A primer pair and kit for identifying or assisting in identifying the hardness of watermelon peel and application thereof

Abstract

The application belongs to the technical field of molecular marker assisted breeding, and particularly relates to a primer pair and a kit for identifying or assisting in identifying watermelon pericarp hardness and application. The application provides a primer pair for identifying or assisting in identifying watermelon pericarp hardness, wherein the primer pair comprises an upstream primer with a nucleotide sequence as shown in SEQ ID No. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 2. The primer pair designed according to the InDel-P159 molecular marker can be used to accurately and rapidly identify the watermelon pericarp hardness trait.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular marker assisted breeding, and particularly relates to a primer pair and kit for identifying or assisting in identifying watermelon fruit peel hardness and application thereof. BACKGROUND

[0002] Watermelon (Citrullus lanatus) is one of the important economic crops, and China is the first country in watermelon production and consumption. The analysis of the market and the development trend of the watermelon industry shows that the consumption of watermelon in China shows a fluctuating upward trend. With the increase of market demand, the demand for diversification and differentiation of watermelon varieties also increases accordingly, and the preference for high quality and storage and transportation resistance is also increasing. Cracking is one of the important factors that reduce the commodity nature of watermelon. Some scholars have carried out molecular level research on cracking resistance genes. For example, Liao et al. (Ethylene-responsive factor 4 is associated with the desirable rind hardness trait conferring cracking resistance in fresh fruits of watermelon, Plant Biotechnology, Journal, 2020, 18: 1066-1077) used high-throughput sequencing method and KASP molecular marker to finely locate the cracking resistance gene of watermelon in the 9732 bp (2,673,328-2,682,700 bp) interval of chromosome 10, and found that the ClERF4 gene in the interval may be related to the cracking resistance of watermelon. Based on the sequence difference of ClERF4 gene, KASP marker was developed, which can genotype 104 germplasm resources, providing technical support for cracking resistance material screening.

[0003] Studies have shown that fruit skin hardness is highly related to cracking resistance, and is the main internal factor of fruit storage resistance (Zhao Zunlian and Wang Ming, Mechanism of fruit storage resistance of watermelon germplasm [J], Acta Horticulturae, 1994, (01): 99-100). Through the improvement of fruit skin hardness traits, it is of great significance to create new watermelon varieties with high quality, high hardness, cracking resistance and storage. At present, the research on watermelon fruit skin hardness mainly focuses on the physiological and biochemical and cellular levels, and it is found that watermelon fruit skin hardness is determined by many factors such as fruit skin cell structure and content of inclusions (Wang Xuezhen et al., Analysis of watermelon fruit skin hardness related traits [J], Journal of Northeast Agricultural University, 2020, 51(02): 35-44). Fruit skin characteristics are mainly determined by fruit skin thickness, cuticle thickness, outer fruit skin cell layer number and stone cell group size (Man Yanping and Zhang Jiannong, Differences in fruit skin microstructure of different storage and transportation watermelons [J]. Journal of Gansu Agricultural University, 2006, (04): 64-67.). Pectin, cellulose and hemicellulose are the main components of cell wall material (Gao Lei, Transcriptional analysis and fine mapping of main genes for watermelon fruit hardness and sourness traits [D]. Chinese Academy of Agricultural Sciences, 2018). Among them, fruit skin thickness, fruit skin toughness and hemicellulose content are closely related to fruit skin hardness (Wang Xuezhen et al., Analysis of watermelon fruit skin hardness related traits, Journal of Northeast Agricultural University [J]. 2020, 51(02): 35-44). The fruit skin hardness of different watermelon varieties is quite different in fruit skin cell structure and content of inclusions. Zhang Jingjing et al. (Zhang Jingjing et al., Transcriptome sequencing and related gene expression analysis of watermelon fruit skin with different hardness [J]. North China Journal of Agricultural Sciences, 2022, 37(03): 44-52.) performed transcriptome sequencing analysis on two materials with significantly different fruit skin hardness, and identified 19 differentially expressed genes related to fruit skin hardness. These genes are all enriched in the phenylpropanoid metabolic pathway. At present, there are relatively few studies on the identification of watermelon fruit skin hardness traits at the molecular level, and InDel molecular markers for identifying watermelon fruit skin hardness traits have not been reported. SUMMARY

[0004] The purpose of the present application is to provide a primer pair for identifying or assisting in identifying watermelon fruit skin hardness. The application of the primer pair can accurately and quickly identify the fruit skin hardness traits of watermelon.

[0005] The present application provides a primer pair for identifying or assisting in identifying watermelon fruit skin hardness, which comprises an upstream primer with a nucleotide sequence as shown in SEQ ID No. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID No. 2.

[0006] Preferably, the primer pair takes InDel-P159 molecular marker as the detection target.

[0007] Preferably, the InDel-P159 molecular marker is located at base 25282282 bp on chromosome 6 of watermelon, and the length of the InDel molecular marker is 199 bp and / or 220 bp, that is, the polymorphism is 21 bp, as determined with reference to the Watermelon (97103) v2.5 Genome.

[0008] This invention provides a kit for identifying or assisting in the identification of watermelon rind hardness, comprising the primer pairs and PCR amplification reagents described in the above technical solution.

[0009] Preferably, the upstream primer concentration in the primer pair is 9–11 μmol / L, and the downstream primer concentration is 9–11 μmol / L.

[0010] This invention provides the application of the primer pairs or kits described in the above technical solutions in identifying or assisting in the identification of watermelon rind hardness.

[0011] This invention provides a method for identifying or assisting in the identification of watermelon rind hardness, comprising the following steps:

[0012] 1) Extract genomic DNA from the watermelon to be identified;

[0013] 2) Using the watermelon genomic DNA to be identified as a template, PCR amplification was performed using the primer pair described in the above technical solution to obtain PCR amplification products;

[0014] 3) Perform polyacrylamide gel electrophoresis on the PCR amplification products. If one band is amplified and the molecular weight of the PCR amplification product is 220 bp, it is determined to be a high-hardness, crack-resistant watermelon. If one band is amplified and the molecular weight of the PCR amplification product is 199 bp, it is determined to be a low-hardness, easily cracked watermelon.

[0015] Preferably, the PCR amplification system, in 15 μL, includes: 7.5 μL 2×T5PCRMix, 4.5 μL ddH2O, 1 μL forward and reverse primers, 1 μL reverse primer, and 1 μL DNA template.

[0016] Preferably, the PCR amplification program is as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 5 min, and storage at 12℃.

[0017] Preferably, the high-hardness, crack-resistant watermelon is a watermelon with a rind hardness value greater than or equal to 9.0N; the low-hardness, easily cracked watermelon is a watermelon with a rind hardness value less than 9.0N.

[0018] The beneficial effects of this invention are as follows: This invention provides a primer pair for identifying or assisting in the identification of watermelon rind hardness. The primer pair includes an upstream primer with the nucleotide sequence shown in SEQ ID No. 1 and a downstream primer with the nucleotide sequence shown in SEQ ID No. 2. Using the primer pair designed with the InDel-P159 molecular marker provided by this invention, the hardness trait of watermelon rind can be accurately and rapidly identified. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 Distribution of pericarp hardness in watermelon parents and RILs population;

[0021] Figure 2 Results of gene mapping for watermelon rind hardness;

[0022] Figure 3 InDel-P159 primers were used to amplify polymorphisms in the parents and RILs population (partial). Lane 1 is the molecular weight marker; lane 2 is the PCR product of the father; lane 3 is the PCR product of the mother; lanes 4-21 are the PCR products of high-hardness, crack-resistant individual plants in the RILs population; lanes 22-35 are the PCR products of low-hardness, easily cracked individual plants in the RILs population.

[0023] Figure 4 Amplification of InDel-P159 in natural populations. Lane 1 is the molecular weight marker; lane 2 is the PCR product of the high-hardness, crack-resistant parent in the natural population; lane 3 is the PCR product of the low-hardness, easily cracked parent in the natural population; lanes 4-40 are the PCR products of 37 natural population materials. Detailed Implementation

[0024] This invention provides a primer pair for identifying or assisting in the identification of watermelon rind firmness. The primer pair includes an upstream primer with the nucleotide sequence shown in SEQ ID No. 1 and a downstream primer with the nucleotide sequence shown in SEQ ID No. 2. The primer pair described in this invention can amplify the InDel-P159 molecular marker via PCR.

[0025] Table 1. Nucleotide sequences of primer pairs

[0026]

[0027] In this invention, when the primer pair is used to identify or assist in the identification of watermelon rind hardness, the InDel-P159 molecular marker is preferably used as the detection target. The InDel-P159 molecular marker of this invention is preferably located at the 25282282 bp position on chromosome 6 of watermelon. The length of the InDel-P159 molecular marker is 199 bp and / or 220 bp, that is, the polymorphism is 21 bp, which is determined with reference to the Watermelon (97103) v2.5 Genome.

[0028] In this invention, the preferred design approach for the InDel-P159 molecular marker is as follows: Using the Watermelon (97103) v2.5 Genome genome as a reference, the watermelon RIL genetic segregating population (2 parents and 69 progeny) was resequencing. The R / qtl software was used to correlate the rind firmness of the watermelon rind in the RIL population, locating the target gene within a 635.33 kb interval of 24650194–25285524 bp on chromosome 6 of the watermelon. An InDel molecular marker was designed targeting the InDel locus within this interval. The InDel gene locus was validated in the paternal and maternal parents and the RIL population, yielding the InDel-P159 molecular marker linked to the watermelon rind firmness gene. This marker is located at position 25282282 bp on chromosome 6 of the watermelon, with a length of 199 bp and / or 220 bp, indicating a polymorphism of 21 bp.

[0029] This invention provides the InDel-P159 molecular marker, which is closely linked to the watermelon rind firmness gene. The InDel-P159 molecular marker is simple and rapid to operate; a single PCR amplification is sufficient for accurate and rapid identification of watermelon rind firmness at the seedling stage. Current research on watermelon rind firmness is relatively limited. Developing molecular markers related to rind firmness for initial screening in the improvement of this trait allows for rapid screening of watermelon rind firmness during the seedling stage, accelerating the improvement of this trait and achieving the goal of marker-assisted selection. This significantly shortens the breeding cycle and improves breeding efficiency.

[0030] In this invention, the method for obtaining the InDel-P159 molecular marker preferably includes the following steps: crossing a low-hardness, easily cracked watermelon variety as the female parent with a high-hardness, crack-resistant watermelon variety as the male parent to obtain the hybrid F1 generation; self-pollinating the F1 generation to obtain a RILs population; using the RILs population as experimental material to determine the hardness of the watermelon rind; performing QTL mapping on the watermelon rind hardness of the RILs population; determining the InDel site within the QTL candidate interval; designing primers for the InDel site; and obtaining the InDel molecular marker after polymorphism verification.

[0031] This invention involves crossing a low-hardness, easily cracked watermelon variety as the female parent with a high-hardness, crack-resistant watermelon variety as the male parent to obtain the F1 hybrid generation. The F1 hybrid generation is then self-crossed for multiple generations to obtain a RILs (Rich Intake Water) population. The preferred female parent in this invention is the low-hardness, easily cracked watermelon variety K3, and the preferred male parent is the high-hardness, crack-resistant watermelon variety PI189225. No special requirements are placed on the hybridization method in this invention; conventional hybridization methods in the art are acceptable. After obtaining the F1 hybrid generation, the F1 generation is self-crossed for multiple generations to obtain the RILs population. The self-crossing method for the F1 generation in this invention is also a conventional self-crossing method in the art.

[0032] This invention preferably uses a RILs (Ripe Hardness Intake) population as experimental material to determine watermelon rind hardness, dividing the RILs population into high-hardness, crack-resistant watermelon materials and low-hardness, crack-prone watermelon materials. Trait associations are performed on the rind hardness of the RILs population, and QTL mapping is conducted. The QTL mapping in this invention preferably utilizes Rqtl software and CIM interval mapping. This invention preferably detects a major-effect QTL on watermelon chromosome 6, with a contribution rate of 41.31%. This major-effect QTL is preferably located in the segment from 24650194 to 25285524 bp, with a physical distance of 635.33 kb.

[0033] This invention preferably identifies InDel sites within QTL candidate intervals, designs primers for the InDel sites, and obtains the InDel molecular marker after polymorphism verification. The InDel-P159 molecular marker of this invention is preferably 199 bp and / or 220 bp in length, i.e., exhibits a polymorphism of 21 bp. In this invention, the polymorphism screening method can employ conventional primer polymorphism screening methods in the art.

[0034] The present invention also provides a kit for identifying or assisting in the identification of watermelon rind hardness, comprising the primer pairs and PCR amplification reagents described in the above technical solution.

[0035] In this invention, the preferred concentration of the upstream primer and the preferred concentration of the downstream primer in the primer pair are 9–11 μmol / L, more preferably 10 μmol / L for both. The preferred concentrations of the upstream and downstream primers in this invention are the concentrations of the primers themselves.

[0036] The present invention does not have any special limitations on the composition and quantity of each component of the PCR amplification reagent in the kit; the composition and quantity of the components of a conventional kit can be used.

[0037] This invention provides the application of the primer pairs or kits described in the above technical solutions in identifying or assisting in the identification of watermelon rind hardness.

[0038] This invention provides a method for identifying or assisting in the identification of watermelon rind hardness, comprising the following steps:

[0039] 1) Extract genomic DNA from the watermelon sample to be identified;

[0040] 2) Using the watermelon genomic DNA to be identified as a template, PCR amplification was performed using the primer pair described in the above technical solution to obtain PCR amplification products;

[0041] 3) Perform polyacrylamide gel electrophoresis on the PCR amplification products. If one band is amplified and the molecular weight of the PCR amplification product is 220 bp, it is determined to be a high-hardness, crack-resistant watermelon. If one band is amplified and the molecular weight of the PCR amplification product is 199 bp, it is determined to be a low-hardness, easily cracked watermelon.

[0042] The high-hardness, crack-resistant watermelon of the present invention is preferably a watermelon with a rind hardness value greater than or equal to 9.0N; the low-hardness, easily cracked watermelon is preferably a watermelon with a rind hardness value less than 9.0N.

[0043] This invention first extracts DNA from watermelon samples. There are no specific limitations on the method used for DNA extraction; conventional methods are acceptable. In practice, this invention preferably utilizes a DNA extraction kit from Beijing Jinsha Biotechnology Co., Ltd., and extracts DNA from the watermelon samples according to the instructions. This invention also preferably extracts DNA from watermelon leaf samples for identifying watermelon rind firmness.

[0044] After obtaining the DNA from the watermelon sample, the present invention uses the DNA from the watermelon sample as a template and performs PCR amplification using the InDel-P159 molecular marker primer pair described in the above technical solution to obtain the PCR amplification product.

[0045] In this invention, the PCR amplification system preferably includes the following per 15 μL: 7.5 μL 2×T5 PCR Mix, 4.5 μL ddH2O, 1 μL upstream primer (primer concentration of 10 μmol / L), 1 μL downstream primer (primer concentration of 10 μmol / L), and 1 μL DNA template (DNA concentration of 10 ng / μL).

[0046] In this invention, the preferred PCR amplification procedure includes: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 58°C annealing for 10 s, 72°C extension for 10 s, 35 cycles; 72°C extension for 5 min, and storage at 12°C.

[0047] In this invention, the PCR amplification products are preferably identified by polyacrylamide gel electrophoresis. The specific bands found in the PCR amplification of the watermelon sample determine whether the variety is a high-hardness, crack-resistant watermelon or a low-hardness, easily cracked watermelon. An 8% polyacrylamide gel is preferably used for electrophoresis identification. If the PCR amplification produces one band with a molecular weight of 220 bp, the watermelon is identified as a high-hardness, crack-resistant watermelon; if the PCR amplification produces one band with a molecular weight of 199 bp, the watermelon is identified as a low-hardness, easily cracked watermelon.

[0048] The high-hardness, crack-resistant watermelon of the present invention is preferably a watermelon with a rind hardness value greater than or equal to 9.0N; the low-hardness, easily cracked watermelon is preferably a watermelon with a rind hardness value less than 9.0N.

[0049] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1: Development of InDel molecular markers for identifying watermelon rind hardness

[0051] 1. Determination of rind hardness of individual watermelon plants in a RILs population

[0052] F1 generation was obtained by crossing the female parent watermelon K3 (a low-hardness, easily cracked variety) and the male parent watermelon PI189225 (a high-hardness, crack-resistant variety). The F1 generation was then self-crossed for several generations to obtain a RILs population, totaling 69 individual plants. This RILs population was used as experimental material to determine rind hardness. The rind hardness of the parents and RILs population was measured using a TMS-Touch texture analyzer (FTC, USA), with three biological replicates, and the average value was taken as the rind hardness value of the samples. Watermelons with a rind hardness value greater than or equal to 9.0 N were classified as high-hardness, crack-resistant watermelons, while those with a rind hardness value less than 9.0 N were classified as low-hardness, easily cracked watermelons. The rind hardness distribution of the two parents and the 69 RILs population is shown below. Figure 1 As shown in Table 2.

[0053] Table 2. Pericarp hardness values ​​of parental lines and RILs population

[0054]

[0055]

[0056] 2. QTL mapping of the watermelon rind hardness gene

[0057] R / QTL software was used to perform trait association on watermelon rind firmness in the RILs population. CIM interval mapping was used to locate rind firmness, and the threshold was set using 1000 PT tests. The LOD threshold (LOD = 5.825) corresponding to a confidence level of 0.99 was used as the localization interval. Analysis showed that a major QTL was detected on chromosome 6, with a contribution rate of 41.31%. This major QTL was located in the segment 24650194–25285524 bp, with a physical distance of 635.33 kb. The localization results are as follows: Figure 2 As shown.

[0058] 3. Development of InDel molecular markers related to watermelon rind hardness

[0059] InDel loci within the major QTL candidate region of watermelon rind hardness on chromosome 6 were screened using the bcftools subroutine vcfutils.pl(varFilter-w5-W10). SNPs within 5 bp of an InDel and adjacent InDel loci within 10 bp were filtered. Fifty pairs of InDel primers were designed using Primer3_core software, and polymorphism screening was performed between two parents. One InDel molecular marker, InDel-P159, was obtained that clearly distinguishes between high hardness and crack resistance and low hardness and crackability. InDel-P159 amplified a 220 bp band in the high hardness and crack resistance parent and a 199 bp band in the low hardness and crackability parent.

[0060] Example 2: Validation of the InDel-P159 molecular marker in a RIL population

[0061] (1) The total DNA of the leaves of the maternal watermelon K3 (low hardness and easy to crack), the paternal watermelon PI189225 (high hardness and crack resistance) and 69 RILs were extracted using the FlaPurePlantDNAExtractionKit (Beijing Jinsha Biotechnology Co., Ltd.).

[0062] (2) Using the DNA from the two parents and the RIL population extracted in step (1) as templates, PCR amplification was performed using InDel-P159 primers. PCR reaction system and procedure: The total PCR reaction system was 15 μL, and the components were as follows: 7.5 μL of 2×T5PCRMix, 1 μL of forward primer (primer concentration 10 μmol / L), 1 μL of reverse primer (primer concentration 10 μmol / L), 1 μL of watermelon leaf DNA (10 ng / μL), and 4.5 μL of ddH2O.

[0063] The PCR reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 10 s, for a total of 35 cycles; 72℃ extension for 5 min, and storage at 12℃ until taken out.

[0064] (3) Take 2 μL of the PCR product from step (2) and perform electrophoresis using an 8% polyacrylamide gel. Figure 3 The results show the amplification of InDel-P159 primers in paternal, maternal, and some RILs single plants. Lane 1 is the molecular weight marker; lane 2 is the paternal PCR product; lane 3 is the maternal PCR product; lanes 4-21 are the PCR products of high-hardness, crack-resistant single plants; and lanes 22-35 are the PCR products of low-hardness, easily cracked single plants. The results indicate that the InDel-P159 marker can amplify a 220 bp fragment in the paternal and high-hardness, crack-resistant single plants; and a 199 bp fragment in the maternal and low-hardness, easily cracked single plants. Figure 3 The results were obtained using SDS-PAGE gel.

[0065] In the RIL population, 22 out of 25 high-hardness, crack-resistant individual plants had genotypes that matched their phenotypes. The primers achieved an accuracy of 88.0% in identifying pericarp hardness in the RIL population. This invention only analyzed the dominant trait, namely, high-hardness, crack-resistant individual plants.

[0066] Example 3: Validation of InDel-P159 in a natural population

[0067] The linkage between the InDel-P159 molecular marker and watermelon rind firmness was further verified using 37 natural population samples. The specific steps are as follows:

[0068] (1) Genomic DNA extraction. Total DNA was extracted from the leaves of 37 natural watermelon populations using the FlaPurePlantDNAExtractionKit (Beijing Jinsha Biotechnology Co., Ltd.). The firmness traits of the 37 natural watermelon populations are shown in Table 3.

[0069] Table 3. Materials and phenotypes of watermelon from natural populations

[0070]

[0071]

[0072] (2) PCR reaction system and procedure. Using the natural population DNA extracted in step (1) as a template, PCR amplification was performed using InDel-P159 primers. The total PCR reaction system was 15 μl, and the components were as follows: 7.5 μl of 2×T5PCRMix, 1 μl of forward primer (primer concentration 10 μmol / L), 1 μl of reverse primer (primer concentration 10 μmol / L), 1 μl of watermelon leaf DNA (10 ng / μl), and 4.5 μl of ddH2O.

[0073] The PCR reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 10 s, for a total of 35 cycles; 72℃ extension for 5 min, and storage at 12℃ until taken out.

[0074] (3) Take 2 μl of the above PCR product and perform electrophoresis detection using 8% polyacrylamide gel. Figure 4 Results of InDel-P159 primer detection for 37 natural population materials. Figure 4 The results were identified using SDS-PAGE gel. M represents the molecular weight marker; P1 is the PCR product of the high-hardness, crack-resistant parent watermelon PI189225 from Example 1; P2 is the PCR product of the low-hardness, easily cracked parent watermelon K3 from Example 1; 1-37 are the PCR products of 37 natural population materials. The results showed that the InDel-P159 marker could amplify a 220 bp fragment in both the high-hardness, crack-resistant parent and individual plants in the natural population; the InDel-P159 marker could amplify a 199 bp fragment in both the low-hardness, easily cracked parent and individual plants in the natural population. In the 37 natural population materials, 9 out of 11 high-hardness, crack-resistant individual plants had genotypes matching their phenotypes, and the accuracy rate of the InDel-P159 primers in identifying the peel hardness of the natural population was 81.82%. This invention only analyzed the dominant trait, namely, high-hardness, crack-resistant individual plants.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer pair for identifying or assisting in the identification of watermelon rind hardness, characterized in that, The primer pair includes an upstream primer with a nucleotide sequence as shown in SEQ ID No. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID No.

2.

2. The primer pair according to claim 1, characterized in that, The primer pair uses the InDel-P159 molecular marker as the detection target. The InDel-P159 molecular marker is located at the 25282282 bp position on chromosome 6 of watermelon. The length of the InDel molecular marker is 199 bp and / or 220 bp, that is, the polymorphism is 21 bp, which is determined with reference to the Watermelon(97103)v2.5 Genome.

3. A kit for identifying or assisting in the identification of watermelon rind hardness, characterized in that, Includes the primer pairs and PCR amplification reagents as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, The upstream primer concentration in the primer pair is 9–11 μmol / L, and the downstream primer concentration is 9–11 μmol / L.

5. The application of the primer pair according to claim 1 or 2 or the kit according to claim 3 or 4 in the identification or auxiliary identification of watermelon rind hardness.

6. A method for identifying or assisting in the identification of watermelon rind hardness, characterized in that, Includes the following steps: 1) Extract genomic DNA from the watermelon to be identified; 2) Using the watermelon genomic DNA to be identified as a template, PCR amplification is performed using the primer pair described in claim 1 or 2 to obtain PCR amplification products; 3) Perform polyacrylamide gel electrophoresis on the PCR amplification products. If one band is amplified and the molecular weight of the PCR amplification product is 220 bp, it is determined to be a high-hardness, crack-resistant watermelon. If one band is amplified and the molecular weight of the PCR amplification product is 199 bp, it is determined to be a low-hardness, easily cracked watermelon.

7. The method according to claim 6, characterized in that, The PCR amplification system, in 15 μL increments, includes: 7.5 μL 2×T5 PCR Mix, 4.5 μL ddH2O, 1 μL forward and reverse primers, 1 μL reverse primer, and 1 μL DNA template.

8. The method according to claim 6, characterized in that, The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 10 s, 35 cycles; 72℃ extension for 5 min, and storage at 12℃.

9. The method according to claim 6, characterized in that, The high-hardness, crack-resistant watermelon is defined as a watermelon with a rind hardness value greater than or equal to 9.0N; the low-hardness, easily cracked watermelon is defined as a watermelon with a rind hardness value less than 9.0N.

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