Gene related to male fertility of capsicum cell nucleus, co-separation molecular marker of gene and application of co-separation molecular marker

Abstract

The invention provides a capsicum annuum cell nucleus male fertility gene Msc-4, a co-separation molecular marker of the gene and application of the capsicum annuum cell nucleus male fertility gene Msc-4. The amino acid sequence of the protein coded by the gene is as shown in SEQ ID NO. 1. The invention further provides a molecular marker SNP-31372133 co-separated with the gene Msc-4, and the molecular marker SNP-31372133 can assist in screening of a hot pepper material with nuclear male sterility. The invention provides a new nucleus sterile germplasm resource and a new nucleus male sterility gene resource for the research on the nucleus male sterility of the pepper, and plays an important role in the application in the field of pepper seed production and breeding.

Description

Technical Field

[0001] The present invention belongs to the field of pepper breeding, and particularly relates to a pepper nuclear male sterility gene, a co-segregating molecular marker of the gene and its application. Background Art

[0002] Pepper (Capsicum annuum L.) is a solanaceous vegetable crop widely cultivated worldwide. The annual planting area of peppers in China exceeds 20 million mu, accounting for 35% of the world's pepper planting area and more than 10% of the national vegetable planting area. The heterosis of peppers is obvious. The pepper varieties cultivated in the Chinese market are generally the first-generation hybrids, and the utilization of pepper heterosis has important economic value and social value.

[0003] Using male sterility to breed first-generation hybrid seeds can eliminate manual emasculation, effectively reduce the production cost of hybrid seeds, and ensure seed purity. It is an ideal way to utilize pepper heterosis. There are mainly two types of male sterility used for the production of first-generation hybrid pepper seeds, namely cytoplasmic male sterility (CMS) and genic male sterility (GMS). The fertility of genic male sterility is relatively stable, the inheritance is simple and easy to utilize, and the fertility of hybrids can be fully restored by ordinary inbred lines. The source of male parents in breeding is extensive, which is convenient for breeding excellent hybrid varieties.

[0004] At present, three nuclear male sterility genes have been cloned in peppers. msc-1 was discovered from a nuclear male sterility mutant of 'Keshan Hot Pepper'. The msc-1 gene is located on chromosome 2, and the candidate gene is Capana02g002096, encoding a bHLH transcription factor. msc-2 was discovered from a nuclear male sterility mutant of 'Ingebite Sweet' pepper. The msc-2 gene is located on chromosome 5, and the candidate gene is Capana05g000766, encoding a transcription factor containing a PHD-finger. msc-3 was discovered from a nuclear male sterility mutant of pepper. The msc-3 gene is located on chromosome 10, and the candidate gene is Capana10g000198, encoding an R2R3-MYB transcription factor.

[0005] In addition, when using the GMS dual-purpose line for the production of the first-generation hybrid pepper seeds at present, the conventional method usually adopted is to start from the initial flowering stage, manually identify the fertility of individual plants, and then remove about 50% of the fertile plants in the population one by one, which greatly limits the large-scale application of the GMS dual-purpose line. By developing co-segregating molecular markers for the GMS gene in pepper, it is possible to achieve early identification of individual fertility at the seedling stage, which is of great significance for the efficient transfer of GMS and the improvement of the seed production efficiency of the first-generation hybrid.

[0006] Therefore, continuously exploring GMS materials in pepper, map-based cloning of the GMS fertility gene in pepper, and developing co-segregating molecular markers for GMS fertility have important theoretical and application values for improving the breeding efficiency of pepper male sterility, reducing the seed production cost of hybrid seeds, and promoting the utilization of heterosis in pepper. Summary of the Invention

[0007] Aiming at the defects of the prior art, one of the purposes of the present invention is to provide a pepper nuclear male fertility gene, a co-segregating molecular marker of this gene, and their applications.

[0008] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a protein related to pepper nuclear male fertility, and the protein is selected from the following a) or b):

[0010] a) Its amino acid sequence is as shown in SEQ ID NO.1 in the sequence listing;

[0011] b) A protein derived from SEQ ID NO.1, in which the amino acid sequence shown in SEQ ID NO.1 in the sequence listing is subjected to substitution and / or deletion and / or addition of one or several amino acid residues, and is related to nuclear male fertility.

[0012] The second aspect of the present invention provides a material related to the protein of the first aspect, and the material includes any one of the following (A1) to (A6):

[0013] (A1) A nucleic acid molecule encoding the protein of the first aspect of the present invention;

[0014] (A2) An expression cassette containing the nucleic acid molecule of (A1);

[0015] (A3) A recombinant vector containing the nucleic acid molecule of (A1);

[0016] (A4) A transgenic plant cell line containing the nucleic acid molecule of (A1);

[0017] (A5) A transgenic plant cell line containing the expression cassette of (A2);

[0018] (A6) A transgenic plant cell line containing the recombinant vector described in (A3);

[0019] Further, the nucleic acid molecule is any one of the following (A11) to (A14):

[0020] (A11) The genomic DNA molecule shown as SEQ ID NO.2 in the sequence listing;

[0021] (A12) The cDNA molecule shown as SEQ ID NO.8 in the sequence listing;

[0022] (A13) A nucleotide sequence that can hybridize with the sequence shown in (A11) under stringent conditions;

[0023] (A14) A nucleotide sequence that can hybridize with the sequence shown in (A12) under stringent conditions.

[0024] The third aspect of the present invention provides the application of the protein related to pepper nuclear male sterility in the first aspect above or the material in the second aspect above in any one of the following (1) to (3):

[0025] (1) Application in regulating pepper male sterility;

[0026] (2) Application in preparing pepper male sterile lines;

[0027] (3) Application in pepper cross breeding.

[0028] The fourth aspect of the present invention provides a primer set for detecting the genotype of a co-segregating molecular marker of a gene related to pepper nuclear male sterility. The co-segregating molecular marker: using Capsicum_annuum_UCD10X_v1 as the reference genome, on chromosome 10, its physical position is 31372133, the nucleotide base at this position is A or G, the genotype of male sterile plants is A:A, and the genotype of male fertile plants is A:G or G:G.

[0029] The gene related to pepper nuclear male sterility refers to the nucleotide sequence encoding the protein described in the first aspect or the nucleic acid molecule involved in the second aspect above.

[0030] Further, the primer set is selected from the following (G1) or (G2):

[0031] (G1):

[0032] The nucleotide sequence of the forward primer is as shown in SEQ ID NO.3 in the sequence listing;

[0033] The nucleotide sequence of the reverse primer is as shown in SEQ ID NO.4 in the sequence listing;

[0034] (G2):

[0035] The nucleotide sequence of the first upstream primer is as shown in SEQ ID NO.10 in the sequence listing;

[0036] The nucleotide sequence of the second upstream primer is as shown in SEQ ID NO.11 in the sequence listing;

[0037] The nucleotide sequence of the downstream primer is as shown in SEQ ID NO.7 in the sequence listing;

[0038] Furthermore, the 5'-ends of the first upstream primer and the second upstream primer of the primer set (G2) respectively further include different fluorescent tag sequences;

[0039] Specifically, the nucleotide sequence of the first upstream primer is as shown in SEQ ID NO.5 in the sequence listing; the nucleotide sequence of the second upstream primer is as shown in SEQ ID NO.6 in the sequence listing.

[0040] The fifth aspect of the present invention provides the application of the primer set of the fourth aspect of the present invention in any one of the following (1) to (3):

[0041] (1) Application in identifying the male fertility of peppers;

[0042] (2) Application in constructing a dual-purpose line of nuclear male sterility in peppers;

[0043] (3) Application in pepper cross-breeding.

[0044] The sixth aspect of the present invention provides a method for identifying the male fertility of peppers, and the method includes:

[0045] Using the primer set of the fourth aspect of the present invention, performing PCR amplification with the genomic DNA of the pepper to be tested as a template; then identifying the amplification product to know the male fertility of the pepper to be tested;

[0046] Furthermore, the PCR amplification program of the primer set (G1) is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 30 s, 38 cycles; extension at 72°C for 5 min;

[0047] The PCR amplification program of the primer set (G2) is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing at 61°C - 55°C for 1 min, amplifying for 10 cycles, and the annealing temperature of each cycle is reduced by 0.6°C relative to the annealing temperature of the previous cycle; denaturation at 94°C for 20 s, extension at 55°C for 1 min, and continuing to amplify for 26 cycles.

[0048] Further, the identification of the primer set (G1) is to sequence the amplification product;

[0049] The identification of the primer set (G2) is as follows: detecting the fluorescence signal of the PCR product by an instrument, and judging the genotype of the chili pepper to be tested according to the fluorescence signal color of the PCR product; preferably, the specific principle for judgment is as follows: if the color shown by the PCR product of the chili pepper to be tested is the same as the fluorescence tag sequence color of the first upstream primer, the genotype of the chili pepper to be tested is G:G; if the color shown by the PCR product of the chili pepper to be tested is the same as the fluorescence tag sequence color of the second upstream primer, the genotype of the chili pepper to be tested is A:A; if the color shown by the PCR product of the chili pepper to be tested is a mixed color of the fluorescence tag sequence color of the first upstream primer and the fluorescence tag sequence color of the second upstream primer, the genotype of the chili pepper to be tested is A:G.

[0050] The seventh aspect of the present invention provides a chili pepper strain carrying the homozygous genotype msc-4 / msc-4 of genic male sterility, which is obtained from the chili pepper seed CaCYPF2 (Capsicum annuum L. CaCYPF2). The chili pepper seed CaCYPF2 is preserved in the China Center for Type Culture Collection (address: Preservation Center of Wuhan University, Wuhan), and the preservation number is CCTCC NO: P202339, and the preservation date is December 8, 2023.

[0051] The eighth aspect of the present invention provides an application of a chili pepper strain carrying the genic male sterility genotype msc-4 / msc-4 in the construction of a chili pepper genic male sterile dual-purpose line, chili pepper cross breeding or seed production.

[0052] The ninth aspect of the present invention provides a method for constructing a chili pepper genic male sterile dual-purpose line, comprising the following steps:

[0053] S1: Using the chili pepper strain carrying the genic male sterility genotype msc-4 / msc-4 provided in the seventh aspect of the present invention as the female parent, and any male-fertile chili pepper inbred line as the male parent, and hybridizing to obtain F1;

[0054] S2: Using the male-fertile chili pepper inbred line selected in S1 as the recurrent parent to hybridize with F1 to obtain BC1 generation seeds, and using the primer set of the fourth aspect of the present invention to identify the genotypes of the BC1 generation plants planted, removing the plants with the genotype G:G, and only planting the plants with the genotype A:G in the field;

[0055] S3: Select a single plant with excellent comprehensive traits from the plants obtained in step S2 and cross it with the recurrent parent to obtain BC2 generation seeds. Use the primer set of the fourth aspect of the present invention to perform genotype identification on the planted BC2 generation plants, remove the plants with the genotype G:G, and only plant the plants with the genotype A:G in the field;

[0056] S4: Select a single plant with excellent comprehensive traits from the plants obtained in step S3, and then repeat step S3 until BC n generation seeds are obtained, where n≥8; use the primer set of the fourth aspect of the present invention to perform genotype identification on the planted BC n generation plants, remove the plants with the genotype G:G, and only plant the plants with the genotype A:G in the field;

[0057] S5: Select a single plant with excellent comprehensive traits from the plants obtained in step S4, self-cross and save seeds to obtain BC n F2 generation seeds; use the primer set of the fourth aspect of the present invention to perform genotype identification on the planted BC n F2 generation plants, remove the plants with the genotype G:G, and plant the plants with the genotype A:A and the plants with the genotype A:G in the field;

[0058] S6: Use the plant with the genotype A:A as the female parent and the plant with the genotype A:G as the male parent for hybridization, and harvest seeds to obtain a pepper genic male sterile dual-purpose line.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The present invention obtained a pepper genic male sterile mutant msc-4 through field investigation, and map-based cloned the gene Capana10g000622 that can control pepper fertility. The CDS nucleotide sequence of this gene is shown in SEQ ID NO.8 in the sequence listing, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.1 in the sequence listing. The sterile mutant has a mutation from G to A at the 429bp of the CDS sequence of the gene Capana10g000622, resulting in premature termination of amino acid coding. The present invention also provides the msc-4 co-segregating molecular marker SNP-31372133, which can assist in screening pepper materials with genic male sterility. The present invention provides new genic sterile germplasm resources and new genic male sterile gene resources for pepper genic male sterility research, and plays an important role in the application of pepper seed production and breeding fields. Description of the Drawings

[0061] Figure 1It is a phenotypic comparison between the pepper inbred line BVRC3790 (referred to as the fertile plant) and the male sterile mutant msc-4 (referred to as the sterile plant); among them: (a): The field phenotype of the fertile plant; (b): The field phenotype of the sterile plant; (c): The anther phenotype of the fertile plant; (d): The anther phenotype of the sterile plant; (e): 1% I2-KI staining of the pollen of the fertile plant; (f): 1% I2-KI staining of the pollen of the sterile plant.

[0062] Figure 2 It is the map-based cloning of the pepper nuclear male sterility gene msc-4. Among them, A: Mining the candidate interval of the sterile gene msc-4 by BSA-seq; B: Fine mapping of msc-4; C: Mining of the candidate gene of msc-4; D: CDS sequence variation between the fertile plant Msc-4 and the sterile plant msc-4.

[0063] Figure 3 It is the result analysis of the PCR amplification product sequencing of the pepper material by the fertility-linked marker SNP-31372133. Among them, A: When the target site (i.e., the mutation site) is a single peak and the base is G, it represents that the measured single plant carries the Msc-4 / Msc-4 genotype and the plant is fertile; B: When the target site is a doublet peak, it represents that the measured single plant carries the Msc-4 / msc-4 genotype and the plant is fertile; C: When the target site is a single peak and the base is A, it represents that the measured single plant carries the msc-4 / msc-4 genotype and the plant is sterile.

[0064] Figure 4 It is a method for constructing the pepper nuclear male sterile dual-purpose line BVRC317AB.

[0065] Figure 5 It is the genotyping result of detecting the genotype of the material to be tested based on the molecular marker SNP-31372133. Specific embodiments

[0066] The technical solutions of the present invention will be described in detail below in combination with specific embodiments and examples. The following examples and embodiments are only for better understanding of the present invention and do not constitute a limitation to the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from regular biochemical reagent stores unless otherwise specified.

[0067] During the process of pepper cultivation, the inventor discovered a pepper male sterile mutant msc-4. By using the above-mentioned mutant msc-4, through map-based cloning, sequence analysis, and expression analysis of genes, it was found that Capana10g000622 specifically expressed in anthers is a strong candidate gene regulating pepper pollen fertility, and Capana10g000622 encodes the CYP703A2 protein. At the 429th bp of the Capana10g000622 CDS sequence in the sterile mutant msc-4, G mutated to A, resulting in premature termination of amino acid coding. The normal expression of Capana10g000622 is necessary for normal pepper male fertility. The male sterile trait exhibited by the mutant belongs to a single-gene recessive trait. Specifically, this mutant is named mutant msc-4, and the genotype corresponding to its male sterile trait is msc-4 / msc-4 or A:A.

[0068] The mutant msc-4 or the inbred line carrying the male sterile homozygous genotype msc-4 / msc-4 bred from this mutant can be used in cross-breeding or seed production. For example, when combined with a fertile pepper inbred line, it can be used to create a dual-purpose line for nuclear male sterility in peppers. The created dual-purpose line for male sterility can be used in cross-breeding or seed production. Typically, non-limitingly, cross-breeding can be carried out by using the inbred line carrying the male sterile homozygous genotype msc-4 / msc-4 as the female parent and a fertile pepper inbred line as the male parent to prepare a hybrid variety.

[0069] The specific embodiment of the present invention also provides a protein related to pepper nuclear male fertility, and the protein is selected from the following a) or b):

[0070] a) Its amino acid sequence is as shown in SEQ ID NO.1 in the sequence listing;

[0071] b) A protein derived from SEQ ID NO.1, in which the amino acid sequence shown in SEQ ID NO.1 in the sequence listing is substituted and / or deleted and / or added by one or several amino acid residues and is related to nuclear male fertility.

[0072] The specific embodiment of the present invention also provides a material related to the above protein, and the material includes any one of the following (A1) to (A6):

[0073] (A1) A nucleic acid molecule encoding the protein related to pepper nuclear male fertility;

[0074] (A2) An expression cassette containing the nucleic acid molecule described in (A1);

[0075] (A3) A recombinant vector containing the nucleic acid molecule described in (A1);

[0076] (A4) A transgenic plant cell line containing the nucleic acid molecule described in (A1);

[0077] (A5) A transgenic plant cell line containing the expression cassette described in (A2);

[0078] (A6) A transgenic plant cell line containing the recombinant vector described in (A3);

[0079] Furthermore, the nucleic acid molecule is any one of the following (A11) to (A14):

[0080] (A11) The genomic DNA molecule shown as SEQ ID NO.2 in the sequence listing;

[0081] (A12) The cDNA molecule shown as SEQ ID NO.8 in the sequence listing;

[0082] (A13) A nucleotide sequence that can hybridize with the sequence shown in (A11) under stringent conditions;

[0083] (A14) A nucleotide sequence that can hybridize with the sequence shown in (A12) under stringent conditions.

[0084] The "stringent conditions" used in the present invention are well-known, including, for example, hybridizing in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4), and 1 mM EDTA at 60 °C for 12 - 16 hours, and then washing with a washing solution containing 0.1% SDS and 0.1% SSC at 65 °C for 15 - 60 minutes.

[0085] In a specific embodiment, the gene related to pepper nuclear male sterility is the pepper nuclear male fertile gene Msc-4, whose nucleotide sequence is shown as SEQ ID NO.2 in the sequence listing, and it encodes the CYP703A2 protein, and the amino acid sequence of this protein is shown as SEQ ID NO.1 in the sequence listing, and the CDS sequence of this gene is shown as SEQ ID NO.8 in the sequence listing. It has been confirmed that this protein is related to male fertility and can be used to regulate the male fertility of peppers. In fertile pepper plants, the genotypes corresponding to the male fertile trait are Msc-4 / msc-4 or G:A, or Msc-4 / Msc-4 or G:G.

[0086] The specific embodiments of the present invention also provide a co-segregating molecular marker SNP of a gene related to nuclear male sterility in pepper (i.e., a nucleotide sequence encoding the protein related to nuclear male sterility in pepper or the above nucleic acid molecule), which is used to identify the male sterility of pepper varieties. Using Capsicum_annuum_UCD10X_v1 as the reference genome, the co-segregating molecular marker is located on chromosome 10, and its physical position is 31372133. The nucleotide base at this position is A or G. The genotype of male sterile plants is A:A, and the genotype of male fertile plants is A:G or G:G.

[0087] The specific embodiments of the present invention also provide a primer set for identifying the male sterility of pepper varieties, which is used to amplify the co-segregating molecular marker of the gene related to nuclear male sterility in pepper. The co-segregating molecular marker: using Capsicum_annuum_UCD10X_v1 as the reference genome, is located on chromosome 10, and its physical position is 31372133. The nucleotide base at this position is A or G. The genotype of male sterile plants is A:A, and the genotype of male fertile plants is A:G or G:G. This primer set can be any feasible forward primer and reverse primer designed for this SNP molecular marker. Specifically, the primer set is selected from any one of the following two groups:

[0088] (G1) A primer set designed for ordinary PCR, including a forward primer and a reverse primer, where

[0089] The nucleotide sequence of the forward primer is as shown in SEQ ID NO.3 in the sequence listing, specifically: 5’-TCACCACCAATGATCCAGAAA-3’;

[0090] The nucleotide sequence of the reverse primer is as shown in SEQ ID NO.4 in the sequence listing, specifically: 5’-CCATTTCACCTTCATCATTATCA-3’.

[0091] (G2) A primer set designed for Kompetitive Allele Specific PCR (KASPar):

[0092] The nucleotide sequence of the first upstream primer is as shown in SEQ ID NO.10 in the sequence listing, specifically: 5’-TGGCTCCTTTAGGTCCAAAATGG-3’;

[0093] The nucleotide sequence of the second upstream primer is as shown in SEQ ID NO.11 in the sequence listing, specifically: 5’-ATTGGCTCCTTTAGGTCCAAAATGA-3’;

[0094] The nucleotide sequence of the downstream primer is shown as SEQ ID NO.7 in the sequence listing, specifically: 5’-TGTCAACAAATGTTCCATACAAATTCTTCTC-3’;

[0095] For facilitating subsequent genotype identification, the 5’ ends of the first upstream primer and the second upstream primer of the primer set (G2) further include different fluorescent tag sequences respectively. The fluorescent linker sequences can be selected from one of FAM, HEX, FITC, RED, TET, JOE, and R110. The color shown by the fluorescent tag sequence of the first upstream primer is different from the color shown by the fluorescent tag sequence of the second upstream primer. Further, the nucleotide sequence of the first upstream primer is shown as SEQ ID NO.5 in the sequence listing; the nucleotide sequence of the second upstream primer is shown as SEQ ID NO.6 in the sequence listing.

[0096] The specific embodiments of the present invention also provide the applications of the above co-segregating molecular markers and the above primer sets in any of the following aspects: (1) Application in identifying pepper male fertility; (2) Application in constructing a pepper genic male sterile dual-purpose line; (3) Application in pepper cross breeding.

[0097] The method for identifying pepper male fertility using the above primer set includes: using the above primer set, performing PCR amplification with the genomic DNA of the pepper to be tested as a template; then identifying the amplification product to obtain the male fertility of the pepper to be tested.

[0098] Specifically, the PCR amplification program of the primer set (G1) is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 30 s, for 38 cycles; extension at 72°C for 5 min;

[0099] The PCR amplification program of the primer set (G2) is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing at 61°C - 55°C for 1 min, for 10 cycles, and the annealing temperature of each cycle is reduced by 0.6°C relative to the annealing temperature of the previous cycle; denaturation at 94°C for 20 s, extension at 55°C for 1 min, and continue to amplify for 26 cycles.

[0100] Specifically, the identification of the primer set (G1) is to sequence the amplification product, thereby determining whether the base at the target site in the amplification product is A or G.

[0101] The identification of the primer set (G2) is as follows: detecting the fluorescence signal of the PCR product by an instrument and judging the genotype of the chili pepper to be tested according to the fluorescence signal color of the PCR product; preferably, when genotyping using the primer set composed of SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, the specific principle for judgment is as follows: if the PCR product of the chili pepper to be tested shows a blue fluorescence signal, the genotype of the chili pepper to be tested is G:G; if the PCR product of the chili pepper to be tested shows a red fluorescence signal, the genotype of the chili pepper to be tested is A:A; if the PCR product of the chili pepper to be tested shows a green fluorescence signal, the genotype of the chili pepper to be tested is A:G.

[0102] The product for detecting the above-mentioned co-segregating molecular marker can be made into a kit; specifically, the kit includes the above-mentioned primer pair and a PCR detection reagent.

[0103] The specific embodiment of the present invention also provides a chili pepper line carrying the nuclear male sterility homozygous genotype msc-4 / msc-4, which is obtained from the chili pepper seed CaCYPF2 (Capsicum annuum L. CaCYPF2). The chili pepper seed CaCYPF2 is preserved in the China Center for Type Culture Collection (address: Wuhan University Preservation Center, Wuhan), and the preservation number is CCTCC NO: P202339, and the preservation date is December 8, 2023.

[0104] The chili pepper seed CaCYPF2 is the F2 generation seed obtained by using a chili pepper nuclear male sterility mutant msc-4 obtained from the inventor's field investigation as the female parent and a chili pepper inbred line Y333 as the male parent to obtain the F1 generation and then self-crossing the F1 generation. The F2 generation seeds are a mixture, including a chili pepper line carrying the nuclear male sterility homozygous genotype msc-4 / msc-4, a chili pepper line carrying the nuclear male fertility homozygous genotype Msc-4 / Msc-4, and a chili pepper line carrying the nuclear male fertility heterozygous genotype Msc-4 / msc-4.

[0105] The method for obtaining a chili pepper line carrying the nuclear male sterility homozygous genotype msc-4 / msc-4 from the chili pepper seed CaCYPF2 (Capsicum annuum L. CaCYPF2):

[0106] Chili seeds can be planted, and then genomic DNA of chili leaves at the seedling stage can be extracted. Subsequently, the above method for identifying chili male fertility can be used to identify the male fertility of chili. For example, the F2 generation plants are genotyped using the KASPar molecular marker SNP-31372133. If the genotype of the chili to be tested is the GG homozygous type or the AG heterozygous type based on the SNP-31372133 locus, then the chili to be tested is a chili line carrying the nuclear male-fertile homozygous genotype Msc-4 / Msc-4 or a chili line carrying the nuclear male-fertile heterozygous genotype Msc-4 / msc-4; if the genotype of the chili to be tested is the AA homozygous type based on the SNP-31372133 locus, then the chili to be tested is a chili line carrying the nuclear male-sterile homozygous genotype msc-4 / msc-4.

[0107] The chili line carrying the nuclear male-sterile genotype msc-4 / msc-4 is the nuclear male-sterile line.

[0108] The present invention also provides an application of a chili line carrying the nuclear male-sterile genotype msc-4 / msc-4 in the construction of a chili nuclear male-sterile dual-purpose line, chili hybrid breeding or seed production.

[0109] The present invention further provides a method for constructing a chili nuclear male-sterile dual-purpose line, comprising the following steps:

[0110] S1: Using the above chili line carrying the nuclear male-sterile genotype msc-4 / msc-4 as the female parent and any male-fertile chili inbred line as the male parent, hybridizing to obtain F1;

[0111] S2: Using the male-fertile chili inbred line selected in S1 as the recurrent parent to hybridize with F1 to obtain BC1 generation seeds. Genotype identification of the planted BC1 generation plants is carried out using the above primer set, and the plants with the genotype G:G are removed, and only the plants with the genotype A:G are planted in the field;

[0112] S3: Selecting a single plant with excellent comprehensive traits from the plants obtained in step S2 and hybridizing it with the recurrent parent to obtain BC2 generation seeds. Genotype identification of the planted BC2 generation plants is carried out using the above primer set, and the plants with the genotype G:G are removed, and only the plants with the genotype A:G are planted in the field;

[0113] S4: Selecting a single plant with excellent comprehensive traits from the plants obtained in step S3, and then repeating step S3 until BC n generation seeds are obtained, where n≥8; Genotype identification of the planted BC n generation plants is carried out using the above primer set, and the plants with the genotype G:G are removed, and only the plants with the genotype A:G are planted in the field;

[0114] S5: Select a single plant with excellent comprehensive traits from the plants obtained in step S4, self-cross and save the seeds to obtain BC n F2 generation seeds; use the above primer set to identify the genotypes of the planted BC n F2 generation plants, remove the plants with the genotype G:G, and plant the plants with the genotype A:A and the plants with the genotype A:G in the field;

[0115] S6: Use the plants with the genotype A:A as the female parent and the plants with the genotype A:G as the male parent for hybridization, and harvest the seeds to obtain the pepper genic male sterile dual-purpose line.

[0116] Any male-fertile pepper variety inbred line selected in the above construction method can be a commercially available or any inbred line with practical application value.

[0117] The technical solution of the present invention will be described in more detail below in conjunction with the embodiments and the accompanying drawings.

[0118] Example 1. Phenotype of pepper male sterile mutant msc-4

[0119] The inventor found a male sterile mutant plant in the pepper inbred line BVRC3790, named msc-4. Compared with normal plants, the anthers of the mutant msc-4 are smaller and not plump. The anthers of the mutant were cut off and stained with 1% I2-KI solution, and no plump and mature pollen grains were observed, showing a complete male sterile phenomenon (see Figure 1 ). This sterility trait is stable, and it can be distinguished by the naked eye in the field whether it is male sterile. Using the mutant plant msc-4 as the female parent and 10 inbred lines with different genetic backgrounds as the male parents for hybridization, all F1 showed male fertility, indicating that the fertility of the mutant plant msc-4 is controlled by nuclear inheritance.

[0120] Example 2. Mapping and determination of male sterile gene msc-4 and male fertile gene Msc-4

[0121] I. Construction of genetic mapping population and genetic analysis of pepper male sterile mutant msc-4

[0122] Using the pepper mutant msc-4 as the female parent and the pepper inbred line Y333 (source: Pepper Genetics and Breeding Group, Institute of Vegetable Sciences, Beijing Academy of Agriculture and Forestry Sciences) as the male parent to produce F1, and all F1 showed male fertility in the field. Then self-cross F1 to obtain the F2 population. Two types of individuals, male fertile and male sterile, can be observed in the F2 population in the field. A total of 538 fertile plants were investigated in the F2 population, and chi-square test showed that fertile:sterile conformed to a 3:1 segregation. It is thus speculated that the phenotype of the pepper mutant msc-4 is controlled by a recessive single gene.

[0123] II. Preliminary mapping of the fertility gene msc-4 for nuclear male sterility in pepper

[0124] The DNA of the F2 population was extracted using the CTAB method. DNA from 50 randomly selected fertile plants in the F2 population was mixed in equal amounts to construct a fertile DNA pool (named mixed pool MF); DNA from 50 randomly selected sterile plants in the F2 population was mixed in equal amounts to construct a sterile DNA pool (named mixed pool MS). Beijing Berry Genomics Co., Ltd. constructed second-generation sequencing libraries for mixed pool MF and mixed pool MS respectively, and sequenced them using the Novaseq6000 platform. The sequencing data are shown in Table 1.

[0125] Table 1. BSA-seq sequencing data

[0126]

[0127]

[0128] Using Capsicum_annuum_UCD10X_v1 (whose genomic sequence can be found at https: / / solgenomics.net) as the reference genome, the effective sequencing data of the two mixed pools were aligned using the Burrows-Wheeler Aligner BWA 0.7.16a software, and then the GATK software was used to detect SNPs in the two mixed pools. A total of 13,539,695 high-quality SNP sites were obtained. Calculate the coverage of the four genotypes of A, T, C, and G of the two offspring mixed pool samples at each SNP site, and filter out the sites with a total coverage of less than 30 in any of the mixed pools. Based on the coverage of the four genotypes of A, T, C, and G, calculate the ED (Euclidean Distance) value of each SNP site. Use 100 SNPs as the window size of the "sliding window", and average the ED values of 100 SNPs. Plot the graph using the ED value and the average frequency value of the sliding window. According to the ED value, the candidate region of the pepper nuclear male sterility gene was determined to be in the range of 19.0 Mb to 44.0 Mb on chromosome 10, approximately a 25 Mb region (see Figure 2 A).

[0129] III. Fine mapping of the fertility gene msc-4 for nuclear male sterility in pepper and determination of candidate genes

[0130] To precisely map the msc-4 gene, eight molecular markers (SNP-19001246, InDel-21755035, InDel-22118539, SNP-30663818, SNP-31055992, SNP-31372133, SNP-34011836, SNP-44368808) with polymorphisms between the two parents (i.e., the pepper mutant msc-4 as the female parent and the pepper inbred line Y333 as the male parent) were developed in the candidate region. The types, primer sequences, and genotype detection methods of these eight polymorphic markers are described in Table 2. Using these eight polymorphic markers to screen for recombinant individuals in the F2 population, the msc-4 gene was finally mapped between the markers SNP-31055992 and SNP-34011836, with the two markers approximately 2.95 Mb apart, which is the candidate interval for the msc-4 gene (see Figure 2 B). Aligning the genomic sequences of the msc-4 candidate interval to the reference genome C.annuum Zunla-1 (whose genomic sequence can be found at https: / / solgenomics.net), there are 11 candidate genes (see Figure 2 C; Table 3), including Capana10g000622, Capana10g000623, Capana10g000624, Capana10g000625, Capana10g000628, Capana10g000629, Capana10g000630, Capana10g000631, Capana10g000632, Capana10g00083, Capana10g000685, among which the candidate gene Capana10g000622 is annotated as CYP703A2, homologous to AT1G01280.1 in Arabidopsis thaliana and is related to male sterility;

[0131] Table 2. Molecular markers for the fine mapping of the male sterility gene msc-4

[0132]

[0133]

[0134]

[0135] Note: The single underlined sequence is the FAM fluorescent label sequence, and the double underlined sequence is the HEX fluorescent label sequence.

[0136] Table 3. Annotations of the 11 genes in the Msc-4 candidate interval

[0137] Gene ID Homologous Arabidopsis ID Functional annotation E-value Capana10g000622 AT1G01280.1 Cytochrome P450 703A2; CYP703A2 0.0 Capana10g000623 AT3G19640.1 Magnesium transporter MRS2-3 1e-26 Capana10g000624 AT5G09400.1 Potassium transporter 7 1e-27 Capana10g000625 AT1G01120.1 3-ketoacyl-CoA synthase 1 0.0 Capana10g000628 AT4G00830.6 Heterogeneous nuclear ribonucleoprotein 2e-136 Capana10g000629 No homologous gene None None Capana10g000630 No homologous gene None None Capana10g000631 No homologous gene None None Capana10g000632 No homologous gene None None Capana10g000683 AT2G46770.1 NAC domain-containing protein 43 2e-112 Capana10g000685 No homologous gene None None

[0138] There is only one SNP between the fertile pool MF and the sterile pool MS at SNPs SNP-31055992 and SNP-34011836, located at position 31372133 bp on chromosome 10 of the Capsicum_annuum_UCD10X_v1 reference genome. The marker SNP-31372133 developed at this locus co-segregates with the fertility phenotype of the F2 population. When the genotype at this locus is A:A, the plants show male sterility in the field. When the genotype at this locus is G:G or A:G, the plants show fertility in the field.

[0139] The flanking sequences of the marker SNP-31372133 on the reference genome Capsicum_annuum_UCD10X_v1 were aligned to the reference genome C.annuum Zunla-1, and it was found that this SNP locus is at position 429 bp of the CDS sequence of the pepper gene Capana10g000622. The genotype of the sterile plants at position 429 bp of this CDS sequence is A:A, and the genotype of the fertile plants at position 429 bp of this CDS sequence is A:G or G:G. The Capana10g000622 gene encodes a cytochrome P450 protein, annotated as CYP703A2. Relative to the fertile plants, the SNP at position 429 bp of the gene Capana10g000622 in the sterile plants is a nonsense mutation, resulting in premature termination of the encoding of the sterile gene msc-4 (see Figure 2 D). The nucleotide sequence of the fertile gene carried by the fertile pepper plant Msc-4 is shown in Sequence Listing SEQ ID NO.2; the CDS sequence is shown in Sequence Listing SEQ ID NO.8, and the amino acid sequence of the protein encoded by it is shown in Sequence Listing SEQ ID NO.1. The amino acid sequence of the protein encoded by the sterile gene carried by the sterile pepper plant msc-4 is shown in Sequence Listing SEQ ID NO.9.

[0140] Example 3 Detection method and verification of detection accuracy of the fertility co-segregating molecular marker SNP-31372133

[0141] 1. Detection method 1

[0142] The genomic DNA of each individual pepper plant to be tested was extracted using the CTAB method. PCR amplification was performed on each genomic DNA using the primer pair of the fertility co-segregating molecular marker SNP-31372133 (the forward primer is shown as SEQ ID NO.3 in the sequence listing, and the reverse primer is shown as SEQ ID NO.4 in the sequence listing). The pepper plants to be tested were selected from the F2 genetic mapping population obtained by self-crossing the F1 generation, which was produced by crossing the pepper mutant strain msc-4 as the female parent and the pepper inbred line Y333 as the male parent. The PCR amplification system was as follows: 10 μl of 2×Phanta Max Master Mix (Nanjing Novoprotein Scientific Inc.), 0.5 μl of each of the forward and reverse primers, 1 μl of DNA, and 8 μl of ddH2O; the PCR amplification program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 30 s, for 38 cycles; extension at 72°C for 5 min. The PCR amplification products were sent to Beijing New Era Zhonghe Technology Co., Ltd. for sequencing. The sequencing results were analyzed using Chromas software. When the PCR sequencing target site is a single peak and the base is G, it represents that the tested individual plant carries the Msc-4 / Msc-4 genotype and the plant is a fertile plant; when the PCR sequencing target site is a doublet peak, it represents that the tested individual plant carries the Msc-4 / msc-4 genotype and the plant is a fertile plant; when the PCR sequencing target site is a single peak and the base is A, it represents that the tested individual plant carries the msc-4 / msc-4 genotype and the plant is a sterile plant (see Figure 3 ).

[0143] 2. Detection method 2: KASPar genotyping method

[0144] KASPar primer-specific PCR was carried out according to the standard experimental procedure provided by LGC (Laboratory of the Government Chemist) Limited in the UK, that is, based on the experimental procedure of competitive allele-specific PCR technology. The reagents were the supporting reagents provided by LGC company except for special instructions. The reagent dosage, usage, and the whole experimental procedure were carried out according to the operation guide Genetyping Assay, Manual Part#15004070Rev.B of LGC company. The reaction was carried out in a 384-well plate or a 1536-well plate (Cat.No.04729749001, Roche), and the reaction system was 3 μl or 1 μl.

[0145] The specific steps are as follows: (1) Use a K-pette liquid dispensing workstation to add 1.5 μL of genomic DNA of the chili pepper to be tested into a microplate and dry it at 60 °C. Then, use a Meridian pipetting workstation under the Kraken operating system to add 1× Master mix (KBS-1016-002 or Cat. No. KBS-1016-011, Laboratory of the Government Chemist) and primer premix (prepared by mixing the upstream primer SNP-31372133-X, upstream primer SNP-31372133-Y, and downstream primer SNP-31372133-C in a molar concentration ratio of 2:2:5, with the final concentration of each primer being 10 μM) into each reaction well. Immediately after the Mix is dispensed, place the microplate on a Kube heat sealer and a Fusion laser film sealer in sequence for film sealing. The PCR reaction is carried out in a high-throughput water bath system Hydrocycler. The specific program is as follows: pre-denaturation at 94 °C for 15 min; denaturation at 94 °C for 20 s, annealing at 61 °C - 55 °C (using the touch-down program, decreasing by 0.6 °C per cycle), extension for 1 min, and amplifying for 10 cycles; denaturation at 94 °C for 20 s, annealing & extension at 55 °C for 1 min, and continuing to amplify for 26 cycles. (2) After completing step (1), when the temperature of the PCR amplification product drops below 40 °C, read the fluorescence values by scanning with the FAM and HEX beams of a BMG PHERAstar instrument (the FAM fluorescence tag sequence is observed and read at an excitation wavelength of 485 nm and an emission wavelength of 520 nm, and the HEX fluorescence tag sequence is observed and read at an excitation wavelength of 528 nm and an emission wavelength of 560 nm). Determine the genotype of the chili pepper to be tested based on the SNP-31372133 locus according to the color of the fluorescence signal. The specific judgment principle is as follows: If the chili pepper to be tested shows a blue fluorescence signal based on the SNP-31372133 locus, then the genotype of the chili pepper to be tested based on the SNP-31372133 locus is the GG homozygous type; if the chili pepper to be tested shows a red fluorescence signal based on the SNP-31372133 locus, then the genotype of the chili pepper to be tested based on the SNP-31372133 locus is the AA homozygous type; if the chili pepper to be tested shows a green fluorescence signal based on the SNP-31372133 locus, then the genotype of the chili pepper to be tested based on the SNP-313721335 locus is the AG heterozygous type. It should be noted that after the amplification is completed, use a BMG PHERAstar instrument to detect the fluorescence signal and view the genotyping situation. If the fluorescence signal is weak after the PCR amplification is completed and affects data analysis, additional cycles (denaturation at 94 °C for 20 s, annealing and extension at 55 °C for 1 min, 3 cycles) can be added until the genotyping is complete.

[0146] The genotype of the chili pepper to be tested at the SNP-31372133 locus is detected using the above method, and then the following judgments are made: If the genotype of the chili pepper to be tested at the SNP-31372133 locus is the GG homozygous type or the AG heterozygous type, then the chili pepper to be tested is male fertile; if the genotype of the chili pepper to be tested at the SNP-31372133 locus is the AA homozygous type, then the chili pepper to be tested is male sterile. The SNP-31372133 locus is located at the 31372133bp of chromosome 10 of the Capsicum_annuum_UCD10X_v1 reference genome. Experiments have proved that the method provided by the present invention can assist in identifying the fertility of chili peppers with an accuracy rate of 100%. See the detection results in Figure 5 。

[0147] 3. Verification of detection accuracy

[0148] Using the chili pepper genic male sterile mutant msc-4 as the female parent and the chili pepper inbred line Y333 as the male parent, the F1 generation was obtained by hybridization. The F1 generation was self-crossed to obtain the F2 population. According to the anther phenotypes in the field, 50 sterile plants and 50 fertile plants were selected, and then the above detection methods 1 and 2 were used to detect these 100 chili pepper plants. The detection results were consistent with the fertility phenotypes in the field. The accuracy rate of identifying fertility using this SNP-31372133 molecular marker was 100%.

[0149] Example 4 Application example of co-segregating molecular markers for chili pepper genic male sterility msc-4

[0150] I. Construction method of the chili pepper genic male sterile dual-purpose line BVRC317AB

[0151] (1) Using the chili pepper genic male sterile mutant msc-4 or a chili pepper line carrying the genic male sterile homozygous genotype msc-4 / msc-4 as the female parent and the chili pepper inbred line BVRC317 (derived from the Vegetable Research Institute of the Beijing Academy of Agriculture and Forestry Sciences) as the male parent, the F1 generation was obtained by hybridization;

[0152] (2) BVRC317 was used as the recurrent parent to hybridize with the F1 generation to obtain the BC1 generation. The genotype of the BC1 generation plants planted in 72-well trays was identified using the detection method of Example 3 with the fertility co-segregating marker SNP-31372133. The single plants with the genotype Msc-4 / Msc-4 were removed, and only the single plants with the genotype Msc-4 / msc-4 were planted in the field;

[0153] (3) Select a single plant with excellent comprehensive traits and cross it with the recurrent parent BVRC317 to obtain BC2 generation seeds. Use the co-segregation molecular marker for fertility SNP-31372133 and the detection method of Example 3 to identify the genotypes of the BC2 generation plants planted in 72-well trays. Remove the single plants with the genotype Msc-4 / Msc-4, and only plant the single plants with the genotype Msc-4 / msc-4 in the field;

[0154] (4) Select a single plant with excellent comprehensive traits and cross it with the recurrent parent BVRC317 to obtain BC3 generation seeds. According to the same method as in step (3), obtain BC8 generation seeds. Use the co-segregation molecular marker for fertility SNP-31372133 and the detection method of Example 3 to identify the genotypes of the BC8 generation plants planted in 72-well trays. Remove the single plants with the genotype Msc-4 / Msc-4, and only plant the single plants with the genotype Msc-4 / msc-4 in the field;

[0155] (5) Select a single plant with excellent comprehensive traits and self-cross to save seeds to obtain BC8F2 generation seeds. Use the co-segregation molecular marker for fertility SNP-31372133 and the detection method of Example 3 to identify the genotypes of the BC8F2 generation plants planted in 72-well trays. Remove the single plants with the genotype Msc-4 / Msc-4, and plant the single plants with the genotype msc-4 / msc-4 (named BVRC317A) and the single plants with the genotype Msc-4 / msc-4 (named BVRC317B) in the field.

[0156] (6) Use BVRC317A as the female parent and BVRC317B as the male parent, and harvest seeds after hybridization to obtain the pepper genic male sterile dual-purpose line BVRC317AB. The construction process of the genic male sterile dual-purpose line is shown in Figure 4 .

[0157] The above method is also applicable to the construction of other inbred line genic male sterile dual-purpose lines, that is, the inbred line BVRC317 can be replaced by other pepper inbred lines.

[0158] II. Identify the fertile single plants in the pepper genic male sterile dual-purpose line at the seedling stage

[0159] Without the technical support of molecular marker-assisted screening, when using the pepper genic male sterile dual-purpose line for the production of hybrid first-generation seeds, it is necessary to identify the fertility of the single plants in the offspring of the dual-purpose line at the flowering stage, and then manually remove about 50% of the fertile plants. However, using the co-segregation marker SNP-31372133 developed in the present invention for the pepper fertility gene msc-4 and the detection method of Example 3 can detect the genotypes of the seedlings of the pepper genic male sterile dual-purpose line planted in trays, determine the fertility of the single plants, and then select the fertile single plants and plant them in the field, thereby reducing the seed production cost.

Claims

1. A protein related to the nuclear male sterility of pepper, said protein being selected from a) or b) as follows: a) Its amino acid sequence is as shown in SEQ ID NO.1 in the sequence listing; b) A protein derived from SEQ ID NO.1, in which the amino acid sequence shown in SEQ ID NO.1 in the sequence listing has been substituted and / or deleted and / or added by one or several amino acid residues and is related to nuclear male sterility.

2. Materials related to the protein described in claim 1, said materials including any one of the following (A1) to (A6): (A1) A nucleic acid molecule encoding the protein described in claim 1; (A2) An expression cassette containing the nucleic acid molecule described in (A1); (A3) A recombinant vector containing the nucleic acid molecule described in (A1); (A4) A transgenic plant cell line containing the nucleic acid molecule described in (A1); (A5) A transgenic plant cell line containing the expression cassette described in (A2); (A6) A transgenic plant cell line containing the recombinant vector described in (A3); Preferably, the nucleic acid molecule is any one of the following (A11) to (A14): (A11) A genomic DNA molecule as shown in SEQ ID NO.2 in the sequence listing; (A12) A cDNA molecule as shown in SEQ ID NO.8 in the sequence listing; (A13) A nucleotide sequence capable of hybridizing with the sequence shown in (A11) under stringent conditions; (A14) A nucleotide sequence capable of hybridizing with the sequence shown in (A12) under stringent conditions.

3. Application of the protein related to the nuclear male sterility of pepper described in claim 1 or the materials described in claim 2 in any one of the following aspects (1) to (3): (1) Application in regulating the male sterility of pepper; (2) Application in preparing pepper male sterile lines; (3) Application in pepper cross breeding.

4. A primer set for detecting the genotype of a co-segregating molecular marker of a gene related to the nuclear male sterility of pepper, said co-segregating molecular marker: using Capsicum_annuum_UCD10X_v1 as the reference genome, on chromosome 10, its physical position is 31372133, the nucleotide base at this position is A or G, the genotype of male sterile plants is A:A, and the genotype of male fertile plants is A:G or G:G.

5. The primer set according to claim 4, characterized in that The primer set is selected from the following (G1) or (G2): (G1)： The nucleotide sequence of the forward primer is as shown in SEQ ID NO.3 in the sequence listing; The nucleotide sequence of the reverse primer is as shown in SEQ ID NO.4 in the sequence listing; (G2)： The nucleotide sequence of the first upstream primer is as shown in SEQ ID NO.10 in the sequence listing; The nucleotide sequence of the second upstream primer is as shown in SEQ ID NO.11 in the sequence listing; The nucleotide sequence of the downstream primer is as shown in SEQ ID NO.7 in the sequence listing; Preferably, the 5' ends of the first upstream primer and the second upstream primer of the primer set (G2) further respectively include different fluorescent tag sequences. More preferably, the nucleotide sequence of the first upstream primer is as shown in SEQ ID NO.5 in the sequence listing; the nucleotide sequence of the second upstream primer is as shown in SEQ ID NO.6 in the sequence listing.

6. The application of the primer set according to claim 4 or 5 in any one of the following aspects (1) to (3): (1) Application in identifying the male fertility of peppers; (2) Application in constructing a two - type line of nuclear male sterility in peppers; (3) Application in pepper cross - breeding.

7. A method for identifying the male fertility of peppers, characterized in that The method includes: Using the primer set according to claim 4 or 5, performing PCR amplification with the genomic DNA of the chili pepper to be tested as a template; then identifying the amplification product to obtain the male fertility of the chili pepper to be tested; Preferably, the PCR amplification program for the primer set (G1) is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 30 s, 38 cycles; extension at 72°C for 5 min; The PCR amplification program for the primer set (G2) is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing at 61°C - 55°C for 1 min, amplifying for 10 cycles, and the annealing temperature for each cycle is reduced by 0.6°C compared to the annealing temperature of the previous cycle; denaturation at 94°C for 20 s, extension at 55°C for 1 min, and continuing to amplify for 26 cycles. Preferably, the identification for the primer set (G1) is to sequence the amplification product; The identification for the primer set (G2) is: using an instrument to detect the fluorescence signal of the PCR product, and judging the genotype of the chili pepper to be tested according to the color of the fluorescence signal of the PCR product; preferably, the specific principle for judgment is as follows: if the color shown by the PCR product of the chili pepper to be tested is the same as the color of the fluorescent tag sequence of the first upstream primer, then the genotype of the chili pepper to be tested is G:G; if the color shown by the PCR product of the chili pepper to be tested is the same as the color of the fluorescent tag sequence of the second upstream primer, then the genotype of the chili pepper to be tested is A:A; if the color shown by the PCR product of the chili pepper to be tested is a mixed color of the fluorescent tag sequence color of the first upstream primer and the fluorescent tag sequence color of the second upstream primer, then the genotype of the chili pepper to be tested is A:G.

8. A pepper strain carrying the homozygous genotype of nuclear male sterility msc - 4 / msc - 4, which is obtained from pepper seeds CaCYPF2, and the pepper seeds CaCYPF2 are preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO:P202339.

9. The application of the pepper strain carrying the nuclear male sterility genotype msc - 4 / msc - 4 according to claim 8 in the construction of a two - type line of nuclear male sterility in peppers, pepper cross - breeding or seed production.

10. A method for constructing a two - type line of nuclear male sterility in peppers, characterized in that It includes the following steps: S1: Using the chili pepper line carrying the genic male sterility genotype msc-4 / msc-4 according to claim 8 as the female parent, and any male-fertile chili pepper inbred line as the male parent, and hybridizing to obtain F1; S2: Using the male-fertile chili pepper inbred line selected in S1 as the recurrent parent to hybridize with F1 to obtain BC1 generation seeds, using the primer set according to claim 4 or 5 to perform genotype identification on the planted BC1 generation plants, removing the single plants with the genotype G:G, and only planting the single plants with the genotype A:G in the field; S3: Select a single plant with excellent comprehensive traits from the plants obtained in step S2 and hybridize it with the recurrent parent to obtain BC2 generation seeds. Use the primer set described in claim 4 or 5 to perform genotype identification on the planted BC2 generation plants, remove the plants with the genotype G:G, and only plant the plants with the genotype A:G in the field; S4: Select a single plant with excellent comprehensive traits from the plants obtained in step S3, and then repeat step S3 until BC n -generation seeds are obtained, where n≥8; use the primer set according to claim 4 or 5 to perform genotype identification on the planted BC n -generation plants, remove the single plants with genotype G:G, and only plant the single plants with genotype A:G in the field; S5: Select a single plant with excellent comprehensive traits from the plants obtained in step S4, self-cross and save seeds to obtain BC n F2 generation seeds; use the primer set described in claim 4 or 5 to perform genotype identification on the planted BC n F2 generation plants, remove the single plants with genotype G:G, and plant the single plants with genotype A:A and the single plants with genotype A:G in the field; S6: Use the plant with the genotype A:A as the female parent and the plant with the genotype A:G as the male parent for hybridization, and harvest seeds to obtain a two - type line of pepper genic male sterility.

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