Tightly linked SNP molecular markers for watermelon seed coat thickness and its application
By developing SNP molecular markers with tightly linked thickness of watermelon seed coat and dCAPS molecular marker N6-32, the problem of fine localization and identification of watermelon seed coat thickness genes was solved, and the rapid and accurate identification of watermelon seed coat thickness genotype was achieved, and the breeding progress of soft-seed watermelons was promoted.
Patent Information
- Application Number
- CN202210957476.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-08-10
- Publication Date
- 2025-03-07
- Estimated Expiration
- 2042-08-10
AI Technical Summary
The prior art is difficult to effectively solve the problems of gene fine localization and molecular identification of watermelon seed coat thickness, which limits the breeding progress of soft-seed watermelons.
A SNP molecular marker with tightly linked thickness of watermelon seed coat was developed. Based on the V2 version of the Watermelon Reference Genomics of Watermelon, the base located at chromosome 7046144bp of chromosome 6 was mutated from T to C. The dCAPS molecular marker primer N6-32 was designed, and the identification of seed coat thickness genotype was achieved through PCR amplification and enzyme cleavage analysis.
It has achieved rapid, accurate and efficient identification of the thickness genotype of watermelon seed coat, improved the accuracy and selection efficiency of breeding, and has important scientific and practical significance.
Smart Images
Figure CN115369183B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of watermelon molecular marker-assisted breeding, and in particular to a watermelon seed coat thickness tightly linked SNP molecular marker and application thereof. Background Art
[0002] watermelon( Citrullus lanatus ) is one of the top ten fruits in the world. It is a seasonal fruit that is deeply loved by people because of its juicy and delicious taste, rich in functional nutrients such as citrulline, carotenoids, and lycopene that are beneficial to the human body. However, the seeds in the watermelon fruit affect the eating experience; therefore, the characteristics of watermelon seeds, such as seed size and number, are the key characteristics to be examined when selecting and breeding new watermelon varieties. Breeding varieties with small seeds and a small number of seeds is conducive to increasing the edible rate of watermelon pulp and making it convenient for consumers to eat. At present, seedless watermelon seeds, which are mainly created based on chromosome doubling technology, have obvious "three lows" problems (Li Han et al., 2014). In addition, their fruits generally mature late and require pollination varieties, which restrict the comprehensive promotion of seedless watermelon in production. If soft-seeded watermelons with edible seeds can be cultivated, like soft-seeded pomegranates, they can be swallowed without obstacles after chewing and without spitting out seeds, which will be a major breakthrough in watermelon breeding.
[0003] On the other hand, watermelon seeds are high-quality protein and vegetable oil resources (Wang Xiaojuan, 2012), which have important health functions for the human body. For fresh watermelon, the thick seed coat is a natural protective barrier for the seeds, but it is also the main reason why the seeds cannot be eaten directly. Citrullus Mucosospermus ) is the population most closely related to modern cultivated watermelon (Guo et al., 2019). Some germplasm seeds are only covered with a light yellow-brown, almost transparent seed coat, and the seed coat thickness is only 1 / 3 of that of ordinary watermelon seed coat. If molecular markers that can be used to assist breeding can be obtained to assist in the selection of thin-skin varieties, and thin-skin seeds and flesh can be used as edible objects, the innovative breeding goal of eating melons without spitting out seeds (soft-seeded watermelon) can be finally achieved, which has important scientific and practical significance. Therefore, it is urgent to develop a molecular marker that is closely linked to the watermelon seed coat thickness, easy to operate, highly efficient, and low-cost, in order to provide efficient technical means for the fine positioning and molecular identification of watermelon seed coat thickness genes.
[0004] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention
[0005] In view of at least one of the above technical problems, the present disclosure provides a SNP site tightly linked to watermelon seed coat thickness (based on the watermelon 97103 reference genome sequence V2 version, the base at 7046144bp of chromosome 6 of the watermelon genome mutated from T to C), and designed dCAPS molecular marker primer N6-32 based on the enzyme cutting information of the SNP site.
[0006] According to one aspect of the present disclosure, a SNP molecular marker tightly linked to the watermelon seed coat thickness gene is provided. Based on the watermelon 97103 reference genome version V2, the base located at 7046144bp on chromosome 6 mutates from T for the thick seed coat trait to C for the thin seed coat trait.
[0007] According to another aspect of the present disclosure, a detection kit is provided, comprising NlaIII restriction endonuclease and the following primer N6-32:
[0008] Forward primer N6-32F: 5′-TAATAGGGAAGAGTAGAGGG -3′;
[0009] Reverse primer N6-32R: 5′- TAAACCAAACACAAGCCAT -3′.
[0010] According to another aspect of the present disclosure, the SNP molecular marker or the detection kit is applied to the breeding of watermelon seed coat thickness trait varieties / lines.
[0011] According to another aspect of the present disclosure, a method for identifying watermelon seed rind thickness genotype is provided, comprising the following steps:
[0012] (1) After extracting the total DNA of the watermelon to be identified, PCR amplification was performed using the primer N6-32:
[0013] (2) Digest the PCR product obtained in the previous step with NlaIII restriction endonuclease;
[0014] (3) The resulting enzyme digestion products were subjected to electrophoresis analysis, and the genotype was determined based on the band size and position relationship of the amplified products. The presence of a single band of 391 bp or two bands of 391 bp and 375 bp indicated a thick seed coat genotype, and the presence of a single band of 375 bp indicated a thin seed coat genotype.
[0015] In some embodiments of the present disclosure, in step (1), the total DNA of the watermelon leaves to be identified is extracted using the plant genomic DNA extraction kit TIANGEN.
[0016] In some embodiments of the present disclosure, in the step (1), the PCR amplification system is: 1 μL of total DNA from watermelon leaves, 1 μL of the forward primer N6-32F, 1 μL of the reverse primer N6-32R, 12.5 μL of 2× Power Taq PCR MasterMix, and 9.5 μL of ddH2O; the amplification program is: 94°C for 5 min, 35 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 30 s, and 72°C for 10 min.
[0017] In some embodiments of the present disclosure, in step (2), the enzyme digestion reaction system is: 2 μl of PCR product, 0.2 μl of NlaIII restriction endonuclease, 1 μl of 10× buffer, and 6.8 μl of ddH2O; the reaction procedure is: constant temperature treatment at 37°C for 10 h and inactivation at 65°C for 20 min.
[0018] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages:
[0019] 1. A molecular marker that is tightly linked to watermelon seed coat thickness was screened and applied to molecular marker-assisted selection breeding, which can achieve directional genetic improvement of watermelon seed coat thickness more quickly and accurately.
[0020] 2. The identification was applied in backcross populations of watermelons with different seed coat thicknesses, and the genotype accuracy was 100%. This can provide a new means for identifying watermelon seed coat thickness and improve breeding accuracy and selection efficiency.
[0021] 3. The watermelon seed coat thickness genotype can be quickly identified through simple experimental operations and analysis, providing a fast, simple, scientific and practical detection and identification technology for the detection of watermelon seed coat thickness traits. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is the QTL positioning result of the watermelon seed coat thickness trait in one embodiment of the present application.
[0023] Figure 2 This is a diagram of the enzyme digestion results of the PCR products of the primers of the molecular marker N6-32 in one embodiment of the present application in watermelon B3 (maternal), B4 (paternal) and part of the BCF2 population; wherein, A: maternal genotype, B: paternal genotype, H: heterozygous genotype, M: marker; the boxes are the maternal and paternal bands respectively. DETAILED DESCRIPTION
[0024] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.
[0025] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; unless otherwise specified, the reagents involved are all commercially available conventional reagents; unless otherwise specified, the test methods involved are all conventional methods.
[0026] Example 1: Determination of SNP loci tightly linked to watermelon seed coat thickness and development of molecular markers
[0027] Simplified genome sequencing was performed on different types of watermelon materials, and the selected regions between populations were determined by combining the differences in population polymorphism, and the chromosome intervals related to the thickness of the watermelon seed coat were obtained. The SNP sites in the target chromosome interval were screened by deep resequencing of the parental materials, and CAPS / dCAPS molecular marker primers were designed. PCR amplification was performed using CAPS / dCAPS molecular marker primers and watermelon genomic DNA, and finally the amplified products were digested and verified by electrophoresis. All the test materials are innovative germplasm materials preserved in the Chinese Watermelon and Muskmelon Medium-term Bank of Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences.
[0028] The specific steps are as follows:
[0029] 1. Preliminary location of the major QTL interval for watermelon seed coat thickness
[0030] The high-density genetic linkage map constructed by hybridizing the female parent B3 (Chinese Watermelon and Muskmelon Mid-term Library No. ZXG01562, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences) of sticky-seed watermelon and the male parent B4 (female parent No. ZXG01562, male parent No. ZXG01625, female parent and male parent are hybridized and backcrossed and self-pollinated to select pure lines) of the hybrid F1 was obtained, and then F1 was backcrossed with B3 to obtain the BC1 population. The seed coat thickness of 99 single melons from the parents, F1 and BC1 populations was visually identified; combined with the genetic linkage map and the phenotypic identification results of the BC1 population, the R / qtl-CIM method was used to perform preliminary QTL positioning, and the major effect QTL of seed coat thickness was identified. The QTL is located on chromosome 6, with a LOD peak of 27.19, explaining 95.96% of the phenotypic variation, and the confidence interval is 4258103bp-12820185bp (see Figure 1 ).
[0031] 2. Development of SNP loci and dCAPS markers for watermelon seed coat thickness
[0032] (1) Total DNA from B3 and B4 watermelon leaves was extracted using a plant genomic DNA extraction kit (TIANGEN). The specific steps are as follows:
[0033] ① Take 100 mg of fresh plant tissue or 20 mg of dry weight tissue, add liquid nitrogen and grind thoroughly. Add 400 μl of buffer FGA and 6 μl of RNase A (10 mg / ml), vortex for 1 min, and leave at room temperature for 10 min.
[0034] ②Add 130 μl buffer LP2, mix thoroughly, and vortex for 1 min.
[0035] ③ Centrifuge at 12,000 rpm (~13,400×g) for 5 min and transfer the supernatant to a new centrifuge tube.
[0036] ④ Add 1.5 times the volume of buffer LP3 (e.g. 500 μl of filtrate plus 750 μl of buffer LP3) (please check whether anhydrous ethanol has been added before use), and immediately shake and mix thoroughly for 15 sec. Flocculent precipitation may appear at this time.
[0037] ⑤ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 sec, pour out the waste liquid, and place the adsorption column CB3 in a collection tube.
[0038] ⑥ Add 600 μl of rinse solution PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the waste liquid, and place the adsorption column CB3 in the collection tube.
[0039] ⑦Repeat step ⑥.
[0040] ⑧Put the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400×g) for 2 min, and discard the waste liquid. Leave the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.
[0041] ⑨ Transfer the adsorption column CB3 into a clean centrifuge tube, add 200 μl of elution buffer TB to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm (~13,400×g) for 2 minutes, and collect the solution into the centrifuge tube.
[0042] ⑩ Determine the concentration of DNA using a UV spectrophotometer and store it in a -20℃ refrigerator for future use.
[0043] (2) Design of dCAPS marker primers for identifying watermelon seed coat thickness
[0044] Deep resequencing of paternal and maternal watermelon materials was used to screen the SNP mutation site in the target chromosome interval. The site was located at 7046144bp on chromosome 6 of the watermelon genome, and its base mutated from T to C.
[0045] The mutation information of the SNP site was analyzed, and the sequence with CAPS / dCAPS mutation was obtained. The specific dCAPS molecular marker of watermelon seed coat thickness was named N6-32, and the dCAPS molecular marker primers were designed:
[0046] Forward primer N6-32F: 5′-TAATAGGGAAGAGTAGAGGG -3′;
[0047] Reverse primer N6-32R: 5′- TAAACCAAACACAAGCCAT -3′;
[0048] The restriction endonuclease used was NlaIII.
[0049] (2) PCR reaction system and procedure
[0050] Reaction system: 1 μL of 100 ng / μL total DNA from watermelon plants, 1 μL of dCAPS molecular marker upstream primer N6-32F, 1 μL of downstream primer N6-32R, 12.5 μL of 2×Power Taq PCR MasterMix, 9.5 μL of ddH2O; reaction program: 94°C for 5 min, 35 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 30 s, and 72°C for 10 min;
[0051] (3) Enzyme digestion reaction system and procedure:
[0052] The reaction system was: PCR product 2 μl, NlaIII restriction enzyme 0.2 μl, 10× buffer 1 μl, ddH2O 6.8 μl;
[0053] The reaction procedure was: constant temperature treatment at 37°C for 10 h, inactivation at 65°C for 20 min;
[0054] (4) Perform 8% polyacrylamide gel electrophoresis, development, staining and band pattern interpretation on the enzyme digestion products.
[0055] A. Reagent Preparation
[0056] ① 5× TBE electrophoresis buffer: weigh 26.95 g Tris, 1.86 g EDTA, 13.75 g boric acid, and dilute to 500 mL with deionized water;
[0057] ② 40% polyacrylamide solution: polyacrylamide 77.34g, methylene bisacrylamide 2.66g, dilute to 200mL with deionized water;
[0058] ③ 8% polyacrylamide gel: 40% polyacrylamide solution 10ml; 5×TBE 5mL; 10% ammonium persulfate (APS) 200μL; tetramethylethylenediamine (TEMED) 80μL; distilled water 22mL;
[0059] ④ Silver staining solution: 1g silver nitrate; 5mL glacial acetic acid; 50mL anhydrous ethanol; add deionized water to make up to 500mL;
[0060] ⑤Developing solution: 15 g of sodium hydroxide; 2.5 mL of formaldehyde (37%); add deionized water to make up to 500 mL.
[0061] (5) Preparation of gel plate: Wash the gel glass plate with distilled water, dry it, wipe it with a cotton ball soaked in anhydrous ethanol, and dry it. Place the concave plate and the flat plate tightly together in the gel maker, press them tightly, and fasten the clamps on both sides, with the concave glass facing inward. Prepare 8% polyacrylamide gel solution in a washing bottle, mix it well, and quickly inject it into the gap between the two plates, and pay attention to prevent bubbles from forming. After filling, quickly insert a toothed comb, and it is best if the bottom of the comb just touches the gel surface, and wait for the solution to fully solidify.
[0062] (6) Electrophoresis: Remove the gel casting stand from the base and place it directly into the matching electrophoresis tank. Pour an appropriate amount of 1×TBE buffer at the bottom of the electrophoresis tank and between the two glass plates on the stand. Add 0.2 times the volume of 6×DNA Loading Buffer to the PCR product, mix well, take 0.8μL and add it to the sample well. Perform electrophoresis at 260V for 35min.
[0063] (7) Staining and development: After electrophoresis is completed, remove the glass plate from the electrophoresis tank and pry off the concave plate. The gel will adhere to the plate. Place the plate with the gel side facing up in the silver staining solution and place it on a destaining shaker and shake gently for 15 minutes. The gel will automatically fall off. After silver staining is completed, take out the gel and wash it in deionized water for 10 seconds. After washing, transfer the gel to the developer and shake the shaker gently. When the bands are clear, take out the gel and place it on a film reader for observation and take a photo for preservation.
[0064] (8) Band pattern interpretation: Place the glass plate that has been naturally dried after development on the reading table to observe the positional differences in the bands of the parental and BC groups.
[0065] The results of genotyping using the N6-32 molecular marker in maternal parent B3 and paternal parent B4 are as follows: Figure 2As shown in the figure, after enzyme digestion, the target fragment size of B3 is 375bp and the target fragment size of B4 is 391bp. The enzyme digestion product band is 375bp (A) for thin seed skin type, the enzyme digestion product band is 391bp (B) for thick seed skin watermelon type, and the product band is 375 and 391bp (H) for thick seed skin watermelon type.
[0066] Example 2: Genotype verification analysis of seed coat thickness in BCF2 population
[0067] Table 1 Identification and validation of N6-32 in the BCF2 population
[0068]
[0069] After the genotype identification of the N6-32 molecular marker in the BCF2 population constructed by B3 and B4 in Example 1, the distribution of the three genotypes of the N6-32 molecular marker in 99 BCF2 populations was checked, and it was found that the N6-32 genotype was completely co-segregated with the watermelon seed coat thickness phenotype. The results are shown in Table 1, where A represents the maternal band type, B represents the paternal band type, and H represents the heterozygous genotype.
[0070] All 28 lines with genotype A of molecular marker N6-32 were thin-skin watermelon types, while 16 lines with genotype B and 55 lines with genotype H were thick-skin watermelon types, and the accuracy of genotype identification was 100%.
[0071] Although some preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.
[0072] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of this application and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. Application of a watermelon seed coat thickness tightly linked SNP molecular marker and primer N6-32 thereof in the breeding of watermelon seed coat thickness varieties / lines, characterized in that: The watermelon seed coat thickness tightly linked SNP molecular marker is based on the watermelon 97103 reference genome version V2, and the base at 7046144bp on chromosome 6 mutates from T of the thick seed coat trait to C of the thin seed coat trait; the primer N6-32 is as follows: Forward primer N6-32F: 5′-TAATAGGGAAGAGTAGAGGG -3′; Reverse primer N6-32R: 5′- TAAACCAAACACAAGCCAT -3′.
2. A method for identifying watermelon seed coat thickness genotype, characterized in that: The steps include: (1) After extracting the total DNA of the watermelon to be identified, PCR amplification was performed using the primer N6-32 described in claim 1: (2) Digest the PCR product obtained in the previous step with restriction endonuclease NlaIII; (3) The resulting enzyme digestion products were subjected to electrophoresis analysis, and the genotype was determined based on the band size and position relationship of the amplified products. The presence of a single band of 391 bp or two bands of 391 bp and 375 bp indicated a thick seed coat genotype, and the presence of a single band of 375 bp indicated a thin seed coat genotype.
3. The method for identifying western seed coat thickness genotype according to claim 2, characterized in that: In the step (1), the total DNA of the watermelon leaves to be identified is extracted using the plant genomic DNA extraction kit TIANGEN.
4. The method for identifying watermelon seed coat thickness genotype according to claim 2, characterized in that: In the step (1), the PCR amplification system is: 1 μL of total DNA from watermelon leaves, 1 μL of the forward primer N6-32F, 1 μL of the reverse primer N6-32R, 12.5 μL of 2× Power Taq PCR MasterMix, and 9.5 μL of ddH2O; the amplification program is: 94°C for 5 min, 35 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 30 s, and 72°C for 10 min.
5. The method for identifying watermelon seed coat thickness genotype according to claim 2, characterized in that: In step (2), the enzyme digestion reaction system is: 2 μl of PCR product, 0.2 μl of NlaIII restriction endonuclease, 1 μl of 10× buffer, and 6.8 μl of ddH2O; the reaction procedure is: constant temperature treatment at 37°C for 10 h and inactivation at 65°C for 20 min.