Molecular marker co-segregated with watermelon short hair male sterility gene and its application

Abstract

The application discloses a molecular marker co-segregated with a watermelon short-hair male sterility gene and application thereof, and belongs to the technical field of genetic engineering. The application can be directly used for molecular marker assisted breeding of the watermelon short-hair male sterility material, improves the selection efficiency of breeding, and speeds up the breeding process. It is worth noting that the short-hair male sterility material is different from the conventional male sterility material. The short-hair trait and the male sterility trait of the short-hair male sterility material are co-segregated, so in actual production, the male sterile plants can be screened from the male sterile maintainer line through the short-hair trait of the plants in the seedling stage, thereby obtaining 100% sterile male sterile plants, and the breeding time is saved. Meanwhile, the application lays a foundation for finally realizing cloning of the watermelon short-hair male sterility gene Clstms and regulation of the anther development and the epidermal hair development of the watermelon, thereby improving the hybrid seed production efficiency, shortening the breeding period, improving the seed purity, and reducing the seed production cost.

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 a short-hairy male sterility trait of watermelon and application thereof. BACKGROUND

[0002] Genotype selection is one of the most important steps in breeding, which refers to selecting genotypes meeting the requirements in a population for subsequent breeding. In traditional breeding, the selection basis is often determined by the appearance of the phenotype, which is time-consuming and may be biased due to ambiguous traits, resulting in selection errors and low efficiency. Molecular marker-assisted breeding can quickly detect target genes or loci co-segregated with target traits from the seedling stage, achieving the purpose of selecting target traits, and has the advantages of rapidness, accuracy, and no interference from environmental conditions.

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

[0004] Watermelon (Citrullus lanatus) belongs to the annual vine herb of Cucurbitaceae and Citrullus, and is known as the king of summer fruits. As an annual plant, it is propagated by seeds. At the same time, due to the obvious heterosis of watermelon, most of the commercial varieties on the market are hybrids. Therefore, a large number of hybrid seeds need to be produced manually for watermelon cultivation every year.

[0005] At present, the production of watermelon hybrid mainly relies on artificial emasculation and pollination, which not only requires high technology and is labor-intensive, but also is time-consuming, labor-intensive and costly, and it is difficult to ensure the purity of hybrid. Using male sterile lines for seed production can significantly reduce the dependence on artificial labor, effectively reduce the cost of seed production, and improve the purity of hybrid. Researchers at home and abroad have excavated a number of excellent male sterile genes in various plant male sterile mutants and applied them in hybrid seed production. At present, it is particularly urgent to explore the use of male sterile lines for watermelon hybrid production, which is the key to improving hybrid seed production efficiency, reducing seed production cost and realizing simple seed production. It is of great significance to accelerate the molecular breeding of watermelon male sterility and simple seed production. However, there are very few male sterile mutants found in watermelon, and the number of male sterile genes that have been finely mapped is very limited. Therefore, it is particularly urgent to excavate and screen excellent male sterile genes with important application value in watermelon and analyze their action mechanism and regulatory network.

[0006] So far, three watermelon male sterile genes have been reported. In 2021, Dong et al. located the gene controlling the G17AB male sterile trait on chromosome 6 in a 0.32 Mb candidate region, and speculated that Cla006625 (ClaPEX1) was the candidate gene. In the same year, Zhang et al. finely mapped the sterile gene in Se18 male sterile line, and confirmed that the gene bHLH transcription factor Cla010576 (ClATM1) regulated watermelon male sterility through gene editing function verification. The next year, Deng et al. identified a dominant male sterile mutant 21SW5304 from a watermelon EMS mutant library and performed fine mapping, and speculated that ClG42_09g0202400 encoding a HSP70 protein was the candidate gene. Although some progress has been made in the study of male sterile genes in watermelon, this field is still in its infancy compared to other crops. Excavating more male sterile genes and performing function verification is crucial for creating excellent watermelon male sterile lines and their application in hybrid seed production.

[0007] In recent years, the research on molecular breeding of watermelon has made rapid progress, and many excellent traits have been located. Some co-segregation markers developed for important traits have also been used in molecular marker-assisted breeding. Through the study of watermelon short hair male sterility, molecular markers co-segregating with watermelon short hair male sterility were developed, which not only can provide effective help for the breeding of watermelon short hair male sterile lines through molecular marker-assisted selection, but also can greatly reduce the cost of watermelon hybrid seed production. SUMMARY

[0008] One of the purposes of the present application is to provide a pair of dCAPS markers co-segregating with watermelon short hair male sterility.

[0009] The second object of the present application is to provide an application of the molecular marker in watermelon molecular breeding.

[0010] The third object of the present application is to provide a method for identifying the trait / genotype of the short-hair male sterility of watermelon.

[0011] The fourth object of the present application is to provide a method for creating a 100% male sterile plant. It is worth noting that the short-hair male sterile material in the present application is different from the conventional male sterile material. The short-hair trait and the male sterile trait of the short-hair male sterile material are co-segregated and controlled by the same gene. In practical application, the short-hair male sterile gene Clstms co-segregated with the above-mentioned dCAPSG marker can be introduced into the core material to prepare a new male sterile line for producing hybrid seeds.

[0012] To achieve the above object, the present application provides the following technical solutions:

[0013] The molecular marker co-segregated with the short-hair male sterile gene Clstms of watermelon is a dCAPS molecular marker, which is named as dCAPSG marker. The polymorphic molecular marker co-segregated with the short-hair male sterile trait of watermelon is developed based on the short-hair male sterile gene Clstms. The sequence of the upstream primer of the primer pair for amplifying the dCAPSG marker is shown in SEQ ID NO. 1, and the sequence of the downstream primer is shown in SEQ ID NO. 2.

[0014] The present application also discloses an application of the molecular marker co-segregated with the short-hair male sterile gene Clstms of watermelon in watermelon molecular breeding. Since the short-hair trait and the male sterile trait are co-segregated, the short-hair trait can be used as a morphological marker to screen the male sterile plants from the maintainer line at the seedling stage, so as to obtain 100% sterile male sterile plants and reduce the cost of land and labor for production. Therefore, by using the characteristic that the molecular marker is co-segregated with the short-hair male sterile trait of watermelon, it can be assisted to identify whether the watermelon plant is the short-hair male sterile phenotype at the molecular level, and the trait of the watermelon plant can be determined at the seedling stage, so as to improve the selection efficiency and speed up the breeding process. Those skilled in the art can understand that, for example, the short-hair male sterile watermelon material can be screened by detecting whether the molecular marker of the present application exists. The detection can be a PCR detection method, and specifically, the primer pair of the molecular marker of the present application can be used. The detection can also be carried out by a sequencing method.

[0015] The present application also discloses a method for identifying the trait / genotype of the short-hair male sterility of watermelon, which adopts a method of PCR amplification and enzyme digestion for detection. The method comprises the following steps:

[0016] (1) extracting the DNA of the watermelon tissue;

[0017] (2) PCR amplification: PCR amplification was performed on the sample extracted in step (1) using a primer pair with the upstream primer sequence shown in SEQ ID NO. 1 and the downstream primer sequence shown in SEQ ID NO. 2;

[0018] (3) The amplified product in step (2) was subjected to enzyme digestion, and then electrophoresis detection was performed;

[0019] (4) The electrophoresis band results of step (3) were determined, and the specific criteria were as follows:

[0020] If the PCR amplification product was a characteristic band of 83 bp in length, the plant to be tested was a homozygous short-hair male sterile watermelon material / genotype; if the PCR amplification product was a characteristic band of 109 bp in length, the plant to be tested was a homozygous long-hair fertile watermelon material / genotype, and if the PCR amplification product was two characteristic bands of 83 bp and 109 bp in length, respectively, the plant to be tested was a heterozygous long-hair fertile watermelon material / genotype;

[0021] Alternatively, the following was used to express:

[0022] If the PCR amplification product had only one characteristic band of 83 bp in length as shown in SEQ ID NO. 3, the watermelon to be tested was a homozygous short-hair male sterile watermelon material / genotype; if the PCR amplification product had only one characteristic band of 83 bp in length as shown in SEQ ID NO. 4, the watermelon to be tested was a homozygous long-hair fertile watermelon material / genotype; and if the PCR amplification product had both a characteristic band of 83 bp in length as shown in SEQ ID NO. 3 and a characteristic band of 109 bp in length as shown in SEQ ID NO. 4, the watermelon to be tested was a heterozygous long-hair fertile watermelon material / genotype.

[0023] Specifically, the reaction system for the PCR amplification was 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 a total volume of 10 μL. The PCR amplification conditions were as follows: 95℃, 5 min; 94℃, 30 s, 56℃, 30 s, 72℃, 50 s, for a total of 35 cycles; 72℃, 10 min; and 4℃ storage.

[0024] The enzyme digestion reaction system was as follows: 5 uL of PCR product, 1 ul of 10X buffer, 0.5 uL of DdeI restriction endonuclease, 3.5 ul of sterilized distilled water, and a total volume of 10 ul. The enzyme digestion conditions were as follows: 37℃, 4 h.

[0025] In addition, the kit containing the primer pair can be used to identify whether the watermelon material is the short-hair male sterile watermelon, and the kit containing the primer pair can be selected in specific application.

[0026] Here, the molecular marker of the present application can also be used to locate the short-hair male sterile gene Clstms, and the above-mentioned applications can be performed according to conventional methods.

[0027] The present application also protects the vector containing the above-mentioned 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-mentioned recombinant vector, the 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 the recombinant cell containing the above-mentioned recombinant vector.

[0028] Advantages of the present application:

[0029] On the one hand, the molecular marker of the present application can ultimately realize the cloning of the Clstms gene, and further lay a foundation for the molecular mechanism research of the anther development and epidermal hair development of watermelon. At the same time, the above-mentioned molecular marker can be directly used for molecular marker assisted breeding of the short-hair male sterile material of watermelon. In the process of watermelon selection breeding, a large number of segregating populations are often produced, and the required plants can be identified in the seedling stage by using the molecular marker, which not only reduces the land area in the breeding process, but also reduces the manpower and material resources required for identification after the plants grow up, 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 the breeding of male sterile lines and hybrid seed production.

[0030] On the other hand, the short-hair male sterile material in the present application separates the short-hair trait and the male sterile trait. By using the co-segregation molecular marker, the fragment thereof can be introduced into the backbone material, and the short-hair trait can be used as a morphological marker to screen the male sterile plants from the male sterile maintainer line in the seedling stage, so as to obtain 100% sterile nuclear male sterile plants and reduce the cost of land and labor for production.

[0031] The application carries out detailed research on the gene related to short hair male sterility of watermelon plant, and finally determines a dCAPS molecular marker co-segregated with the short hair male sterility gene Clstms of watermelon plant by using molecular marker and chromosome walking method and other related research methods. The positioning result shows that the short hair male sterility gene Clstms is located between two molecular markers dCAPS2 and dCAPS3, and the physical distance is 658Kb; the dCAPS G molecular marker in the interval is co-segregated with the short hair male sterility trait of watermelon. This finding lays a foundation for the cloning of the Clstms gene, the establishment of the molecular marker assisted breeding system, and also helps to lay a foundation for the research on the regulation network of anther development and epidermal hair development of watermelon. In general, since the molecular marker has very important significance for the final functional gene positioning, and in addition, the molecular marker has the advantages of simplicity, rapidness and high throughput in the establishment of the molecular marker breeding system, so that the application has very important application value, and has very important significance for the breeding of new type male sterile line of watermelon and hybrid production. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Fig. A, C, E, G are phenotype photos of normal watermelon material G42 (Fig. A, C, E, G) and watermelon short hair male sterile material 23ww043 (Fig. B, D, F, H);

[0033] Figure 2 Fig. 3 is a fine mapping diagram of the gene Clstms for the short hair male sterility trait of watermelon;

[0034] Figure 3 Fig. 4 is a gel electrophoresis diagram of the co-segregation marker dCAPSG in the parent and F2 population;

[0035] In the figure, M is DL2000 Marker, and 100bp and 250bp bands are shown in the figure; P1 represents watermelon normal material G42, and P2 represents watermelon short hair male sterile material 23ww043.

[0036] Figure 4 Fig. 5 is a gel electrophoresis diagram of the co-segregation marker dCAPSG in the parent and 40 natural populations;

[0037] In the figure, M is DL2000 Marker, and 100bp and 250bp bands are shown in the figure; P1 represents watermelon normal material G42, and P2 represents watermelon short hair male sterile material 23ww043. DETAILED DESCRIPTION

[0038] The following further explanation and description are made in combination with the application, and before introducing the specific embodiments, the biological materials, experimental reagents and related experimental background situations in the following embodiments are briefly introduced as follows.

[0039] The materials, reagents, instruments and methods used in the following examples are conventional materials, reagents, instruments and methods in the art, which are commercially available, unless otherwise specified. The watermelon germplasm materials (watermelon short hair male sterile material 23ww043 and watermelon normal material G42) involved in the present application can also be obtained by purchase.

[0040] Biological material:

[0041] Watermelon material G42 (father) is a pure inbred line after multiple generations of selection, which is a watermelon normal material (long hair fertile material) and can be stably inherited Figure 1 A, C, E, G) and is characterized by long hair and fertility, which is referred to as normal phenotype hereinafter.

[0042] Watermelon short hair male sterile material 23ww043 (mother) is obtained by EMS mutagenesis of G42, which has abnormal development of epidermal hair and exhibits less and short epidermal hair, and is male sterile Figure 1 B, D, F, H); wherein the short hair is relative to the long hair of the normal watermelon plant, and the plant phenotype is the same as that of G42 except for the abnormal development of epidermal hair and male sterility. Since 23ww043 is a male sterile trait, it cannot be selfed for seed saving. It is saved in the form of a male sterile maintainer line (F1 generation of cross between 23ww043 and G42 and backcross to 23ww043), which is currently saved in the applicant's laboratory. The long hair fertile and short hair male sterile plants are separated at a ratio of 1:1 in the offspring, indicating that the short hair male sterile mutant is a single gene recessive inheritance, and the fertility of the sterile line is stable and is not affected by environmental factors, which has potential application value in variety improvement and hybrid seed production.

[0043] The above watermelon material G42 is consistent with the normal ordinary material G42 used in the Chinese invention patent “Watermelon male sterile gene ClMS1 and its application” with publication number CN115385994B.

[0044] 23ww043 is obtained by EMS mutagenesis of G42, and the specific method is referred to in the published article “Atelomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding”.

[0045] The above materials are saved in the applicant's laboratory (Guacrops Genetic Breeding Group, Henan Agricultural University).

[0046] 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 management was adopted. After 30 days of planting, the related phenotypic traits were preliminarily investigated and counted, and after 45 days of planting, the related phenotypic traits were investigated and confirmed again.

[0047] The primers for PCR amplification and gene sequencing were provided and completed by Beijing Nosy Genomics Research Center Co., Ltd.

[0048] Experimental reagents:

[0049] PCR 2x 3G Taq Master Mix for PCR amplification was purchased from Nanjing Novozyme Biotech Co., Ltd.

[0050] Restriction endonuclease Ddel was purchased from Guangzhou Yisen Biological Technology Co., Ltd.

[0051] Electrophoresis and silver staining related reagents such as acrylamide, methylene acrylamide, AgNO3, NaOH and formaldehyde were purchased from Beijing Solaybao Technology Co., Ltd.

[0052] Experimental equipment:

[0053] PCR instrument, Zhuhai Heima Medical Instrument Co., Ltd. Hema9600 type gene amplification instrument

[0054] JY300HC universal electrophoresis instrument, produced by Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.

[0055] HT-SCZ04A high-throughput vertical electrophoresis tank, produced by Beijing Hongtao Jiye Technology Development Co., Ltd.

[0056] Examples

[0057] This example mainly introduces the fine mapping process of the watermelon short hair male sterile gene Clstms, including the construction of genetic segregation population, preliminary positioning, fine positioning and other processes. In this process, the development, design and testing process of the dCAPSG molecular marker finally co-segregated with the watermelon short hair male sterile trait are involved. The related experimental process is briefly introduced as follows.

[0058] I. Construction of genetic segregation population

[0059] The watermelon short hair male sterile material 23ww043 was used as the female parent, and the normal watermelon material G42 was used as the male parent (it needs to be explained that during the experiment, considering the availability of the material and the convenience of operation, the inventors used G42 as the male parent. When other homozygous normal materials are used, the related experiments can also be carried out). The hybrid combination was configured by using the two parents, and the results showed that the F1 generation was fertile and the epidermal hair was normal.

[0060] Ten single plants were selected from F1 generation plants, self-crossed to obtain F2 generation seeds for genetic analysis and gene location. The phenotypes of these F2 generation individuals were identified, and verified by chi-square test.

[0061] The results showed that:

[0062] A F2 population containing 174 plants was planted, including 123 plants with normal phenotype, 51 plants with short hair and male sterile phenotype, χ 2 =1.6169<3.84, which is consistent with the segregation ratio of 3:1. Analysis shows that the short hair trait and male sterile trait in G42stms are controlled by the same 1 pair of recessive genes, which is named as Clstms, and the normal phenotype (STMS) is completely dominant to the short hair and male sterile phenotype (stms).

[0063] II. Preliminary location of the gene

[0064] The mutmap method commonly used for mutant location was used for preliminary location, and the specific experimental process was as follows:

[0065] (1) First, prepare the gene pool, specifically:

[0066] In the F2 population of step one above, 20 normal phenotype single plants and 20 short hair and male sterile phenotype single plants were randomly selected, and the relatively tender leaves were collected from each single plant 45 days after planting. The CTAB method was used to extract the genomic DNA, and the normal gene pool and short hair and male sterile gene pool were mixed respectively (normal phenotype and normal phenotype were mixed, and short hair and male sterile phenotype and short hair and male sterile phenotype were mixed).

[0067] (2) Whole genome resequencing of parents and mixed pools, specifically:

[0068] The normal gene pool, short hair and male sterile gene pool and the genomes of the parents constructed in the above (1) were subjected to >40x whole genome resequencing using the Illumina Hi-seq2000 high-throughput sequencing platform. Mainly by the following 4 steps: (i) library construction: physical fragmentation method (ultrasonic oscillation) to break the qualified genomic DNA to the desired fragment (350 bp), and then through the steps of end repair, A addition, adapter addition, target fragment selection and PCR to construct a small fragment sequencing library; (ii) library quality control: using Qseq400 and Qubit to detect library fragment size and library quantification to determine whether the library meets the sequencing standard; (iii) chip fixation: fixing the library to the sequencing chip through bridge PCR; (iv) sequencing on machine: using Illumina sequencer to perform double-end 150bp (PE 150) sequencing on the library, and the data generated by sequencing is used for information analysis after quality control.

[0069] (3) Mutmap analysis, specifically:

[0070] Firstly, the sequencing data was subjected to sequencing adapter removal using trim_galore, and the adapter-removed data was analyzed using mutmap software. The watermelon T2T reference genome G42 (http: / / www.watermelondb.cn / # / geneInfo?group=G42.nogap.v4) was used as the reference genome, and the G42 resequencing data was used as the wild-type sequence. The short-male sterile gene pool sequencing data was used as the mutant pool data, and the normal gene pool sequencing data was used for correction.

[0071] According to the mutmap software analysis results, a significant peak appeared on chromosome 1, and the gene was preliminarily located in the 2.1 MB candidate interval on chromosome 1 (SNP-index value) according to the SNP-index value. Figure 2 A).

[0072] III. Fine mapping of the gene

[0073] Based on the preliminary location in step two, the inventors further fine-mapped the watermelon short-male sterile trait gene Clstms gene. The specific process is briefly introduced as follows.

[0074] (1) Development of molecular markers:

[0075] The sequence in the 2.1 MB candidate interval in the preliminary location was used as the reference sequence, and the publicly available free software package BWA (http: / / bio-bwa.sourceforge.net / ) was used to align the resequencing sequences of the two parents and the candidate segment, respectively, to find the difference sites of the two parents in the candidate segment. Then, the SNPs in the candidate interval were screened out, and the appropriate restriction enzymes were screened out by dCAPSFinder 2.0 software, and the SNP site was converted into a dCAPS marker.

[0076] (2) Linkage analysis

[0077] To verify the candidate interval of the preliminary location and further narrow the location interval for fine mapping, the dCAPS markers designed were used for genotype analysis of 106 strains in the F2 population. The obtained same band shape as the normal parent G42 was marked as "1", the obtained same band shape as the short-male sterile parent 23ww043 was marked as "2", and the obtained heterozygous band shape was marked as "3".

[0078] The final molecular marker typing result is used for linkage analysis by using JoinMap4.0 software to finely map the short hairy male sterility trait gene of watermelon, and finally the short hairy male sterility trait gene is mapped between dCAPS2 and dCAPS3 (the related code is coded by the inventor himself during the research process and does not have special meaning), and the physical distance of the two molecular markers is 658Kb Figure 2 B) in the middle.

[0079] The PCR reaction system is carried out according to the standard process of PCR reaction program, and the PCR reaction system is shown in Table 1.

[0080] Table 1 PCR reaction system

[0081]

[0082] The PCR amplification program is: 94℃, 5min; 94℃, 30s, 55℃, 30s, 72℃, 30s, 35 cycles; 72℃, 5min.

[0083] The enzyme digestion reaction system is shown in Table 2:

[0084] Table 2 Enzyme digestion reaction system

[0085]

[0086]

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

[0088] 8% non-denaturing polyacrylamide gel electrophoresis is carried out for detection. When electrophoresis detection is carried out, the polyacrylamide gel electrophoresis buffer is 0.6×TBE, and the constant voltage electrophoresis is 200V for 1N1.5h. After electrophoresis, silver staining is carried out for observation and detection, and the silver staining method is as follows:

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

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

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

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

[0093] E. Finally, tap water is added and repeatedly rinsed several times;

[0094] F. Dry at room temperature, then take a photo.

[0095] IV. Development of co-segregation dCAPS marker

[0096] In its 658Kb candidate segment, we found only one SNP mutation in mutant material 23ww043 relative to wild type material G42, and developed a dCAPS G molecular marker for this SNP mutation. By randomly selecting single plants in F2 generation for verification, we found that the dCAPS G molecular marker co-segregated with the short hairy male sterile trait of watermelon Figure 3 ).

[0097] wherein the molecular marker dCAPSG is as shown in SEQ ID NO. 3 or SEQ ID NO. 4, the sequence of the upstream primer (dCAPSG-F) of the primer pair for amplifying the molecular marker is as shown in SEQ ID NO. 1, and the sequence of the downstream primer (dCAPSG-R) is as shown in SEQ ID NO. 2, specifically:

[0098] dCAPSG-F: 5'-GATACTAACCCGCCGCTTCT-3' (SEQ ID NO. 1) ;

[0099] dCAPSG-R: 5'-GATAGAGAGCACAATTATCGAGCTG-3' (SEQ ID NO. 2) ;

[0100] The amplification product of the primer in the maternal (short hairy male sterile material) is 83bp in size, and the amplification product in the paternal (normal material, long hairy fertile material) is 109bp in size. Specifically:

[0101] The 83bp characteristic band obtained by PCR amplification has the following specific base sequence:

[0102]

[0103] The 109bp characteristic band obtained by PCR amplification has the following specific base sequence:

[0104]

[0105] V. Application of molecular marker

[0106] (1) DNA extraction, PCR amplification and electrophoresis detection

[0107] DNA extraction was performed by CTAB method, and PCR reaction program 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.

[0108] (2) Watermelon sample short hair male sterile material / genotype detection

[0109] The primer pair is used for PCR amplification of the watermelon sample (parent and F2 population) to be tested, and the results show that if the product after enzyme digestion is a characteristic band of 83 bp in length, the plant to be tested is a homozygous short hair male sterile watermelon material; if the product after enzyme digestion is a characteristic band of 109 bp in length, the plant to be tested is a homozygous long hair fertile watermelon material, and if the product after enzyme digestion is two characteristic bands of 83 bp and 109 bp in length, the plant to be tested is a heterozygous long hair fertile watermelon material.

[0110] Therefore, according to the amplified band, the material / genotype / character of a single plant can be quickly distinguished; further, the inventors use dCAPSG molecular markers to perform PCR amplification gel electrophoresis detection on two parents and a natural population (randomly selected 40 watermelon materials). Figure 4 Combined with the phenotype data, it is found that the phenotype of the parents and the randomly selected 40 watermelon materials is consistent with the actual determination result (Table 3), indicating that the dCAPSG marker of the application has an assisted selection rate of 100% for short hair male sterile molecular markers in watermelon natural population. Based on this result, it is shown that the molecular marker dCAPSG of the application can effectively distinguish whether a watermelon plant is a short hair male sterile material.

[0111] Table 3: Number and phenotype of 40 watermelon natural materials for verification

[0112]

[0113]

[0114] In summary, the derived enzyme digestion amplification sequence polymorphism marker of the application can identify a large number of watermelon short hair male 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 applied on a large scale in production.

[0115] The above only describes the preferred embodiments of the application and is not intended to limit the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

1. A method for determining a short-haired male sterility trait of watermelon, characterized by, The method comprises the following steps: (1) extracting the genomic DNA of the watermelon sample to be tested; (2) using the extracted genomic DNA in step (1) as a template, using a primer pair to perform PCR amplification on the sample extracted in step (1); the sequence of the upstream primer of the primer pair is shown in SEQ ID NO. 1, and the sequence of the downstream primer is shown in SEQ ID NO. 2; (3) using DdeI restriction endonuclease to perform enzyme cutting treatment on the amplification product in step (2), and then performing polyacrylamide gel electrophoresis detection; (4) determining according to the electrophoresis band result of step (3), and the specific standard is: if the enzyme cutting product only has one characteristic band with a length of 83 bp as shown in SEQ ID NO. 3, then the watermelon to be tested is a homozygous short-hair male sterile watermelon material; if the enzyme cutting product only has one characteristic band with a length of 109 bp as shown in SEQ ID NO. 4, then the watermelon to be tested is a homozygous long-hair fertile watermelon material; if the PCR amplification product has both a characteristic band with a length of 83 bp as shown in SEQ ID NO. 3 and a characteristic band with a length of 109 bp as shown in SEQ ID NO. 4, then the watermelon to be tested is a hybrid long-hair fertile watermelon material, and the short-hair male sterile watermelon material is "23ww043".

2. The method of claim 1, characterized in that: The DNA of the test material is extracted by using the CTAB method.

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

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