Molecular markers closely linked to pepper seed male fertility gene and application thereof

Abstract

The application belongs to the field of plant genetic breeding, and discloses a molecular marker closely linked to a pepper nuclear male sterility gene and application thereof, and specifically discloses a molecular marker closely linked to a pepper nuclear male sterility gene, wherein the molecular marker comprises Ind146 and / or Ind147, the sequence of the Ind146 consists of the nucleotide sequence shown in SEQ ID NO:1 and SEQ ID NO:2, and the sequence of the Ind147 consists of the nucleotide sequence shown in SEQ ID NO:5 and SEQ ID NO:6. The molecular marker provided by the application realizes fine positioning of the pepper nuclear male sterility gene, and lays a foundation for final cloning of the pepper nuclear male sterility gene and establishment of a molecular marker assisted breeding system. The molecular marker of the application can be simply, rapidly and high-throughput applied to pepper seed production and breeding practice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetics and breeding technology, specifically involving molecular markers closely linked to the male fertility gene of pepper kernels and their applications. Background Technology

[0002] Chili peppers are an important vegetable crop in my country, with a planting area that has remained stable at over 2.1 million hectares in recent years, making it the most widely planted vegetable crop. Because chili peppers are typically cross-pollinated, they exhibit outstanding hybrid vigor, and over 90% of the chili pepper varieties currently promoted and used in China are F1 hybrids. Currently, chili pepper hybrid seed production includes two methods: artificial emasculation hybridization and male-sterile line hybridization. However, artificial emasculation is slow, labor-intensive, and often incomplete, leading to high seed production costs, unstable yields, and difficulty in guaranteeing hybrid purity. The discovery and application of the male-sterile trait in chili peppers can not only solve the problem of artificial emasculation and reduce production costs, but also improve the purity and yield of first-generation hybrids.

[0003] Heritable male sterility in plants can be classified into two types based on genotypic composition: genicmale sterility (GMS) and cytoplasmic male sterility (CMS). Pepper is one of the few crops that possesses both GMS and CMS systems. Currently, both systems are widely used in hybrid pepper seed production in China. In peppers, genicmale sterility has its own advantages compared to cytoplasmic male sterility: the vast majority of genicmale sterility traits are controlled by a single recessive gene, resulting in a simple and stable inheritance pattern that is easy to transfer; furthermore, genicmale sterility leads to complete abortion and is less affected by environmental factors, allowing for wider adaptability; and genicmale sterility has a wide range of restorer sources, reducing limiting factors in hybrid breeding and overcoming the problem of a lack of restorer sources in sweet peppers and large-fruited peppers.

[0004] Compared to model plants and other crops, molecular research on nuclear male sterility in pepper started relatively late. Although more than 20 nuclear male sterility genes in pepper have been identified, only 4 have been finely mapped or cloned, namely... msc-1 , msc-2 ( ms1 and ms w (It is the same gene as it) msc-3 and msc-4 The molecular mechanism underlying male sterility in pepper kernels remains unclear. Therefore, the discovery and cloning of the male sterility gene in pepper kernels is not only of great significance for pepper seed production and other production practices, but also provides theoretical support for elucidating the molecular mechanism of male sterility in pepper kernels, which is of great scientific importance. Summary of the Invention

[0005] This invention aims to solve at least one of the technical problems existing in the prior art. This invention provides molecular markers closely linked to the male fertility gene in pepper kernels and their applications.

[0006] The first aspect of this invention is to provide a molecular marker that is closely linked to the male fertility gene in pepper kernels.

[0007] The second aspect of the present invention aims to provide a primer set for amplifying the molecular markers of the first aspect of the present invention that are closely linked to the male fertility gene of pepper nucleus.

[0008] A third aspect of the present invention is to provide a reagent kit.

[0009] The fourth aspect of this invention aims to provide the application of the molecular markers of the first aspect of this invention, the primer sets of the second aspect of this invention, or the kits of the third aspect of this invention in the preparation of products for assisted breeding.

[0010] The fifth aspect of this invention aims to provide an auxiliary breeding method.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a molecular marker closely linked to a male fertility gene in pepper kernels, the molecular marker comprising Ind146 and / or Ind147, wherein the sequence of Ind146 consists of the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2, and the sequence of Ind147 consists of the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:6.

[0012] In some embodiments of the present invention, the sequence fragment shown in SEQ ID NO:1 or SEQ ID NO:5 is associated with the male fertility gene of pepper kernel. Gms1 Closely linked, the sequence fragments shown in SEQ ID NO:2 or SEQ ID NO:6 are associated with the male sterility gene in pepper nuclei. gms1 Closely linked.

[0013] In some embodiments of the present invention, the nuclear male fertility gene Gms1 and nuclear male sterility gene gms1 It is located on chromosome 4 of pepper, between 146219579 and 147712678 bp.

[0014] A second aspect of the present invention provides a primer set for amplifying the molecular markers described in the first aspect of the present invention that are closely linked to the male fertility gene of pepper nucleus.

[0015] In some embodiments of the present invention, the primer set for Ind146 includes Ind146-F and Ind146-R; the nucleotide sequences of the primer set are shown below: Ind146-F: 5'-GAAAACTGGGTGTGGGAC-3'; Ind146-R: 5'-TCCTTGGCTCTCGCTATC-3'.

[0016] In some embodiments of the present invention, the primer set for Ind147 includes Ind147-F and Ind147-R; the nucleotide sequences of the primer set are shown below: Ind147-F: 5'-GATAAATTACAACAGAGATCATAGAGAGG-3'; Ind147-R: 5'-TAATATAAGTTCCACATAAGGTATAGCGT-3'.

[0017] A third aspect of the present invention provides a kit comprising the primer set described in the second aspect of the present invention.

[0018] In some embodiments of the present invention, the kit further includes at least one of polymerase, dNTP, PCR buffer, and MgCl2.

[0019] In some embodiments of the present invention, the kit further includes positive and negative control reaction systems.

[0020] A fourth aspect of the present invention provides the use of any one of (1) to (3) in the preparation of products for assisted breeding; (1) Molecular markers of the first aspect of the present invention; (2) The primer set of the second aspect of the present invention; (3) The reagent kit of the third aspect of the present invention.

[0021] The fifth aspect of this invention aims to provide an assisted breeding method, wherein the fertility of the pepper is determined by detecting the genotype of the molecular marker closely linked to the male fertility gene of pepper in the first aspect of this invention in the genome of the plant to be tested.

[0022] In some embodiments of the present invention, if the genome of the plant to be tested contains the fragments shown in SEQ ID NO:1 and / or SEQ ID NO:5, the plant is nuclear male fertile; if the genome of the plant to be tested contains only the fragments shown in SEQ ID NO:2 and / or SEQ ID NO:6, the plant is nuclear male sterile.

[0023] In some embodiments of the present invention, the method includes the following steps: using the DNA of the sample to be tested as a template, performing PCR amplification using the primer set described in the second aspect of the present invention or the kit described in the third aspect of the present invention to obtain PCR amplification products; detecting the PCR amplification products to determine the genotype of the sample to be tested that corresponds to the molecular marker.

[0024] In some embodiments of the present invention, the PCR amplification reaction program is as follows: 93-95℃, 4-6 min, 1 cycle; 93-95℃, 20-30 s, 54-56℃, 20-30 s, 71-73℃, 1 min, 30-35 cycles; 71-73℃, 5 min, 1 cycle.

[0025] The beneficial effects of this invention are: This invention discloses two closely linked molecular markers, Ind146 and Ind147, for a novel nuclear male fertility gene in chili peppers. These two markers locate the novel nuclear male fertility gene within a 1.49 Mb range between 146219579 and 147712678 bp on chromosome 4 of chili peppers, achieving fine localization of the chili pepper nuclear male fertility gene.

[0026] The molecular markers closely linked to the male fertility gene of pepper kernel in this invention lay the foundation for the final cloning of the male fertility gene of pepper kernel and the establishment of a molecular marker-assisted breeding system; the molecular markers of this invention can be applied to pepper seed production and breeding practices in a simple, rapid and high-throughput manner. Attached Figure Description

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This image shows the BSA-seq association analysis and fine mapping of the nuclear male sterility gene. In image a, BSA-seq association analysis results are shown; the red dashed line represents the 99th percentile threshold, and the black line represents the fitted ΔSNP-index value. Regions where the ΔSNP-index value exceeds the threshold are the target gene association regions. Image b shows the nuclear male sterility gene. gms1 In the fine mapping results, the gray cylinders represent chromosomes, the lower part of the cylinders are linkage molecular markers, and the numbers between the markers above them indicate the number of exchanged individuals between adjacent markers.

[0028] Figure 2 Genotypic analysis of a partial population of the C120AB dual-use line using two markers. In the figures, a is the electrophoresis diagram of the amplified product of marker Ind146, and b is the electrophoresis diagram of the amplified product of marker Ind147. In the figures, A represents the genotype of the C120A sterile line, B represents the genotype of the C120B maintainer line, H represents the heterozygous genotype, F represents fertile, and S represents sterile.

[0029] Figure 3 Genotypic analysis of a portion of the F2 population using two markers. In the figures, a is the electrophoresis diagram of the amplified product of marker Ind146, and b is the electrophoresis diagram of the amplified product of marker Ind147. In the figures, A represents the genotype of the maternal C120A sterile line, B represents the M601 genotype, H represents the heterozygous genotype, F represents fertile, and S represents sterile. Detailed Implementation

[0030] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0031] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0032] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0033] Example 1: Development of molecular markers closely linked to the male fertility gene in pepper kernels C120AB is a naturally mutant nuclear male-sterile dual-purpose breeding material bred by the Vegetable and Flower Research Institute of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences. Using the C120A male-sterile line as the female parent and the C120B maintainer line (fertile) as the male parent, BC1 and F2 segregating populations were constructed through sister backcrosses and self-pollination, respectively. Genetic pattern surveys showed that in the 81 BC1 plants, the number of fertile and male-sterile plants were 41 and 40, respectively. Chi-square tests (…) χ 2 The test results showed that the number of fertile and sterile plants segregated in a 1:1 ratio. In the F2 population of 221 plants, the number of fertile and sterile plants were 163 and 58, respectively, as determined by chi-square test. χ 2 The test results showed that the number of fertile and sterile plants segregated at a ratio of 3:1 (Table 1). Therefore, the sterility trait of the C120AB dual-purpose line is controlled by a pair of recessive genes, and it was thus named... gms1 Its allele (male fertility gene) is named Gms1.

[0034] Table 1. Inheritance patterns of sterility traits in the C120A sterile line.

[0035] Thirty fertile plants and 30 sterile plants were randomly selected from the F2 segregating population. DNA was extracted using the CTAB method to construct fertile DNA pools and sterile DNA pools, respectively. BSA-seq technology was then used to analyze the DNA. gms1 The nuclear sterility gene was preliminarily located. The BSA-seq experiment was commissioned to Beijing Biomarker Biotechnology Co., Ltd.

[0036] The results are as follows Figure 1 As shown in Figure a, gms1 The nuclear sterility gene was initially located in a region of approximately 7.75 Mb on chromosome 4 of the pepper plant.

[0037] Based on the initial mapping using BSA-seq, the inventors screened out polymorphic markers Ind116 and Ind147 flanking the initial mapping interval. These two markers were then used to analyze a segregating population of approximately 3000 BC1 plants, identifying 17 exchanged individuals. Further analysis of the genotypes of these exchanged individuals was conducted using encrypted markers. Combined with phenotypic data, the final... gms1 The gene locus was finely mapped to a 1.49 Mb interval between markers Ind146 and Ind147, such as... Figure 1 As shown in b.

[0038] Among them, the marker Ind146 consists of the sequence 5'-GAAAACTGGGTGTGGGACCCATATTTAGTCAAAAGGACCTCGAGTAGAAATTTTGAATGTGCTATTTAGTTTGAAATATCGAATTAAGTGTTGTTATAAAGTTTGTCTGCATATAGAGGATTACTGATGAATCTCGAGAGCTTGGCGTGAT ATTTTAGGTACATCATGATCGCGACAATTTAAGTCATAAACATAACTCCTTGTTCAAACCCTAGTACGCAATCATTAGTGGTGATGTGACAATAGCGACTCATGACTTTGAGGCTTTGTACCACGATCACAAATATCGAGTAGTGATAGCGAGAGCCAAGGA-3' (SEQ ID The sequence consists of two nucleotide fragments: NO:1 and sequence 5'-GAAAACTGGGTGTGGGACCCATATTTAGTCAAAAGGACCTCGAGTAGAAATTTTGAATGTGCTATTTAGTTTGAAATATCGAATTAAGTGTTGTTATAAAGTTTGTCTGCATATAGAGGATTACTGATGAATCTCGAGAGCTTGGCGTGATATTTTAGGTACATCATGATCGCGATGTCTTTGGCCGTGATTGTGACTCCTTGACAAGGGTAGTAAGTGTTGTGATCGTGACAATTTAAGTCATAAACATAACTCCTTGTTCAAACCCTAGTACGCAATCATTAGTGGTGATGTGACAATAGCGACTCATGACTTTGAGGCTTTGTACCACGATCACAAATATCGAGTAGTGATAGCGAGAGCCAAGGA-3' (SEQ ID NO:2). The fragment shown in SEQ ID NO:1 corresponds to the male fertility gene of the pepper kernel. Gms1 Closely linked, the fragment shown in SEQ ID NO:2 is associated with the male sterility gene in pepper nuclei. gms1 Tight linkage. Primers were designed targeting the molecular marker Ind146, which was amplified by the upstream primer sequence Ind146-F: 5'-GAAAACTGGGTGTGGGAC-3' (SEQ ID NO:3) and the downstream primer sequence Ind146-R: 5'-TCCTTGGCTCTCGCTATC-3' (SEQ ID NO:4).

[0039] The marker Ind147 consists of two nucleotide fragments: 5'-GATAAATTACAACAGAGATCATAGAGAGGTGCTGCGTCACCCCCTCTAGGTTTCTCCTTTATATATATATATATATATATATATATATATATATGCATAACCGCGCGATGCGCGGATAATATTAAACAATGTAATTACGCTATACCTTATGTGGAACTTATATTA-3' (SEQ ID NO:5) and 5'-GATAAATTACAACAGAGATCATAGAGAGGTGCTGCGTCACCCCCTCTAGGTTTCTCCTTTATTTATATATATATATATATATATATATATATATATATATATATATATATGCATAACCGCGCGATGCGCGGATAATATTAAACAATGTAATTACGCTATACCTTATGTGGAACTTATATTA-3' (SEQ ID NO:6). The fragment shown in SEQ ID NO:5 corresponds to the male fertility gene in pepper kernels. Gms1 Closely linked, the fragment shown in SEQ ID NO:6 is associated with the male sterility gene in pepper nuclei. gms1 Tight linkage. Primers were designed targeting the molecular marker Ind147, which was amplified by the upstream primer sequence Ind147-F: 5'-GATAAATTACAACAGAGATCATAGAGAGG-3' (SEQ ID NO:7) and the downstream primer sequence Ind147-R: 5'-TAATATAAGTTCCACATAAGGTATAGCGT-3' (SEQ ID NO:8).

[0040] Example 2 Field Trial Verification 200 seeds of the C120AB dual-purpose line (sister backcross population BC1) obtained in Example 1 were sown in 50-cell seedling trays. 186 seedlings emerged. At the 2-3 true leaf stage, leaves of the seedlings were picked and DNA was extracted using the CTAB method. After passing the quality test, the genotype of the 186 plants was detected using primers of Ind146 and Ind147, molecular markers closely linked to the male fertility gene of pepper kernels in Example 1.

[0041] The specific steps involved in genotyping are as follows: (1) Genomic DNA of the peppers to be tested was extracted using the CTAB method.

[0042] (2) Using the genomic DNA from step (1) as a template, PCR amplification was performed using primer pairs consisting of primers Ind146-F and Ind146-R, and Ind147-F and Ind147-R from Example 1 to obtain the PCR amplification products. The specific PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3. 2×Taq PCR StarMix for PAGE was purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.

[0043] Table 2 PCR reaction system

[0044] Table 3 RCR reaction conditions

[0045] (3) PAGE gel electrophoresis: The PCR amplification products obtained in step (2) are subjected to PAGE gel electrophoresis.

[0046] 1) Select two clean glass plates without any stains or residue. Clamp them together at the center of both sides of the glass plates with clips. Insert a 68-tooth comb halfway into the top of the glass plates. 2) Add 50 mL of 1×8% polyacrylamide gel working solution to a beaker, transfer it to a fume hood, add 20 μL of LTEMED and 500 μL of ammonium persulfate solution, mix quickly, pour it between two glass plates, remove air bubbles, then fully insert the comb and let it stand for more than 30 minutes. 3) After the 1×8% polyacrylamide gel has completely solidified, remove the comb, fix the glass plate in the vertical electrophoresis tank, and slowly add 1×TBE solution above and below the electrophoresis tank until the iron wire is completely submerged. 4) Add 1.0-1.4 μL of PCR product to the sample well using a 2 μL pipette, and perform electrophoresis at 160V for 2 hours; 5) After electrophoresis, gently remove the gel and place it in a stainless steel tray, then rinse twice with pure water; 6) After cleaning, add 200mL of pure water and 10mL of 0.1% AgNO3 staining solution (the volume ratio of pure water to staining solution is 20:1), transfer to a shaker in a fume hood, and stain for 10 minutes at a speed of 125-145rpm / min. 7) After staining, rinse twice with pure water, add 200mL of pure water and 10mL of 20×NaOH colorimetric solution, transfer to a shaker in a fume hood and add 800μL of formaldehyde for color development (colorimetric solution: formaldehyde = 1mL: 80μL), and develop the color at a speed of 125-145rpm / min for 10min. 8) After development, rinse twice with tap water and place the cleaned gel on an X-ray film viewing lamp for photographing to facilitate subsequent strip reading and analysis.

[0047] (4) Based on the staining results obtained in step (3), make the following judgments: If the amplification product contains the fragments shown in SEQ ID NO:1 and / or SEQ ID NO:5, the plant is nuclear male fertile; if the amplification product contains only the fragments shown in SEQ ID NO:2 and / or SEQ ID NO:6, the plant is nuclear male sterile.

[0048] The results showed that fragments SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:6 were detected in 92 plants (hereinafter referred to as population 1), indicating that population 1 consisted of nucleomale fertile plants; while only fragments SEQ ID NO:2 and SEQ ID NO:6 were detected in the other 94 plants (hereinafter referred to as population 2), indicating that population 1 consisted of nucleomale sterile plants. Some results are shown below. Figure 2 As shown in the electrophoresis diagram of the Ind146-labeled amplification product ( Figure 2 In section a), the amplification product of the C120A sterile line genotype is fragment (A) shown in SEQ ID NO:2, representing a homozygous genotype of nuclear male sterility; the amplification product of the C120B maintainer line genotype is fragment (B) shown in SEQ ID NO:1, representing a homozygous genotype of nuclear male fertility; the amplification product of the heterozygous genotype (H) is fragments shown in SEQ ID NO:1 and SEQ ID NO:2, representing a heterozygous genotype of nuclear male fertility. Electrophoresis diagram of the amplification products labeled Ind147 (…) Figure 2 In section b), the amplification product of the C120A sterile line genotype is fragment (A) shown in SEQ ID NO:6, representing a homozygous genotype of nuclear male sterility; the amplification product of the C120B maintainer line genotype is fragment (B) shown in SEQ ID NO:5, representing a homozygous genotype of nuclear male fertility; and the amplification product of the heterozygous genotype (H) is fragments shown in SEQ ID NO:5 and SEQ ID NO:6, representing a heterozygous genotype of nuclear male fertility.

[0049] When the plants have 6-8 true leaves, these 186 plants will be transplanted to the field. During the flowering period, the pollen fertility of each plant will be investigated.

[0050] The results showed that all plants in group 1 were fertile, while all plants in group 2 were sterile. Therefore, the plant phenotype and genotype in this group were 100% identical.

[0051] Example 3: Field Trial Verification In order to remove the sterility gene carried by the C120A sterile line gms1In this example, the C120A male-sterile line was used as the female parent, and the superior chili inbred line M812, independently bred by the inventors' team, was used as the male parent for hybridization to obtain F1 offspring. F1 offspring were then self-pollinated to obtain an F2 segregating population of 192 plants. DNA was extracted from the leaves of the seedlings, and after passing quality testing, the genotypes of the 192 plants were detected using primers for the molecular markers Ind146 and Ind147, which are closely linked to the male fertility gene in chili peppers, obtained in Example 1. The genotype detection method described in Example 2 was also used to detect the plant genotypes.

[0052] The results are as follows Figure 3 As shown, SEQ ID NO:1 and SEQ ID NO:5 fragments were detected in 49 plants (hereinafter referred to as population 1), indicating that population 1 is a homozygous nucleomale fertile plant; SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:6 fragments were detected simultaneously in 95 plants (hereinafter referred to as population 2), indicating that population 2 is a heterozygous nucleomale fertile plant; and only SEQ ID NO:2 and SEQ ID NO:6 fragments were detected in 48 plants (hereinafter referred to as population 3), indicating that population 3 is a nucleomale sterile plant. Some results are shown below. Figure 3 As shown in the electrophoresis diagram of the Ind147-labeled amplification product ( Figure 3 In section a), the amplification product of the C120A sterile line genotype is fragment (A) shown in SEQ ID NO:2, representing a homozygous genotype of nuclear male sterility; the amplification product of the M812 genotype is fragment (B) shown in SEQ ID NO:1, representing a homozygous genotype of nuclear male fertility; the amplification product of the heterozygous genotype (H) is fragments shown in SEQ ID NO:1 and SEQ ID NO:2, representing a heterozygous genotype of nuclear male fertility. Electrophoresis diagram of the amplification product labeled Ind147 (…) Figure 3 In section b), the amplification product of the C120A sterile line genotype is fragment (A) shown in SEQ ID NO:6, representing a homozygous genotype of nuclear male sterility; the amplification product of the M812 genotype is fragment (B) shown in SEQ ID NO:5, representing a homozygous genotype of nuclear male fertility; and the amplification product of the heterozygous genotype (H) is fragments shown in SEQ ID NO:5 and SEQ ID NO:6, representing a heterozygous genotype of nuclear male fertility.

[0053] According to the experimental objectives, population 1 was eliminated during the seedling stage, while populations 2 and 3 were transplanted to the field. During the flowering period, population 3 was used as the female parent and population 2 as the male parent for sister backcrossing to conduct subsequent breeding experiments.

[0054] Pollen fertility surveys during the flowering period showed that all plants in group 2 were heterozygous and fertile, while all plants in group 3 were sterile. This indicates that using the two molecular markers Ind146 and Ind147 of this invention for auxiliary selection is accurate and reliable, and the plant phenotype and genotype achieved 100% match.

[0055] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A molecular marker closely linked to a male fertility gene in pepper kernels, characterized in that, The molecular markers include Ind146 and / or Ind147, wherein the sequence of Ind146 consists of the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2, and the sequence of Ind147 consists of the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:

6.

2. The molecular marker according to claim 1, characterized in that, The sequence fragment shown in SEQ ID NO:1 or SEQ ID NO:5 is related to the male fertility gene in pepper kernels. Gms1 Closely linked, the sequence fragments shown in SEQ ID NO:2 or SEQ ID NO:6 are associated with the male sterility gene in pepper nuclei. gms1 Closely linked.

3. A primer set for amplifying the molecular markers of claim 1 or 2 that are closely linked to the male fertility gene of pepper nucleus.

4. The primer set according to claim 3, characterized in that, The primer set for Ind146 includes Ind146-F and Ind146-R; the nucleotide sequences of the primer set are shown below: Ind146-F: 5'-GAAAACTGGGTGTGGGAC-3'; Ind146-R: 5'-TCCTTGGCTCTCGCTATC-3'.

5. The primer set according to claim 3, characterized in that, The primer set for Ind147 includes Ind147-F and Ind147-R; the nucleotide sequences of the primer set are shown below: Ind147-F: 5'-GATAAATTACAACAGAGATCATAGAGAGG-3'; Ind147-R: 5'-TAATATAAGTTCCACATAAGGTATAGCGT-3'.

6. A kit comprising the primer set according to any one of claims 3 to 5.

7. The reagent kit according to claim 6, characterized in that, The kit also includes at least one of polymerase, dNTP, PCR buffer, and MgCl2.

8. Any one of (1) to (3) in the preparation of products for assisted breeding; (1) The molecular marker according to claim 1; (2) The primer set according to any one of claims 3 to 5; (3) The kit according to claim 6 or 7.

9. A method for assisted breeding, characterized in that, The method detects the genotype of the molecular marker closely linked to the male fertility gene of pepper as described in claim 1 in the genome of the plant to be tested, and determines the fertility of pepper based on the genotype; if the genome of the plant to be tested contains the fragment shown in SEQ ID NO:1 and / or SEQ ID NO:5, it indicates that the plant is male fertile, and if the genome of the plant to be tested contains only the fragment shown in SEQ ID NO:2 and / or SEQ ID NO:6, it indicates that the plant is male sterile.

10. The method according to claim 8, characterized in that, The method includes the following steps: using the DNA of the sample to be tested as a template, performing PCR amplification using the primer set of any one of claims 3 to 5 or the kit of claim 6 or 7 to obtain PCR amplification products; detecting the PCR amplification products to determine the genotype of the sample to be tested that corresponds to the molecular marker.

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