Molecular marker co-segregated with watermelon short-vine and few-lateral-branch gene and application thereof

Abstract

The invention discloses a molecular marker co-segregated with a watermelon short-vine and few-lateral-branch gene and application of the molecular marker, and belongs to the technical field of gene engineering. The molecular marker dCAPS2 is co-separated from the watermelon short-vine and few-lateral-branch gene Cldbl, and can be directly used for genotype identification of watermelon short-vine and few-lateral-branch materials. According to the invention, on the basis of a watermelon material G42 and a mutant material 21SW281, fine positioning is carried out on the watermelon short-vine few-lateral-branch gene Cldbl, and a molecular marker co-separated from the watermelon short-vine few-lateral-branch character is developed. According to the result, the molecular marker can be used for molecular marker-assisted breeding of watermelon short-vine and few-lateral-branch materials, and the molecular marker has important application value in watermelon plant type improvement and cultivation of new varieties suitable for simplified cultivation.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a dCAPS marker co-segregated with a short vine and few lateral branches 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 selection errors and low efficiency. Molecular marker-assisted breeding can quickly detect the target gene or the site that co-segregates 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, and the obtained amplification product is digested with a specific restriction endonuclease, and finally polyacrylamide gel electrophoresis is used to detect the digested fragments, and the polymorphism between samples is judged 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, and 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 herb belonging to the genus Citrullus of the Cucurbitaceae family. It occupies a very important position in the world's horticultural crops. In recent years, facility cultivation has gradually become the mainstream watermelon cultivation method in my country. In facility cultivation, the planting method of hanging vine cultivation is usually adopted. Therefore, the length of watermelon vines and the characteristics of side branches have become one of the main considerations for variety selection. Among the existing main cultivated varieties, the main vine length can reach 4-5m, and it also shows strong branching characteristics. Excessive side branches not only occupy more space, but also require sufficient plant spacing and row spacing during watermelon cultivation, thereby reducing the planting density. In actual production, in order to increase ventilation and light transmission and reduce the spread and spread of pests and diseases, frequent pruning and branching are required during planting; after fruiting, in order to reduce the competition for nutrients between vegetative growth and reproductive growth and improve fruit quality, it is also necessary to regulate plant growth by removing growth points and pruning and branching. However, this process is time-consuming and labor-intensive and requires high labor costs. It can be seen that the characteristics of plant height and multiple side branches have become a bottleneck restricting the industrialization and large-scale development of watermelon. Therefore, cultivating new dwarf watermelon varieties with few side branches that are suitable for light and simplified cultivation will effectively promote the industrialization, large-scale and standardized development of watermelon.

[0005] With the completion of the whole genome sequencing of watermelon, the research on the gene positioning of important traits of watermelon has developed rapidly in recent years, and some important and excellent traits have been genetically positioned. For example, the short-vine watermelon material was first reported in 1956. Mohr of the University of Kentucky in the United States found a short-vine watermelon mutant with short vines and few branches in the long-vine watermelon variety "WB-2" that can be stably inherited. The study found that the shortening of internodes is caused by the shortening of cell length. Sun et al. reported that the vines of the short-vine mutant w106 of watermelon were shorter than those of the control Charleston Gray, and the cell length of the longitudinal section of the internode was also significantly shortened; Dou et al. located a gene Clbl that controls the development of watermelon lateral branches through the watermelon forkless inbred line WCZ (forkless early); Deng et al. used EMS to induce G42 to obtain a watermelon short-vine or short internode mutant. Preliminary genetic studies showed that its dwarfing trait was controlled by a pair of recessive genes.

[0006] By using the short vine and few lateral branches trait for genetic improvement and new variety creation, plant height and the number of lateral branches can be significantly reduced, thereby reducing labor costs, increasing field ventilation and light transmission, and reducing the spread of pests and diseases. The present invention develops molecular markers that are co-separated with the short vine and few lateral branches trait of watermelon plants through research on the short vine and few lateral branches of watermelon plants, which can not only provide effective assistance for the molecular marker-assisted selection and breeding of new watermelon varieties with few lateral branches, but also lay a good technical foundation for the breeding of new watermelon varieties with short vines and few lateral branches. Summary of the invention

[0007] One of the objectives of the present invention is to provide a pair of dCAPS markers that co-segregate with the short vine and few lateral branches trait of watermelon.

[0008] The second object of the present invention is to provide the application of the above molecular markers in watermelon molecular breeding.

[0009] The third object of the present invention is to provide a method for identifying watermelon varieties / genotypes with short vines and few side branches.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] A pair of molecular markers co-segregated with the watermelon short vine and few lateral branches gene Cldbl, the molecular markers are dCAPS molecular markers, named dCAPS2 markers, and the polymorphic molecular markers co-segregated with the watermelon short vine and few lateral branches trait are developed based on the short vine and few lateral branches gene Cldbl. The upstream primer sequence of the primer pair for amplifying the dCAPS2 marker is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2.

[0012] The present invention also discloses the application of a molecular marker co-segregated with a watermelon short vine and few lateral branches gene Cldbl in watermelon molecular breeding. The molecular marker co-segregates with the short vine and few lateral branches trait of the watermelon plant, and can assist in identifying whether the watermelon plant is a phenotype of short vine and few lateral branches at the molecular level, and can determine the watermelon plant trait at the seed or 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, a watermelon material with short vine and few lateral branches 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.

[0013] The present invention also discloses a method for determining watermelon short vines and few side branches material / genotype, which is detected by enzyme cutting after PCR amplification. The method comprises the following steps:

[0014] (1) Extract DNA from watermelon tissue;

[0015] (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);

[0016] (3) digesting the amplified product in step (2) with enzymes, followed by electrophoresis detection;

[0017] (4) Determine according to the electrophoresis band results of step (3), the specific criteria are:

[0018] If the PCR amplification product is a characteristic band of 109 bp in length, the plant to be tested is a homozygous watermelon material with short vines and few side branches; if the PCR amplification product is a characteristic band of 129 bp, the plant to be tested is a homozygous watermelon material with long vines and many side branches; if the PCR amplification product is two characteristic bands of 109 bp and 129 bp in length, the plant to be tested is a heterozygous watermelon material with long vines and many side branches;

[0019] Or, expressed as follows:

[0020] If the PCR amplification product has only one characteristic band with a length of 109 bp as shown in SEQ ID NO.3, then the watermelon to be tested is a watermelon material / genotype with homozygous short vines and few side branches; if the PCR amplification product has only one characteristic band with a length of 129 bp as shown in SEQ ID NO.4, then the watermelon to be tested is a watermelon material / genotype with homozygous long vines and many side branches; if the PCR amplification product has both a characteristic band with a length of 109 bp as shown in SEQ ID NO.3 and a characteristic band with a length of 129 bp as shown in SEQ ID NO.4, then the watermelon to be tested is a watermelon material / genotype with heterozygous long vines and many side branches.

[0021] Specifically, the reaction system of the PCR amplification is: DNA 1 μL, 2×PCR Mix 5 μL, upstream primer 1 μL, downstream primer 1 μL, sterile distilled water 2 μL, total volume 10 μL. PCR amplification conditions are: 95°C, 5 min; 94°C, 30 s, 56°C, 30 s, 72°C, 50 s, 35 cycles in total; 72°C, 10 min; stored at 4°C.

[0022] The enzyme digestion reaction system was: PCR product 4uL, 10X buffer 1ul, DdeI restriction endonuclease 0.5uL, sterile distilled water 4.5ul, total volume 10ul, and the enzyme digestion conditions were 37°C and 4h.

[0023] In addition, the kit containing the above primer pair can be used to identify whether the watermelon material has the trait of short vines and few side branches. In specific applications, the kit containing the primer pair can be selected.

[0024] Here, the molecular markers of the present invention can also be used to locate the watermelon short vine and few lateral branches gene Cldbl. The above applications can be carried out according to conventional methods.

[0025] 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.

[0026] Advantages of the present invention:

[0027] The molecular marker can be directly used for molecular marker-assisted breeding of watermelon plant materials with short vines and few lateral branches. In the process of watermelon selection breeding, a large number of segregating populations are often produced. By using the molecular marker, the required plants can be quickly 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 marker provided by the present invention has good application value in the cultivation of new varieties.

[0028] The present invention carries out genetic analysis on a watermelon plant short vine and few lateral branches material, finds that the short vine and few lateral branches trait is controlled by a pair of recessive single genes, and uses molecular markers and chromosome walking method to finely locate the gene, and finally determines a dCAPS molecular marker co-segregated with the short vine and few lateral branches of watermelon. The positioning result shows that the short vine and few lateral branches gene Cldbl is located between dCAPS4 and dCAPS6, with a physical distance of 0.89Mb, wherein the molecular marker dCAPS2 is completely co-segregated with the short vine and few lateral branches trait. This discovery lays a solid foundation for the final cloning of the Cldbl gene and the establishment of a molecular marker-assisted breeding system, and also lays a good working foundation for the molecular mechanism analysis of the formation of the short vine and few lateral branches trait of watermelon. In short, since molecular markers are of great significance for the final functional gene positioning, and they have the advantages of being simple, fast and high-throughput in the molecular marker-assisted breeding system, the present application has a very important application value, and also has a very important protection significance for the cultivation of new watermelon varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Phenotypic photos of normal watermelon material G42 (A left) and watermelon short-vine and few-lateral-branch material 21SW281 (A right), as well as plant height survey (B) and lateral-branch number survey (C) 3-7 weeks after planting;

[0030] Figure 2 To map the fine positioning of the gene Cldbl for the short vine and few lateral branches trait of watermelon;

[0031] Figure 3 The gel electrophoresis diagram of the co-segregation marker dCAPS2 in the parental and F2 generation populations;

[0032] In the figure, M is DM5000 Marker, and the 100bp and 250bp bands are shown in the figure; P1 represents the normal watermelon material G42, and P2 represents the watermelon material 21SW281 with short vines and few side branches.

[0033] Figure 4 The gel electrophoresis diagram of the co-segregation marker dCAPS2 in the parent and 40 natural populations;

[0034] In the figure, M is DM5000 Marker, and the 100bp and 250bp bands are shown in the figure; P1 represents the normal watermelon material G42, and P2 represents the watermelon material 21SW281 with short vines and few side branches. DETAILED DESCRIPTION

[0035] 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.

[0036] Biomaterials:

[0037] 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 vines and multiple side branches) and can be stably inherited ( Figure 1 A left), characterized by long vines and many side branches, hereinafter referred to as the normal phenotype.

[0038] The watermelon short vine and few side branches material 21SW281 (female parent) was obtained from G42 by EMS mutagenesis. Compared with G42, the plant height is reduced and the number of side branches is reduced ( Figure 1 A right); short vines and few side branches are relative to the long vines and many side branches of normal watermelon materials, and the obtained 21SW281 material can be stably inherited during the later planting process. The above 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.

[0039] The 21SW281 is derived from the seeds of the M2 generation watermelon mutant mutated from the G42 material (the seeds were donated by Deng Yun of the Modern Agricultural Research Institute of Peking University, and the seeds of the M1 generation watermelon mutant were obtained by EMS mutagenesis. The specific method refers to the publicly 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 and identification, we identified homozygous mutants in the M2 and M3 generation seeds, among which the materials with reduced plant height and reduced number of lateral branches have excellent traits for breeding watermelon materials in production. Therefore, we conducted gene positioning research on this material and developed molecular markers based on it. The material is stored in the melon crop genetic breeding research group of Henan Agricultural University.

[0040] The above materials are all kept in the applicant's laboratory (Henan Agricultural University, Melon Crop Genetic Breeding Project). They can be distributed to the public for verification tests within 20 years from the date of application, or the public can purchase them.

[0041] 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. Normal watermelon cultivation and management methods were adopted, and the plant height and the number of lateral branches were investigated and counted 21d, 28d, 35d, and 45d after transplanting.

[0042] The primers for PCR amplification and gene sequencing were provided by Beijing Nosai Genome Research Center Co., Ltd.

[0043] Experimental reagents:

[0044] PCR 2x 3G Taq Master Mix for PCR amplification was purchased from Nanjing Novozyme Biotech Co., Ltd.;

[0045] Restriction endonuclease Dde1 was purchased from Guangzhou Yisheng Biotechnology Co., Ltd.;

[0046] Electrophoresis and silver staining related reagents such as acrylamide, methylene acrylamide, AgNO3, NaOH, and formaldehyde were purchased from Beijing Solebow Technology Co., Ltd.;

[0047] Experimental equipment:

[0048] PCR instrument, Hema9600 gene amplification instrument from Zhuhai Heima Medical Instrument Co., Ltd.;

[0049] JY300HC universal electrophoresis apparatus, produced by Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.;

[0050] HT-SCZ04A high-throughput vertical electrophoresis tank is produced by Beijing Hongtao Jiye Technology Development Co., Ltd.

[0051] Example

[0052] This example mainly introduces the fine positioning process of the watermelon short vine gene Cldbl, including the construction of genetic segregation populations, preliminary positioning, fine positioning and other processes. In this process, the development, design and testing process of the dCAPS2 molecular marker that is ultimately co-segregated with the short vine and few lateral branches trait of watermelon is involved. The relevant experimental process is briefly introduced as follows.

[0053] 1. Construction of genetically isolated populations

[0054] With normal watermelon material G42 ( Figure 1 A left) as the male parent, with the watermelon short vine and few side branches material 21SW281 ( Figure 1 A right) as the female parent (it should be explained that during the experiment, considering the availability of materials and the convenience of operation, the inventors 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 plant height and the number of side branches of the F1 generation were basically consistent with those of the female parent G42.

[0055] 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.

[0056] The results show that:

[0057] The F2 population of 1236 plants was planted, of which 930 were normal phenotype plants, 306 were short-vine and few-branch phenotype plants, and χ 2 =0.046<3.84, which is consistent with the segregation ratio of 3:1. Analysis shows that the short vines and few lateral branches in 21SW281 are controlled by the same pair of recessive genes, which are named Cldbl. The normal phenotype (DBL) is completely dominant over the short vines and few lateral branches phenotype (dbl).

[0058] 2. Preliminary location of the gene

[0059] The mutmap method for localizing EMS-induced mutants was used for preliminary localization. The specific experimental process is as follows:

[0060] (1) First, prepare a gene pool, specifically:

[0061] Randomly select 25 normal phenotype plants and 25 short vines and few side branches phenotype plants from the F2 generation population in the above step 1, collect relatively tender leaves from each plant 30 days after planting, extract their genomic DNA using the CTAB method, and mix them to make a normal gene pool and a short vine and few side branches gene pool (normal phenotypes are mixed with normal phenotypes, and short vines and few side branches phenotypes are mixed with short vines and few side branches phenotypes).

[0062] (2) Whole genome resequencing of parents and pools, specifically:

[0063] The normal gene pool, short vine and few lateral branches 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 a small fragment sequencing library was constructed through end repair, A addition, linker addition, target fragment selection and PCR; (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 step of information analysis after quality control.

[0064] (3) Mutmap analysis, specifically:

[0065] 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, and the short vine and few side branches gene pool sequencing data was the mutation mixed pool data.

[0066] According to the analysis results of mutmap software, a significant peak appeared at the end of chromosome 7. According to the SNP-index value, the gene was initially located in the 2.4MB candidate interval of chromosome 7 ( Figure 2 in the figure above).

[0067] 3. Fine positioning of genes

[0068] Based on the preliminary positioning in step 2, the inventors further carried out fine positioning of the Cldbl gene related to the short vine and few lateral branches trait of watermelon. The specific process is briefly described as follows.

[0069] (1) Development of molecular markers:

[0070] The sequences in the 2.4Mb candidate interval in the preliminary positioning were used as reference sequences, and the resequencing sequences of the two parents and the candidate segments 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 segments. Subsequently, the SNPs in the candidate interval were screened out, and the appropriate restriction enzymes were selected by dCAPSFinder 2.0 software and the SNP sites were converted into dCAPS markers.

[0071] (2) Linkage analysis

[0072] 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 400 plants in the F2 population. The bands with the same shape as the normal parent G42 were recorded as "1", the bands with the same shape as the short vine and few side branches parent 21SW281 were recorded as "2", and the heterozygous bands were recorded as "3".

[0073] The typing results of the final molecular markers were analyzed by linkage analysis using JoinMap4.0 software to finely locate the gene for the short vine and few lateral branches trait of watermelon. Finally, the gene for the short vine and few lateral branches trait was located between dCAPS4 and dCAPS6 (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 0.89 Mb ( Figure 2 The PCR reaction system was carried out according to the standard process. The PCR reaction system is shown in Table 1.

[0074] Table 1 PCR reaction system

[0075]

[0076] 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.

[0077] The enzyme digestion reaction system is shown in Table 2:

[0078] Table 2 Enzyme digestion reaction system

[0079]

[0080] The enzyme digestion conditions were 37°C and 4h.

[0081] Perform 8% non-denaturing polyacrylamide gel electrophoresis. During electrophoresis, the polyacrylamide gel electrophoresis buffer is 0.6×TBE, and the electrophoresis is performed at a constant voltage of 200V for 1 to 1.5 hours. After the electrophoresis, silver staining is performed for observation and detection. The silver staining method is as follows:

[0082] 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.

[0083] B. Wash with ultrapure water for 1 to 3 minutes;

[0084] C. Place the washed plastic plate in the staining solution and shake for 1-2 minutes. The staining solution is 0.2% silver nitrate aqueous solution.

[0085] 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 15mL formaldehyde to 1L distilled water;

[0086] E. Finally, rinse repeatedly in tap water several times;

[0087] F. Dry at room temperature and then take photos.

[0088] (3) Development of co-segregation dCAPS markers

[0089] In the 0.89Mb candidate segment, we found that there was only one SNP mutation in the mutant material 21SW281 relative to the wild-type material G42. We developed the dCAPS2 molecular marker for this SNP mutation. By randomly selecting individual plants in the F2 generation, we found that the dCAPS2 molecular marker co-segregated with the short vine trait of watermelon ( Figure 3 ).

[0090] The molecular marker dCAPS2 is shown in SEQ ID NO.3 or SEQ ID NO.4, and the upstream primer (dCAPS2-F) and the downstream primer (dCAPS2-R) of the primer pair for amplifying the molecular marker are respectively:

[0091] dCAPS2-F: 5'-ATCCTTGTTAAAGCGGATGCTA-3' (SEQ ID NO. 1);

[0092] dCAPS2-R: 5'-TCCCAGTAAGTTTTCATGCAAGT-3' (SEQ ID NO. 2);

[0093] The size of the amplified product of this primer in the male parent (normal material) is 129 bp, and the size of the amplified product in the female parent (short vine and few side branches material) is 109 bp. Specifically:

[0094] 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:

[0095]

[0096] A characteristic band of 129 bp was obtained by PCR amplification. The specific base sequence of the characteristic band of 129 bp is as follows:

[0097]

[0098] IV. Application of Molecular Markers

[0099] (1) DNA extraction, PCR amplification and electrophoresis detection

[0100] 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 Cldbl, Henan Agricultural University, 2021.

[0101] (2) Detection of short vines and few side branches varieties / genotypes of watermelon samples

[0102] PCR amplification was performed on the watermelon samples to be tested (parents and F2 population) using the primers. The results showed that if the product after enzyme cleavage was a characteristic band with a length of 109 bp, the plant to be tested was a homozygous watermelon short vine and few side branches material; if the product after enzyme cleavage was a characteristic band with a length of 129 bp, the plant to be tested was a homozygous watermelon normal material; if the product after enzyme cleavage was two characteristic bands with lengths of 109 bp and 129 bp respectively, the plant to be tested was a heterozygous watermelon normal material.

[0103] Therefore, according to the amplified bands, the variety / genotype / trait of a single plant can be quickly distinguished; further, the inventors used the dCAPS2 molecular marker 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 dCAPS2 marker of the present invention for the short vine and few side branches molecular marker in the natural population of watermelon was 100%. Based on this result, it is shown that the molecular marker dCAPS2 of the present invention can effectively distinguish whether the watermelon plant is a short vine and few side branches material.

[0104] Table 3 Numbers and phenotypes of 40 natural watermelon materials used for verification

[0105] Material No. Germplasm resource bank number Phenotype Material No. Germplasm resource bank number Phenotype wm002 PI 386018 02 SD Long vines with many side branches wm240 PI 295843 Long vines with many side branches wm003 PI 388770 01 SD Long vines with many side branches wm241 PI 296335 Long vines with many side branches wm019 PI 244018 01 SD Long vines with many side branches wm250 PI 482286 Long vines with many side branches wm025 PI 296342 01 SD Long vines with many side branches wm252 PI 482303 Long vines with many side branches wm100 PI 432337 01 SD Long vines with many side branches wm253 PI 482307 Long vines with many side branches wm101 PI 537300 02 SD Long vines with many side branches wm254 PI 482309 Long vines with many side branches wm103 PI 635598 01 SD Long vines with many side branches wm266 PI 494817 Long vines with many side branches wm106 PI 635702 01 SD Long vines with many side branches wm268 PI 500332 Long vines with many side branches wm108 PI 635730 01 SD Long vines with many side branches wm269 PI 504519 Long vines with many side branches wm110 PI 482326 01 SD Long vines with many side branches wm270 PI 505604 Long vines with many side branches wm154 PI 271773 01 SD Long vines with many side branches wm271 PI 512854 Long vines with many side branches wm220 PI 482303-02SD Long vines with many side branches wm274 PI 532667 Long vines with many side branches wm223 PI 171392 Long vines with many side branches wm275 PI 532668 Long vines with many side branches wm224 PI 179240 Long vines with many side branches wm276 PI 532669 Long vines with many side branches wm228 PI 225557 Long vines with many side branches wm282 PI 549163 Long vines with many side branches wm229 PI 254624 Long vines with many side branches wm284 PI 596658 Long vines with many side branches wm233 PI 270565 Long vines with many side branches wm288 PI 596675 Long vines with many side branches wm234 PI 271767 Long vines with many side branches wm290 PI 606135 Long vines with many side branches wm235 PI 271770 Long vines with many side branches wm291 PI 632751 Long vines with many side branches wm238 PI 274035 Long vines with many side branches wm292 PI 635670 Long vines with many side branches

[0106] In summary, the derived enzyme-cut amplified sequence polymorphism marker of the present invention can be used for large-scale identification of the short vine and few lateral branches traits of watermelon. It is not only fast and effective, but also can be identified at the seedling stage, greatly shortening the breeding cycle, and can be used on a large scale in production.

[0107] 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 short vine and few lateral branches gene of watermelon, 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 short vine and few lateral branches gene of watermelon according to claim 1, characterized in that: The molecular marker is a dCAPS marker.

3. Use of the molecular marker co-segregated with the short vine and few lateral branches gene of watermelon according to claim 1 in identifying or assisting in identifying the short vine and few lateral branches trait / genotype of watermelon.

4. A method for determining a watermelon variety / genotype with short vines and few side branches, 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 109 bp as shown in SEQ ID NO.3, then the watermelon to be tested is a homozygous watermelon material / genotype with short vines and few side branches; if the enzyme cleavage product has only one characteristic band with a length of 129 bp as shown in SEQ ID NO.4, then the watermelon to be tested is a homozygous watermelon material / genotype with long vines and many side branches; if the PCR amplification product has both a characteristic band with a length of 109 bp as shown in SEQ ID NO.3 and a characteristic band with a length of 129 bp as shown in SEQ ID NO.4, then the watermelon to be tested is a heterozygous watermelon material / genotype with long vines and many side branches.

5. The method according to claim 4, characterized in that: The DNA of the test materials was extracted using the CTAB method.

6. The method according to claim 4, characterized in that The enzyme digestion reaction system described in step (3) is: 4uL of PCR product, 1ul of 10X buffer, 0.5uL of DdeI restriction endonuclease, 4.5ul of sterile distilled water, total volume 10ul, and the enzyme digestion conditions are 37°C and 4h.

7. A kit for identifying the short vine and few side branches trait of watermelon, characterized in that: Comprising the primer pair described in claim 1.

8. Use of a reagent for detecting the presence of a dCAPS marker in localizing the short vine and few lateral branches gene Cldbl in watermelon, characterized in that: The upstream primer sequence of the primer pair for amplifying the dCAPS marker is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2.

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