Molecular Markers Tightly Linked to the Watermelon Pericarp Intermittent Stripe Gene ClIS and Their Applications

Abstract

The present invention discloses two pairs of molecular markers closely linked to the watermelon pericarp intermittent stripe gene ClIS and their applications, belonging to the technical field of molecular breeding. The present invention has for the first time finely mapped the watermelon pericarp intermittent stripe trait and obtained two pairs of Indel markers that are closely linked to the intermittent stripe trait, have stable amplification and good polymorphism. These two pairs of Indel markers are closely linked to the watermelon pericarp intermittent stripe gene ClIS and can be used for molecular marker-assisted breeding of the watermelon intermittent stripe trait, which helps to improve the breeding efficiency, accelerate the breeding improvement process, and provides a theoretical basis for molecular breeding of watermelon appearance quality.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular breeding, and particularly relates to a molecular marker closely linked to the watermelon pericarp intermittent stripe gene ClIS and its application. Background Art

[0002] The appearance quality of fruits has always been one of the most important traits that consumers are most concerned about. Unique and novel pericarp traits are more attractive and can determine consumers' choices. As an important appearance trait, the pericarp stripe has various variation types. For watermelons, according to the background color of the pericarp, it can be divided into yellow, dark green, light green, white, etc.; according to the stripe type, the watermelon pericarp can be divided into strip-shaped, reticulate, intermittent, spotted, etc. In addition, there are some other special watermelon stripe types, such as pericarp yellow spots, yellow belly on the pericarp, irregular stripes on the pericarp, etc. For watermelon breeders, different pericarp morphologies have always been a research hotspot.

[0003] In previous studies, the research on watermelon pericarp mainly focused on genetic analysis and preliminary mapping. Genetic analysis showed that the light green pericarp of watermelon is controlled by a single gene g, and light green is recessive inheritance to dark green pericarp color. However, it has also been reported that there are two loci jointly regulating dark green and light green pericarp traits in watermelons. For watermelon stripes, researchers have proved that wide-toothed thick stripes are dominant to thin stripes and are regulated by a single gene; the yellow color on the abdomen of the watermelon pericarp and the intermittent stripes are both controlled by a single gene; the yellow spots on the watermelon pericarp are controlled by two genes. So far, few genes controlling watermelon pericarp traits have been mapped. Currently, researchers have obtained BC 1 populations from the cross between dark green pericarp and light green pericarp watermelons and identified a gene locus controlling the dark green pericarp of watermelon on chromosome 8; and further identified the watermelon dark green pericarp regulatory gene ClCGMenG through fine mapping. Another study showed that the insertion / deletion of 3 bases in the ClGS gene determined the formation of watermelon dark green stripes. In addition, the yellow pericarp of watermelon is controlled by a gene locus at the forefront of chromosome 4; for the watermelon yellow spot trait, two loci located on chromosomes 1 and 8 play a major role. For the watermelon pericarp stripe type, comprehensive multiple research results show that there are multiple gene loci related to watermelon pericarp stripes on chromosome 6. However, the fine mapping and cloning research on some special watermelon pericarp stripe types, especially intermittent stripes, have not been reported, and molecular markers closely linked to them have not been developed. Summary of the Invention

[0004] The object of the present invention is to provide two pairs of molecular markers closely linked to the watermelon pericarp intermittent stripe gene ClIS, namely Indel2 and Indel8 markers. By using these two markers alone or simultaneously for assisted selection of the watermelon pericarp stripe gene ClIS, the breeding efficiency of watermelon pericarp stripes can be significantly improved.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In the present invention, two pairs of molecular markers closely linked to the watermelon pericarp stripe gene ClIS. One pair of molecular markers is Indel2, and the upstream primer sequence of the primer pair for amplifying the molecular marker is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2.

[0007] Another pair of Indel8 molecular markers closely linked to the watermelon pericarp stripe gene ClIS in the present invention, the upstream primer sequence of the primer pair for amplifying the molecular marker is shown in SEQ ID NO.5, and the downstream primer sequence is shown in SEQ ID NO.6.

[0008] The most important inventive point of the present invention lies in the application of the molecular markers closely linked to the above-mentioned watermelon pericarp intermittent stripe gene ClIS in watermelon molecular breeding, including using the molecular markers of the present invention to identify whether they exist to determine the genotype of watermelon pericarp intermittent stripes.

[0009] Among them, the method for determining watermelon pericarp intermittent stripe varieties or genotypes using the Indel2 molecular marker, the determination method includes the following steps:

[0010] (1) Extract the genomic DNA of the watermelon to be tested;

[0011] (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer pair of the Indel2 molecular marker, and perform electrophoresis detection and / or sequencing on the PCR amplification product;

[0012] (3) Make a determination according to the electrophoresis band and / or sequencing result of step (2), and the specific criteria are as follows:

[0013] If the PCR amplification product has only one characteristic band with a length of 87 bp as shown in SEQ ID NO.3, then the test material is the homozygous genotype of watermelon peel intermittent stripes (ClIS / ClIS); if the PCR amplification product has only one characteristic band with a length of 95 bp as shown in SEQ ID NO.4, then the test watermelon material is the homozygous genotype of light green peel stripes (Clis / Clis); if the PCR amplification product has both a characteristic band with a length of 87 bp as shown in SEQ ID NO.3 and a characteristic band with a length of 95 bp as shown in SEQ ID NO.4, then the test watermelon material is the heterozygous genotype of peel intermittent stripes (ClIS / Clis).

[0014] A method for determining watermelon peel intermittent stripe varieties or genotypes using the Indel8 molecular marker, the determination method comprising the following steps:

[0015] (1) Extract genomic DNA of the test watermelon;

[0016] (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer pair of the Indel8 molecular marker, and perform electrophoresis detection and / or sequencing on the PCR amplification product;

[0017] (3) Make a determination based on the electrophoresis band and / or sequencing result of step (2), and the specific criteria are as follows:

[0018] If the PCR amplification product has only one characteristic band with a length of 99 bp as shown in SEQ ID NO.7, then the test watermelon material is the homozygous genotype of watermelon peel intermittent stripes (ClIS / ClIS); if the PCR amplification product has only one characteristic band with a length of 94 bp as shown in SEQ ID NO.8, then the test watermelon material is the homozygous genotype of light green peel stripes (Clis / Clis); if the PCR amplification product has both a characteristic band with a length of 99 bp as shown in SEQ ID NO.7 and a characteristic band with a length of 94 bp as shown in SEQ ID NO.8, then the test watermelon material is the heterozygous genotype of peel intermittent stripes (ClIS / Clis).

[0019] In addition, a kit containing the above primer pair can be used to identify the peel stripe traits of watermelon materials. Specifically, when applying, a reagent containing the above molecular marker primer pair can be selected to make a kit.

[0020] Furthermore, the application of a reagent for detecting the presence of two pairs of Indel markers in the mapping of the watermelon pericarp stripe gene ClIS. Using the molecular markers of the present invention, the watermelon pericarp stripe gene ClIS can be mapped, and the above applications can all be carried out according to conventional methods.

[0021] It should be noted that in this application, the watermelon pericarp intermittent stripe gene ClIS and the watermelon pericarp light green stripe gene Clis are alleles. Among them, the watermelon pericarp intermittent stripe gene ClIS is dominantly controlled (that is, the pericarp stripes of watermelons containing this gene are intermittent stripes, that is, the corresponding gene pair is ClIS / ClIS or ClIS / Clis), while the watermelon pericarp light green stripe gene Clis is recessively controlled (that is, only when the homozygous gene pair is Clis / Clis, the pericarp of this watermelon variety shows a light green stripe phenotype).

[0022] The present invention also protects a vector containing the above molecular markers. The recombinant vector can be an expression vector or a cloning vector inserted with the molecular markers of the present invention. After obtaining the above recombinant vector, those skilled in the art can, according to different needs, transform the recombinant vector into a suitable cell to obtain a recombinant cell containing the recombinant vector. Therefore, the present invention also protects a recombinant cell containing the recombinant vector.

[0023] Advantages of the present invention:

[0024] In this study, the watermelon intermittent stripe homozygous inbred line WT20 and the light green stripe homozygous inbred line WCZ were used as materials to construct F 2 and BC 1 segregating populations. The molecular marker technology was used to finely map the watermelon intermittent stripe trait, and two pairs of Indel markers tightly linked to the intermittent stripe trait were found. These molecular markers have stable amplification, good polymorphism, and convenient and efficient detection, so they can be directly used for molecular marker-assisted breeding of watermelon pericarp intermittent stripe materials. Since molecular markers have the advantages of simplicity, rapidity, and high efficiency in the assisted selection breeding system, the molecular markers provided by the present invention have good application value in the cultivation of new watermelon pericarp intermittent stripe varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 are the watermelon pericarp intermittent stripe material WT20 (left) and the light green stripe material WCZ (right);

[0026] Figure 2 is the fine mapping diagram of the watermelon pericarp intermittent stripe gene ClIS;

[0027] Figure 3 is the Indel2 marker used in the present invention in the parents WT20 and WCZ, F 1 and F2 The polymorphic electrophoresis pattern in the progeny plants;

[0028] Figure 4 The Indel8 marker used in the present invention in the parents WT20 and WCZ, F 1 and F 2 The polymorphic electrophoresis pattern in the progeny plants. Detailed implementation manners

[0029] The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0030] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.

[0031] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are only for demonstration purposes.

[0032] Plant materials:

[0033] The watermelon material WT20 is a high-generation inbred line selected by the inventor. Its fruit peel background color is light green with intermittent stripes;

[0034] The watermelon material WCZ is a high-generation inbred line selected by the inventor. The fruit peel background color is light green with light green fine stripes. This material can be obtained through commercial channels or provided by the Melon Crop Genetic Breeding Research Group of Henan Agricultural University (it should be noted that using this material as the research basis is only for the convenience of obtaining experimental materials and should not be understood that the implementation of the relevant technical solutions of this application must rely on this experimental material);

[0035] The above-mentioned material WCZ is the same as the watermelon plant without tendril material "Wucha Zao" used in the Chinese invention patent "Molecular Marker Tightly Linked to the Clnt Gene Without Tendrils in Watermelon Plants and Its Application" with the publication number CN1 10938706A.

[0036] The above biological materials are all stored in the laboratory of the applicant's unit and can be distributed to the public for verification tests within twenty years from the filing date, or the public can also obtain them by purchasing.

[0037] During the experiment, both parents and the constructed population were planted in the solar greenhouse of Maozhuang Science and Education Park, Henan Agricultural University. The planting process was as follows: After germination, plug seedlings were raised, and normal watermelon cultivation management methods were adopted. The statistical analysis of the fruit peel stripe type per plant was started on the 10th day after pollination.

[0038] Experimental reagents and equipment:

[0039] During the experiment, the PCR Taq-Mix for PCR amplification was purchased from Nanjing Novozymes Gene Technology Co., Ltd.; other electrophoresis and silver staining related reagents such as acrylamide, methylene bisacrylamide, AgNO 3 , NaOH, and formaldehyde were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0040] The primers (synthesized manually) for PCR amplification and gene sequencing during the experiment were provided and completed by Qingke Genome Research Center Co., Ltd.

[0041] The PCR instrument was the Hema9600 type gene amplifier from Zhuhai Hema Medical Instrument Co., Ltd.;

[0042] The electrophoresis instrument was the JY300HC general electrophoresis instrument, produced by Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.;

[0043] The electrophoresis tank was the HT-SCZ04A high-throughput vertical electrophoresis tank, produced by Beijing Hongtao Jiye Technology Development Co., Ltd.

[0044] Example 1 Mapping of the ClIS gene

[0045] (I) Construction of the genetic segregation population

[0046] Using the watermelon fruit peel intermittent stripe material WT20 as the male parent and the watermelon fruit peel light green stripe material WCZ as the female parent, a watermelon stripe trait segregation population was constructed using WT20 (P 1 ) and WCZ (P 2 ). WT20 was crossed with WCZ to obtain F 1 , F 1 was self-crossed to obtain the F 2 population, and F 1 was backcrossed with each parent to obtain BC 1 P 1 (F 1 ×P 1 ) and BC 1 P 2 (F 1 ×P 2 ). The results showed that all the fruit epidermis types of the obtained F 1 showed intermittent stripes. (The phenotypes of the parental materials are as Figure 1 shown.)

[0047] Select 10 individual plants from F 1 plants and self-cross them to obtain F 2 seeds for genetic analysis and gene mapping.

[0048] Identify the pericarp stripe phenotypes of F 2 and BC 1 individuals, and verify them using the chi-square test. The results showed that: In the spring of 2020, among the 130 F 2 plants in the population, 100 plants showed intermittent pericarp stripes, and 30 plants showed light green stripes. The chi-square test conformed to the segregation ratio of 3:1; all 59 BC 1 P 1 plants all showed intermittent pericarp stripes; among the 59 BC 1 P 2 plants, 33 plants showed intermittent pericarp stripes, and 26 plants showed light green stripes. The chi-square test conformed to the segregation ratio of 1:1.

[0049] For the 217 F 2 plants planted in the autumn of 2020, the phenotypic survey showed that 164 plants showed intermittent pericarp stripes and 53 plants showed light green stripes. The chi-square test conformed to the segregation ratio of 3:1.

[0050] For the 849 F 2 plants planted in the spring of 2021, the stripe survey results showed that 622 plants showed intermittent pericarp stripes and 227 plants showed light green stripes. The chi-square test also conformed to the segregation ratio of 3:1.

[0051] In summary, it can be known that the intermittent pericarp stripe trait of watermelon is controlled by a dominant single gene, and this gene is named ClIS. Intermittent pericarp stripes (ClIS) are completely dominant over light green pericarp stripes (Clis).

[0052] (2) Preliminary mapping of the intermittent pericarp stripes of watermelon

[0053] (1) First, prepare gene pools, specifically:

[0054] Randomly select 20 watermelon plants with intermittent pericarp stripes and 20 plants with light green pericarp stripes from the F 2 population in the above step (1), collect young leaves, extract genomic DNA using the CTAB method, and mix them separately to make a gene pool for watermelon plants with intermittent pericarp stripes and a gene pool for plants with light green pericarp stripes (mix intermittent stripes with intermittent stripes, and mix light green pericarp stripes with light green pericarp stripes).

[0055] (2) Polymorphism screening and analysis, specifically:

[0056] Using 1,257 pairs of SSR primers developed from the watermelon whole genome by the inventors in the early stage (for the specific primer sequences, refer to "Genome wide characterization of simple sequence repeats in watermelon genome and their application in comparative mapping and genetic diversity analysis", Zhu et al., BMC Genomics, 2016, 17: 557 - 573.), markers with polymorphisms between the parents were screened. Among them, a total of 532 markers showed polymorphisms between the parents. Then, the two gene pools prepared in step (1) were subjected to a second polymorphism screening using 532 markers with polymorphisms between the parents, and 9 pairs of polymorphic markers with good amplification effects and clear and distinguishable bands were obtained, which were distributed on chromosome 6 of watermelon.

[0057] Furthermore, the 9 pairs of polymorphic SSR markers screened were used for genotype analysis of 217 F 2 populations in the autumn of 2020. Those with the same band pattern as the light green peel stripe parent were denoted as 2, those with the same band pattern as the intermittent stripe parent were denoted as 1, and those with heterozygous band patterns were denoted as 3. Finally, joinmap 4.0 was used for linkage analysis of the typing results, and ultimately the ClIS gene was mapped to chromosome 6 of watermelon.

[0058] During the polymorphism screening and analysis process, when performing PCR amplification, the 10 μL amplification system was designed as follows:

[0059] Gene pool sample (genomic DNA, 30 ng / μL), 1 μL (about 30 ng);

[0060] F and R primers, 0.5 μL each (primer concentration is 5 μmol / L)

[0061] PCR Taq - Mix, 5.0 μL;

[0062] dd H 2 O, 3.0 μL;

[0063] The PCR amplification program was: 94 °C, 5 min; 94 °C, 30 s, 55 °C, 30 s, 72 °C, 30 s, 35 cycles; 72 °C, 5 min.

[0064] It should be noted that the F and R primers (1,257 pairs of SSR primers) in the above - mentioned PCR amplification system represent the forward primer and the reverse primer in a pair of primers respectively. Since these primers are not directly related to the subject matter to be protected in this application, for the sake of brevity of the text, no further details will be provided.

[0065] The PCR amplification products were detected by 8% non-denaturing polyacrylamide gel electrophoresis. During electrophoresis, the polyacrylamide gel electrophoresis buffer was 1×TBE, and the electrophoresis was performed at a constant voltage of 200V for 1 to 1.5 hours. After the electrophoresis, silver staining was performed for observation and detection. The silver staining method was as follows:

[0066] A. Wash with ultrapure water for 1 to 3 minutes;

[0067] B. Place the washed film in the staining solution and shake it for 2 minutes using a decolorizing shaker. The staining solution is a 0.2% silver nitrate aqueous solution.

[0068] C. Soak the stained plastic plate in ultrapure water for 30 seconds, put it in a plastic box containing developer, and shake it gently until the bands are clearly visible. The developer is obtained by adding 15g NaOH and 15mL formaldehyde to 1L distilled water;

[0069] D. Finally, add ddH 2 O Rinse repeatedly several times;

[0070] E. Dry at room temperature, then take a photo, wrap with plastic wrap and store.

[0071] (3) Fine positioning of intermittent stripes on watermelon peel, specifically:

[0072] The two parents were resequenced at 20× depth using the Illumina Hi-seq2000 high-throughput sequencing platform. Combined with the results in (2), more SSR markers were developed to expand the F 2 Based on the number of individual plants in the population, the watermelon rind intermittent stripe gene ClIS was located within a genetic distance of 230Kb between ClSSR18071 and C1SSR180298 on chromosome 6. In order to further narrow the candidate interval and obtain more other molecular markers for fine positioning, based on the resequencing data between the two parents, we developed two pairs of dCAPS markers and two pairs of Indel markers between C1SSR18071 and C1SSR180298. These four pairs of markers were only used for polymorphism analysis of recombinant plants. The final candidate gene ClIS was located between the two markers dCAPS-C and dCAPS-E. Based on the physical position of the markers, the ClIS gene was ultimately precisely located within a 160Kb interval between 25.92Mb and 26.08Mb on chromosome 6 of watermelon ( Figure 2 ).

[0073] (4) Development of Indel markers in candidate regions

[0074] The present invention utilizes the Indel2 molecular marker to perform PCR amplification differentiation, and the primer sequence is designed as follows:

[0075]

[0076] An 87-bp specific band was obtained by PCR amplification. The specific base sequence of the 87-bp specific band is shown as follows:

[0077]

[0078] An 95-bp specific band was obtained by PCR amplification. The specific base sequence of the 95-bp specific band is shown as follows:

[0079]

[0080] The amplification product size of this primer in the male parent (intermittent stripes) is 87 bp, and the amplification product size in the female parent (light green stripes) is 95 bp.

[0081] The present invention uses the Indel8 molecular marker for PCR amplification, and the primer sequence is designed as:

[0082]

[0083] An 99-bp specific band was obtained by PCR amplification. The specific base sequence of the 99-bp specific band is shown as follows:

[0084]

[0085] An 94-bp specific band was obtained by PCR amplification. The specific base sequence of the 94-bp specific band is shown as follows:

[0086]

[0087] The amplification product size of this primer in the male parent (intermittent stripes) is 99 bp, and the amplification product size in the female parent (light green stripes) is 94 bp.

[0088] Example 2 Application of Molecular Markers in Breeding

[0089] (1) DNA extraction, PCR amplification and electrophoresis detection

[0090] The CTAB method, a conventional method, was used for DNA extraction. The PCR reaction procedure and the polyacrylamide gel electrophoresis process refer to the master's thesis of Yang Huihui: Fine Mapping and Functional Verification of the Few Lateral Branches Gene Clbl in Watermelon, Henan Agricultural University, 2021.

[0091] Using the Indel2 and Indel8 molecular markers of the present invention, respectively, for F 2Verify the genotype of the distribution in the population.

[0092] Specifically, the application of the Indel2 molecular marker in breeding is as follows:

[0093] Use the Indel2 molecular marker for PCR reaction to determine the genotype of this molecular marker in the F 2 population. Detect the distribution of the two genotypes of the Indel2 molecular marker in 1066 F 2 populations (see part of the detection results in Figure 3 ). According to the amplified band situation, the genotype of a single plant can be quickly distinguished; if the PCR amplification product has only one band with a size of 87 bp, the fruit of this single plant is the homozygous genotype of the intermittent stripes on the watermelon peel; if the PCR amplification product has only one characteristic band with a length of 95 bp, the fruit of this single plant is the homozygous genotype of the light green stripes on the watermelon; if the PCR amplification product has both a characteristic band with a length of 87 bp and a characteristic band with a length of 95 bp, the fruit of this single plant is the heterozygous genotype of the intermittent stripes on the watermelon peel.

[0094] The application of the Indel8 molecular marker in breeding is as follows:

[0095] Use the Indel8 molecular marker for PCR reaction to determine the genotype of this molecular marker in the F 2 population. Detect the distribution of the two genotypes of the Indel8 molecular marker in 1066 F 2 populations (see part of the detection results in Figure 4 ). If the PCR amplification product has only one characteristic band with a length of 99 bp as shown in SEQ ID NO.7, the fruit of this single plant is the homozygous genotype of the intermittent stripes on the watermelon peel; if the PCR amplification product has only one characteristic band with a length of 94 bp as shown in SEQ ID NO.8, the fruit of this single plant is the homozygous genotype of the light green stripes on the watermelon; if the PCR amplification product has both a characteristic band with a length of 99 bp as shown in SEQ ID NO.7 and a characteristic band with a length of 94 bp as shown in SEQ ID NO.8, the fruit of this single plant is the heterozygous genotype of the intermittent stripes on the watermelon peel.

[0096] Therefore, according to the amplified band situation, the genotype of a single plant can be quickly distinguished; further, the inventor combined with the phenotypic data and found that the phenotype of the natural material is consistent with the genotype. Based on this result, it shows that the molecular markers Indel2 and Indel8 of the present invention can effectively distinguish the light green stripe material of watermelon and the intermittent stripe material of watermelon peel.

[0097] In summary, combined with the electronic BSA analysis and verification in natural populations, the molecular markers Indel2 and Indel8 of the present invention can accurately perform molecular marker-assisted selection in the early stage of watermelon growth, greatly improving the process of selection and breeding. It provides theoretical and technical support for the cultivation of new high-quality watermelon varieties.

Claims

1. Indel2 molecular marker tightly linked to the intermittent stripe gene of watermelon pericarp ClIS and It is characterized in that the molecular marker is as shown in SEQ ID NO.3 or SEQ ID NO.4, the upstream primer sequence of the primer pair for amplifying the molecular marker is as shown in SEQ ID NO.1, and the downstream primer sequence is as shown in SEQ ID NO.

2.

2. Indel8 molecular marker tightly linked to the intermittent stripe gene of watermelon pericarp ClIS and tightly linked Indel8 molecular marker It is characterized in that the molecular marker is as shown in SEQ ID NO.7 or SEQ ID NO.8, the upstream primer sequence of the primer pair for amplifying the molecular marker is as shown in SEQ ID NO.5, and the downstream primer sequence is as shown in SEQ ID NO.

6.

3. Use of the molecular marker tightly linked to the watermelon pericarp intermittent stripe gene as claimed in claim 1 or claim 2 ClIS in identifying or assisting in the selection of watermelon pericarp intermittent stripe traits.

4. A method for determining watermelon pericarp stripe varieties / genotypes It is characterized in that the determination method comprises the following steps: (1) Extract the genomic DNA of the watermelon to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification with the primer pair of the molecular marker described in claim 1, and perform electrophoresis detection and / or sequencing on the PCR amplification product; (3) Make a determination according to the electrophoresis band and / or sequencing result of step (2), and the specific criteria are as follows: If the PCR amplification product has only one characteristic band with a length of 87 bp as shown in SEQ ID NO.3, then the watermelon material to be tested is a homozygous variety / genotype of pericarp intermittent stripes; if the PCR amplification product has only one characteristic band with a length of 95 bp as shown in SEQ ID NO.4, then the watermelon material to be tested is a homozygous variety / genotype of pericarp light green stripes; if the PCR amplification product has both a characteristic band with a length of 87 bp as shown in SEQ ID NO.3 and a characteristic band with a length of 95 bp as shown in SEQ ID NO.4, then the watermelon material to be tested is a heterozygous variety / genotype of pericarp intermittent stripes.

5. A method for determining watermelon pericarp stripe varieties / genotypes It is characterized in that the determination method comprises the following steps: (1) Extract the genomic DNA of the watermelon to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification with the primer pair of the molecular marker described in claim 2, and perform electrophoresis detection and / or sequencing on the PCR amplification product; (3) Make a determination according to the electrophoresis band and / or sequencing result of step (2), and the specific criteria are as follows: If the PCR amplification product has only one characteristic band with a length of 99 bp as shown in SEQ ID NO.7, then the watermelon material to be tested is a homozygous variety / genotype of pericarp intermittent stripes; if the PCR amplification product has only one characteristic band with a length of 94 bp as shown in SEQ ID NO.8, then the watermelon material to be tested is a homozygous variety / genotype of pericarp light green stripes; if the PCR amplification product has both a characteristic band with a length of 99 bp as shown in SEQ ID NO.7 and a characteristic band with a length of 94 bp as shown in SEQ ID NO.8, then the watermelon material to be tested is a heterozygous variety / genotype of pericarp intermittent stripes.

6. Use of a reagent for detecting the presence of the Indel2 marker according to claim 1 in the watermelon pericarp stripe gene ClIS in gene mapping It is characterized in that The upstream primer sequence of the primer pair for amplifying the marker is shown as SEQ ID NO.1, and the downstream primer sequence is shown as SEQ ID NO.2; if the amplification product has only one characteristic band with a length of 87 bp as shown in SEQ ID NO.3, then the watermelon material to be tested is a homozygous variety / genotype of intermittent peel stripes; if the PCR amplification product has only one characteristic band with a length of 95 bp as shown in SEQ ID NO.4, then the watermelon material to be tested is a homozygous variety / genotype of light green peel stripes; if the PCR amplification product has both a characteristic band with a length of 87 bp as shown in SEQ ID NO.3 and a characteristic band with a length of 95 bp as shown in SEQ ID NO.4, then the watermelon material to be tested is a heterozygous variety / genotype of intermittent peel stripes.

7. Use of a reagent for detecting the presence of the Indel8 marker according to claim 2 in mapping the watermelon pericarp stripe gene ClIS positioning It is characterized in that The upstream primer sequence of the primer pair for amplifying the marker is shown as SEQ ID NO.5, and the downstream primer sequence is shown as SEQ ID NO.

6. If the amplification product has only one characteristic band with a length of 99 bp as shown in SEQ ID NO.7, then the watermelon material to be tested is a homozygous variety / genotype of intermittent peel stripes; if the PCR amplification product has only one characteristic band with a length of 94 bp as shown in SEQ ID NO.8, then the watermelon material to be tested is a homozygous variety / genotype of light green peel stripes; if the PCR amplification product has both a characteristic band with a length of 99 bp as shown in SEQ ID NO.7 and a characteristic band with a length of 94 bp as shown in SEQ ID NO.8, then the watermelon material to be tested is a heterozygous variety / genotype of intermittent peel stripes.

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