A SNP molecular marker for detecting cucumber fruit top shape, a KASP primer and application thereof

Abstract

The application provides a SNP molecular marker for detecting cucumber fruit top shape, a KASP primer and application thereof. The SNP molecular marker is closely linked to the cucumber fruit top shape, is located at the position of 25221828 of a Chinese Long v3 genome Chr01 chromosome, and is a C to G SNP mutation, and is related to the phenotype of the cucumber fruit top shape. A KASP primer group is designed through the SNP marker, and the KASP primer group comprises CsFAS-FAM, CsFAS-HEX and CsFAS-COMMON, and the cucumber fruit top shape can be accurately identified through PCR amplification and genotyping technology. The molecular marker can be widely applied to molecular assisted selection of the fruit top shape in cucumber breeding, and has high accuracy and efficiency. In the cucumber breeding, the technology can be screened through genotyping at an early stage, avoids uncertainty and time cost problems existing in traditional phenotype identification, and provides a simple and efficient tool for the cucumber breeding. The technical method of the application is simple and rapid, can be popularized and applied in the cucumber breeding, and has important economic value and social value.

Description

Technical Field

[0001] This invention relates to the field of biological identification technology, and more specifically, to an SNP molecular marker for detecting the shape of cucumber fruit tops, a KASP primer, and their applications. Background Technology

[0002] cucumber( Cucumis sativus L. Cucumbers are one of the most widely cultivated vegetable crops globally, and the appearance and quality of their fruits directly affect market demand and commercial value. Among the appearance traits of cucumbers, the shape of the fruit apex is a key characteristic. Different apex shapes (such as round or pointed) not only affect the commercial value of cucumbers but also directly influence consumer purchasing preferences. Therefore, accurate identification of the fruit apex shape plays a crucial role in cucumber breeding.

[0003] Currently, the identification of cucumber fruit apex shape mainly relies on phenotypic observation in the field. This method is not only time-consuming and labor-intensive, but also easily affected by environmental factors, thus limiting the accuracy and stability of the identification results. In addition, phenotypic observation usually requires a long growth cycle to determine trait expression, therefore, the breeding process is often slow and inefficient.

[0004] With the development of molecular biology techniques, marker-assisted selection (MAS) technology has provided a new approach to improve the accurate identification of cucumber fruit apex shape. Currently, commonly used molecular marker methods mainly include Indel markers. These markers require electrophoretic analysis after PCR amplification, a cumbersome and time-consuming process. Furthermore, due to limitations in electrophoresis technology, the throughput is low, and the operation is complex and costly. Although Indel markers can improve the efficiency of molecular breeding to some extent, their cumbersome operation makes it difficult to meet the demands of modern agricultural production for efficient and rapid breeding, especially in large-scale screening.

[0005] Therefore, existing molecular marker methods have obvious defects and shortcomings, including high cost, low throughput, and long cycle, which make it impossible to achieve rapid, accurate, and efficient screening of cucumber fruit top shape. This restricts the improvement of cucumber varieties and the enhancement of breeding efficiency.

[0006] Against this backdrop, developing a novel molecular marker method capable of rapidly and accurately identifying the shape of cucumber fruit apex has become crucial for solving existing technological problems. This method should be able to accurately identify the shape of cucumber fruit apex at the seedling stage and possess advantages such as high throughput, low cost, and ease of operation, thereby promoting the improvement of cucumber breeding efficiency. Summary of the Invention

[0007] The first technical problem solved by the present application 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 large time consumption in the prior art.

[0008] To overcome the defects of the prior art, the present application provides a SNP molecular marker for detecting the shape of cucumber fruit top, which is a SNP site closely linked to the shape of cucumber fruit top, located at the SNP mutation of C to G at the position of 25221828 of the Chinese Long v3 genome Chr01 chromosome of cucumber, and the SNP site is related to the phenotype of the shape of cucumber fruit top.

[0009] Compared with the prior art, the SNP molecular marker for detecting the shape of cucumber fruit top of the present application has the following advantages: improved accuracy and efficiency: the fruit top shape phenotype screening method in the prior art relies on manual observation, and has high error and subjectivity. The SNP molecular marker provided by the present application is closely linked to the fruit top shape, can accurately detect the genotype of the fruit top shape of cucumber at the molecular level, avoids phenotype error, and greatly improves the accuracy and efficiency of screening; simplifies the breeding process: the traditional fruit top shape screening method often needs long time field observation and a large amount of manual labor, and the result is difficult to confirm in early stage. The SNP molecular marker of the present application can be rapidly detected by PCR technology in the early growth stage of cucumber, greatly shortens the breeding period, and reduces the labor input; can be applied to large-scale breeding: compared with traditional phenotype screening, the SNP molecular marker provided by the present application can be used for high-throughput molecular marker detection, is suitable for application in large-scale breeding, can rapidly screen individuals meeting the target fruit top shape, and greatly improves the overall efficiency of breeding work; has precise molecular assisted selection ability: the SNP molecular marker provided by the present application can not only be used for genetic research of the shape of cucumber fruit top, but also be widely applied in cucumber breeding, as an important tool for molecular assisted selection, and realizes precise control of variety traits.

[0010] In a possible implementation, the nucleotide sequence of the SNP molecular marker for detecting the shape of cucumber fruit top is as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0011] When the 120th position of the sequence is C, i.e. SEQ ID NO:1, the corresponding cucumber fruit top is round fruit top;

[0012] When the 120th position of the sequence is G, i.e. SEQ ID NO:2, the corresponding cucumber fruit top is pointed fruit top.

[0013] Compared with the prior art, by adopting the 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 judged by detecting the specific SNP site (C to G mutation), and the C or G mutation of the SNP site directly corresponds to the shape (round fruit top or pointed fruit top) of the cucumber fruit top. The molecular marker is closely related to the phenotype of the fruit top shape, and the grouping information of the SNP site can be used to accurately infer the morphology of the cucumber fruit top without relying on environmental factors and manual intervention, thereby significantly improving the accuracy and efficiency of the cucumber fruit top shape screening, and making the breeding process more efficient and controllable. The technology provides a more advanced molecular assisted selection tool for cucumber breeding, which helps to optimize the breeding strategy and cultivate more cucumber varieties meeting market demand.

[0014] The second technical problem to be solved by the present application is to provide a KASP primer for detecting the shape of the cucumber fruit top, so as to solve the problem of inaccurate marker design and inability to efficiently realize molecular assisted selection of the shape of the cucumber fruit top in the prior art.

[0015] In order to overcome the defects of the prior art, the present application provides a KASP primer for detecting the shape of the cucumber fruit top, wherein the KASP primer is designed according to the SNP molecular marker, and the KASP molecular marker comprises a CsFAS primer group, the sequence of which is shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and comprises:

[0016] CsFAS-FAM: 5'-GAAGGTGACCAAGTTCATGCTGCCACATCATCGATTGCGACAC-3';

[0017] CsFAS-HEX: 5'-GAAGGTCGGAGTCAACGGATTGCCACATCATCGATTGCGACAG-3';

[0018] CsFAS-COMMON: 5'-GGAAGAAGAGGGGACATGTCCTT-3'.

[0019] Compared with the prior art, the KASP primer for detecting the cucumber fruit top shape has the following advantages: the KASP primer of the present application detects the genotype of the SNP site by designing specific primers (FAM, HEX, COMMON). These primers can specifically recognize the SNP mutation (C to G) in the cucumber genome, and quickly and accurately obtain the genetic information of the cucumber fruit top shape through PCR amplification and fluorescence detection method, providing an efficient and accurate tool for molecular assisted breeding, avoiding the limitations of phenotype identification in traditional technology, and the KASP primer provided by the present application can significantly improve the detection accuracy and efficiency of the cucumber fruit top shape; through the high-specificity and high-efficiency KASP molecular marker technology, breeders can accurately screen cucumber varieties with target fruit top shape, thereby accelerating the breeding process and improving the overall efficiency and quality of cucumber breeding. In addition, the molecular assisted selection based on the technology can eliminate the errors caused by environmental factors and manual operation in phenotype selection, so that the genetic improvement of the cucumber fruit top shape is more accurate and controllable.

[0020] The third technical problem to be solved by the present application is the 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.

[0021] To overcome the defects of the above prior art, the present application provides the application of the KASP molecular marker, which is used for fruit top shape molecular assisted selection in cucumber breeding.

[0022] In a possible implementation, the fruit top shape molecular assisted selection in cucumber breeding includes the following steps:

[0023] S1: extracting genomic DNA in the cucumber sample to be tested;

[0024] S2: using the KASP primer of claim 3 for PCR amplification;

[0025] S3: detecting the genotype of the SNP site by fluorescence reading and genotyping;

[0026] S4: judging the fruit top shape according to the genotype: if the genotype is CC, it is round fruit top; if the genotype is GG, it is sharp fruit top; if the genotype is CG, it is intermediate fruit top.

[0027] Compared with the prior art, the application of the KASP primer for detecting the shape of the cucumber fruit top has the following advantages: the application is based on the SNP molecular marker of the shape of the cucumber fruit top, and the marker is rapidly detected through the KASP technology, and the KASP technology detects the genotype by using FAM and HEX fluorescent probes, and in combination with the genotyping data, the shape of the cucumber fruit top can be accurately judged, the time required for phenotype screening is reduced, the interference of the external environment on the evaluation of the shape of the fruit top is avoided, and the accuracy of molecular selection is ensured; the application of the KASP molecular marker provided in the application provides a precise, rapid and efficient solution for the molecular assisted selection of the shape of the cucumber fruit top in cucumber breeding, through the genotyping detection, breeders can make accurate breeding decisions at an early stage, the uncertainty caused by phenotype observation in the traditional method is avoided, and therefore the success rate and efficiency of breeding are greatly improved.

[0028] In a possible implementation, in the step S1, the genomic DNA is extracted by using a TPS method, and the extraction process includes the following steps:

[0029] A: 2g of a new cucumber leaf is sampled, placed in a centrifuge tube containing steel balls, and ground by using a sample tissue grinder at a frequency of 60Hz for 30 seconds;

[0030] B: 700ul of TPS solution preheated to 65 DEG C is added to the ground centrifuge tube, and after being fully mixed, the centrifuge tube is treated in a 65 DEG C water bath for 30 minutes;

[0031] C: After water bath, the centrifuge tube is cooled to room temperature, centrifuged at a speed of 12000rpm for 10 minutes, and then 400ul of supernatant is taken and transferred to a 1.5ml centrifuge tube;

[0032] D: 400ul of pre-cooled isopropanol is added to the centrifuge tube, and the centrifuge tube is centrifuged again at a speed of 12000rpm for 10 minutes, and the supernatant is discarded;

[0033] E: The precipitate is rinsed with 75% ethanol, dried in air, and then dissolved with ddH2O to obtain a genomic DNA solution.

[0034] Compared with the prior art, by using the above technical scheme, high-quality cucumber genomic DNA can be obtained in a short time by using the TPS method to extract DNA. 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 thus improves the effect of fruit top shape molecular assisted selection in cucumber breeding, effectively overcomes the problems of low efficiency and low purity in the existing DNA extraction method, and ultimately enables the present application to more accurately perform molecular marker detection, improves the accuracy and efficiency of breeding work, and meets the requirements of modern breeding for high efficiency, simplicity and speed.

[0035] In one possible implementation, in the step S2, the primers for PCR amplification include, per 100 μL: 12 μL of primer CsFAS-FAM at a concentration of 100 μM, 12 μL of primer CsFAS-HEX at a concentration of 100 μM, 30 μL of primer CsFAS-COMMON at a concentration of 100 μM, and 46 μL of ddH2O.

[0036] Compared with the prior art, by using the above technical scheme, the CsFAS primer set (including CsFAS-FAM, CsFAS-HEX and CsFAS-COMMON) can be used for efficient amplification of specific SNP sites, and multiplex detection can be achieved through fluorescent labeling (FAM and HEX dyes), ensuring the specificity of PCR amplification and enabling each sample to accurately detect the genotype related to cucumber fruit top shape, thereby improving the sensitivity and accuracy of detection. By setting the appropriate concentration of primers (12 μL of CsFAS-FAM and 12 μL of CsFAS-HEX), and a specific amount of CsFAS-COMMON primer (30 μL at 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 and DNA template), effectively enhancing the sensitivity of PCR reaction, enabling efficient genotyping under low DNA content, and avoiding non-specific amplification.

[0037] In one possible implementation, in the step S2, the PCR reaction system used for PCR amplification includes:

[0038] 2.5 μL of 2×KASP Master Mix;

[0039] 2.5 μL of template DNA;

[0040] 0.07 μL of KASP primer;

[0041] The remaining solution volume is supplemented with ddH2O to the required volume.

[0042] Compared with the prior art, by adopting the technical scheme, through setting the primers with appropriate concentration (12 muL CsFAS-FAM and 12 muL CsFAS-HEX) and the specific amount of CsFAS-COMMON primer (30 muL with a concentration of 100 muM), the balance and stability of the PCR reaction system can be optimized, the concentration of each primer is accurately matched with other components (such as KASP MasterMix and DNA template) in the PCR reaction system, the reaction condition is more stable, the sensitivity of the PCR reaction is significantly enhanced, and then the efficiency of the PCR amplification can be effectively improved, especially in the case that the DNA template concentration is low, the high typing accuracy can still be maintained, through optimizing the primer configuration, the occurrence of non-specific amplification phenomenon is avoided, and it is ensured that only the target SNP site is amplified, so that the accuracy of the genotype analysis is improved.

[0043] In a possible implementation, the reaction procedure of the PCR amplification in the step S2 comprises:

[0044] Step one: 94 DEG C, 15 minutes;

[0045] Step two: 94 DEG C, 20 seconds; 61-55 DEG C, -0.6 DEG C / cycle, 60 seconds, for 10 cycles;

[0046] Step three: 94 DEG C, 20 seconds; 55 DEG C, 60 seconds, for 26 cycles;

[0047] Step four: 4 DEG C, 60 seconds, end.

[0048] Compared with the prior art, by adopting the technical scheme, the SNP typing of the cucumber fruit top shape can be completed under precise temperature control, the 94 DEG C preheating treatment (15 minutes) of step one helps the denaturation of DNA, and ensures that the template DNA is completely denatured, which provides a stable basis for subsequent PCR amplification, step two optimizes the binding conditions of the primers and the template by setting the temperature reduction program (61 DEG C to 55 DEG C, -0.6 DEG C / cycle), and enough annealing time (60 seconds) is maintained, so that the primers can be accurately combined with the target sequence, thereby improving the amplification specificity; and the 94 DEG C short-term treatment (20 seconds) of step three ensures that the DNA is completely denatured again, and the annealing temperature of 55 DEG C provides the best conditions for the combination of the primers, so that the amplification efficiency and specificity are further improved, in addition, the amplification step of 26 cycles ensures enough amplification times to obtain enough PCR product amount, thereby improving the sensitivity and accuracy of the genotype detection.

[0049] A fourth technical problem to be solved by the present application is to provide a reagent or kit to solve the problem that there is a lack of a convenient tool suitable for cucumber fruit top shape typing in the prior art.

[0050] To overcome the defects of the prior art, the present application provides a reagent or kit containing the KASP primer for detecting cucumber fruit top shape.

[0051] Compared with the prior art, the reagent or kit of the present application has the following advantages: through the reagent or kit, the user can quickly and accurately detect the SNP site without additional primer preparation and complex operation, greatly improving the convenience and accuracy of the typing process, greatly improving the work efficiency of SNP typing in the laboratory and breeding process, and reducing the possible operation differences in the experiment. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 Figure 1 is a phenotype diagram 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;

[0053] Figure 2 Figure 2 is a ΔSNP-index analysis diagram of cucumber fruit top shape;

[0054] Figure 3 Figure 3 is the SNP marker typing of different single plants in the F2 separation population by CsFAS primer; the blue point indicates that the FAM signal is detected, which is the CC genotype, and is a homozygous round fruit top shape cucumber; the red point indicates that the HEX signal is detected, which is the GG genotype, and is a homozygous pointed fruit top shape cucumber; the green point indicates that the FAM signal and the HEX signal are detected at the same time, which is the CG genotype, and is a heterozygous intermediate fruit top shape cucumber; the black point is NTC. DETAILED DESCRIPTION

[0055] First of all, those skilled in the art should understand that these embodiments 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 adjust them as needed in order to adapt to specific application occasions.

[0056] The present application provides a SNP molecular marker for detecting cucumber fruit top shape, which is a SNP site closely linked to cucumber fruit top shape, located at the SNP mutation of C to G at the position of 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.

[0057] As Figure 1 shown, Figure 1Figure 1 is a phenotype diagram 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.

[0058] As a preferred solution, the nucleotide sequence of the SNP molecular marker for detecting cucumber fruit top shape is as shown in SEQ ID NO: 1, SEQ ID NO: 2;

[0059] When the 120th position of the sequence is C, i.e. SEQ ID NO: 1, the corresponding cucumber fruit top is round fruit top;

[0060] When the 120th position of the sequence is G, i.e. SEQ ID NO: 2, the corresponding cucumber fruit top is pointed fruit top.

[0061] The application also provides a KASP primer for detecting cucumber fruit top shape, wherein the KASP primer is designed according to the SNP molecular marker, and the KASP molecular marker comprises a CsFAS primer group, and the sequences of the CsFAS primer group are as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and the CsFAS primer group comprises:

[0062] CsFAS-FAM: 5'-GAAGGTGACCAAGTTCATGCTGCCACATCATCGATTGCGACAC-3';

[0063] CsFAS-HEX: 5'-GAAGGTCGGAGTCAACGGATTGCCACATCATCGATTGCGACAG-3';

[0064] CsFAS-COMMON: 5'-GGAAGAAGAGGGGACATGTCCTT-3'.

[0065] Compared with the prior art, the KASP primer provided by the application has the following advantages:

[0066] High specificity and accuracy: the KASP primer group of the application is designed based on the SNP site closely linked to cucumber fruit top shape, and can accurately detect the genotype of cucumber fruit top shape, while some molecular markers in the prior art may be non-specific due to the design of the primer, resulting in inaccurate genotyping results, which in turn affects the breeding decision. The application strictly screens and designs the primer to ensure the specificity and reliability of the marker, thereby improving the accuracy of genotyping.

[0067] Improve screening efficiency: the traditional marker method often needs complex experimental steps and a long time to obtain results, while the KASP primer set provided by the application uses FAM and HEX fluorescent probes for labeling in PCR amplification, combined with KASP technology to complete genotyping in a shorter time, the method of the application is simple to operate, consumes less time, and is suitable for large-scale breeding screening.

[0068] No need to rely on phenotype observation: compared with the phenotype observation method in the prior art, the application realizes direct detection of genotype through KASP molecular markers, is not affected by environmental factors, seasonal changes and observer differences, thereby avoiding subjective errors that may occur in phenotype identification, and the genetic information of cucumber fruit top shape can be directly obtained from the gene level, greatly improving the accuracy and efficiency of breeding selection.

[0069] Convenient molecular assisted selection: the KASP primer set provided by the application can accurately mark the SNP site controlling the shape of the cucumber fruit top, thereby providing an effective tool for molecular assisted selection in cucumber breeding, and through this technology, breeders can predict the fruit top shape at an early stage of cucumber, without waiting for the plant to mature completely, thereby accelerating the breeding process.

[0070] The application also provides an application of the KASP molecular marker, which is used for molecular assisted selection of fruit top shape in cucumber breeding.

[0071] As a preferred scheme, the molecular assisted selection of fruit top shape in cucumber breeding comprises the following steps:

[0072] S1: extracting genomic DNA in the cucumber sample to be tested;

[0073] S2: using the KASP primer of claim 3 for PCR amplification;

[0074] S3: detecting the genotype of the SNP site by fluorescence reading and genotyping;

[0075] S4: judging the fruit top shape according to the genotype: if the genotype is CC, it is a round fruit top; if the genotype is GG, it is a pointed fruit top; and if the genotype is CG, it is an intermediate fruit top.

[0076] Compared with the prior art, the application of the KASP primer for detecting the shape of the cucumber fruit top has the following advantages:

[0077] Improve the efficiency and accuracy of molecular assisted selection: Traditional fruit top shape screening methods rely on phenotypic observation, which is greatly influenced by environmental conditions and human factors, and cannot accurately predict fruit top shape at early breeding stages. The present application uses KASP technology based on SNP markers to detect at the molecular level, avoiding the limitations of phenotypic observation and enabling accurate prediction at an early stage, thereby greatly improving breeding efficiency.

[0078] Simplify the breeding process: The molecular marker technology in the prior art may require a complex operation process, while the KASP technology of the present application can obtain genotype information in a short time through fluorescence labeling and genotyping. The application of the present application can realize high-throughput screening in breeding work, making the genotyping of cucumber fruit top shape more convenient and fast.

[0079] Reduce experimental errors: Traditional phenotypic identification methods rely on manual observation and are easily influenced by subjective judgment of the experimenter and environmental factors, which may result in errors. The KASP molecular marker technology of the present application uses genotype detection, which has high precision and is not affected by external environment and human intervention, ensuring the accuracy of the results.

[0080] Suitable for large-scale breeding screening: KASP technology can directly detect SNP sites at the molecular level and combine with automated equipment for efficient genotyping, which has strong adaptability and can support molecular assisted selection in large-scale cucumber breeding, improving the overall breeding efficiency and production benefit.

[0081] As a preferred scheme, in the step S1, the extraction method of the genomic DNA is TPS method, which includes:

[0082] A: 2g of young cucumber new leaves were taken and placed in a centrifuge tube containing steel balls, and a sample tissue grinder was used for grinding at a frequency of 60Hz for 30 seconds;

[0083] B: 700μL of TPS solution preheated to 65℃ was added to the ground centrifuge tube, mixed thoroughly, and then treated in a 65℃ water bath for 30 minutes;

[0084] C: After water bath cooling to room temperature, centrifugation was performed at 12000rpm for 10 minutes, then 400μL of supernatant was aspirated and transferred to a 1.5ml centrifuge tube;

[0085] D: 400μL of pre-cooled isopropanol was added to the centrifuge tube and centrifuged again at 12000rpm for 10 minutes, and the supernatant was discarded;

[0086] E: The precipitate was rinsed with 75% ethanol, dried in air, and then dissolved with ddH2O to obtain a genomic DNA solution.

[0087] The DNA extraction method adopted by the present application has at least the following technical advantages:

[0088] Improve DNA extraction efficiency and purity: TPS method effectively removes impurities and contaminants in the sample through sufficient cell wall disruption and multiple centrifugation steps, thereby obtaining high-purity genomic DNA. Compared with traditional extraction methods, TPS method has better DNA recovery rate and lower impurity pollution, which can provide more reliable template DNA for subsequent PCR amplification and molecular marker detection.

[0089] Ensure high-quality DNA samples: TPS method uses high temperature and isopropanol precipitation steps, which help remove intracellular proteins and lipids, thereby ensuring better quality of the final DNA, suitable for high-precision molecular detection. Lower impurities and DNA degradation risk make the method more stable and reliable in long-term storage or high-throughput screening.

[0090] Simple operation and short time: By using a grinder and a single solution system, the number of complex steps in traditional methods is reduced, and the efficiency of processing is improved. The water bath and centrifugation steps of the above method have lower requirements for experimental operators and have good universality.

[0091] As a preferred solution, in the step S2, the primers for PCR amplification include, per 100 μL: 12 μL of primer CsFAS-FAM with a concentration of 100 μM, 12 μL of primer CsFAS-HEX with a concentration of 100 μM, 30 μL of primer CsFAS-COMMON with a concentration of 100 μM, and 46 μL of ddH2O.

[0092] As a preferred solution, in the step S2, the PCR reaction system used for PCR amplification includes:

[0093] 2.5 μL, 2×KASP Master Mix;

[0094] 2.5 μL of template DNA;

[0095] 0.07 μL of KASP primer;

[0096] The remaining solution volume is supplemented with ddH2O to the required volume.

[0097] As a preferred solution, in the step S2, the reaction program of the PCR amplification includes:

[0098] Step one: 94℃, 15 minutes;

[0099] Step two: 94°C, 20 seconds; 61-55°C, -0.6°C / cycle, 60 seconds, for a total of 10 cycles;

[0100] Step three: 94°C, 20 seconds; 55°C, 60 seconds, for a total of 26 cycles;

[0101] Step four: 4°C, 60 seconds, end.

[0102] The application also provides a reagent or kit containing the KASP primer for detecting the fruit top shape of cucumber.

[0103] The following provides specific examples in combination with specific data and operations to further explain the above technical solutions of the application: Embodiment

[0104] The embodiment provides a SNP molecular marker, a KASP primer and application thereof for detecting the fruit top shape of cucumber, and specifically comprises the following steps:

[0105] Obtaining of the SNP molecular marker related to the fruit top shape of cucumber

[0106] The F2 separation population is constructed by taking the round fruit top cucumber inbred line '1CU-X' and the pointed fruit top cucumber inbred line '1CU-Y' as parents. The fruit top shape is obviously separated in the F2 separation population. 20 single plants of extreme pointed fruit top and round fruit top are selected from the F2 separation population, and an equal amount of tender leaves of each plant is taken to construct a pointed fruit top pool and a round fruit top pool, which are sent to Beijing Nuowoziyuan Technology Co., Ltd. together with the tender leaves of the parents for sequencing.

[0107] After the sequencing data is filtered and controlled, the sequencing data is compared to the cucumber Chinese Long v3 (http: / / cucurbitgenomics.org / organism / 20) genome, and then BSA-seq analysis is performed, and ΔSNP-index algorithm is used for analysis, as shown in Figure 2 Figure 2 ​The ΔSNP-index analysis graph of cucumber fruit top shape is shown in Figure 1. According to the analysis results of BSA-seq, the gene controlling cucumber fruit top shape is located in the physical interval of 22469440 - 25198607 bp on chromosome 1, with an interval size of 2.72 Mb. KASP primers are developed near the candidate interval according to the resequencing data of the parent genomes. In order to ensure the KASP typing effect, the primer design follows the GC content of 40% to 50%, and avoids special structures, and prime-blast is performed to ensure the specificity of the primer. Bioinformatics analysis found that the SNP mutation of C to G at 25221828 on chromosome 1 is significantly related to cucumber fruit top shape, and KASP primer CsFAS is designed according to the SNP site.

[0108] The molecular marker for detecting the presence or absence of cucumber fruit top shape is the CsFAS primer set, which consists of single-stranded DNA with names CsFAS-FAM, CsFAS-HEX and CsFAS-COMMON, respectively, and the nucleotide sequences thereof are shown in SEQ ID NO: 3-5, specifically as follows:

[0109] CsFAS-FAM: 5'-GAAGGTGACCAAGTTCATGCTGCCACATCATCGATTGCGACAC-3';

[0110] CsFAS-HEX: 5'-GAAGGTCGGAGTCAACGGATTGCCACATCATCGATTGCGACAG-3'

[0111] CsFAS-COMMON: 5'-GGAAGAAGAGGGGACATGTCCTT-3'.

[0112] The application provides the use of the above-mentioned molecular marker for molecular assisted selection of fruit top shape in cucumber breeding, specifically including:

[0113] Verification of SNP markers related to cucumber fruit top shape traits:

[0114] The KASP molecular marker primer set CsFAS obtained in Example 1 is used to detect the genotype of cucumber materials in the F2 population of 1CU-X and 1CU-Y, and the phenotype of cucumber fruit top shape of each single cucumber is statistically analyzed in the field to determine the accuracy of CsFAS for molecular marker assisted selection:

[0115] The steps include: using the DNA sample of the F2 separation population as a template, preparing a KASP system, and performing PCR amplification and fluorescence reading, genotyping; namely S1: extracting genomic DNA in the cucumber sample to be tested; S2: using the KASP primer in claim 3 to perform PCR amplification; S3: detecting the genotype of the SNP site by fluorescence reading and genotyping; S4: judging the fruit top shape according to the genotype: if the genotype is CC, it is a round fruit top; if the genotype is GG, it is a pointed fruit top; and if the genotype is CG, it is an intermediate fruit top.

[0116] In step S1, the DNA extraction adopts the TPS method, and the specific operation includes: about 2 g of young cucumber new leaves are placed in a 2 ml centrifuge tube containing steel balls, and are ground by a sample tissue grinder (frequency: 60 Hz, time: 30 s). After grinding, 700 μL of TPS solution preheated at 65℃ is added to the centrifuge tube, and after being fully mixed, it is placed in a 65℃ water bath for 30 min. After the water bath is cooled to room temperature, centrifugation is performed (speed: 12000 rpm, time: 10 min), and 400 μL of supernatant is taken and placed in a 1.5 ml centrifuge tube. 400 μL of pre-cooled isopropanol is added and centrifuged (speed: 12000 rpm, time: 10 min), and the supernatant is discarded. After rinsing with 75% ethanol and air drying, the DNA aqueous solution is obtained by dissolving with ddH2O.

[0117] In step S2, the KASP primer preparation includes: 12 μL of primer CsFAS-FAM with a concentration of 100 μM, 12 μL of primer CsFAS-HEX with a concentration of 100 μM, 30 μL of primer CsFAS-COMMON with a concentration of 100 μM, and 46 μL of ddH2O.

[0118] The KASP-PCR reaction system includes: 2.5 μL of (2×KASP Master Mix, 2.5 μL of template DNA, 0.07 μL of KASP primer.

[0119] The KASP-PCR reaction program includes:

[0120]

[0121] After the PCR in step S2 is completed, step S3 is performed by using the SNP typing detector Omega F to perform fluorescence reading, and the Csustercaller software is used to cluster and genotype the fluorescence data.

[0122] In step S4, the results are determined by comparing the results of the KASP genotyping with the phenotype identification of the cucumber fruit top shape in the field when the cucumber matures.

[0123] The results of the detection are as follows Figure 3As shown, the typing results show that there are 24 blue dot single plants, indicating that FAM signal is detected, which is CC genotype, and is a homozygous round fruit top shape cucumber; there are 42 green dot single plants, indicating that FAM signal and HEX signal are detected simultaneously, which is CG genotype, and is a heterozygous intermediate fruit top shape cucumber; there are 28 red dot single plants, indicating that HEX signal is detected, which is GG genotype, and is a homozygous sharp fruit top shape cucumber, which is consistent with the field phenotype; the black dot represents NTC.

[0124] The above detection results prove that the SNP molecular marker and the KASP primer set provided by the application for the fruit top shape of cucumber can accurately and quickly genotype the fruit top shape of cucumber, and realize the molecular assisted selection of the fruit top shape. By comparing with the field phenotype, the KASP molecular marker is highly related to the phenotype of the fruit top shape of cucumber, which verifies the feasibility and effectiveness of the application in the molecular marker assisted selection of the fruit top shape in cucumber breeding. Compared with the traditional phenotype identification method, the molecular marker detection method of the application has higher accuracy and efficiency, and can effectively overcome the uncertainty and time cost problem in the traditional breeding method. By using the KASP molecular marker, the breeder can accurately genotype at an early stage, which provides a simple and efficient tool for the innovation and improvement of cucumber breeding, and has a wide application prospect.

[0125] In the description of the present application, the description of the terms "one embodiment", "some embodiments", "in this embodiment", "specific examples" or "some examples" means that the specific features, mechanisms, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, mechanisms, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0126] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any skilled person in the art can easily think of changes or replacements within the technical scope disclosed in the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A KASP primer for detecting the shape of the fruit apex of Cucumis sativus, characterized in that, The KASP primer comprises a CsFAS primer group, and the sequence is shown as SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:

5.

2. Use of a KASP primer according to claim 1, characterized in that, The primer is used for molecular assisted selection of fruit top shape in cucumber breeding.

3. Use of a KASP primer according to claim 2, wherein, The molecular assisted selection of fruit top shape in cucumber breeding comprises the following steps: S1: extracting genomic DNA in a to-be-tested cucumber sample; S2: performing PCR amplification by using the KASP primer in claim 3; S3: detecting the genotype of the SNP site by fluorescence reading and genotyping; S4: judging the fruit top shape according to the genotype: if the genotype is CC, it is a round fruit top; if the genotype is GG, it is a pointed fruit top; and if the genotype is CG, it is an intermediate fruit top.

4. Use of a KASP primer according to claim 3, wherein, In the step S1, the genomic DNA is extracted by using a TPS method, which comprises the following steps: A: 2g of a young cucumber new leaf is sampled and placed in a centrifuge tube containing steel balls, and a sample tissue grinder is used for grinding at a frequency of 60Hz for 30 seconds; B: 700μL of TPS solution preheated to 65℃ is added to the centrifuge tube after grinding, and after being fully mixed, it is treated in a 65℃ water bath for 30 minutes; C: After water bath cooling to room temperature, centrifugation is performed at a speed of 12000rpm for 10 minutes, and then 400μL of supernatant is aspirated and transferred to a 1.5ml centrifuge tube; D: 400μL of pre-cooled isopropanol is added to the centrifuge tube, and centrifugation is performed again at a speed of 12000rpm for 10 minutes, and the supernatant is discarded; E: The precipitate is rinsed with 75% ethanol, and after air drying, it is dissolved with ddH2O to obtain a genomic DNA solution.

5. Use of a KASP primer according to claim 3, wherein, In the step S2, the primer for PCR amplification comprises, per 100μL: 12μL of primer CsFAS-FAM with a concentration of 100μM, 12μL of primer CsFAS-HEX with a concentration of 100μM, 30μL of primer CsFAS-COMMON with a concentration of 100μM, and 46μL of ddH2O.

6. Use of a KASP primer according to claim 3, wherein, In the step S2, the PCR reaction system used in the PCR amplification comprises: 2.5μL, 2×KASP Master Mix; 2.5μL of template DNA; 0.07μL of KASP primer; The remaining solution volume is supplemented with ddH2O to the required volume.

7. Use of a KASP primer according to claim 5, wherein, In the step S2, the reaction program of the PCR amplification comprises: Step one: 94℃, 15 minutes; Step two: 94℃, 20 seconds; 61–55℃, -0.6℃ / cycle, 60 seconds, for a total of 10 cycles; Step three: 94℃, 20 seconds; 55℃, 60 seconds, for a total of 26 cycles; Step four: 4℃, 60 seconds, end.

8. An agent, characterized in that, The reagent contains the KASP primer in claim 1, and the reagent is used for detecting the fruit top shape of cucumber.

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