Molecular marker co-separated from watermelon short hair male sterility gene and application thereof

Abstract

The invention discloses a molecular marker co-segregated with a watermelon short hair male sterility gene and application of the molecular marker, and belongs to the technical field of gene engineering. The molecular marker can be directly used for molecular marker-assisted breeding of the watermelon short-hair male sterility material, the breeding selection efficiency is improved, and the breeding process is accelerated; it is worthy of noting that the short-hair male sterile material is different from a conventional male sterile material. The short hair character and the male sterility character of the short hair male sterility material are co-separated, so that in actual production, a male sterility plant can be screened from a male sterility maintainer line according to the short hair character of the plant in the seedling stage, the male sterility plant with 100% sterility is obtained, and the breeding time is saved. Meanwhile, a foundation can be laid for finally realizing cloning of the watermelon short-hair male sterility gene Clstms and a regulation and control network of watermelon anther development and epidermal hair development, so that the hybrid seed production efficiency is improved, the breeding period is shortened, the seed purity is improved, and the seed production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a dCAPS marker co-segregating with a short-haired male sterile trait of watermelon and an application thereof. Background Art

[0002] The selection of genotypes is one of the most important links in breeding. It refers to the selection of genotypes that meet the requirements in a group for subsequent cultivation. In the traditional breeding process, the basis for selection is often determined by the appearance phenotype of the plant. This selection is time-consuming and may deviate from the required genotype when the trait is ambiguous, resulting in wrong selection and low efficiency. Molecular marker-assisted breeding can quickly detect the target gene or the site co-segregated with the target trait from the seedling stage to achieve the purpose of selecting the target trait. It has the advantages of being fast, accurate, and not affected by environmental conditions.

[0003] The dCAPS (derivative amplified polymorphic sequence) marker is an improvement on the CAPS (enzyme-assisted amplified polymorphic sequence) marker. The basic principle is: when designing the proposed amplification primer, mismatched bases are introduced to generate new restriction endonuclease action sites, and then the primer is used for PCR amplification. The obtained amplified product is digested with a specific restriction endonuclease, and finally polyacrylamide gel electrophoresis is used to detect the digested fragments. The polymorphism between samples is determined based on whether the fragments are cut. The dCAPS marker is a co-dominant marker that can distinguish between heterozygous and homozygous genotypes. It can be directly analyzed by polyacrylamide gel electrophoresis, and the operation is simple and fast. Compared with the CAPS marker, the dCAPS marker does not need to consider whether the SNP is on the restriction endonuclease site. It can maximize the conversion of SNPs into markers, making the utilization rate of SNPs on the genome higher.

[0004] Watermelon (Citrullus lanatus) is an annual vine herbaceous plant belonging to the genus Citrullus of the Cucurbitaceae family, and is known as the king of summer fruits. As an annual plant, seeds are the basic means of reproduction. At the same time, due to the obvious hybrid vigor of watermelons, most of the commercial varieties on the market are hybrids. Therefore, a large amount of artificial hybrid seeds are needed every year for watermelon cultivation.

[0005] At present, the production of watermelon hybrids in my country mainly relies on artificial emasculation and pollination. This process is not only technically demanding and labor-intensive, but also time-consuming, labor-intensive, and costly, and it is difficult to ensure the purity of hybrids. Using male sterile lines to produce seeds can significantly reduce dependence on labor, effectively reduce seed production costs, and improve hybrid purity. Domestic and foreign researchers have discovered multiple 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 the efficiency of hybrid seed production, reducing seed production costs, and realizing simplified seed production. It is of great significance to accelerate the molecular breeding of male sterility in watermelon and simplified seed production. However, there are very few male sterile mutants found in watermelon, and the male sterile genes that have been finely located are very limited. Therefore, it is particularly urgent to explore and screen excellent male sterile genes with important application value in watermelon and analyze their mechanism of action and regulatory network.

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

[0007] In recent years, research on molecular breeding of watermelon has progressed rapidly. Many excellent traits have been genetically located, and some co-segregating markers with important traits have also begun to be used in the molecular marker-assisted breeding process. Through the study of the short-haired male sterility trait of watermelon, molecular markers that co-segregate with the short-haired male sterility trait of watermelon have been developed. This can not only provide effective assistance for the molecular marker-assisted selection of watermelon short-haired male sterile lines, but also greatly reduce the cost of watermelon hybrid seed production. Summary of the invention

[0008] One of the objects of the present invention is to provide a pair of dCAPS markers co-segregating with the pubescent male sterility trait of watermelon.

[0009] The second object of the present invention is to provide the application of the above molecular markers in watermelon molecular breeding.

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

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

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

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

[0014] The present invention also discloses the application of a molecular marker co-segregated with a watermelon short-haired male sterile gene Clstms in watermelon molecular breeding. Since the short-haired trait is co-segregated with the male sterile trait, the short-haired trait can be used as a morphological marker to screen male sterile plants from a maintenance line at the seedling stage, thereby obtaining 100% sterile male sterile plants and reducing the production land and labor costs. Therefore, by utilizing the co-segregation characteristics of the molecular marker and the short-haired male sterile trait of the watermelon, it is possible to assist in identifying whether the watermelon plant is a short-haired male sterile phenotype at the molecular level, and the watermelon plant trait can be determined at the seedling stage, thereby improving the selection efficiency and accelerating the breeding process. Those skilled in the art can understand that, for example, by detecting whether the molecular marker of the present invention exists, short-haired male sterile watermelon materials can be screened. The detection can be a PCR detection method, specifically, the primer pair of the molecular marker of the present invention can be used, and the detection can also be performed by a sequencing method.

[0015] The present invention also discloses a method for determining the short-haired male sterile trait / genotype of watermelon, which is detected by enzyme cutting after PCR amplification. The method comprises the following steps:

[0016] (1) Extract DNA from watermelon tissue;

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

[0018] (3) digesting the amplified product in step (2) with enzymes, followed by electrophoresis detection;

[0019] (4) Determine according to the electrophoresis band results of step (3), the specific criteria are:

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

[0021] Or, expressed as follows:

[0022] If the PCR amplification product has only one characteristic band with a length of 83 bp as shown in SEQ ID NO.3, then the watermelon to be tested is a watermelon material / genotype with a homozygous short-haired male sterile trait; if the PCR amplification product has only one characteristic band with a length of 83 bp as shown in SEQ ID NO.4, then the watermelon to be tested is a watermelon material / genotype with a homozygous long-haired fertile trait; 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 watermelon material / genotype with a heterozygous long-haired fertile trait.

[0023] Specifically, the reaction system of the PCR amplification is: DNA 1μL, 2×PCR Mix 5μL, upstream primer 0.5μL, downstream primer 0.5μL, sterile distilled water 3μL, total volume 10μL. PCR amplification conditions are: 95°C, 5min; 94°C, 30s, 56°C, 30s, 72°C, 50s, a total of 35 cycles; 72°C, 10min; 4°C storage.

[0024] The enzyme digestion reaction system is: 5uL of PCR product, 1ul of 10X buffer, 0.5uL of DdeI restriction endonuclease, 3.5ul of sterile distilled water, total volume 10ul, and the enzyme digestion conditions are 37°C and 4h.

[0025] In addition, the kit containing the above primer pair can be used to identify whether the watermelon material is watermelon short-haired male sterile. In specific applications, the kit containing the primer pair can be selected.

[0026] Here, the molecular markers of the present invention can also be used to locate the watermelon short-haired male sterility gene Clstms, and the above applications can be carried out according to conventional methods.

[0027] The present invention also protects vectors containing the above-mentioned molecular markers. 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-mentioned recombinant vector, those skilled in the art can transform the recombinant vector into suitable cells according to different needs to obtain recombinant cells containing the recombinant vector. Therefore, the present invention also protects recombinant cells containing the recombinant vector.

[0028] Advantages of the present invention:

[0029] On the one hand, the molecular markers of the present invention can ultimately realize the cloning of the Clstms gene, and then lay the foundation for the study of the molecular mechanism of watermelon anther development and epidermal hair development. At the same time, the above molecular markers can be directly used for molecular marker-assisted breeding of watermelon short-haired male sterile materials. In the process of watermelon selection breeding, a large number of separation groups are often produced. By using the molecular markers, the required plants can be identified in the seedling stage, which not only reduces the floor space 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 markers provided by the present invention have good application value in male sterile line breeding and hybrid seed production.

[0030] On the other hand, the short-hair trait in the short-hair male sterile material of the present invention is co-segregated with the male sterile trait. By utilizing this characteristic and introducing its fragment into the backbone material through the co-segregated molecular marker, the short-hair trait can be used as a morphological marker to screen male sterile plants from the male sterile maintenance line at the seedling stage, thereby obtaining 100% sterile nuclear male sterile plants and reducing the production land and labor costs.

[0031] The present invention conducts a detailed study on genes related to short-haired male sterility in watermelon plants, and finally determines a dCAPS molecular marker co-segregated with the short-haired male sterility gene Clstms in watermelon plants by using molecular markers and chromosome walking methods and other related research methods. The positioning results show that the short-haired male sterility gene Clstms is located between two molecular markers dCAPS2 and dCAPS3, with a physical distance of 658Kb; the dCAPSG molecular marker co-segregates with the short-haired male sterility trait of watermelon in the interval. This discovery lays the foundation for the final cloning of the Clstms gene and the establishment of a molecular marker-assisted breeding system, and also helps to lay the foundation for the final research on the regulatory network of anther development and epidermal hair development in watermelon. In short, since molecular markers are of great significance for the final functional gene positioning, and molecular markers have the advantages of simplicity, rapidity and high throughput in the establishment of molecular marker breeding systems, the present invention has very important application value, and is also of great significance for the selection and breeding of new male sterile lines of watermelon and the production of hybrids. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Phenotypic photos of normal watermelon material G42 (Figures A, C, E, G) and watermelon short-haired male sterile material 23ww043 (Figures B, D, F, H);

[0033] Figure 2 To map the fine-grained location of the gene Clstms for the short-haired male sterility trait in watermelon;

[0034] Figure 3 The gel electrophoresis diagram of the co-segregation marker dCAPSG in the parental and F2 generation populations;

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

[0036] Figure 4 The gel electrophoresis diagram of the co-separation marker dCAPSG in the parent and 40 natural populations;

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

[0038] The following is a further explanation in conjunction with the application. Before introducing the specific embodiments, the biological materials, experimental reagents and related experimental background 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 unless otherwise specified, and can be obtained through commercial channels. The watermelon germplasm materials (watermelon short-haired male sterile material 23ww043 and watermelon normal material G42) involved in the present invention can also be obtained by purchase.

[0040] Biomaterials:

[0041] Watermelon material G42 (male parent) is a homozygous inbred line that has been selected for many generations. This material is a normal watermelon material (long-haired fertile material) and can be stably inherited ( Figure 1 A, C, E, G), showing long hair and fertility, hereinafter referred to as normal phenotype.

[0042] The watermelon short-haired male sterile material 23ww043 (female parent) was obtained from GG42 by EMS mutagenesis. The epidermal hairs of the whole plant developed abnormally, showing fewer and shorter epidermal hairs, and were male sterile ( Figure 1 B, D, F, H); short hairs refer to the long hairs of normal watermelon plants. Except for the abnormal development of epidermal hairs and male sterility, other plant phenotypes are the same as G42. Since 23ww043 is a male sterile trait, it cannot be self-pollinated and preserved, so it is preserved as a male sterile maintenance line (obtained by backcrossing 23ww043 with the F1 generation of 23ww043 and G42) (currently preserved in the applicant's laboratory). The ratio of long-haired fertile plants to short-haired male sterile plants separated from the offspring is 1:1, indicating that the short-haired 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. It has potential application value in variety improvement and hybrid seed production.

[0043] The above-mentioned watermelon material G42 is consistent with the normal common material G42 used in the Chinese invention patent "Watermelon male sterility gene ClMS1 and its application" with publication number CN115385994B.

[0044] 23ww043 was obtained by EMS mutagenesis of G42. For specific methods, please refer to the publicly published article "Atelomere-to-telomere gap-free reference genome of watermelon and its mutation libraryprovide important resources for gene discovery and breeding".

[0045] The above materials are all preserved in the applicant's laboratory (Henan Agricultural University, melon crop genetic breeding project).

[0046] During the experiment, the watermelon materials were planted in the solar greenhouse of Maozhuang Science and Education Park of Henan Agricultural University. During the planting process, plug tray seedlings were used after germination, and normal watermelon cultivation and management methods were adopted. A preliminary investigation and statistics of relevant phenotypic traits were conducted 30 days after planting, and the relevant phenotypic traits were investigated and confirmed again 45 days after planting.

[0047] The primers for PCR amplification and gene sequencing were provided by Beijing Nosai Genome 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 Yisheng Biotechnology Co., Ltd.;

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

[0052] Experimental equipment:

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

[0054] JY300HC universal electrophoresis apparatus, produced by Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.;

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

[0056] Example

[0057] This example mainly introduces the fine positioning process of the watermelon short-haired male sterile gene Clstms, including the construction of genetic segregation populations, preliminary positioning, fine positioning and other processes. This process involves the development, design and testing of the dCAPSG molecular marker that is ultimately co-segregated with the watermelon short-haired male sterile trait. The relevant experimental process is briefly introduced as follows.

[0058] 1. Construction of genetically isolated populations

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

[0060] Ten individual plants were selected from the F1 generation and self-pollinated to obtain F2 generation seeds for genetic analysis and gene positioning. The phenotypes of these F2 generation individuals were identified and verified using the chi-square test.

[0061] The results show that:

[0062] The F2 population of 174 plants was planted, of which 123 plants had normal phenotype, 51 plants had short hair and male sterile phenotype, and 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 sterility trait in G42stms are controlled by the same pair of recessive genes, which are named Clstms. The normal phenotype (STMS) is completely dominant over the short-hair and male sterility phenotype (stms).

[0063] 2. Preliminary location of the gene

[0064] The mutmap method commonly used for EMS-induced mutant localization was used for preliminary localization. The specific experimental process is as follows:

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

[0066] Randomly select 20 normal phenotype plants and 20 short-haired male sterile phenotype plants from the F2 generation population in the above step 1, collect relatively tender leaves from each plant 45 days after planting, extract their genomic DNA using the CTAB method, and mix them to make a normal gene pool and a short-haired male sterile gene pool (normal phenotypes are mixed with normal phenotypes, and short-haired male sterile phenotypes are mixed with short-haired male sterile phenotypes).

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

[0068] The normal gene pool, short-haired male sterile gene pool and the genomes of the parents constructed in (1) were subjected to >40x whole genome resequencing using the Illumina Hi-seq2000 high-throughput sequencing platform. The process mainly consists of the following four steps: (i) Library construction: Physical fragmentation (ultrasonic vibration) was used to fragment qualified genomic DNA into target fragments (350 bp), and then end repair, A addition, linker addition, target fragment selection and PCR were performed to construct a small fragment sequencing library; (ii) Library quality control: Qseq400 and Qubit were used to detect the library fragment size and library quantification to determine whether the library met the sequencing standards; (iii) Chip fixation: The library was fixed to the sequencing chip by bridge PCR; (iv) On-machine sequencing: The library was subjected to double-end 150 bp (PE 150) sequencing using an Illumina sequencer, and the data generated by the sequencing was used for the next information analysis after quality control.

[0069] (3) Mutmap analysis, specifically:

[0070] First, trim_galore was used to remove the sequencing adapters from the sequencing data, and the data without adapters was analyzed by mutmap software. The watermelon T2T reference genome G42 (http: / / www.watermelondb.cn / # / geneInfo?group=G42.nogap.v4) was used as the reference genome, the G42 resequencing data was the wild-type sequence, the short-haired male sterile gene pool sequencing data was the mutant mixed pool data, and the normal gene pool sequencing data was used for correction.

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

[0072] 3. Fine positioning of genes

[0073] Based on the preliminary positioning in step 2, the inventors further carried out fine positioning of the Clstms gene related to the short-haired male sterility trait of watermelon. The specific process is briefly described as follows.

[0074] (1) Development of molecular markers:

[0075] The sequences in the 2.1MB candidate interval in the preliminary positioning were used as reference sequences, and the resequencing sequences of the two parents and the candidate segment were compared using the free software package BWA (http: / / bio-bwa.sourceforge.net / ) available online to find the difference sites between the two parents in the candidate segment. Subsequently, the SNPs in the candidate interval were screened out, and the appropriate restriction endonucleases were selected by dCAPSFinder 2.0 software and the SNP sites were converted into dCAPS markers.

[0076] (2) Linkage analysis

[0077] In order to verify the candidate interval of the preliminary positioning and further narrow the positioning interval for fine positioning, the designed dCAPS marker was used to analyze the genotype of 106 strains in the F2 population. The same band shape as the normal parent G42 was recorded as "1", the same band shape as the short-haired male sterile parent 23ww043 was recorded as "2", and the heterozygous band shape was recorded as "3".

[0078] The typing results of the final molecular markers were used for linkage analysis using JoinMap4.0 software to finely locate the short-haired male sterile trait gene in watermelon. The short-haired male sterile trait gene was finally located between dCAPS2 and dCAPS3 (the relevant codes were coded by the inventors during the research process and have no special meaning). The physical distance between the two molecular markers is 658Kb ( Figure 2 (middle B).

[0079] PCR reaction system The PCR reaction procedure was carried out according to the standard process. The PCR reaction system is shown in Table 1.

[0080] Table 1 PCR reaction system

[0081]

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

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

[0084] Table 2 Enzyme digestion reaction system

[0085]

[0086]

[0087] The enzyme digestion conditions were 37°C and 4h.

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

[0089] A. Place the glass plate with glue in the fixing solution and gently shake it on a shaker until the indicator loses its color. The fixing solution consists of glacial acetic acid: anhydrous ethanol: distilled water in a volume ratio of 0.5:10:100.

[0090] B. Wash with ultrapure water for 1 to 3 minutes;

[0091] C. Place the washed plastic plate in the staining solution and shake for 10 minutes. The staining solution is 0.2% silver nitrate aqueous solution.

[0092] D. Rinse the stained plastic plate in ultrapure water for 30 seconds, place it in a plastic box containing developer, and shake it gently until the bands are clearly visible. The developer is prepared by adding 15g NaOH and 3mL formaldehyde to 1L distilled water;

[0093] E. Finally, rinse repeatedly in tap water several times;

[0094] F. Dry at room temperature and then take photos.

[0095] 4. Development of co-segregating dCAPS markers

[0096] In the 658Kb candidate segment, we found that there was only one SNP mutation in the mutant material 23ww043 relative to the wild-type material G42. We developed the dCAPSG molecular marker for this SNP mutation. By randomly selecting individual plants in the F2 generation, we found that the dCAPSG molecular marker co-segregated with the short-haired male sterility trait of watermelon ( Figure 3 ).

[0097] The molecular marker dCAPSG is shown in SEQ ID NO.3 or SEQ ID NO.4, the upstream primer (dCAPSG-F) sequence of the primer pair for amplifying the molecular marker is shown in SEQ ID NO.1, and the downstream primer (dCAPSG-R) is 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 size of the amplified product of this primer in the female parent (short-haired male sterile material) is 83 bp, and the size of the amplified product in the male parent (normal material, long-haired fertile material) is 109 bp. Specifically:

[0101] The characteristic band of 83 bp was obtained by PCR amplification. The specific base sequence of the characteristic band of 83 bp is as follows:

[0102]

[0103] A characteristic band of 109 bp was obtained by PCR amplification. The specific base sequence of the characteristic band of 109 bp is as follows:

[0104]

[0105] 5. Application of Molecular Markers

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

[0107] The conventional CTAB method was used for DNA extraction, and the PCR reaction procedure and polyacrylamide gel electrophoresis process referred to Yang Huihui's master's thesis: Fine positioning and functional verification of the watermelon lateral branch gene Clbl, Henan Agricultural University, 2021.

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

[0109] The primers were used to perform PCR amplification on the watermelon samples to be tested (parents and F2 population). The results showed that if the product after enzyme cleavage was a characteristic band of 83 bp in length, the plant to be tested was a homozygous short-haired male sterile watermelon material; if the product after enzyme cleavage was a characteristic band of 109 bp, the plant to be tested was a homozygous long-haired fertile watermelon material; if the product after enzyme cleavage was two characteristic bands with lengths of 83 bp and 109 bp respectively, the plant to be tested was a heterozygous long-haired fertile watermelon material.

[0110] Therefore, according to the amplified bands, the material / genotype / trait of a single plant can be quickly distinguished; further, the inventors used dCAPSG molecular markers to perform PCR amplification gel electrophoresis detection on the two parents and the natural population (40 watermelon materials were randomly selected) ( Figure 4 ). Combined with the phenotypic data, it was found that the phenotypes of the parents and 40 randomly selected watermelon materials were consistent with the actual measurement results (Table 3), indicating that the auxiliary selection rate of the dCAPSG marker of the present invention for the short-haired male sterile molecular marker in the natural population of watermelon was 100%. Based on this result, it is shown that the molecular marker dCAPSG of the present invention can effectively distinguish whether the watermelon plant is a short-haired male sterile material.

[0111] Table 3 Numbers and phenotypes of 40 natural watermelon materials used for verification

[0112]

[0113]

[0114] In summary, the derived enzyme-cut amplified sequence polymorphism marker of the present invention can be used for large-scale identification of the short-haired male trait of watermelon. It is not only fast and effective but also can be used for identification at the seedling stage, which greatly shortens the breeding cycle and can be used on a large scale in production.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A molecular marker co-segregating with the watermelon short-haired male sterile gene, characterized in that: The molecular marker is shown as SEQ ID NO.3 or SEQ ID NO.4, the upstream primer sequence of the primer pair for amplifying the molecular marker is shown as SEQ ID NO.1, and the downstream primer sequence is shown as SEQ ID NO.

2.

2. The molecular marker co-segregating with the watermelon short-haired male sterile gene according to claim 1, characterized in that: The molecular marker is a dCAPS marker.

3. Use of the molecular marker co-segregating with the short-haired male sterile trait of watermelon as described in claim 1 in identifying or assisting in identifying the short-haired male sterile trait / genotype of watermelon.

4. The use according to claim 3, characterized in that: The short-hair trait is co-segregated with the male sterility trait. At the seedling stage, male sterile plants can be screened out from the maintenance line through the short-hair trait expressed by the plants.

5. A method for determining the short-haired male sterility trait / genotype of watermelon, characterized in that: The method comprises the following steps: (1) extracting genomic DNA of watermelon samples to be tested; (2) using the genomic DNA extracted in step (1) as a template and the primer pair described in claim 1 to perform PCR amplification on the sample extracted in step (1); (3) digesting the amplified product in step (2) and then detecting it by polyacrylamide gel electrophoresis; (4) Determine according to the electrophoresis band results of step (3), the specific criteria are: If the enzyme cleavage product has only one characteristic band with a length of 83 bp as shown in SEQ ID NO.3, the watermelon to be tested is a homozygous short-haired male sterile watermelon material / genotype; if the enzyme cleavage product has only one characteristic band with a length of 109 bp as shown in SEQ ID NO.4, the watermelon to be tested is a homozygous long-haired fertile watermelon material / genotype; 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, the watermelon to be tested is a heterozygous long-haired fertile watermelon material / genotype.

6. The method according to claim 5, characterized in that: The DNA of the test materials was extracted using the CTAB method.

7. The method according to claim 5, characterized in that The enzyme digestion reaction system described in step (3) is: 5uL of PCR product, 1ul of 10X buffer, 0.5uL of DdeI restriction endonuclease, 3.5ul of sterile distilled water, total volume 10ul, and the enzyme digestion conditions are 37°C and 4h.

8. A kit for identifying the short-haired male sterility trait, characterized in that: Comprising the primer pair described in claim 1.

9. Application of a reagent for detecting the presence of a dCAPS marker in localizing the short-haired male sterility gene Clstms in watermelon, characterized in that: The upstream primer sequence of the primer pair for amplifying the molecular marker described in claim 1 is shown as SEQ ID NO.1, and the downstream primer sequence is shown as SEQ ID NO.2.

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