Molecular marker, primer, kit for breaking linkage of smooth glabrous male sterility and co-separation of watermelon fertility genes and application thereof

Abstract

This invention discloses a molecular marker and its application that breaks the linkage between smooth, hairless male sterility and co-segregation of watermelon fertility genes, aiming to solve the technical problem of the current lack of methods for identifying and characterizing major watermelon fertility genes. This application develops a molecular marker that breaks the linkage between smooth, hairless male sterility and co-segregation of watermelon fertility genes (based on the watermelon 97103 reference genome V2 version, which contains a genomic region related to male sterility in the 27.94-27.95 Mb region on chromosome 8; watermelons with deletions or partial deletions of this genomic region exhibit male sterility), and designs the corresponding primer pair BY2-3. Applying this molecular marker for marker-assisted selection breeding enables faster and more accurate targeted genetic improvement of watermelon fertility, thereby shortening the breeding cycle.

Description

Technical Field

[0001] This invention application relates to the field of molecular marker-assisted breeding technology, specifically to a molecular marker, primer, kit, and its application for breaking the linkage between smooth, hairless male sterility and watermelon fertility genes. Background Technology

[0002] Since the 1980s, when my country began to promote and apply watermelon hybrid breeding technology, the use of male-sterile lines has greatly reduced seed production costs and improved seed quality, thus gaining widespread application. Types of male-sterile traits include smooth, hairless male-sterile lines, G17AB male-sterile lines, short-vine male-sterile lines, and translocation male-sterile lines (Ma Shuangwu et al., 2006, Progress in Research and Utilization of Watermelon-Specific Germplasm Resources in my country). Smooth, hairless mutants are usually sterile lines. The National Watermelon and Melon Germplasm Resource Mid-term Bank preserves three materials from which smooth, hairless male-sterile materials can be isolated (due to sterility, seeds can only be preserved as F1).

[0003] Currently, with the rapid development and cost reduction of third-generation sequencing technology, as well as the advancement of related bioinformatics technologies, a large number of molecular marker loci can be identified in both model and non-model species. Molecular markers such as Indels and SNPs can be widely used in the construction of genetic maps for horticultural crops, QTL analysis, and marker-assisted selection breeding.

[0004] However, despite the high economic value and market demand of watermelon, an important horticultural crop, the methods for identifying and characterizing major genes related to watermelon fertility remain insufficient. This has, to some extent, hindered the progress of watermelon breeding, making it difficult to improve and select watermelon varieties with superior fertility traits through precise means. Therefore, developing a molecular marker closely related to watermelon fertility that is easy to operate, has high identification efficiency, and is relatively inexpensive has become an urgent task in current watermelon genetic breeding research. This will not only help reveal the genetic mechanisms of watermelon fertility but also greatly accelerate the breeding process of superior watermelon varieties and promote the sustainable development of the watermelon industry.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The inventors of this application (Ma Shuangwu et al.) discovered a hairless fertile mutant while conducting basic work on germplasm resources. The mutant has smooth stems and leaves without hairs, and its male and female flowers are normal. It can produce fruit normally after pollination. The discovery of this mutant provides the possibility of breaking the linkage of smooth, hairless male sterility and directly identifying fertility genes.

[0007] Based on the above research findings, this disclosure provides a molecular marker that breaks the linkage between smooth, hairless male sterility and watermelon fertility genes, and designs corresponding identification / detection primers BY2-3 based on this molecular marker, aiming to solve the current technical problem of lacking effective methods for identifying and characterizing major watermelon fertility genes.

[0008] According to the first aspect of this disclosure, a molecular marker co-segregating with a watermelon fertility gene is provided, based on the watermelon 97103 reference genome version V2, which contains a genomic deletion region in the 27.94-27.95 Mb region of chromosome 8 that is associated with male sterility and is not linked to the smooth, hairless trait. Watermelons with deletion or partial deletion of this genomic region exhibit male sterility.

[0009] According to a second aspect of this disclosure, a primer is provided for identifying or detecting the molecular marker, wherein the upstream primer sequence is located in the genomic deletion region associated with male infertility, and the downstream primer sequence is located within 5000 bp downstream of the genomic region associated with male infertility.

[0010] In some embodiments of this disclosure, the primers include the primer pair BY2-3: BY2-3F: 5'- TCTGACTTGCCTGGAACAT -3'; BY2-3R: 5'-AAATCCAACCGTCTATCGT-3'.

[0011] According to the third aspect of this disclosure, the above-mentioned molecular marker, its detection reagent or the above-mentioned primers are used in any one of the following (1) to (7): (1) Screening or identifying watermelon fertility traits; (2) Prepare reagents / kits for screening or identifying watermelon fertility traits; (3) Selection of watermelon fertility traits or preparation of reagents / kits for selection of watermelon fertility traits; (4) Molecular marker-assisted breeding related to watermelon fertility; (5) Variety improvement related to watermelon fertility; (6) Watermelon fertility genotyping; (7) Screening or identifying hairless sterile, hairless fertile, or / and hairy fertile watermelon materials / single lines.

[0012] According to a fourth aspect of this disclosure, a method for selecting / identifying watermelon fertility is provided, comprising the following steps: (1) Extract genomic DNA from watermelon tissue; (2) The genomic DNA was amplified using the primers described above, and the amplification products were detected by electrophoresis to determine the banding pattern; (3) The fertility of watermelon is determined according to the banding pattern. If the amplified product has no band (genotype A), it is a male-sterile watermelon; if the amplified product band size is 1180bp (genotype B), it is a fertile watermelon.

[0013] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: 1. The molecular markers in this application break the linkage between smooth, hairless watermelon and male sterility, and can identify three types of materials and single lines of watermelon: hairless sterile, hairless fertile, and hairy fertile. The genotypic accuracy rate is 100%, which provides a new means for identifying watermelon fertility and breeding, and improves the accuracy and selection efficiency of breeding.

[0014] 2. This application provides a rapid, simple, scientific and practical detection and identification technique for watermelon fertility traits through simple experimental operation and analysis.

[0015] 3. Applying the molecular markers of this application for marker-assisted selection breeding can enable faster and more accurate directional genetic improvement of watermelon fertility, thereby shortening the breeding cycle. Attached Figure Description

[0016] Figure 1 This is the QTL localization result of the smooth, hairless trait of watermelon in one embodiment of this application.

[0017] Figure 2 This is a visual representation of the key genetic variations (IGV) of three types of materials—hairless sterile, hairless fertile, and hairy fertile—in one embodiment of this application.

[0018] Figure 3 The BY2-3 markers in one embodiment of this application represent the banding patterns and material types they represent; where A: sterile genotype, B: fertile genotype, and M: marker. Detailed Implementation

[0019] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the detection methods involved are all conventional methods unless otherwise specified.

[0020] The three types of watermelon materials involved in the following examples are: hairy fertile watermelon material 8R121 (national watermelon and melon germplasm resource mid-term bank number ZXG00140, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences); hairless fertile mutant watermelon material WMX (national watermelon and melon germplasm resource mid-term bank number ZXG00303 mutation, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences); hairless sterile watermelon material G325 (national watermelon and melon germplasm resource mid-term bank number ZXG00325 mutation, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences); and G1368 (national watermelon and melon germplasm resource mid-term bank number ZXG01368 mutation, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences).

[0021] The specific implementation of this application will be described below with reference to the embodiments. However, the following embodiments are only used to illustrate this application in detail and do not limit the scope of this application in any way.

[0022] Example 1: Identification of molecular markers and primers co-segregating with watermelon fertility using resequencing F1 was obtained by crossing 8R121 and WMX, and BC1 population was obtained by backcrossing F1 with WMX. Genetic linkage map was constructed, and 30 monoline seedlings from the 8R121, WMX, F1, and BC1 populations were visually identified. Based on the constructed genetic linkage map and phenotypic identification results, genome-wide QTL mapping was performed to obtain a major QTL associated with the hairless trait. Figure 1 The QTL is located in the 26.2-26.7 Mb region of chromosome 8 (based on the Watermelon 97103 reference genome version V2), with a peak LOD of 9.86, explaining 16.8% of the phenotypic variation, and a confidence interval of 168.12-174.73 cM.

[0023] IGV analysis of resequencing results from three different watermelon materials (8R121, WMX, G325, and G1368) revealed large deletions near QTL peaks. Based on the genomic characteristics of hairy / hairy and male-sterile / fertile traits, these deletions were identified as genomic deletion regions associated with hairlessness (based on Watermelon 97103 Reference Genome V2, Chr8: 27.90-27.94 Mb) and male-sterile genomic deletion regions (based on Watermelon 97103 Reference Genome V2, Chr8: 27.94-27.95 Mb). Figure 2 Molecular markers were developed based on genomic deletion regions associated with male infertility. The upstream primer sequence is located in the genomic deletion region associated with male infertility, and the downstream primer sequence is located downstream of the genomic region associated with male infertility. This marker was named BY2-3. BY2-3F: 5'- TCTGACTTGCCTGGAACAT -3'; BY2-3R: 5'-AAATCCAACCGTCTATCGT-3'.

[0024] Example 2: Genotyping analysis of the BY2-3 marker in different types of materials This example demonstrates the accuracy of the BY2-3 molecular marker and its primers developed in Example 1: 1. Total DNA was extracted from leaves of different types of watermelon materials using a plant genomic DNA extraction kit (TIANGEN). The specific steps are as follows: ① Take 100 mg of fresh plant tissue or 20 mg of dry 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 incubate at room temperature for 10 min; ② Add 130 μl of buffer LP2, mix thoroughly, and vortex for 1 min; ③ Centrifuge at 12,000 rpm (~13,400×g) for 5 min, and transfer the supernatant to a new centrifuge tube; ④ Add 1.5 times the volume of buffer LP3 (e.g., 500 μl of filtrate plus 750 μl of buffer LP3) (please check before use whether anhydrous ethanol has been added), and immediately vortex thoroughly for 15 minutes. 5. Add the solution and flocculent precipitate obtained in the previous step to the adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube; 6. Add 600 μl of washing buffer 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 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube; 7. Repeat step 6; 8. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400×g) for 2 minutes, discard the waste liquid, and then place the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual washing liquid in the adsorption material; 9. Transfer the adsorption column CB3 to a clean centrifuge tube, and add 200 μl of the solution to the middle of the adsorption membrane. Add μl of elution buffer TB, incubate at room temperature for 2-5 min, centrifuge at 12,000 rpm (~13,400×g) for 2 min, and collect the solution in a centrifuge tube; ⑩ Determine the DNA concentration using a UV spectrophotometer and store at -20℃ for later use.

[0025] 2. PCR amplification: DNA extracted using primers designed in Example 1 was amplified by PCR. The reaction system and procedure for PCR amplification are as follows: (1) Reaction system: 100 ng / μL total DNA from watermelon plants 1 μL, upstream primer BY2-3F of BY2-3 molecular marker 1 μL, downstream primer BY2-3R 1 μL, 2×Power Taq PCR MasterMix 12.5 μL, ddH2O 9.5 μL; (2) Reaction procedure: 94℃ for 5 min, 35 cycles of 94℃ for 30 s, 55℃ for 1 min, 72℃ for 60 s, 72℃ for 10 min.

[0026] 3. Perform 1% agarose gel electrophoresis on the PCR products (using a 120mm*120mm tray as an example) to determine the banding pattern and determine the genotype based on the banding pattern. The specific steps are as follows: (1) Gel preparation: Place the gel preparation tray in a horizontal gel preparation rack and insert the required comb. Weigh 1g of agarose (Bio9109) and put it into an Erlenmeyer flask. Add 100mL of 1×TAE buffer (dilute 50×TAE buffer (Solepro) to 1× as the working solution), heat it in a microwave oven until completely dissolved, and then take it out and cool it. When the temperature drops to 50-60℃, add 10μL of non-toxic nucleic acid dye (4S Green Plus non-toxic nucleic acid dye, Sangon Biotech, A616696), mix well, and slowly pour it into the prepared gel tray. After the gel has completely solidified, remove the comb and place the tray and gel together into the horizontal electrophoresis tank. The side of the gel with the hole should be at the negative electrode of the electrophoresis tank, i.e. the black end. Then add 1×TAE buffer until the gel is completely submerged. (2) Spotting: Take 5 μL of PCR product and add it into the spotting well. Finally, spot the appropriate DNA marker. (3) Electrophoresis: Connect the positive and negative electrodes of the electrophoresis tank to the electrophoresis apparatus respectively. Stabilize the voltage at 150V and electrophore for about 20 minutes, then turn off the power. (4) Band interpretation: Take out the agarose gel and place it in the UV gel cutter. Observe the bands under the UV lamp and take pictures to record the results.

[0027] The results of genotyping using the BY2-3 molecular marker in different fertility monolines are shown in Table 1 and 2. Figure 3 As shown, the PCR products exhibited two band patterns: blank (A) and 1180bp (B). Analysis revealed that all sterile monolines (1-7, 26-29) were blank (A), while all fertile monolines (8-24, 30-48) were 1180bp (B). Among all fertile monolines, both hairy (8-19, 30-40) and hairless (17-24, 41-48) showed fertile genotypes, indicating that the molecular marker BY2-3 broke the linkage of smooth, hairless male sterility and is a co-segregating molecular marker with the watermelon fertility gene. Furthermore, the accuracy of genotype identification using this molecular marker and its primers was 100%.

[0028] Table 1 Genotyping of different fertile materials .

[0029] Although some preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0030] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of its inventive concept. Therefore, if these modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include these modifications and variations.

Claims

1. A molecular marker co-segregating with a watermelon fertility gene, characterized in that, Based on the reference genome version V2 of watermelon 97103, there is a genomic region associated with male sterility in the 27.94-27.95 Mb region of chromosome 8. Watermelons with deletions or partial deletions of this genomic region exhibit male sterility.

2. A primer for identifying or detecting the molecular marker of claim 1, characterized in that, Its upstream primer sequence is located in the genomic deletion region associated with male infertility, and its downstream primer sequence is located within 5000 bp downstream of the genomic region associated with male infertility.

3. The primer according to claim 2, characterized in that, Including the following primer pair BY2-3: BY2-3F: 5'- TCTGACTTGCCTGGAACAT -3'; BY2-3R: 5'-AAATCCAACCGTCTATCGT-3'.

4. The molecular marker of claim 1, its detection reagent, or the primer of claim 2 may be used in any one of the following (1) to (7): (1) Screening or identifying watermelon fertility traits; (2) Prepare reagents / kits for screening or identifying watermelon fertility traits; (3) Selection of watermelon fertility traits or preparation of reagents / kits for selection of watermelon fertility traits; (4) Molecular marker-assisted breeding related to watermelon fertility; (5) Variety improvement related to watermelon fertility; (6) Watermelon fertility genotyping; (7) Screening or identifying hairless sterile, hairless fertile, or / and hairy fertile watermelon materials / single lines.

5. A method for selecting / identifying the fertility of watermelons, characterized in that, Includes the following steps: (1) Extract genomic DNA from watermelon tissue; (2) The genomic DNA is amplified using the primers described in claim 2, and the amplification products are detected by electrophoresis to determine the banding pattern; (3) The fertility of watermelon is determined according to the band pattern. If there is no band, it is a male-sterile watermelon; if the band size is 1180bp, it is a fertile watermelon.

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