2.5 genome. The polymorphism of the SNP site is closely linked to the dark yellow / light yellow color trait of watermelon flesh, and its polymorphism is G / A.

[0025] As a preferred embodiment, the SNP site Chr06:24308944 is located within a watermelon nucleotide sequence, and is numbered starting from the 5′ end of the nucleotide sequence: When the nucleotide sequence is as shown in SEQ ID NO:4, and the base at the corresponding SNP site is G, the watermelon exhibits a deep yellow flesh texture. When the nucleotide sequence is as shown in SEQ ID NO:5, and the base corresponding to the SNP site is A, the watermelon exhibits a light yellow flesh appearance.

[0026] This invention provides a KASP primer set for detecting the SNP molecular marker, the primer set comprising: The first allele-specific forward primer named ClCyf-FAM has the nucleotide sequence shown in SEQ ID NO:1; The second allele-specific forward primer named ClCyf-HEX has the nucleotide sequence shown in SEQ ID NO:2; The reverse primer named ClCyf-COMMON has the nucleotide sequence shown in SEQ ID NO:3; Among them, SEQ ID NO:1 is: 5′-GAAGGTGACCAAGTTCATGCTAAACAGAGTCGTCTCACGAAG-3′; SEQ ID NO:2 is: 5′-GAAGGTCGGAGTCAACGGATTAAACAGAGTCGTCTTCACGAAA-3′; SEQ ID NO:3 is: 5′-TGTCGAATATGAACTCACAAACCCA-3′.

[0027] The KASP molecular marker developed in this invention is closely linked to the dark yellow / light yellow flesh color trait of watermelon and can be used for marker-assisted breeding of watermelon. Based on SNP loci highly consistent with the target trait, this molecular marker reflects the genetic basis of dark yellow and light yellow flesh through allele differences, enabling accurate differentiation of the phenotypic trait of flesh color at the molecular level. By combining it with KASP competitive allele-specific PCR technology, this molecular marker has the advantages of small reaction system, low reagent consumption, simple operation procedure, no need for electrophoresis separation, and suitability for high-throughput genotyping. It can rapidly detect a large number of seedling samples in the seedling stage, avoiding the problems of long cycle and low efficiency caused by waiting for fruit maturity for phenotypic identification in traditional breeding, and greatly accelerating the screening and breeding process of dark yellow and light yellow flesh watermelon materials.

[0028] This invention provides a method for detecting the color traits of watermelon flesh using the aforementioned primer set, comprising the following steps: (a) Extracting genomic DNA from the watermelon sample to be tested; (b) Using the DNA extracted in step (a) as a template, perform KASP-PCR amplification using the primer set described above; (c) Detect the fluorescence signal of the PCR product and perform genotyping; (d) Determine the flesh color trait of the watermelon to be tested based on the genotyping results: If the genotype is GG, it is determined to be a dark yellow flesh type; If the genotype is AA, it is determined to be a light yellow flesh type; If the genotype is AG, it is determined to be a light yellow flesh type.

[0029] As a preferred embodiment, the DNA extraction in step (a) is performed using the TPS method, which includes: grinding young watermelon leaves, adding TPS solution, incubating in a 65°C water bath, centrifuging, collecting the supernatant, precipitating the DNA with isopropanol, and dissolving it with an aqueous solution.

[0030] As a preferred embodiment, the KASP reaction system in step (b) comprises: 2.5 μL of 2× KASPMaster Mix, 2.5 μL of template DNA, and 0.07 μL of KASP primers, wherein the KASP primers are prepared from the primer set, and the remaining volume is made up by ddH2O.

[0031] As a preferred embodiment, the PCR amplification procedure in step (b) includes: Step 1: Pre-denaturation at 94 ℃ for 15 minutes; Step 2: 10 cycles of landing PCR, each cycle consisting of denaturation at 94 °C for 20 seconds, and annealing / extension at 61 °C starting at 0.6 °C and holding for 60 seconds each cycle. Step 3: 29 cycles of standard PCR, each cycle consisting of denaturation at 94 °C for 20 seconds and annealing / extension at 55 °C for 60 seconds.

[0032] This invention provides a watermelon flesh color detection method based on the aforementioned molecular markers. This method involves genotyping the genomic DNA of the watermelon sample and determining the flesh color based on the stable correspondence between the alleles of the SNP loci and the flesh color trait. When the detection result is GG genotype, it indicates that the plant carries an allele associated with the deep yellow flesh trait, and its fruit will have deep yellow flesh. When the detection result is AA or AG genotype, it indicates that the plant corresponds to the light yellow flesh trait, and its fruit will have light yellow flesh. By directly converting genotype information into a judgment result for the flesh color trait, this method can accurately predict the deep yellow or light yellow flesh trait in the early stages of watermelon growth, providing a scientific basis for subsequent breeding selection.

[0033] The present invention provides a kit for detecting the color of watermelon flesh, the kit comprising a primer set of the KASP molecular marker, and at least one of a DNA extraction reagent, a PCR reaction buffer, or a fluorescent detection reagent.

[0034] In a preferred embodiment, the molecular marker detection kit of the present invention, in addition to containing KASP primers for detecting the SNP sites, may further contain conventional components known to those skilled in the art, such as DNA extraction reagents, buffers required for PCR amplification reactions, fluorescent detection reagents, positive controls or negative controls; the components in the kit can be combined or provided individually according to actual detection needs.

[0035] As a preferred embodiment, the primer set is provided in the kit as a premixed KASP primer mixture and is prepared by dilution with ddH2O.

[0036] This invention provides an application of the aforementioned SNP molecular markers in watermelon breeding, used to select watermelon flesh with the aid of molecular markers during the seedling stage for the trait of dark yellow or light yellow color. This includes using KASP technology to genotype watermelon F2 or F3 segregating populations and comparing the results with phenotypic data to screen individual plants with dark yellow or light yellow flesh.

[0037] The following specific embodiments, incorporating data, are provided to further elaborate on the technical solution of the present invention: Example 1: Obtaining SNP markers associated with the dark yellow / light yellow trait of watermelon flesh A cross was conducted using the dark yellow flesh watermelon inbred line "Tianyi 2054" as the female parent and the light yellow flesh watermelon inbred line "Huangti 1804" as the male parent to obtain F1 (variety name "Huangti No. 1", registration number: GPD Watermelon (2023) 330063). The F1 population was then self-crossed to construct the F2 segregating population, such as... Figure 1 As shown, the maternal parent 'Tianyi 2054' has dark yellow flesh, the paternal parent 'Huangti 1804' has light yellow flesh, and the F1 generation ('Huangti No. 1') has light yellow flesh, which is used to illustrate the phenotypic characteristics of flesh color of the parents and F1 generation materials used in this study. Flesh color segregation occurred in the F2 population. From the F2 population, 20 plants each with light yellow and dark yellow flesh were selected, and equal amounts of young leaves were taken from each plant to construct light yellow and dark yellow mixed pools, respectively. These pools, along with the young leaves from both parents, were sent to Beijing Novogene Technology Co., Ltd. for sequencing.

[0038] Sequencing data from both parents and the two pooled sequencing datasets were filtered and quality-controlled before alignment to the watermelon 97103 v2.5 reference genome (http: / / cucurbitgenomics.org / v2 / organism / 16). BSA-seq analysis was then performed using the ΔSNP-index algorithm. The candidate region associated with the deep yellow / light yellow flesh color trait of watermelon was found to be located on chromosome 6, Chr06:24709139, bp to 25408664 bp, with a candidate region size of 699.5 kb. The localization results based on the BSA pooled sequencing method are as follows: Figure 2 As shown, where Figure 2 a is a distribution map of the SNP-index of the dark yellow flesh-colored pool across the entire genome. Figure 2 b is the distribution map of the SNP-index of the light yellow flesh-colored pool across the entire genome. Figure 2 c is the distribution map of ΔSNP-index across the entire genome, based on Figure 2 The results show that the candidate interval is located in Chr06:24709139 bp~25408664 bp.

[0039] Based on the BSA-seq analysis results, KASP primers were designed from SNP sites in both parents that exhibited polymorphism within the candidate regions. To ensure the effectiveness of KASP genotyping, primer design followed principles such as a GC content of 40%–50% and avoidance of special structures, and primer specificity was verified using Primer-BLAST.

[0040] Genotyping of F2 segregating population plants was performed using the designed KASP primers. Recombinant plants were screened based on the individual plant's flesh color phenotype (dark yellow or light yellow), and these recombinant plants were self-pollinated to obtain F3. Genotyping of F3 plants was then performed using KASP primers. Based on the flesh color phenotype data of the F3 families, the fine mapping results of the F3 families are as follows: Figure 3 As shown, by combining the genotypes of recombinant single plants and F3 families with the phenotypes of fruit flesh color, the target gene was further limited to the range of Chr06:23732428 bp~24308944 bp.

[0041] Within this interval, the genotype detected at the SNP locus at Chr06:24308944 bp was completely consistent with the flesh color phenotype, confirming that this SNP locus was closely linked to the deep yellow / light yellow trait of watermelon flesh. The KASP molecular marker corresponding to this SNP locus was named ClCyf. After PCR amplification, fluorescence reading, and genotyping, the plants clustered with the maternal parent "Tianyi 2054" were identified as having deep yellow flesh, while the plants clustered with the paternal parent "Huangti 1804" were identified as having light yellow flesh.

[0042] The primer set for the ClCyf molecular marker consists of three primers: ClCyf-FAM, ClCyf-HEX, and ClCyf-COMMON, and their nucleotide sequences are shown in SEQ ID NO:1-3, as follows: ClCyf-FAM: 5′-GAAGGTGACCAAGTTCATGCTAAACAGAGTCGTCTCACGAAG-3′; ClCyf-HEX: 5′-GAAGGTCGGAGTCAACGGATTAAACAGAGTCGTCTTCACGAAA-3′; ClCyf-COMMON: 5′-TGTCGAATATGAACTCACAAACCCA-3′.

[0043] Example 2: This example is based on the SNP molecular marker ClCyf and its primer set that are closely linked to the trait obtained in Example 1. The F2 segregating population is detected according to steps (a) to (d) of claim 4 and compared with the mature phenotype.

[0044] Step (a) Extract genomic DNA from the watermelon sample to be tested: Take approximately 2 g of young watermelon leaves and place them in a 2 mL centrifuge tube containing steel balls. Grind the leaves using a tissue homogenizer (frequency: 60 Hz, time: 30 s). After grinding, add 700 μL of TPS solution preheated to 65 ℃, mix thoroughly, and incubate at 65 ℃ for 30 min. After cooling to room temperature, centrifuge (12000 rpm, 10 min), and transfer 400 μL of the supernatant to a 1.5 mL centrifuge tube. Add 400 μL of pre-cooled isopropanol and centrifuge (12000 rpm, 10 min). Discard the supernatant, wash with 75% ethanol, air dry, and dissolve in ddH2O to obtain an aqueous DNA solution.

[0045] Step (b) Using the DNA from step (a) as a template, perform a KASP-PCR amplification reaction: 1) KASP primer preparation: Take 12 μL (100 μM) ClCyf-FAM primer, 12 μL (100 μM) ClCyf-HEX primer, 30 μL (100 μM) ClCyf-COMMON primer, add 46 μL ddH2O to prepare KASP primer mixture.

[0046] 2) KASP-PCR reaction system: 2.5 μL 2×KASP Master Mix, 2.5 μL template DNA, 0.07 μL KASP primers (provided by the above primer mixture), and the remaining volume is made up with ddH2O.

[0047] 3) The KASP-PCR reaction procedure is shown in Table 1: Table 1: KASP-PCR reaction procedure: Step (c) Detect the fluorescence signal of the PCR product and perform genotyping. After PCR, fluorescence was read using the Omega F SNP genotyping instrument, and the fluorescence data were clustered and genotyped using ClusterCaller software.

[0048] Step (d) Determine the flesh color trait based on the genotyping results and compare it with the phenotype. The flesh color of watermelons was identified in the field during the ripening period and compared with the KASP genotyping results to determine the accuracy of the genotyping results.

[0049] Example 3: Validation of the F2 segregating population based on the method of Example 2 and statistical consistency between genotyping and phenotype. In Example 3, based on Example 2, to verify the accuracy of ClCyf molecular markers in marker-assisted selection, the F2 segregating population obtained from “Tianyi 2054 × Huangti 1804” was genotyped, and the consistency between genotype and the flesh color phenotype at maturity was statistically analyzed.

[0050] Step (a) Extract genomic DNA from the sample to be tested: Take approximately 2 g of young, tender watermelon leaves and place them in a 2 mL centrifuge tube containing steel balls. Grind the leaves using a tissue homogenizer (frequency: 60 Hz, time: 30 s). After grinding, add 700 μL of TPS solution preheated to 65 ℃, mix thoroughly, and incubate at 65 ℃ for 30 min. After cooling to room temperature, centrifuge (12000 rpm, 10 min), and transfer 400 μL of the supernatant to a 1.5 mL centrifuge tube. Add 400 μL of pre-cooled isopropanol and centrifuge (12000 rpm, 10 min). Discard the supernatant, wash with 75% ethanol, air dry, and dissolve in ddH2O to obtain an aqueous DNA solution.

[0051] Step (b) KASP-PCR amplification reaction: KASP primer preparation: 12 μL (100 μM) ClCyf-FAM primer, 12 μL (100 μM) ClCyf-HEX primer, 30 μL (100 μM) ClCyf-COMMON primer, and 46 μL ddH2O.

[0052] KASP-PCR reaction system: 2.5 μL 2×KASP Master Mix, 2.5 μL template DNA, 0.07 μL KASP primers, and the remaining volume is made up with ddH2O.

[0053] The KASP-PCR reaction procedure was the same as in Example 2: 94 °C for 15 min; 10 cycles of landing PCR (starting at 61 °C, -0.6 °C per cycle, 60 s); 29 cycles of conventional PCR (55 °C for 60 s), ending at 4 °C for 60 s.

[0054] Step (c) Fluorescence signal detection and genotyping: After PCR, fluorescence was read using the Omega F SNP genotyping instrument, and the fluorescence data were clustered and genotyped using ClusterCaller software.

[0055] Step (d) Phenotypic identification and consistency statistics: The clustering results of KASP typing of the F2 segregating population using ClCyf primers are as follows... Figure 4As shown, blue dots indicate the detection of FAM signal, indicating the GG genotype; red dots indicate the detection of HEX signal, indicating the AA genotype; green dots indicate the simultaneous detection of FAM and HEX signals, indicating the AG genotype; and black dots indicate NTC. When watermelons matured, the phenotypic characteristics of the flesh color of individual plants were assessed in the field and compared with the results of KASP genotyping. Statistical results showed: The blue dots represent 93, indicating that the FAM signal was detected, which is the GG genotype, and the corresponding field phenotype is a dark yellow flesh watermelon. The green dots represent 185, indicating that both FAM and HEX signals were detected simultaneously, which is the AG genotype and corresponds to a light yellow flesh watermelon in the field. The red dots represent 102, indicating that the HEX signal was detected, which is the AA genotype, and the corresponding field phenotype is light yellow flesh watermelon. The above genotyping results are consistent with the field phenotypes.

[0056] The above embodiments further demonstrate that the SNP locus Chr06:24308944 identified in this invention is closely linked to the dark yellow / light yellow color trait of watermelon flesh, and the KASP molecular marker ClCyf constructed from this locus can stably and accurately genotype the segregating population and predict its phenotype. Example 1 confirmed that the genotype of the SNP locus at Chr06:24308944 bp was completely consistent with the flesh color phenotype, establishing ClCyf as a molecular marker highly consistent with the target trait. Examples 2 and 3 further completed KASP genotyping in the F2 segregating population and compared it with the field phenotype at maturity. The results showed that the GG genotype corresponds to dark yellow flesh, while the AA and AG genotypes correspond to light yellow flesh. The genotyping and clustering results were clear and consistent with the phenotypic statistics, indicating that the marker of this invention is suitable for marker-assisted selection and related breeding applications.

[0057] This invention uses SNP loci highly consistent with the target trait as detection targets. Utilizing the mechanism of KASP competitive allele-specific PCR, two allele-specific primers selectively amplify different alleles, and genotypes are distinguished by fluorescence signals. This transforms the flesh color phenotype, which cannot be directly observed at the seedling stage, into a molecular typing result that can be detected early. Based on this principle, the ClCyf marker of this invention is closely linked to the dark yellow / light yellow flesh color trait, and the typing result has a stable correspondence with the phenotype. It can accurately identify individual plants at the seedling stage, improving the reliability of selection. Furthermore, the KASP detection system is small, consumes low reagents, has a simple operation procedure, requires no electrophoresis, and is suitable for high-throughput detection. This enables rapid screening of a large number of breeding materials, significantly shortening the breeding cycle, reducing field identification costs, and improving breeding efficiency, thereby accelerating the breeding process of new dark yellow or light yellow flesh watermelon materials.

[0058] In the description of this invention, the references to "one embodiment," "some embodiments," "in this embodiment," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0059] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A SNP molecular marker for detecting the color of watermelon flesh, characterized in that, The molecular marker corresponds to watermelon Citrullus lanatus 97103 v2.5 The SNP site Chr06:24308944 on chromosome 6 of the genome, wherein the polymorphism of the SNP site is closely linked to the dark yellow / light yellow color trait of watermelon flesh, and its polymorphism is G / A.

2. The SNP molecular marker according to claim 1, characterized in that: The SNP site Chr06:24308944 is located in a watermelon nucleotide sequence, and is numbered starting from the 5′ end of the nucleotide sequence: When the nucleotide sequence is as shown in SEQ ID NO:4, and the base corresponding to the SNP site is G, the watermelon exhibits a dark yellow flesh texture. When the nucleotide sequence is as shown in SEQ ID NO:5, and the base corresponding to the SNP site is A, the watermelon exhibits a light yellow flesh appearance.

3. A KASP primer set for detecting the SNP molecular marker of claim 1, characterized in that, The primer set includes: The first allele-specific forward primer named ClCyf-FAM has the nucleotide sequence shown in SEQ ID NO:1; The second allele-specific forward primer named ClCyf-HEX has the nucleotide sequence shown in SEQ ID NO:2; The reverse primer named ClCyf-COMMON has the nucleotide sequence shown in SEQ ID NO:3; Among them, SEQ ID NO:1 is: 5′-GAAGGTGACCAAGTTCATGCTAAACAGAGTCGTCTCACGAAG-3′; SEQ ID NO:2 is: 5′-GAAGGTCGGAGTCAACGGATTAAACAGAGTCGTCTTCACGAAA-3′; SEQ ID NO:3 is: 5′-TGTCGAATATGAACTCACAAACCCA-3′.

4. A method for detecting the color trait of watermelon flesh using the primer set described in claim 3, characterized in that, Includes the following steps: (a) Extracting genomic DNA from the watermelon sample to be tested; (b) Using the DNA extracted in step (a) as a template, perform KASP-PCR amplification reaction using the primer set described in claim 3; (c) Detect the fluorescence signal of the PCR product and perform genotyping; (d) Determine the flesh color trait of the watermelon to be tested based on the genotyping results: If the genotype is GG, it is determined to be a dark yellow flesh type; If the genotype is AA, it is determined to be a light yellow flesh type; If the genotype is AG, it is determined to be a light yellow flesh type.

5. The method according to claim 4, characterized in that, The DNA extraction in step (a) uses the TPS method, which includes: grinding young watermelon leaves, adding TPS solution, incubating in a 65°C water bath, centrifuging, collecting the supernatant, precipitating the DNA with isopropanol, and dissolving it with an aqueous solution.

6. The method according to claim 4, characterized in that, The KASP reaction system in step (b) comprises: 2.5 μL of 2× KASP Master Mix, 2.5 μL of template DNA, and 0.07 μL of KASP primers, wherein the KASP primers are prepared from the primer set described in claim 3, and the remaining volume is made up by ddH2O.

7. The method according to claim 4, characterized in that, The PCR amplification procedure in step (b) includes: Step 1: Pre-denaturation at 94 ℃ for 15 minutes; Step 2: 10 cycles of landing PCR, each cycle consisting of denaturation at 94 °C for 20 seconds, and annealing / extension at 61 °C starting at 0.6 °C and holding for 60 seconds each cycle. Step 3: 29 cycles of standard PCR, each cycle consisting of denaturation at 94 °C for 20 seconds and annealing / extension at 55 °C for 60 seconds.

8. A reagent kit for detecting the color of watermelon flesh, characterized in that, The kit comprises the primer set of the KASP molecular marker as described in claim 3, and at least one of DNA extraction reagent, PCR reaction buffer or fluorescent detection reagent.

9. The reagent kit according to claim 8, characterized in that, The primer set is provided in the kit as a premixed KASP primer mixture and is prepared by dilution with ddH2O.

10. An application of the SNP molecular marker according to claim 1 in watermelon breeding, characterized in that, This method is used to select watermelon seedlings for dark yellow or light yellow flesh color traits using molecular markers. It involves using KASP technology to genotype watermelon F2 or F3 segregating populations and comparing the results with phenotypic data to screen individual plants with dark yellow or light yellow flesh.

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