Molecular markers co-segregated with watermelon fruit size gene and its application

Abstract

The application discloses a molecular marker co-segregated with a watermelon fruit size gene Cllf and application thereof, and belongs to the field of molecular breeding. The application can be directly used for molecular marker assisted breeding of the small fruit trait of the watermelon, improves the selection efficiency of breeding, and speeds up the breeding process. The application of the marker to molecular marker assisted selection breeding can quickly and accurately realize directional genetic improvement of the fruit size of the watermelon, and provides a fast and simple identification technology for molecular detection of the small fruit trait of the watermelon. Meanwhile, the application lays a theoretical foundation for cloning the fruit size gene Cllf of the watermelon and analyzing a molecular regulation network of the fruit size.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a dCAPS marker co-segregated with the fruit size trait of watermelon and application thereof. BACKGROUND

[0002] The selection of genotypes is one of the most important steps in breeding, which refers to selecting genotypes meeting the requirements for subsequent breeding in a population. In the traditional breeding process, the selection is usually based on the appearance phenotype of plants, which is time-consuming and may be inaccurate due to ambiguous traits, resulting in selection errors and low efficiency. The use of molecular marker-assisted breeding can quickly detect target genes or loci co-segregated with target traits from seedlings, achieving the purpose of selecting target traits, and having the advantages of rapidness, accuracy and non-interference by environmental conditions.

[0003] The dCAPS (derived cleaved amplified polymorphic sequences) marker is improved on the basis of the CAPS (cleaved amplified polymorphic sequences) marker. The basic principle is that a mismatched base is introduced when designing the proposed amplification primer, so that a new restriction endonuclease site can be generated. Then, the amplification product obtained by PCR amplification with the primer is subjected to enzyme digestion with a specific restriction endonuclease, and finally the enzyme digestion fragments are detected by polyacrylamide gel electrophoresis, and the polymorphism between samples is determined according to whether the fragments are cut or not. The dCAPS marker is a co-dominant marker that can distinguish between heterozygous and homozygous genotypes, and can be directly analyzed by polyacrylamide gel electrophoresis, which is simple and fast. The dCAPS marker does not need to consider whether the SNP is on the restriction endonuclease site, and can maximize the conversion of SNPs into markers, making the utilization rate of SNPs on the genome higher.

[0004] Watermelon (Citrullus lanatus) belongs to an annual vine herb of the Cucurbitaceae family and the Citrullus genus, and is widely planted worldwide. With the development of the watermelon industry, small fruit gift watermelons suitable for facility cultivation are increasingly popular in the market, and people have put forward new requirements for high-quality small fruit watermelon varieties. Therefore, it is particularly urgent to mine and screen excellent fruit size control genes with important application value in watermelon, and to analyze their action mechanisms and regulatory networks.

[0005] Fruit size, as an important agronomic trait, is also an important index affecting the economic value of horticultural crops. Currently, in horticultural crops such as citrus, apple, loquat and tomato, a series of progress has been made in the study of fruit size. In citrus, Li found that R2R3-MYB transcription factor CsMYB77 negatively regulates fruit ripening in citrus and tomato, and also plays a role in regulating citrus fruit size; in loquat, Su first found that cell size is the main factor affecting loquat fruit size, and analyzed the mechanism of EjBZR1 regulating loquat cell swelling and fruit size by affecting brassinosteroid biosynthesis pathway; in apple, Wang et al. found a MdAP2-like transcription factor that can regulate fruit softening and fruit size, providing valuable gene resources for breeding new varieties of large fruit type apple resistant to storage and transportation; in watermelon, Li constructed three populations from a small fruit new germplasm and different inbred lines with medium, large and giant fruit, respectively, and whole genome QTL mapping revealed a significant QTL (fs-chr8) on chromosome 8 related to fruit size. Compared with other crops, the study of fruit size genes in watermelon is still relatively weak, and this field is still in its infancy. Mining more fruit size genes and performing functional verification will be crucial for creating excellent small fruit type watermelon and its application in new variety breeding.

[0006] In recent years, the research on molecular breeding of watermelon has developed rapidly, and many excellent traits have been located. Some co-segregation markers developed with the regulatory trait genes have also been used in the process of molecular marker-assisted breeding. Through the study of small fruit traits in watermelon, molecular markers co-segregated with small fruit traits in watermelon are developed, which not only can provide effective help for molecular marker-assisted selection of watermelon to breed new varieties with small fruit, but also can greatly shorten the breeding process and improve the accuracy of selection. SUMMARY

[0007] One of the purposes of the present application is to provide a pair of dCAPS markers co-segregated with the fruit size gene of watermelon.

[0008] The second purpose of the present application is to provide the application of the above-mentioned molecular markers in watermelon molecular breeding.

[0009] The third purpose of the present application is to provide a determination method for identifying the fruit size trait / genotype of watermelon.

[0010] To achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0011] The molecular marker co-segregated with the watermelon fruit size gene Cllf is a dCAPS molecular marker, named lf-dCAPS marker, which is developed based on the fruit size gene Cllf, and the sequence of the upstream primer of the primer pair for amplifying the lf-dCAPS marker is shown in SEQ ID NO. 1, and the sequence of the downstream primer is shown in SEQ ID NO. 2.

[0012] The application also discloses application of the molecular marker co-segregated with the watermelon fruit size gene Cllf in watermelon molecular breeding, and the molecular marker co-segregated with the watermelon fruit size trait can assist in identifying whether a watermelon plant is of a small fruit phenotype at a molecular level, and the watermelon plant trait can be determined at a seed or seedling stage, so that selection efficiency is improved and breeding process is accelerated.

[0013] The application also discloses a method for determining a watermelon fruit size trait / genotype, which adopts a method of PCR amplification and enzyme digestion, and the method comprises the following steps:

[0014] (1) extracting DNA of watermelon tissue;

[0015] (2) PCR amplification: using the primer pair with the sequence of the upstream primer shown in SEQ ID NO. 1 and the sequence of the downstream primer shown in SEQ ID NO. 2, the sample extracted in step (1) is subjected to PCR amplification;

[0016] (3) performing enzyme digestion on the amplification product in step (2), and then performing electrophoresis detection;

[0017] (4) determining according to the electrophoresis band result of step (3), and the specific standard is that:

[0018] if the enzyme digestion product is a characteristic band with a length of 118 bp, the plant to be determined is a homozygous small fruit watermelon material; if the enzyme digestion product is a characteristic band with a length of 95 bp, the plant to be determined is a homozygous large fruit watermelon material, and if the enzyme digestion product is two characteristic bands with lengths of 95 bp and 118 bp respectively, the plant to be determined is a heterozygous large fruit watermelon material;

[0019] or, expressed as follows:

[0020] If the enzyme digestion product has only one characteristic band of 118bp as shown in SEQ ID NO. 3, the watermelon to be tested is a homozygous small fruit trait watermelon material / genotype; if the enzyme digestion product has only one characteristic band of 95bp as shown in SEQ ID NO. 4, the watermelon to be tested is a homozygous large fruit trait watermelon material / genotype; if the enzyme digestion product has both a characteristic band of 118bp as shown in SEQ ID NO. 3 and a characteristic band of 95bp as shown in SEQ ID NO. 4, the watermelon to be tested is a heterozygous large fruit trait watermelon material / genotype.

[0021] Specifically, the reaction system of the PCR amplification is as follows: 1 μL of DNA, 5 μL of 2X PCR Mix, 0.5 μL of the upstream primer, 0.5 μL of the downstream primer, 3 μL of sterilized distilled water, and the total volume is 10 μL. The PCR amplification conditions are as follows: 95℃, 5 min; 94℃, 30 s, 56℃, 30 s, 72℃, 50 s, for a total of 35 cycles; 72℃, 10 min; 4℃ preservation.

[0022] The enzyme digestion reaction system is as follows: 5 μL of PCR product, 1 μL of 10X buffer, 0.5 μL of TaqI restriction endonuclease, 3.5 μL of sterilized distilled water, and the total volume is 10 μL. The enzyme digestion conditions are as follows: 37℃, 4 h.

[0023] In addition, the kit containing the above primer pair can be used to identify whether the watermelon material is a small fruit watermelon. In specific applications, a kit containing the primer pair can be selected.

[0024] Here, the molecular marker of the present application can also be used to map the watermelon fruit size gene Cllf. The above applications can be performed according to conventional methods.

[0025] The present application also protects a vector containing the above molecular marker. The recombinant vector can be an expression vector or a cloning vector into which the molecular marker of the present application is inserted. After obtaining the above recombinant vector, those 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 present application also protects a recombinant cell containing the recombinant vector.

[0026] Advantages of the present application:

[0027] The molecular marker of the present application can lay a foundation for cloning the Cllf gene and further researching the molecular mechanism of the fruit size of watermelon. Meanwhile, the above-mentioned molecular marker can be directly used for molecular marker assisted breeding of the small fruit material of watermelon. In the process of selection breeding of watermelon, a large number of segregating populations are often produced. By using the molecular marker, the required plants can be identified in the seedling stage, so that the land area in the breeding process is reduced, and the human and material resources required for identification after the plants grow up are also reduced, thereby greatly improving the efficiency of breeding and shortening the selection period. Therefore, the molecular marker provided by the present application has good application value in new variety breeding.

[0028] The present application has carried out a detailed study on the fruit size related gene of watermelon plant, and finally determines a dCAPS molecular marker co-segregated with the fruit size gene Cllf of watermelon by using BSA-seq and molecular marker and other related research methods. The lf-dCAPS molecular marker in the interval is co-segregated with the fruit size trait of watermelon. This finding lays a foundation for cloning the Cllf gene, establishing a molecular marker assisted breeding system, and also helps to lay a foundation for studying the regulation network of the fruit size of watermelon. In summary, since the molecular marker has very important significance for the final functional gene positioning, and the molecular marker has the advantages of simplicity, rapidness, high throughput in the establishment of a molecular marker breeding system, the present application has very important application value, and also has very important protection significance for the breeding of new varieties of watermelon. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a phenotype photo (Fig. A, B) and statistical chart (Fig. A, B) of related traits (Fig. C, D, E, F) of the normal material G42 and the small fruit material G42lf of watermelon (in Fig. A, B, left is G42, right is G42lf, the scale of Fig. A is 5mm, and the scale of Fig. B is 5cm);

[0030] Figure 2 It is a fine mapping diagram of the fruit size trait gene Cllf of watermelon;

[0031] Figure 3 It is a gel electrophoresis diagram of the co-segregation marker lf-dCAPS in the parent and F2 population; in the diagram, M is DL2000 Marker, and the bands of 100bp and 250bp are shown in the diagram; P1 represents the normal material G42 of watermelon, and P2 represents the small fruit material G42lf of watermelon;

[0032] Figure 4 It is a gel electrophoresis diagram of the co-segregation marker lf-dCAPS in the parent and 30 natural populations; in the diagram, M is DL2000 Marker, and the bands of 100bp and 250bp are shown in the diagram; P1 represents the normal material G42 of watermelon, and P2 represents the small fruit material G42lf of watermelon. Detailed Implementation

[0033] The following is a further explanation based on the application. Before introducing the specific embodiments, the biological materials, experimental reagents and related experimental background of the following embodiments are briefly introduced as follows.

[0034] Biomaterials:

[0035] Watermelon material G42 (male parent) is a homozygous inbred line bred through multiple generations. This material produces normal watermelon fruits and can be stably inherited. Figure 1 (A left) The fruit is large, which is referred to as the normal phenotype below.

[0036] The small watermelon fruit material G42lf (mother parent) was obtained from G42 through EMS mutagenesis, and its ovary size ( Figure 1 C) Fruit size Figure 1 A right, Figure 1 D) Fruit weight Figure 1 E) was significantly smaller than G42, and the fruit sugar content was not affected. Figure 1 F); The small fruit phenotype is relative to the large fruit of normal materials, and the obtained G42lf material can be stably inherited during later planting.

[0037] The watermelon material G42 mentioned above is the same as the normal ordinary material G42 used in the Chinese invention patent "Watermelon Male Sterility Gene ClMS1 and Its Application" with publication number CN115385994B.

[0038] G42lf was derived from seeds of the M1 generation watermelon mutant of the G42 material (the seeds were donated by Deng Yun of the Institute of Modern Agriculture, Peking University; the M1 generation watermelon mutant seeds were obtained using EMS mutagenesis, the specific method of which is described in the published article "A telomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding"). After several generations of planting and screening, homozygous mutants were identified in the M2 and M3 generation seeds. Among them, the material phenotyped as having fruits smaller than the normal watermelon material G42 possesses excellent traits for breeding watermelon materials in production. Therefore, gene mapping studies were conducted on this material, and molecular markers were developed based on this. The material is deposited at the Cucurbit Crop Genetics and Breeding Research Group of Henan Agricultural University.

[0039] The above materials are preserved in the applicant's laboratory (Guacrops Genetic Breeding Group of Henan Agricultural University) and can be released to the public for verification tests or obtained by purchase within twenty years from the application date.

[0040] During the experiment, the watermelon materials were planted in the sunlight greenhouse of Maozhuang Science and Education Park of Henan Agricultural University. During the planting process, after germination, plug seedling was carried out, and normal watermelon cultivation and management methods were adopted. After planting, the related phenotypic traits were preliminarily investigated and counted when the female flowers opened, and the related phenotypic traits were investigated and confirmed again 30 days after pollination.

[0041] The primer for PCR amplification and gene sequencing work were provided and completed by Beijing Nosy Genomics Research Center Co., Ltd.

[0042] Experimental reagents:

[0043] PCR 2x 3G Taq Master Mix for PCR amplification was purchased from Nanjing Novozyme Biotech Co., Ltd.

[0044] Restriction endonuclease TaqI was purchased from Guangzhou Yisen Biological Technology Co., Ltd.

[0045] Electrophoresis and silver staining related reagents such as acrylamide, methylene acrylamide, AgNO3, NaOH and formaldehyde were purchased from Beijing Solaybao Technology Co., Ltd.

[0046] Experimental equipment:

[0047] PCR instrument, Zhuhai Hema Medical Instrument Co., Ltd. Hema9600 type gene amplification instrument

[0048] JY300HC universal electrophoresis instrument produced by Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.

[0049] HT-SCZ04A high-throughput vertical electrophoresis tank produced by Beijing Hongtao Jiye Technology Development Co., Ltd.

[0050] Examples

[0051] This example mainly introduces the fine mapping process of the watermelon fruit size gene Cllf, including the construction of genetic segregation population, preliminary positioning and other processes. In this process, the development, design and testing process of the lf-dCAPS molecular marker finally co-segregated with the watermelon small fruit trait are involved. The related experimental process is briefly introduced as follows.

[0052] I. Construction of genetic segregation population

[0053] The watermelon small fruit material G42lf( Figure 1 A right) was used as the female parent, and the watermelon fruit normal material G42 (Figure 1 A left) as the father (need to explain that during the experiment, from the material availability and easy to operate, the inventor with G42 as the father, in the use of other homozygous normal material, can also be related experiments), the use of these two parents configured hybrid combination, the results show that the F1 generation fruit size is normal.

[0054] From the F1 generation plants, 10 single plants were selected, selfed to obtain F2 generation seeds for genetic analysis and gene mapping. The phenotypes of these F2 individuals were identified, and verified by chi-square test.

[0055] The results show that:

[0056] Planting F2 population containing 308 plants, including 250 plants with normal phenotype, 67 plants with small fruit phenotype, χ 2 = 0.216 < 3.84, consistent with the segregation ratio of 3:1. Analysis shows that the small fruit trait in G42lf is controlled by a pair of recessive genes, which is named as Cllf, and the normal phenotype (LF) is completely dominant over the small fruit phenotype (lf).

[0057] II. Preliminary mapping of the gene

[0058] Mutmap method for mutant mapping was used for preliminary mapping, and the specific experimental process was as follows:

[0059] (1) First, two fruit size pools, specifically:

[0060] In the F2 population of step one above, 20 normal phenotype single plants and 20 small fruit phenotype single plants were randomly selected, and the relatively tender leaves were collected from each single plant 30 days after pollination. The CTAB method was used to extract the genomic DNA, and the normal gene pool and small fruit gene pool were mixed respectively (normal phenotype mixed with normal phenotype, small fruit phenotype mixed with small fruit phenotype).

[0061] (2) Whole genome resequencing of parents and pools, specifically:

[0062] The normal fruit gene pool, small fruit gene pool and genome of the parents constructed in (1) above are subjected to >40x whole genome resequencing using an Illumina Hi-seq2000 high-throughput sequencing platform. The process mainly comprises the following four steps: (i) library construction: the qualified genomic DNA is physically broken (ultrasonic oscillation) to the desired fragment (350 bp), and then subjected to end repair, A addition, adapter addition, target fragment selection and PCR to construct a small fragment sequencing library; (ii) library quality inspection: the Qseq400 and Qubit are used to detect the library fragment size and library quantification to determine whether the library meets the sequencing standard; (iii) chip fixation: the library is fixed to the sequencing chip through bridge PCR; (iv) sequencing: the library is subjected to double-end 150 bp (PE 150) sequencing using an Illumina sequencer, and the data generated by sequencing are subjected to quality control and then used for information analysis in the next step.

[0063] (3) Mutmap analysis, specifically:

[0064] Firstly, the sequencing data are subjected to sequencing adapter removal using trim_galore, and the data after adapter removal are analyzed using mutmap software. The watermelon T2T reference genome G42 (http: / / www.watermelondb.cn / # / geneInfo?group=G42.nogap.v4) is used as the reference genome, and the G42 resequencing data are wild type sequences, and the small fruit gene pool sequencing data are mutant mixed pool data.

[0065] According to the mutmap software analysis results, a significant peak appears on chromosome 8, and the gene is preliminarily located in a 2.7 Mb candidate interval at the end of chromosome 8 according to the SNP-index value. Figure 2 ).

[0066] III. Development and linkage verification of co-segregation molecular markers

[0067] On the basis of the preliminary location in step two, the inventors further develop and verify the linkage of co-segregation molecular markers for the watermelon fruit size gene Cllf, and the specific process is briefly introduced as follows.

[0068] (1) Development of molecular markers:

[0069] The sequence in the 2.7 Mb candidate interval in the preliminary positioning was taken as a reference sequence, and the resequencing sequences of the two parents and the candidate interval were respectively aligned by using the free software package BWA (http: / / bio-bwa.sourceforge.net / ) disclosed on the network, and the difference sites of the two parents in the candidate interval were found. Then, the SNPs in the candidate interval were screened out, and the appropriate restriction endonuclease was screened out by dCAPS Finder 2.0 software, and the SNP site was converted into a dCAPS marker.

[0070] (2) Linkage verification of co-segregation molecular markers:

[0071] The designed dCAPS marker was used for genotype analysis of 308 strains in the F2 population. The obtained band shape same as the normal parent G42 was recorded as "1", the obtained band shape same as the small fruit parent G42lf was recorded as "2", and the obtained heterozygous band shape was recorded as "3".

[0072] The typing results of the above molecular markers were analyzed, and the results showed that the SNP site mutation from C to T of the small fruit material G42lf in the candidate interval led to the mutation from arginine to tryptophan, and the dCAPS marker developed based on the site co-segregated with the small fruit trait in the F2 population ( Figure 3 ), which was named lf-dCAPS.

[0073] The molecular marker lf-dCAPS is shown in SEQ ID NO. 3 or SEQ ID NO. 4, and the upstream primer (lf-dCAPS-F) and the downstream primer (lf-dCAPS-R) of the primer pair for amplifying the molecular marker are as follows:

[0074]

[0075] The enzyme digestion product size of the primer in the father (normal material) is 95 bp, and the enzyme digestion product size in the mother (small fruit material) is 118 bp. Specifically:

[0076] The 118 bp characteristic band obtained by enzyme digestion is as follows:

[0077]

[0078] The 95 bp characteristic band obtained by enzyme digestion is as follows:

[0079]

[0080] The PCR reaction system was carried out according to the standard process of PCR reaction program, and the PCR reaction system is shown in Table 1.

[0081] Table 1 PCR reaction system

[0082]

[0083] The PCR amplification program was: 94℃, 5min; 94℃, 30s, 55℃, 30s, 72℃, 30s, 35 cycles; 72℃, 5min.

[0084] The enzyme digestion reaction system is shown in Table 2:

[0085] Table 2 Enzyme digestion reaction system

[0086]

[0087] The enzyme digestion condition was 37℃, 4h.

[0088] 8% non-denaturing polyacrylamide gel electrophoresis was carried out for detection. When the electrophoresis detection was carried out, the polyacrylamide gel electrophoresis buffer was 0.6xTBE, and the constant voltage electrophoresis was carried out at 200V for 1-1.5h. After the electrophoresis was completed, silver staining was carried out for observation and detection, and the silver staining method was as follows:

[0089] A, the glass plate with glue was put into the fixing liquid, and gently shaken on the shaking table until the indicator faded, wherein the composition of the fixing liquid was that the volume ratio of glacial acetic acid: anhydrous ethanol: distilled water was 0.5:10:100;

[0090] B, washed with ultrapure water for 1-3min;

[0091] C, the washed gel plate was put into the staining liquid and shaken for 10min, and the staining liquid was 0.2% silver nitrate aqueous solution;

[0092] D, the stained gel plate was put into ultrapure water for 30s, put into a plastic box containing developing liquid, and gently shaken until the bands were clear, and the developing liquid was obtained by mixing 15g NaOH and 3mL formaldehyde in 1L distilled water;

[0093] E, finally put into tap water and rinse several times;

[0094] F, dry at room temperature, and then take a picture.

[0095] Four, application of molecular markers

[0096] (1) DNA extraction, PCR amplification and electrophoresis detection

[0097] The DNA extraction was performed by the conventional CTAB method, and the PCR reaction procedure and polyacrylamide gel electrophoresis process were referred to Yang Hui's master thesis: Fine mapping and functional verification of watermelon less lateral branch gene Clbl, Henan Agricultural University, 2021.

[0098] (2) Watermelon small fruit material / genotype detection

[0099] The primers are used for PCR amplification of the watermelon samples (parents and F2 population) to be tested, and the results show that if the product after enzyme digestion is a characteristic band with a length of 118 bp, the plant to be tested is a homozygous watermelon small fruit material; if the product after enzyme digestion is a characteristic band with a length of 95 bp, the plant to be tested is a homozygous watermelon large fruit material, and if the product after enzyme digestion is two characteristic bands with lengths of 95 bp and 118 bp, respectively, the plant to be tested is a heterozygous watermelon large fruit material.

[0100] Therefore, according to the amplified band, the material / genotype / character of a single plant can be quickly distinguished; further, the inventors use the lf-dCAPS molecular marker to perform PCR amplification and gel electrophoresis detection on two parents and a natural population (randomly selected 30 watermelon materials) Figure 4 Combined with the phenotype data, it is found that the phenotype of the parents and the randomly selected 30 watermelon materials is consistent with the actual determination result (Table 3), indicating that the lf-dCAPS marker of the present application has a 100% auxiliary selection rate for small fruit molecular markers in the natural population of watermelon. Based on this result, it is shown that the molecular marker lf-dCAPS of the present application can effectively distinguish whether a watermelon plant is a small fruit material.

[0101] Table 3 Number and phenotype of 30 watermelon natural materials for verification

[0102]

[0103]

[0104] In summary, the derived restriction enzyme digestion and amplification sequence polymorphism marker of the present application can be used for large-scale identification of watermelon small fruit traits, which is not only fast and effective, but also can be identified at the seedling stage, greatly shortening the breeding cycle and can be widely used in production.

[0105] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A dCAPS molecular marker co-segregating with the watermelon fruit size gene, characterized in that, The nucleotide sequence of the dCAPS molecular marker is shown as SEQ ID NO. 3, wherein a C / T mutation exists at the 96th position of the sequence shown in SEQ ID NO. 3; the sequence of the upstream primer of the primer pair for amplifying the molecular marker is shown as SEQ ID NO. 1, and the sequence of the downstream primer is shown as SEQ ID NO. 2, wherein the watermelon material is G42lf and its offspring.

2. Use of the dCAPS molecular marker co-segregating with the watermelon fruit size gene of claim 1 for identifying or aiding in the identification of the watermelon small fruit trait, wherein, The watermelon material is G42lf and its offspring.

3. A method for determining the size of a watermelon fruit, characterized by, The method comprises the following steps: (1) extracting the genomic DNA of the watermelon sample to be tested; (2) using the genomic DNA extracted in step (1) as a template, and using the primer pair in claim 1, performing PCR amplification on the sample extracted in step (1); (3) performing TaqI restriction endonuclease enzyme cutting treatment on the amplification product in step (2), and then performing polyacrylamide gel electrophoresis detection; (4) determining according to the electrophoresis band results of step (3), and the specific standard is as follows: if the enzyme cutting product only has one characteristic band with a length of 118 bp as shown in SEQ ID NO. 3, then the watermelon to be tested is a homozygous small-fruit watermelon material; if the enzyme cutting product only has one characteristic band with a length of 95 bp as shown in SEQ ID NO. 4, then the watermelon to be tested is a homozygous large-fruit watermelon material; if the enzyme cutting product has both one characteristic band with a length of 118 bp as shown in SEQ ID NO. 3 and one characteristic band with a length of 95 bp as shown in SEQ ID NO. 4, then the watermelon to be tested is a heterozygous large-fruit watermelon material, wherein the watermelon material is G42lf and its offspring.

4. The method of claim 3, wherein, The DNA of the test material is extracted by using the CTAB method.

5. The method of claim 3, wherein, The enzyme cutting reaction system in step (3) is as follows: 5 μL of PCR product, 1 μL of 10X buffer, 0.5 μL of TaqI restriction endonuclease, 3.5 μL of sterilized distilled water, and a total volume of 10 μL; the enzyme cutting conditions are 37℃ and 4 h.

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