SNP molecular marker for detecting cucumber top shape, KASP primer and application of SNP molecular marker and KASP primer

Abstract

The invention provides an SNP (Single Nucleotide Polymorphism) molecular marker for detecting the shape of a cucumber top, a KASP primer and application of the SNP molecular marker and the KASP primer. The SNP molecular marker is closely linked with the shape of the cucumber top, and is located at the 25221828 position of the chromosome Chr01 of the cucumber Chine Long v3 genome, and the SNP mutation from C to G is related to the phenotype of the shape of the cucumber top. A KASP primer group is designed through the SNP marker, the KASP primer group comprises CsFAS-FAM, CsFAS-HEX and CsFAS-COMMON, and accurate identification of the shape of the fruit top of the cucumber can be achieved through PCR amplification and genetic typing technologies. The molecular marker can be widely applied to fruit top shape molecule-assisted selection in cucumber breeding, and has relatively high accuracy and efficiency. In the cucumber breeding process, the technology can avoid the problems of uncertainty and time cost existing in traditional phenotype identification through genotype screening in the early stage, and a simple, convenient and efficient tool is provided for cucumber breeding. The technical method provided by the invention is simple, convenient and rapid, can be popularized and applied in cucumber breeding, and has important economic value and social value.

Description

Technical Field

[0001] The invention relates to the technical field of biological identification, and in particular to a SNP molecular marker for detecting the shape of cucumber fruit top, a KASP primer and an application thereof. Background Art

[0002] cucumber( Cucumis sativus L. ) is one of the important vegetable crops widely cultivated in the world. The appearance quality of its fruit directly affects the market demand and commodity value. Among the appearance traits of cucumber, the shape of the fruit top is a key feature. Different fruit top shapes (such as dome, pointed top, etc.) not only affect the commodity value of cucumber, but also have a direct impact on consumers' purchasing preferences. Therefore, the accurate identification of the fruit top shape occupies an important position in cucumber breeding.

[0003] At present, the identification of cucumber fruit top shape mainly relies on phenotypic observation in the field, which is not only time-consuming and laborious, but also easily interfered by environmental factors, which limits the accuracy and stability of the identification results. In addition, phenotypic observation usually requires a long growth cycle to determine the trait performance, so the breeding process is often slow and inefficient.

[0004] With the development of molecular biology technology, molecular marker-assisted selection (MAS) technology has provided a new way to improve the accurate identification of cucumber fruit top shape. At present, the commonly used molecular marker methods mainly include Indel markers, which require electrophoresis analysis after PCR amplification. The steps are cumbersome and time-consuming. Due to the limitations of electrophoresis technology, the throughput is low, the operation is complicated and the cost is high. Although Indel markers can improve the efficiency of molecular breeding to a certain extent, the cumbersome operation makes it difficult for this technology to meet the needs of efficient and rapid breeding in modern agricultural production, especially in large-scale screening.

[0005] Therefore, the molecular marker methods in the existing technology have obvious defects and shortcomings, including high cost, low throughput, long cycle and other problems, and cannot achieve rapid, accurate and efficient screening of cucumber fruit top shape, which restricts the improvement of cucumber varieties and the improvement of breeding efficiency.

[0006] In this context, developing a new molecular marker method that can quickly and accurately identify the shape of cucumber fruit tops has become the key to solving the problems of existing technologies. This method should be able to accurately identify the shape of cucumber fruit tops at the seedling stage and have the advantages of high throughput, low cost, and simple operation, thereby promoting the improvement of cucumber breeding efficiency.

[0007] The first technical problem to be solved by the present invention is to provide a SNP molecular marker for detecting the shape of cucumber fruit top, so as to solve the problems of low screening efficiency, poor accuracy and high time consumption in the prior art.

[0008] In order to overcome the defects of the above-mentioned prior art, the present invention provides a SNP molecular marker for detecting the shape of cucumber fruit top, wherein the SNP molecular marker is a SNP site tightly linked to the shape of cucumber fruit top, which is a SNP mutation from C to G at position 25221828 of the Chr01 chromosome of the cucumber Chinese Long v3 genome, and the SNP site is related to the phenotype of cucumber fruit top shape.

[0009] Compared with the prior art, the SNP molecular marker for detecting the shape of cucumber fruit tops of the present invention has the following advantages: improved accuracy and efficiency: the fruit top shape phenotypic screening method in the prior art relies on manual observation, which has high errors and subjectivity. The SNP molecular marker provided by the present invention is closely linked to the fruit top shape, and can accurately detect the genotype of the cucumber fruit top shape at the molecular level, avoiding phenotypic errors, thereby greatly improving the accuracy and efficiency of screening; and simplifying the breeding process: the traditional fruit top shape screening method often requires long-term field observation and a large amount of manual labor, and the results are difficult to confirm in the early stage. The SNP molecular markers of the present invention can be quickly detected by PCR technology in the early stage of cucumber growth, which greatly shortens the breeding cycle and reduces manpower investment; can be applied to large-scale breeding: compared with traditional phenotypic screening, the SNP molecular markers provided by the present invention can perform high-throughput molecular marker detection, which is suitable for application in large-scale breeding, and can quickly screen out individuals that meet the target fruit top shape, greatly improving the overall efficiency of breeding work; have precise molecular assisted selection capabilities: the SNP molecular markers provided by the present invention can not only be used for genetic research on cucumber fruit top shape, but also can be widely used in cucumber breeding, as an important tool for molecular assisted selection, to achieve precise control of variety traits. .

[0010] In a possible embodiment, the nucleotide sequence of the SNP molecular marker for detecting the cucumber fruit top shape is shown in SEQ ID NO: 1 and SEQ ID NO: 2; When the 120th position of the sequence is C, that is, SEQ ID NO: 1, the corresponding cucumber fruit top is a round fruit top; When the 120th position of the sequence is G, that is, SEQ ID NO: 2, the corresponding cucumber fruit top is a pointed fruit top.

[0011] Compared with the prior art, the present invention adopts the above technical scheme. Based on the close linkage relationship between the cucumber fruit top shape trait and the SNP site, the phenotype of the cucumber fruit top shape can be determined by detecting a specific SNP site (C to G mutation). The C or G mutation of the SNP site directly corresponds to the shape of the cucumber fruit top (round fruit top or pointed fruit top). The molecular marker is closely related to the phenotype of the fruit top shape. The typing information of the SNP site can be used to accurately infer the morphology of the cucumber fruit top without relying on environmental factors and human intervention, thereby significantly improving the accuracy and efficiency of the cucumber fruit top shape screening, and making the breeding process more efficient and controllable. This technology provides a more advanced molecular assisted selection tool for cucumber breeding, which helps to optimize breeding strategies and cultivate more cucumber varieties that meet market demand.

[0012] The second technical problem to be solved by the present invention is to provide a KASP primer for detecting the shape of cucumber fruit top, so as to solve the problem of inaccurate marker design and inability to efficiently realize molecular assisted selection of cucumber fruit top shape in the prior art.

[0013] In order to overcome the defects of the above prior art, the present invention provides a KASP primer for detecting the shape of cucumber fruit top, wherein the KASP primer is designed according to the SNP molecular marker, and the KASP molecular marker includes a CsFAS primer set, whose sequence is shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and comprises: CsFAS-FAM: 5'-GAAGGTGACCAAGTTCATGCTGCCACATCATCGATTGCGACAC-3'; CsFAS-HEX: 5'-GAAGGTCGGAGTCAACGGATTGCCACATCATCGATTGCGACAG-3'; CsFAS-COMMON: 5'-GGAAGAAGAGGGGACATGTCCTT-3'.

[0014] Compared with the prior art, the KASP primers for detecting the shape of cucumber fruit tops of the present invention have the following advantages: the KASP primers of the present invention detect the genotype of the SNP site by designing specific primers (FAM, HEX, COMMON). These primers can specifically identify the SNP mutation (C to G) in the cucumber genome, and quickly and accurately obtain the genetic information of the shape of cucumber fruit tops through PCR amplification and fluorescence detection methods, providing an efficient and accurate tool for molecular assisted breeding, avoiding the limitations of phenotypic identification in traditional technologies, and the KASP primers provided by the present invention can significantly improve the detection accuracy and efficiency of the shape of cucumber fruit tops; through the highly specific and efficient KASP molecular marker technology, breeders can accurately screen cucumber varieties with target fruit top shapes, thereby accelerating the breeding process and improving the overall efficiency and quality of cucumber breeding. In addition, molecular assisted selection based on this technology can eliminate the errors caused by environmental factors and manual operations in phenotypic selection, making the genetic improvement of cucumber fruit top shape more accurate and controllable.

[0015] The third technical problem to be solved by the present invention is an application of the KASP molecular marker to solve the problems of low efficiency and insufficient accuracy in the molecular-assisted selection of cucumber fruit top shape in the prior art.

[0016] In order to overcome the above defects of the prior art, the present invention provides an application of the KASP molecular marker, which is used for molecular-assisted selection of fruit top shape in cucumber breeding.

[0017] In a possible embodiment, the molecular-assisted selection of fruit top shape in cucumber breeding comprises the following steps: S1: Extract genomic DNA from the cucumber sample to be tested; S2: performing PCR amplification using the KASP primers described in claim 3; S3: Detect the genotype of the SNP locus through fluorescence reading and genotyping; S4: Determine the shape of the fruit top based on the genotype: if the genotype is CC, it is a round fruit top; if the genotype is GG, it is a pointed fruit top; if the genotype is CG, it is an intermediate fruit top.

[0018] Compared with the prior art, the application of KASP primers for detecting the shape of cucumber fruit tops has the following advantages: the application of the present invention is based on the SNP molecular marker of the shape of cucumber fruit tops, and the marker is quickly detected by the KASP technology, and the KASP technology uses FAM and HEX fluorescent probes to perform genotype detection, combined with genotyping data, and can accurately determine the shape of the cucumber fruit top, which not only reduces the time required for phenotypic screening, but also avoids the interference of the external environment on the evaluation of the fruit top shape, and ensures the accuracy of molecular selection; the application of the KASP molecular marker provided by the present invention provides an accurate, fast and efficient solution for the molecular assisted selection of the fruit top shape in cucumber breeding, and through the typing detection of genotypes, breeders can make accurate breeding decisions at an early stage, avoiding the uncertainty caused by phenotypic observation in traditional methods, thereby greatly improving the success rate and efficiency of breeding.

[0019] In a possible implementation, in step S1, the genomic DNA is extracted by TPS method, which includes: A: Take 2g of sample from young cucumber leaves, place in a centrifuge tube filled with steel balls, and grind using a sample tissue grinder at a frequency of 60Hz for 30 seconds; B: Add 700 μL TPS solution preheated to 65°C into the ground centrifuge tube, mix thoroughly and place in a 65°C water bath for 30 minutes; C: Cool to room temperature after water bath, centrifuge at 12000 rpm for 10 minutes, then aspirate 400 μL of supernatant and transfer to a 1.5 ml centrifuge tube; D: Add 400 μL of pre-cooled isopropanol to the centrifuge tube and centrifuge again at 12,000 rpm for 10 minutes, and discard the supernatant; E: Rinse the precipitate with 75% ethanol, air-dry it, and then dissolve it in ddH2O to obtain a genomic DNA solution.

[0020] Compared with the prior art, the present invention adopts the technical scheme and the TPS method to extract DNA, and can obtain high-quality cucumber genomic DNA in a short time. This improved DNA extraction method provides an efficient and high-quality DNA template for subsequent KASP molecular marker analysis, ensures the accuracy and reliability of the genotype, and further improves the effect of molecular-assisted selection of fruit top shape in cucumber breeding, effectively overcomes the problems of low efficiency and low purity in the existing DNA extraction method, and finally enables the present invention to perform molecular marker detection more accurately, improves the accuracy and efficiency of breeding work, and meets the requirements of modern breeding that are efficient, simple and fast.

[0021] In a possible embodiment, in step S2, the primers for PCR amplification include, per 100 μL, 12 μL of 100 μM primer CsFAS-FAM, 12 μL of 100 μM primer CsFAS-HEX, 30 μL of 100 μM primer CsFAS-COMMON, and 46 μL of ddH2O.

[0022] Compared with the prior art, the above technical scheme adopts a CsFAS primer set (including CsFAS-FAM, CsFAS-HEX and CsFAS-COMMON) to efficiently amplify specific SNP sites, and realizes multiple detection through fluorescent labeling (FAM and HEX dyes), thereby ensuring the specificity of PCR amplification, and enabling each sample to accurately detect the genotype related to the cucumber fruit top shape, thereby improving the sensitivity and accuracy of the detection, and by setting appropriate concentrations of primers (12 μL CsFAS-FAM and 12 μL CsFAS-HEX), and a specific amount of CsFAS-COMMON primers (30 μL with a concentration of 100 μM), the concentration of each primer is properly matched with other components in the PCR reaction system (such as KASP Master Mix, DNA template), effectively enhancing the sensitivity of the PCR reaction, enabling efficient genotyping in the case of low DNA content, and avoiding nonspecific amplification.

[0023] In a possible implementation, in step S2, the PCR reaction system used in the PCR amplification includes: 2.5μL, 2×KASP Master Mix; 2.5 μL template DNA; 0.07 μL KASP primer; The remaining volume of the solution was made up to the required volume with ddH2O.

[0024] Compared with the prior art, the above technical solution can optimize the balance and stability of the PCR reaction system by setting appropriate concentrations of primers (12 μL CsFAS-FAM and 12 μL CsFAS-HEX) and a specific amount of CsFAS-COMMON primers (30 μL with a concentration of 100 μM). The concentration of each primer is precisely matched with other components in the PCR reaction system (such as KASP MasterMix and DNA template), making the reaction conditions more stable, thereby significantly enhancing the sensitivity of the PCR reaction, and thus effectively improving the efficiency of PCR amplification, especially when the DNA template concentration is low, it can still maintain a high typing accuracy. By optimizing the primer configuration, the occurrence of nonspecific amplification is avoided, ensuring that only the target SNP site is amplified, thereby improving the accuracy of genotype analysis.

[0025] In a possible implementation, in step S2, the reaction procedure of the PCR amplification includes: Step 1: 94°C, 15 minutes; Step 2: 94°C, 20 seconds; 61–55°C, -0.6°C / cycle, 60 seconds, 10 cycles in total; Step 3: 94°C, 20 seconds; 55°C, 60 seconds, 26 cycles in total; Step 4: 4℃, 60 seconds, end.

[0026] Compared with the prior art, the above technical solution can complete the SNP typing of cucumber fruit top shape under precise temperature control. The 94°C preheating treatment (15 minutes) in step one helps the denaturation of DNA and ensures that the template DNA is completely unzipped, which provides a stable basis for subsequent PCR amplification. Step two optimizes the binding conditions of primers and templates by setting a cooling program (61°C to 55°C, -0.6°C / cycle), and maintains a sufficient annealing time (60 seconds) so that the primers can accurately bind to the target sequence, thereby improving the amplification specificity; and the short 94°C treatment (20 seconds) in step three ensures that the DNA is completely denatured again, and the annealing temperature of 55°C provides the best conditions for the binding of primers, further improving the amplification efficiency and specificity. In addition, a total of 26 cycles of amplification steps ensure sufficient amplification times to obtain sufficient PCR product amounts, thereby improving the sensitivity and accuracy of genotype detection.

[0027] The fourth technical problem to be solved by the present invention is to provide a reagent or a kit to solve the problem that the prior art lacks a convenient tool suitable for cucumber fruit top shape classification.

[0028] In order to overcome the above defects of the prior art, the present invention provides a reagent or a kit, wherein the reagent and the kit contain the KASP primers, and the reagent and the kit are used to detect the shape of cucumber fruit tops.

[0029] Compared with the prior art, the reagent or kit of the present invention has the following advantages: through the reagent or kit, the user can quickly and accurately detect SNP sites without the need for additional primer preparation and complicated operations, which greatly improves the convenience and accuracy of the typing process, greatly improves the work efficiency of SNP typing in the laboratory and breeding process, and reduces the possible operational differences in the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Phenotypic diagrams of different cucumber fruit top shapes; 1CU-X: round fruit top cucumber inbred line; 1CU-Y: pointed fruit top cucumber inbred line; 1CU-F1: intermediate fruit top cucumber; Figure 2 This is the ΔSNP-index analysis diagram of cucumber fruit top shape; Figure 3 The SNP marker typing results of different individual plants in the F2 segregation population using CsFAS primers; blue dots indicate that FAM signals were detected, which is the CC genotype, and is a homozygous round-fruit-top cucumber; red dots indicate that HEX signals were detected, which is the GG genotype, and is a homozygous pointed-fruit-top cucumber; green dots indicate that both FAM and HEX signals were detected, which is the CG genotype, and is a heterozygous cucumber with an intermediate fruit-top shape; black dots indicate NTC. DETAILED DESCRIPTION

[0031] First, those skilled in the art should understand that these implementations are only used to explain the technical principles of the embodiments of the present application, and are not intended to limit the protection scope of the embodiments of the present application. Those skilled in the art can make adjustments to them as needed to adapt to specific application scenarios.

[0032] The present invention provides a SNP molecular marker for detecting the shape of cucumber fruit top. The SNP molecular marker is a SNP site tightly linked to the shape of cucumber fruit top, which is located at a SNP mutation from C to G at position 25221828 of the Chr01 chromosome of the cucumber Chinese Long v3 genome. The SNP site is associated with the phenotype of the shape of cucumber fruit top.

[0033] like Figure 1 As shown, Figure 1 These are the phenotypes of different cucumber fruit top shapes; 1CU-X: round fruit top cucumber inbred line; 1CU-Y: pointed fruit top cucumber inbred line; 1CU-F1: intermediate fruit top cucumber.

[0034] As a preferred solution, the nucleotide sequences of the SNP molecular markers for detecting the shape of cucumber fruit tops are shown in SEQ ID NO: 1 and SEQ ID NO: 2; When the 120th position of the sequence is C, that is, SEQ ID NO: 1, the corresponding cucumber fruit top is a round fruit top; When the 120th position of the sequence is G, that is, SEQ ID NO: 2, the corresponding cucumber fruit top is a pointed fruit top.

[0035] The present invention also provides a KASP primer for detecting the shape of cucumber fruit tops, wherein the KASP primer is designed according to the SNP molecular marker, and the KASP molecular marker comprises a CsFAS primer set, whose sequence is shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and comprises: CsFAS-FAM: 5'-GAAGGTGACCAAGTTCATGCTGCCACATCATCGATTGCGACAC-3'; CsFAS-HEX: 5'-GAAGGTCGGAGTCAACGGATTGCCACATCATCGATTGCGACAG-3'; CsFAS-COMMON: 5'-GGAAGAAGAGGGGACATGTCCTT-3'.

[0036] Compared with the prior art, the KASP primer provided by the present invention has the following advantages: High specificity and accuracy: The KASP primer set of the present invention is designed based on the SNP loci that are closely linked to the cucumber fruit top shape, and can accurately detect the genotype of the cucumber fruit top shape. However, some molecular markers in the prior art may lead to inaccurate genotyping results due to the non-specificity of primer design, thereby affecting breeding decisions. The present invention ensures the specificity and reliability of the markers by strictly screening and designing primers, thereby improving the accuracy of genotyping.

[0037] Improve screening efficiency: Traditional labeling methods often require complex experimental steps and a long time to obtain results, while the KASP primer set provided by the present invention uses FAM and HEX fluorescent probes for labeling in PCR amplification, and combined with KASP technology, genotyping can be completed in a shorter time. The method of the present invention is easy to operate, less time-consuming, and suitable for large-scale breeding screening.

[0038] No need to rely on phenotypic observation: Compared with the phenotypic observation methods in the prior art, the present invention realizes direct detection of genotypes through KASP molecular markers, which is not affected by environmental factors, seasonal changes and observer differences, thereby avoiding subjective errors that may occur in phenotypic identification. The genetic information of cucumber fruit top shape can be obtained directly at the gene level, greatly improving the accuracy and efficiency of breeding selection.

[0039] Facilitates molecular assisted selection: The KASP primer set provided by the present invention can accurately mark the SNP site that controls the shape of the cucumber fruit top, thereby providing an effective tool for molecular assisted selection in cucumber breeding. Through this technology, breeders can predict the shape of the fruit top in the early stage of cucumber without waiting until the plant is fully mature, thereby accelerating the breeding process.

[0040] The present invention also provides an application of the KASP molecular marker, wherein the molecular marker is used for molecular-assisted selection of fruit top shape in cucumber breeding.

[0041] As a preferred solution, the molecular-assisted selection of fruit top shape in cucumber breeding comprises the following steps: S1: Extract genomic DNA from the cucumber sample to be tested; S2: performing PCR amplification using the KASP primers described in claim 3; S3: Detect the genotype of the SNP locus through fluorescence reading and genotyping; S4: Determine the shape of the fruit top based on the genotype: if the genotype is CC, it is a round fruit top; if the genotype is GG, it is a pointed fruit top; if the genotype is CG, it is an intermediate fruit top.

[0042] Compared with the prior art, the application of the KASP primer for detecting the shape of cucumber fruit tops in the present invention has the following advantages: Improve the efficiency and accuracy of molecular-assisted selection: Traditional fruit top shape screening methods rely on phenotypic observations, which are greatly affected by environmental conditions and human factors, and cannot accurately predict fruit top shape in the early breeding stage. The present invention uses KASP technology based on SNP markers to perform detection at the molecular level, avoiding the limitations of phenotypic observations and enabling accurate predictions at an early stage, thereby greatly improving breeding efficiency.

[0043] Simplify the breeding process: The molecular marker technology in the prior art may require complex operating procedures, while the KASP technology of the present invention can obtain genotype information in a relatively short time through fluorescent labeling and genotyping. The application of the present invention can realize high-throughput screening in breeding work, making the genotyping of cucumber fruit top shape simpler and faster.

[0044] Reducing experimental errors: Traditional phenotypic identification methods rely on manual observation, which is easily affected by the subjective judgment of the experimenter and environmental factors, and may result in errors. The KASP molecular marker technology of the present invention uses genotype detection, which has high precision and is not affected by the external environment and human intervention, thus ensuring the accuracy of the results.

[0045] Suitable for large-scale breeding screening: Since KASP technology can directly detect SNP sites at the molecular level and combine with automated equipment for efficient typing, it has strong adaptability and can support molecular-assisted selection in large-scale cucumber breeding, thereby improving overall breeding efficiency and production benefits.

[0046] As a preferred solution, in step S1, the genomic DNA is extracted by TPS method, which includes: A: Take 2g of sample from young cucumber leaves, place in a centrifuge tube filled with steel balls, and grind using a sample tissue grinder at a frequency of 60Hz for 30 seconds; B: Add 700 μL TPS solution preheated to 65°C into the ground centrifuge tube, mix thoroughly and place in a 65°C water bath for 30 minutes; C: Cool to room temperature after water bath, centrifuge at 12000 rpm for 10 minutes, then aspirate 400 μL of supernatant and transfer to a 1.5 ml centrifuge tube; D: Add 400 μL of pre-cooled isopropanol to the centrifuge tube and centrifuge again at 12,000 rpm for 10 minutes, and discard the supernatant; E: Rinse the precipitate with 75% ethanol, air-dry it, and then dissolve it in ddH2O to obtain a genomic DNA solution.

[0047] The DNA extraction method adopted by the present invention has at least the following technical advantages: Improve DNA extraction efficiency and purity: The TPS method effectively removes impurities and contaminants in the sample through sufficient cell wall fragmentation and multiple centrifugation steps, thereby obtaining high-purity genomic DNA. Compared with traditional extraction methods, the TPS method has a better DNA recovery rate and lower impurity contamination, and can provide more reliable template DNA for subsequent PCR amplification and molecular marker detection.

[0048] Ensure high-quality DNA samples: The TPS method uses high temperature and isopropanol precipitation steps, which help remove proteins and lipids in cells, thereby ensuring that the final DNA obtained is of better quality and suitable for high-precision molecular detection. The lower risk of impurities and DNA degradation makes this method more stable and reliable in long-term storage or high-throughput screening.

[0049] Easy operation and short time: By using a grinder and a single solution system, not only are multiple complex steps in the traditional method reduced, but the processing efficiency is also improved. The water bath and centrifugation steps of the above method have lower requirements for experimental operators and have good versatility.

[0050] As a preferred solution, in step S2, the primers for PCR amplification include: 12 μL of 100 μM primer CsFAS-FAM, 12 μL of 100 μM primer CsFAS-HEX, 30 μL of 100 μM primer CsFAS-COMMON, and 46 μL of ddH2O per 100 μL.

[0051] As a preferred solution, in step S2, the PCR reaction system used in the PCR amplification includes: 2.5μL, 2×KASP Master Mix; 2.5 μL template DNA; 0.07 μL KASP primer; The remaining volume of the solution was made up to the required volume with ddH2O.

[0052] As a preferred solution, in step S2, the reaction procedure of the PCR amplification includes: Step 1: 94°C, 15 minutes; Step 2: 94°C, 20 seconds; 61–55°C, -0.6°C / cycle, 60 seconds, 10 cycles in total; Step 3: 94°C, 20 seconds; 55°C, 60 seconds, 26 cycles in total; Step 4: 4℃, 60 seconds, end.

[0053] The invention also provides a reagent or a kit, wherein the reagent or the kit contains the KASP primer, and the reagent or the kit is used for detecting the shape of cucumber fruit top.

[0054] The following provides specific embodiments in combination with specific data and operations to further illustrate the above technical solution of the present invention: Example

[0055] This embodiment provides a SNP molecular marker for detecting the shape of cucumber fruit top, KASP primers and their application, which specifically include: Obtaining SNP molecular markers related to cucumber fruit top shape traits The F2 segregation population was constructed with the inbred line '1CU-X' of round-fruited cucumber and the inbred line '1CU-Y' of pointed-fruited cucumber as parents. There was obvious separation in the shape of the fruit top in the F2 segregation population. Twenty plants with extreme pointed fruit tops and round fruit tops were selected from the F2 segregation population, and an equal amount of young leaves from each plant were taken to construct the mixed pool of pointed fruit tops and round fruit tops, which were sent together with the young leaves of the parents to Beijing Novogene Technology Co., Ltd. for sequencing.

[0056] The sequencing data were filtered and quality controlled and aligned to the cucumber Chinese Long v3 ( http: / / cucurbitgenomics.org / organism / 20 ) genome, and then subjected to BSA-seq analysis using the ΔSNP-index algorithm, as Figure 2 As shown, Figure 2 This is the ΔSNP-index analysis diagram of cucumber fruit top shape. According to the analysis results of BSA-seq, the gene controlling the shape of cucumber fruit top was located in the physical interval range of 22469440-25198607bp on chromosome 1, with an interval size of 2.72 Mb. KASP primers were developed near the candidate interval based on the parental genome resequencing data. To ensure the KASP typing effect, the primer design followed the GC content of 40% to 50%, avoided special structures, and prime-blast was performed to ensure primer specificity. Bioinformatics analysis found that the SNP mutation from C to G at 25221828 on chromosome 1 was significantly associated with the shape of cucumber fruit top, and the KASP primer CsFAS was designed based on this SNP site.

[0057] The molecular marker for detecting the presence or absence of the cucumber fruit top shape is a CsFAS primer set, which is composed of single-stranded DNA named CsFAS-FAM, CsFAS-HEX and CsFAS-COMMON, and the nucleotide sequences thereof are shown in SEQ ID NOs: 3-5, specifically: CsFAS-FAM: 5'-GAAGGTGACCAAGTTCATGCTGCCACATCATCGATTGCGACAC-3'; CsFAS-HEX: 5'-GAAGGTCGGAGTCAACGGATTGCCACATCATCGATTGCGACAG-3' CsFAS-COMMON: 5'-GGAAGAAGAGGGGACATGTCCTT-3'.

[0058] This embodiment provides an application of the above molecular markers for molecular-assisted selection of fruit top shape in cucumber breeding, specifically including: Validation of SNP markers associated with cucumber fruit top shape traits: The primer set CsFAS of the KASP molecular marker of the SNP marker closely linked to the cucumber fruit top shape obtained in Example 1 was used to detect the genotype of the cucumber materials of the F2 population of 1CU-X and 1CU-Y, and the phenotype of the fruit top shape of each individual cucumber plant was counted in the field to determine the accuracy of CsFAS for molecular marker-assisted selection: The steps include: using a DNA sample of an F2 segregation group as a template, preparing a KASP system, and performing PCR amplification, fluorescence reading, and genotyping; namely, S1: extracting genomic DNA from a cucumber sample to be tested; S2: performing PCR amplification using the KASP primers described in claim 3; S3: detecting the genotype of a SNP site through fluorescence reading and genotyping; and S4: judging the shape of the fruit top according to the genotype: if the genotype is CC, the fruit top is round; if the genotype is GG, the fruit top is pointed; and if the genotype is CG, the fruit top is intermediate.

[0059] Among them, the DNA extraction in step S1 adopts the TPS method, and the specific operation includes: about 2g of young cucumber leaves are placed in a 2 ml centrifuge tube filled with steel balls and ground with a sample tissue grinder (frequency: 60Hz, time: 30s). After grinding, 700μL of TPS solution preheated to 65℃ is added to the centrifuge tube, and after sufficient mixing, it is placed in a 65℃ water bath for 30min. After cooling to room temperature in a water bath, centrifuge (speed: 12000rpm, time: 10min), and 400μL of supernatant is taken and placed in a 1.5ml centrifuge tube. 400μL of precooled isopropanol is added and centrifuged (speed: 12000rpm, time: 10min), the supernatant is discarded, rinsed with 75% ethanol, air-dried, and dissolved with ddH2O to obtain a DNA aqueous solution.

[0060] In step S2, the KASP primer preparation includes: 12 μL of 100 μM primer CsFAS-FAM, 12 μL of 100 μM primer CsFAS-HEX, 30 μL of 100 μM primer CsFAS-COMMON, and 46 μL of ddH2O.

[0061] The KASP-PCR reaction system includes: 2.5 μL of (2×KASP Master Mix, 2.5 μL of template DNA, and 0.07 μL of KASP primers.

[0062] The KASP-PCR reaction procedure includes:

[0063] After the PCR in step S2 is completed, the fluorescence reading is performed using the SNP typing detector Omega F to proceed to step S3, and the fluorescence data are clustered and genotyped using Csustercaller software.

[0064] When the cucumbers are mature, the shape of the cucumber fruit top is phenotyped in the field and compared with the results of KASP genotyping to determine the results of step S4: The test results are as follows Figure 3 As shown, the typing results show that there are 24 blue dot plants, indicating that the FAM signal was detected, which is the CC genotype, and it is a homozygous round fruit top cucumber; there are 42 green dot plants, indicating that both FAM signal and HEX signal were detected, which is the CG genotype, and it is a heterozygous intermediate fruit top cucumber; the total number of AA genotype and AG genotype plants is 59, which is consistent with the field phenotype. There are 28 red dot plants, indicating that the HEX signal was detected, which is the GG genotype, and it is a homozygous pointed fruit top cucumber, which is consistent with the field phenotype; black dots represent NTC.

[0065] The above test results prove that the SNP molecular markers of cucumber fruit top shape and the KASP primer set thereof provided by the present invention can accurately and quickly identify the genotype of cucumber fruit top shape, and realize the molecular assisted selection of fruit top shape. Through comparison with the field phenotype, the KASP molecular marker is highly correlated with the cucumber fruit top shape phenotype, which verifies the feasibility and effectiveness of the molecular marker assisted selection of fruit top shape in cucumber breeding by the present invention. Compared with the traditional phenotypic identification method, the molecular marker detection method of the present invention has higher accuracy and efficiency, and can effectively overcome the uncertainty and time cost problems in the traditional breeding method. By using the KASP molecular marker, breeders can perform accurate genotype screening at an early stage, which provides a simple and efficient tool for the innovation and improvement of cucumber breeding, and has broad application prospects.

[0066] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "in the present embodiment", "specific example", or "some examples" etc. means that the specific features, mechanisms, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, mechanisms, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0067] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A SNP molecular marker for detecting cucumber fruit top shape, characterized in that: The SNP molecular marker is a SNP site closely linked to the cucumber fruit top shape, which is located at the SNP mutation from C to G at position 25221828 of the Chr01 chromosome of the cucumber Chinese Long v3 genome. The SNP site is associated with the phenotype of the cucumber fruit top shape.

2. The SNP molecular marker according to claim 1, characterized in that: The nucleotide sequences of the SNP molecular markers for detecting the shape of cucumber fruit tops are shown in SEQ ID NO: 1 and SEQ ID NO: 2; When the 120th position of the sequence is C, that is, SEQ ID NO: 1, the corresponding cucumber fruit top is a round fruit top; When the 120th position of the sequence is G, that is, SEQ ID NO: 2, the corresponding cucumber fruit top is a pointed fruit top.

3. A KASP primer for detecting the shape of cucumber fruit top, characterized in that: The KASP primers are designed according to the SNP molecular markers according to any one of claims 1 to 2, and the KASP molecular markers include a CsFAS primer set, whose sequences are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, including: CsFAS-FAM: 5'-GAAGGTGACCAAGTTCATGCTGCCACATCATCGATTGCGACAC-3'; CsFAS-HEX: 5'-GAAGGTCGGAGTCAACGGATTGCCACATCATCGATTGCGACAG-3'; CsFAS-COMMON: 5'-GGAAGAAGAGGGGACATGTCCTT-3'.

4. An application of the KASP molecular marker according to claim 3, characterized in that: The molecular marker is used for molecular-assisted selection of fruit top shape in cucumber breeding.

5. The use of KASP molecular marker according to claim 4, characterized in that: The molecular assisted selection of fruit top shape in cucumber breeding comprises the following steps: S1: Extract genomic DNA from the cucumber sample to be tested; S2: performing PCR amplification using the KASP primers described in claim 3; S3: Detect the genotype of the SNP locus through fluorescence reading and genotyping; S4: Determine the shape of the fruit top based on the genotype: if the genotype is CC, it is a round fruit top; if the genotype is GG, it is a pointed fruit top; if the genotype is CG, it is an intermediate fruit top.

6. The use of KASP molecular marker according to claim 5, characterized in that: In step S1, the genomic DNA is extracted by TPS method, which includes: A: Take 2g of sample from young cucumber leaves, place in a centrifuge tube filled with steel balls, and grind using a sample tissue grinder at a frequency of 60Hz for 30 seconds; B: Add 700 μL TPS solution preheated to 65°C into the ground centrifuge tube, mix thoroughly and place in a 65°C water bath for 30 minutes; C: Cool to room temperature after water bath, centrifuge at 12000 rpm for 10 minutes, then aspirate 400 μL of supernatant and transfer to a 1.5 ml centrifuge tube; D: Add 400 μL of pre-cooled isopropanol to the centrifuge tube and centrifuge again at 12,000 rpm for 10 minutes, and discard the supernatant; E: Rinse the precipitate with 75% ethanol, air-dry it, and then dissolve it in ddH2O to obtain a genomic DNA solution.

7. The use of KASP molecular marker according to claim 5, characterized in that: In step S2, the primers for PCR amplification include: 12 μL of 100 μM primer CsFAS-FAM, 12 μL of 100 μM primer CsFAS-HEX, 30 μL of 100 μM primer CsFAS-COMMON, and 46 μL of ddH2O per 100 μL.

8. The use of KASP molecular marker according to claim 5, characterized in that: In step S2, the PCR reaction system used in the PCR amplification includes: 5 μL, 2× KASP Master Mix; 5 μL template DNA; 07 μL KASP primer; The remaining volume of the solution was made up to the required volume with ddH2O.

9. The use of KASP molecular marker according to claim 5, characterized in that: In step S2, the reaction procedure of the PCR amplification includes: Step 1: 94°C, 15 minutes; Step 2: 94°C, 20 seconds; 61–55°C, -0.6°C / cycle, 60 seconds, 10 cycles in total; Step 3: 94°C, 20 seconds; 55°C, 60 seconds, 26 cycles in total; Step 4: 4℃, 60 seconds, end.

10. A reagent or a kit, characterized in that: The reagent and the kit contain the KASP primers as claimed in claim 3, and the reagent and the kit are used for detecting the shape of cucumber fruit top.

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