CN115772578A - Molecular marker closely linked with watermelon peel intermittent stripe gene ClIS and application - Google Patents
Molecular marker closely linked with watermelon peel intermittent stripe gene ClIS and application Download PDFInfo
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Abstract
The invention discloses two pairs of molecular markers closely linked with a watermelon peel intermittent stripe gene ClIS and application thereof, belonging to the technical field of molecular breeding. The method finely positions the intermittent stripe character of the watermelon peel for the first time, and obtains two pairs of Indel markers which are closely linked with the intermittent stripe character, stable in amplification and good in polymorphism. The two pairs of Indel markers are closely linked with the intermittent watermelon peel stripe gene ClIS, can be used for molecular marker-assisted breeding of intermittent watermelon stripe characters, is beneficial to improving breeding efficiency, accelerates breeding improvement progress and provides a theoretical basis for molecular breeding of watermelon appearance quality.
Description
Technical Field
The invention belongs to the technical field of molecular breeding, and particularly relates to a molecular marker closely linked with an intermittent stripe gene ClIS of watermelon peel and application thereof.
Background
The appearance quality of fruit has always been one of the most important traits of interest to consumers. The unique and novel peel profile is more attractive and can determine consumer choice. The peel stripe is an important appearance character, and the variation types of the peel stripe are various. For watermelons, yellow, dark green, light green, white and the like can be distinguished according to the bottom color of the peel; according to the type of the stripe, the watermelon peel can be divided into a strip shape, a net shape, an intermittent shape, a spot shape and the like. In addition, there are other special watermelon streak types, such as yellow peel, yellow belly peel, irregular peel, etc. For watermelon breeders, different peel morphologies have been the focus of research.
In previous studies, the research on watermelon peel has focused mainly on genetic analysis and preliminary localization. Genetic analysis shows that the light green peel of the watermelon is controlled by a single gene g, and the color of the light green peel to the dark green peel is recessive inheritance. However, it has also been reported that two loci co-regulate the dark green and light green pericarp traits in watermelon. For watermelon streaks, researchers have demonstrated that wide-toothed thick streaks are dominant over thin streaks and are under monogenic regulation; the yellow belly and intermittent stripes of the watermelon peel are controlled by a single gene; the yellow spots of watermelon peel are controlled by two genes. To date, few genes have been targeted to control the watermelon peel trait. BC obtained by hybridization of dark green peel and light green peel watermelons by researchers 1 Identifying a gene locus for controlling the dark green peel of the watermelon on chromosome 8 by a colony; and further identifying the watermelon dark green peel regulatory gene ClCGmenG through fine positioning. Another study showed that the 3 base insertion/deletion in the ClGS gene determines the formation of the dark green streaks in watermelon. In addition, the watermelon yellow peel is controlled by the foremost gene locus of chromosome 4; for the yellow spot trait of watermelon, two loci located on chromosome 1 and 8 respectively play a roleIt has the following effects. For the watermelon peel stripe type, a plurality of research results are combined to show that a plurality of gene sites related to the watermelon peel stripe exist on the No.6 chromosome. However, the fine positioning and cloning studies of some specific watermelon peel streak types, particularly intermittent streaks, have not been reported, nor have molecular markers closely linked thereto been developed.
Disclosure of Invention
The invention aims to provide two pairs of molecular markers which are closely linked with an intermittent stripe gene ClIS of watermelon peel and are Indel2 markers and Indel8 markers respectively. The two intermittent stripe genes ClIS with the watermelon peel are independently or simultaneously used for auxiliary selection, so that the efficiency of watermelon peel stripe breeding can be obviously improved.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention relates to two pairs of molecular markers which are tightly linked with a watermelon peel streak gene ClIS, wherein one pair of molecular markers is Indel2, the sequence of an upstream primer of a primer pair for amplifying the molecular markers is shown as SEQ ID NO.1, and the sequence of a downstream primer is shown as SEQ ID NO. 2.
In the invention, another pair of Indel8 molecular markers closely linked with the watermelon peel streak gene ClIS is provided, the upstream primer sequence of the primer pair for amplifying the molecular markers is shown as SEQ ID NO.5, and the downstream primer sequence is shown as SEQ ID NO. 6.
The invention mainly aims to adopt the molecular marker tightly linked with the watermelon peel intermittent stripe gene ClIS to be applied to molecular breeding of watermelon, and the molecular marker is used for identifying whether the molecular marker exists or not to judge the genotype of the watermelon peel intermittent stripe.
The method for judging the variety or genotype of the watermelon peel intermittent stripes by using Indel2 molecular markers comprises the following steps:
(1) Extracting the watermelon genome DNA to be detected;
(2) Taking the genomic DNA extracted in the step (1) as a template, performing PCR amplification by using the primer pair of the molecular marker in claim 1, and performing electrophoresis detection and/or sequencing on the PCR amplification product;
(3) And (3) judging according to the electrophoresis band and/or the sequencing result in the step (2), wherein the specific standard is as follows:
if the PCR amplification product only has a characteristic band with the length of 87bp as shown in SEQ ID NO.3, the material to be detected is the watermelon peel intermittent stripe homozygous genotype (ClIS/ClIS); if the PCR amplification product only has a characteristic strip with the length of 95bp as shown in SEQ ID NO.4, the watermelon material to be detected is a peel light green stripe homozygous genotype (Clis/Clis); if the PCR amplification product has a characteristic strip with the length of 87bp shown as SEQ ID NO.3 and a characteristic strip with the length of 95bp shown as SEQ ID NO.4, the watermelon material to be detected is a pericarp intermittent stripe heterozygous genotype (ClIS/Clis).
The method for judging the variety or the genotype of the watermelon peel intermittent stripes by using Indel8 molecular markers comprises the following steps:
(1) Extracting the genomic DNA of the watermelon to be detected;
(2) Performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primer pair of the molecular marker of claim 2, and performing electrophoresis detection and/or sequencing on the PCR amplification product;
(3) And (3) judging according to the electrophoresis band and/or the sequencing result in the step (2), wherein the specific standard is as follows:
if the PCR amplification product only has a characteristic strip with the length of 99bp as shown in SEQ ID NO.7, the watermelon material to be detected is a peel intermittent stripe homozygous genotype (ClIS/ClIS); if the PCR amplification product only has a characteristic strip with the length of 94bp as shown in SEQ ID NO.8, the watermelon material to be detected is the homozygous genotype of the light green stripes on the peel (Clis/Clis); if the PCR amplification product has a characteristic strip with the length of 99bp shown as SEQ ID NO.7 and a characteristic strip with the length of 94bp shown as SEQ ID NO.8, the watermelon material to be detected is a pericarp intermittent stripe heterozygous genotype (ClIS/Clis).
In addition, the kit comprising the primer pair can be used for identifying the peel streak character of the watermelon material, and in the specific application, the reagent containing the molecular marker primer pair can be selected to be made into the kit.
Moreover, the application of the reagent for detecting whether two pairs of Indel markers exist in the positioning of the watermelon peel streak gene ClIS can position the watermelon peel streak gene ClIS by utilizing the molecular marker of the invention, and the applications can be carried out according to the conventional method.
It should be noted that, in the present application, the watermelon peel intermittent stripe gene ClIS and the watermelon peel light green stripe gene ClIS are alleles, wherein the watermelon peel intermittent stripe gene ClIS is dominant controlled (i.e. the watermelon peel stripe containing the gene is intermittent stripe, i.e. the corresponding gene pair is ClIS/ClIS or ClIS/ClIS), and the watermelon peel light green stripe gene ClIS is recessive controlled (i.e. only when the homozygous gene pair ClIS/ClIS, the peel of the watermelon variety shows the light green stripe phenotype).
The invention also protects a carrier containing the molecular marker. The recombinant vector may be an expression vector or a cloning vector into which the molecular marker of the present invention is inserted. After obtaining the above recombinant vector, one skilled in the art can transform the recombinant vector into a suitable cell according to different needs to obtain a recombinant cell containing the recombinant vector. Therefore, the invention also protects a recombinant cell containing the recombinant vector.
The invention has the advantages that:
in the research, an intermittent watermelon stripe homozygous inbred line WT20 and a light green stripe homozygous inbred line WCZ are used as materials to construct F 2 And BC 1 Separating the population, and finely positioning the intermittent stripe character of the watermelon by using a molecular marker technology to find two pairs of Indel markers which are closely linked with the intermittent stripe character, wherein the molecular markers have the advantages of stable amplification, good polymorphism and convenient and efficient detection, so the molecular markers can be directly used for molecular marker assisted breeding of the intermittent stripe material of the watermelon peel. The molecular marker has the advantages of simplicity, convenience, rapidness and high efficiency in an auxiliary selective breeding system, so the molecular marker provided by the invention has a good application value in the cultivation of new varieties of watermelon peel intermittent stripes.
Drawings
FIG. 1 shows watermelon peel intermittent streaking WT20 (left) and light green streaking WCZ (right);
FIG. 2 is a fine positioning diagram of intermittent stripe gene ClIS of watermelon peel;
FIG. 3 shows the Indel2 markers used in the present invention on the parent WT20 and WCZ, F 1 And F 2 Generating polymorphism electrophoresis pattern in plant;
FIG. 4 shows the Indel8 markers used in the present invention on the parent WT20 and WCZ, F 1 And F 2 Polymorphism electrophoresis pattern in generation plants.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
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 indicated, all reagents and materials used are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Moreover, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Plant material:
the watermelon material WT20 is a high-generation inbred line bred by an inventor, the bottom color of the peel of the watermelon material WT is light green, and intermittent stripes are arranged on the peel;
the watermelon material WCZ is a high-generation inbred line bred by an inventor, the bottom color of the peel is light green, and light green thin stripes are arranged on the watermelon material WCZ, and the material can be obtained commercially or provided by a genetic breeding subject group of melon crops of the university of agriculture in Henan province (it needs to be explained that the material is used as a research basis and is only a reason for convenience of obtaining experimental materials, and the realization of the related technical scheme of the application is not understood to be necessarily dependent on the experimental materials);
the WCZ material is consistent with the molecular marker closely linked with the watermelon plant non-tendril gene Clnt and the application thereof adopted in the Chinese invention patent with the publication number of CN 110938706A.
The above biological materials are stored in the laboratories of the applicant units and can be distributed to the public for verification tests within twenty years from the filing date or can be obtained by the public by purchase.
In the experimental process, the two parents and the constructed group are planted in a sunlight greenhouse of a maozhuang scientific education park of Henan university, and the planting process comprises the following steps: and (4) performing plug seedling after pregermination, and counting the stripe type of the individual plant peel on the 10 th day after pollination by adopting a normal watermelon cultivation management mode.
Experimental reagents and equipment:
in the experimental process, PCR Taq-Mix for PCR amplification is purchased from Nanjing Novozam Gene technology Co., ltd; other electrophoresis and silver staining related reagents such as acrylamide, methylene acrylamide, agNO 3 Reagents such as NaOH and formaldehyde were purchased from Beijing Solaibao science and technology, inc.;
the primers for PCR amplification (artificial synthesis) and gene sequencing in the experimental process were provided by Oncorks genome research center, inc.
The PCR instrument is Hema9600 type gene amplification instrument of Zhuhai Black horse medical instrument, inc.;
the electrophoresis apparatus is JY300HC general-purpose electrophoresis apparatus produced by Beijing Junyi Oriental electrophoresis apparatus Co., ltd;
the electrophoresis tank is a HT-SCZ04A high-flux vertical electrophoresis tank and is produced by the company with limited liability to the development of science and technology of great waves in Beijing.
Example 1 localization of ClIS Gene
(ii) construction of genetically isolated populations
Taking intermittent watermelon peel strip material WT20 as male parent, taking light green watermelon peel strip material WCZ as female parent, and utilizing WT20 (P) 1 ) And WCZ (P) 2 ) And constructing a watermelon stripe character segregation population. Hybridization of WT20 with WCZ to obtain F 1 ，F 1 Selfing to obtain F 2 Group of people，F 1 Backcrossing with each parent to obtain BC 1 P 1 (F 1 ×P 1 ) And BC 1 P 2 (F 1 ×P 2 ). The results show that F is obtained 1 The fruit cuticle types all appear as intermittent streaks. (parent material phenotype is shown in FIG. 1.)
From F 1 Selecting 10 individual plants from the plants, and selfing to obtain F 2 Seeds for genetic analysis and gene mapping.
To F 2 And BC 1 The individual's pericarp streak phenotype was identified and verified using the chi-square test. The results show that: spring 2020, 130 strains F 2 100 plants in the population are represented by intermittent stripes of pericarp, 30 plants are represented by light green stripes, and chi-square test accords with the separation ratio of 3: 1; all 59 BC 1 P 1 Plants are all expressed as intermittent stripes of pericarp; 59 BC 1 P 2 Of the plants, 33 showed intermittent streaks of pericarp and 26 showed greenish streaks, with a chi-square test consistent with a 1: 1 split ratio.
For 217 plants F planted in autumn of 2020 2 Population phenotype studies showed that 164 lines appeared as intermittent streaks in the pericarp and 53 lines appeared as pale green streaks, with the chi-square test consistent with a 3: 1 split ratio.
2021 spring planted 849F 2 Stripe survey of the population showed that 622 plants showed intermittent stripes in the pericarp, 227 plants showed greenish stripes, and chi-square test also met a 3: 1 separation ratio.
In conclusion, the intermittent peel streak character of the watermelon peel is controlled by a dominant single gene, the gene is named as ClIS, and the intermittent peel streak (ClIS) is completely dominant to light green peel streak (Clis).
Preliminary positioning of intermittent strips of watermelon peel
(1) Firstly, preparing a gene pool, specifically:
f in the above step (one) 2 Randomly selecting 20 watermelon peel intermittent stripe single plants and 20 light green peel stripe single plants in a group, collecting young leaves, extracting genome DNA by a CTAB method, and respectively mixingAnd (3) combining the watermelon peel intermittent stripe gene pool and the light green peel stripe gene pool (the intermittent stripes are mixed with the intermittent stripes, and the light green peel stripes are mixed with the light green peel stripes).
(2) Polymorphism screening analysis, specifically:
the inventors previously developed 1,257 pairs of SSR primers from the whole Genome of watermelon (specific primer sequences are referred to in Genome and sequence reproduction in molecular mapping and genetic diversity analysis, zhu et al, BMC Genomics,2016, 17 557-573). Of which a total of 532 markers show polymorphisms between parents. And then carrying out second polymorphism screening on the two gene pools prepared in the step (1) by 532 markers with polymorphism between parents to obtain 9 pairs of polymorphism markers which are distributed on the No.6 watermelon chromosome, have good amplification effect and have clear and easily-distinguished bands.
Further, the screened 9 pairs of polymorphic SSR markers are combined with 217 strain F in the 2020 autumn 2 And (3) carrying out genotype analysis on the population, recording the obtained banding pattern which is the same as that of the light green peel streak parent as 2, obtaining the banding pattern which is the same as that of the intermittent streak parent as 1, and obtaining the banding pattern of the heterozygous band as 3. And finally, performing linkage analysis on the typing result by using the joinmap 4.0, and finally positioning the ClIS gene to the watermelon No.6 chromosome.
In the polymorphism screening analysis process, during PCR amplification, a 10-mu L amplification system is designed as follows:
pool samples (genomic DNA,30 ng/. Mu.L), 1. Mu.L (about 30 ng);
F. 0.5. Mu.L of each of the R primers (the primer concentrations are 5. Mu. Mol/L)
PCR Taq-Mix，5.0μL；
dd H 2 O，3.0μL；
The PCR amplification procedure was: 94 ℃ for 5min;94 ℃ C., 30s,55 ℃ C., 30s,72 ℃ C., 30s,35 cycles; 72 deg.C, 5min.
It should be noted that the F and R primers (1257 SSR primers) in the PCR amplification system represent the front and back primers of a pair of primers, respectively, and for the sake of brevity, these primers are not described in detail.
And (3) carrying out 8% non-denaturing polyacrylamide gel electrophoresis detection on the PCR amplification product. During electrophoresis detection, the polyacrylamide gel electrophoresis buffer is 1 × TBE, and electrophoresis is carried out for 1-1.5 h at a constant voltage of 200V. And (3) silver staining is carried out after the electrophoresis is finished so as to facilitate observation and detection, and the silver staining method comprises the following steps:
A. washing with ultrapure water for 1-3 min;
B. placing the washed film into a staining solution which is 0.2% silver nitrate aqueous solution by using a decoloring shaking table, and shaking for 2 min;
C. placing the dyed rubber plate into ultrapure water for bleaching for 30s, placing the dyed rubber plate into a plastic box filled with a developing solution, and slightly shaking until a strip is clearly presented, wherein the developing solution is obtained by adding 15g of NaOH and 15mL of formaldehyde into 1L of distilled water and uniformly mixing;
D. finally put into ddH 2 Repeatedly rinsing for several times;
E. drying at room temperature, taking a picture, wrapping with a preservative film, and storing.
(3) The fine positioning of the intermittent stripes of the watermelon peel specifically comprises the following steps:
both parents were re-sequenced 20 x deep using Illumina Hi-seq2000 high throughput sequencing platform. Combining the results in (2), expanding F by developing more SSR markers 2 The number of individual plants in the population and the watermelon peel intermittent streak gene ClIS are positioned in the genetic distance of 230Kb between the ClSSR18071 and the ClSSR180298 of No.6 chromosomes. To further narrow the candidate interval and obtain more additional molecular markers for fine localization, we developed two pairs of dCAPS markers and two pairs of Indel markers between ClSSR18071 and ClSSR180298 based on the re-sequencing data between the two parents, and these four pairs of markers were only used for polymorphism analysis of the recombinant individual. The final candidate gene ClIS was mapped between the two markers dCAPS-C and dCAPS-E, and the ClIS gene was finally mapped precisely to an interval of 160Kb between 25.92Mb and 26.08Mb on chromosome 6 of watermelon (FIG. 2), depending on the physical location of the markers.
(4) Development of candidate region Indel markers
The invention utilizes the Indel2 molecular marker to carry out PCR amplification distinction, and the primer sequence is designed as follows:
Indel2-F：5’-GATGGGTGACTGCAGGAGTT-3’(SEQ ID NO.1)；
Indel2-R：5’-AAGAGGAGCTGTCGATCCAA-3’(SEQ ID NO.2)。
obtaining an 87bp characteristic band through PCR amplification, wherein the specific base sequence of the 87bp characteristic band is as follows:
obtaining a 95bp characteristic band through PCR amplification, wherein the specific base sequence of the 95bp characteristic band is as follows:
the size of the amplification product of the primer in a male parent (intermittent stripe) is 87bp, and the size of the amplification product in a female parent (light green stripe) is 95bp.
The invention utilizes the Indel8 molecular marker to carry out PCR amplification, and the primer sequence is designed as follows:
Indel8-F：5’-TGTTTAACTCGGCGTTCTTG-3’(SEQ ID NO.5)；
Indel8-R：5’-GGATACCATGATTAAAATCCCTCTG-3’(SEQ ID NO.6)。
obtaining a 99bp characteristic band through PCR amplification, wherein the specific base sequence of the 99bp characteristic band is as follows:
obtaining a 94bp characteristic band through PCR amplification, wherein the specific base sequence of the 94bp characteristic band is as follows:
the size of the amplification product of the primer in the male parent (intermittent stripe) is 99bp, and the size of the amplification product in the female parent (light green stripe) is 94bp.
Example 2 application of molecular markers in Breeding
(1) DNA extraction, PCR amplification and electrophoresis detection
DNA extraction is carried out by adopting a conventional method CTAB method, and PCR reaction procedures and polyacrylamide gel electrophoresis procedures are referred to a Master of the Yankee society: the detailed location and functional verification of the gene Clbl of the watermelon few lateral branches, henan university of agriculture, 2021.
Respectively aligning F with Indel2 and Indel8 molecular markers of the invention 2 The distribution in the population was genotyped.
Specifically, the application of Indel2 molecular markers in breeding comprises the following steps:
PCR reaction Using Indel2 molecular marker to confirm the molecular marker at F 2 Genotype in a population. The two genotypes of the Indel2 molecular marker are detected at 1066F 2 The distribution condition in the population (partial detection results are shown in figure 3), and the genotypes of the individual plants can be rapidly distinguished according to the amplified strip condition; if only one PCR amplification product has the size of 87bp, the single-plant fruit is the watermelon peel intermittent stripe homozygous genotype; if the PCR amplification product only has a characteristic strip with the length of 95bp, the single-plant fruit is the watermelon light green stripe homozygous genotype; if the PCR amplification product has a characteristic strip with the length of 87bp and a characteristic strip with the length of 95bp, the single fruit plant is the heterozygous genotype of the watermelon peel intermittent stripes.
The application of Indel8 molecular marker in breeding comprises the following steps:
PCR reaction Using Indel8 molecular marker to confirm the molecular markerF 2 Genotype in the population. The two genotypes of the Indel8 molecular marker are detected at 1066F 2 The distribution of the population (partial detection results refer to fig. 4), if the PCR amplification product only has a characteristic strip with the length of 99bp as shown in SEQ ID NO.7, the single plant fruit is the watermelon peel intermittent stripe homozygous genotype; if the PCR amplification product only has a characteristic strip with the length of 94bp as shown in SEQ ID NO.8, the single plant fruit is the homozygous genotype of the watermelon light green stripes; if the PCR amplification product has a characteristic strip with the length of 99bp as shown in SEQ ID NO.7 and a characteristic strip with the length of 94bp as shown in SEQ ID NO.8, the single fruit plant is the watermelon peel intermittent stripe heterozygous genotype.
Therefore, according to the amplified band condition, the genotype of the individual plant can be rapidly distinguished; further, the inventors have combined phenotypic data to find that the phenotype of natural material is consistent with the genotype. Based on the result, the molecular markers Indel2 and Indel8 can effectively distinguish the watermelon light green stripe material and the watermelon peel intermittent stripe material.
In conclusion, the molecular markers Indel2 and Indel8 can accurately perform molecular marker-assisted selection in the early stage of watermelon growth by combining electronic BSA analysis and verification in natural populations, and greatly improve the processes of selection and breeding. Provides theoretical support and technical support for the cultivation of high-quality new species of watermelon.
Claims (9)
1. The Indel2 molecular marker closely linked with the intermittent stripe gene ClIS of the watermelon peel is characterized in that an upstream primer sequence of a primer pair for amplifying the molecular marker is shown as SEQ ID No.1, and a downstream primer sequence is shown as SEQ ID No. 2.
2. The Indel8 molecular marker tightly linked with the intermittent stripe gene ClIS of the watermelon peel is characterized in that an upstream primer sequence of a primer pair for amplifying the molecular marker is shown as SEQ ID No.5, and a downstream primer sequence is shown as SEQ ID No. 6.
3. The use of the molecular marker tightly linked with the intermittent streak gene ClIS of watermelon peel as claimed in claim 1 or claim 2 in molecular breeding of watermelon.
4. The use of claim 3, wherein the molecular marker is used for identifying or assisting in the selection of the intermittent streaking trait of watermelon peel.
5. The method for judging the variety/genotype of the watermelon peel stripe is characterized by comprising the following steps of:
(1) Extracting the genomic DNA of the watermelon to be detected;
(2) Taking the genomic DNA extracted in the step (1) as a template, performing PCR amplification by using the primer pair of the molecular marker in claim 1, and performing electrophoresis detection and/or sequencing on the PCR amplification product;
(3) And (3) judging according to the electrophoresis band and/or the sequencing result in the step (2), wherein the specific standard is as follows:
if the PCR amplification product only has a characteristic strip with the length of 87bp as shown in SEQ ID NO.3, the watermelon material to be detected is a peel intermittent stripe homozygous variety/genotype; if the PCR amplification product only has a characteristic strip with the length of 95bp as shown in SEQ ID NO.4, the watermelon material to be detected is a peel light green stripe homozygous variety/genotype; if the PCR amplification product has a characteristic strip with the length of 87bp shown as SEQ ID NO.3 and a characteristic strip with the length of 95bp shown as SEQ ID NO.4, the watermelon material to be detected is a pericarp intermittent stripe heterozygous variety/genotype.
6. The method for judging the variety/genotype of the watermelon peel stripe is characterized by comprising the following steps of:
(1) Extracting the genomic DNA of the watermelon to be detected;
(2) Performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primer pair of the molecular marker of claim 2, and performing electrophoresis detection and/or sequencing on the PCR amplification product;
(3) And (3) judging according to the electrophoresis band and/or the sequencing result in the step (2), wherein the specific standard is as follows:
if the PCR amplification product only has a characteristic strip with the length of 99bp as shown in SEQ ID NO.7, the watermelon material to be detected is a peel intermittent stripe homozygous variety/genotype; if the PCR amplification product only has a characteristic strip with the length of 94bp as shown in SEQ ID NO.8, the watermelon material to be detected is a pure variety/genotype with light green peel stripes; if the PCR amplification product has a characteristic strip with the length of 99bp as shown in SEQ ID NO.7 and a characteristic strip with the length of 94bp as shown in SEQ ID NO.8, the watermelon material to be detected is a peel intermittent stripe heterozygous variety/genotype.
7. A kit for identifying intermittent stripe traits of watermelon, which comprises the primer pair as claimed in claim 1 or claim 2.
8. The application of the reagent for detecting the existence of Indel2 marker in the positioning of watermelon peel streak gene ClIS is characterized in that the sequence of the upstream primer of the primer pair for amplifying the marker is shown as SEQ ID NO.1, and the sequence of the downstream primer is shown as SEQ ID NO. 2.
9. The application of the reagent for detecting whether Indel8 marker exists in the positioning of the watermelon peel streak gene ClIS is characterized in that the sequence of an upstream primer of a primer pair for amplifying the marker is shown as SEQ ID No.5, and the sequence of a downstream primer is shown as SEQ ID No. 6.
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