A molecular marker co-segregated with pepper core male sterile gene msc-4 and application thereof

Abstract

The present application relates to the technical field of vegetable molecular breeding, and discloses a molecular marker co-segregated with a pepper nuclear male sterility gene msc-4 and application thereof.The molecular marker CaMSC4 according to the present application is a dCAPS marker, which is composed of specific primers CaMSC4-F and CaMSC4-R and is used in cooperation with a nucleic acid restriction endonuclease Taq I.According to the molecular marker and the application method thereof, male sterile and fertile plants can be accurately distinguished through the band type of the enzyme digestion product of the target sequence.The co-segregated marker provided by the present application can improve the transformation efficiency and reduce the cost in the process of transformation of nuclear male sterile pepper varieties, and can help to remove fertile plants in advance in the process of pepper hybrid seed production, thereby saving land and labor costs.In summary, the molecular marker provided by the present application has important application value in pepper breeding and seed production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of vegetable molecular breeding, and particularly relates to a molecular marker co-segregated with a pepper nuclear male sterility gene msc-4 and application thereof. BACKGROUND

[0002] Pepper is an important economic crop widely cultivated in the world. Pepper has very obvious heterosis, and using male sterile lines for seed production is an important way to efficiently utilize the heterosis of pepper. Pepper male sterility includes cytoplasmic male sterility (CMS) and genic male sterility (GMS). GMS has the advantages of stable sterility, no constraints from restorer lines, and simple transformation procedures. Therefore, GMS is one of the important traits in pepper breeding.

[0003] Marker assisted selection (MAS) is a technique that uses the characteristics of close linkage between molecular markers and target genes to select offspring or individuals carrying target genotypes by genotyping hybrid offspring. This technique has been widely used in food crops such as rice, corn, and wheat, and horticultural crops such as tomato and cucumber, greatly improving breeding efficiency, ensuring human food security, and improving human living standards.

[0004] The breeding or transformation of pepper GMS lines usually uses the method of saturated backcross. However, GMS is usually a recessive trait, and each backcross requires selfing and selecting sterile individuals as female parents after flowering, resulting in long breeding time, high land cost, and heavy workload. The application of molecular markers in the breeding or transformation of pepper GMS lines can select genotypes at the seedling stage, plant only individuals with heterozygous marker genotypes each season, and eliminate the selfing procedure required for identifying the genotype of backcross female parents in traditional breeding, greatly saving land cost, shortening breeding time, and reducing workload. In addition, during hybrid seed production using GMS lines, the use of GMS gene co-segregation molecular markers can help to remove fertile plants in advance, eliminating the need for manual removal of fertile plants, thereby saving land and labor costs. Therefore, the development of molecular markers co-segregated with pepper GMS genes has important application value for pepper hybrid breeding and seed production. SUMMARY

[0005] The primary object of the present application is to provide a molecular marker co-segregated with a GMS gene in pepper. The GMS gene is found in a cluster of bell pepper, and its chromosomal location is different from the related reports in pepper. The co-segregated marker CaMSC4 of the GMS gene is applicable to the nuclear male sterile two-purpose line MSC4AB and its derived F2 fertility separation population and fertile natural population, and can accurately identify the male sterile and male fertile plants in the fertility separation population.

[0006] Another object of the present application is to provide a primer pair of the molecular marker CaMSC4, comprising two single-stranded nucleotide primers CaMSC4-F and CaMSC4-R, as SEQ1 and SEQ2 in the sequence listing.

[0007] Still another object of the present application is to provide a kit comprising the above-mentioned primer. The length of the small fragment DNA obtained after PCR amplification of the genomic DNA of the pepper seedling to be tested using the kit provided by the present application is 81-82 bp, which is the target fragment of the molecular marker CaMSC4, and its sequence is SEQ3 in the sequence listing.

[0008] The fourth object of the present application is to provide the application of the above-mentioned molecular marker, primer pair and kit in the identification of fertility phenotype or assisted selection breeding of pepper. The CaMSC4 target fragment obtained using the above-mentioned primer can be clearly distinguished by electrophoresis band type and the genotype of the plant after restriction enzyme Taq I digestion and polyacrylamide gel electrophoresis, so as to accurately predict the pollen fertility phenotype of the pepper plant.

[0009] The fifth object of the present application is to provide an identification method of male sterile and male fertile plants of pepper.

[0010] The specific scheme adopted by the present application is as follows:

[0011] In the first aspect, a molecular marker co-segregated with a nuclear male sterility gene msc-4 in pepper is provided, wherein the molecular marker is a target sequence as shown in SEQ ID NO: 3 or a gene sequence comprising the target sequence; and the 24th-27th bases of the target sequence are Taq I restriction enzyme recognition sites, and the A base at the 27th position is deleted in sterile plants.

[0012] In the second aspect, a primer pair for amplifying the above-mentioned molecular marker is provided. Further, the primer pair comprises specific primers CaMSC4-F and CaMSC4-R as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0013] In the third aspect, a kit comprising the above-mentioned primer pair is provided. Further, the kit further comprises a PCR buffer containing magnesium ions, dNTP, Taq enzyme and ddH2O.

[0014] In a fourth aspect, the molecular marker, primer pair and kit are applied in pepper fertility phenotype identification or assisted selection breeding.

[0015] In a fifth aspect, a method for identifying pepper male sterile and male fertile plants, the method comprising the steps of: amplifying the above-mentioned molecular marker from the genomic DNA of a single pepper plant to be tested, performing restriction enzyme digestion and electrophoresis on the amplified target sequence, and finally identifying the fertility of the single pepper plant to be tested according to the genotype.

[0016] The method specifically comprises the following steps:

[0017] Step 1: Extracting the genomic DNA of the pepper plant to be tested;

[0018] Step 2: Using the genomic DNA of the pepper plant extracted in Step 1 as a template, and amplifying the target sequence using the above-mentioned primer pair;

[0019] Step 3: Performing restriction enzyme digestion on the target sequence amplified in Step 2 using Taq I restriction enzyme, and then identifying the fertile and sterile plants according to the banding pattern after electrophoresis.

[0020] Further, in Step 3, the specific identification method is as follows:

[0021] When the plant to be tested is derived from the MSC4AB dual line, if two bands with sizes of 81 bp and 58 bp appear after electrophoresis, it is a fertile plant; if only one band with a size of 81 bp appears after electrophoresis, it is a sterile plant.

[0022] When the plant to be tested is derived from the F2 population, if only one band with a size of 58 bp appears after electrophoresis, it is a homozygous fertile plant; if two bands with sizes of 81 bp and 58 bp appear after electrophoresis, it is a heterozygous fertile plant; if only one band with a size of 81 bp appears after electrophoresis, it is a sterile plant.

[0023] Beneficial effects: The application develops a molecular marker CaMSC4 co-segregated with pepper nuclear male sterility gene msc-4 by BSA-seq analysis and verification. The CaMSC4 belongs to dCAPS (derived cleaved amplified polymorphic sequences) marker, which is composed of specific primers CaMSC4-F and CaMSC4-R and is used in combination with nucleic acid restriction endonuclease Taq I. According to the molecular marker and the application method thereof provided by the application, male sterile and fertile plants can be accurately distinguished through the band type of the enzyme digestion product of the target sequence. The co-segregation marker provided by the application can help to remove fertile plants in advance during pepper hybrid seed production, thereby saving land and labor costs; and can improve the transformation efficiency and reduce the cost during the transformation of nuclear male sterile pepper varieties. In summary, the molecular marker provided by the application has important application value in pepper breeding and seed production. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure (a) shows that there is no obvious pollen visible to the naked eye on the anther of the open flower of the sterile plant in the pepper nuclear male sterile dual-purpose line MSC4AB of Example 1, and figure (b) shows that the surface of the anther of the open flower of the fertile plant is covered with a large amount of pollen visible to the naked eye. The scale length is 5.0 mm.

[0025] Figure 2 Figure is the Δ (SNP-index) whole genome distribution diagram obtained by BSA-seq analysis in Example 1, the black fold line is the fitting average value of Δ (SNP-index) of each site, and the blue and red fold lines are 95% and 99% confidence threshold lines, respectively.

[0026] Figure 3 Figure is a schematic diagram of the development principle of the co-segregation marker CaMSC4. Figure (a) shows the alignment results of the sequence near the candidate co-segregation site, figure (b) shows the amplification of the target sequence of the (homozygous) fertile plant (Msc-4Msc-4) by the co-segregation marker CaMSC4 and the cleavability of the matching restriction endonuclease Taq I, and figure (c) shows the amplification of the target sequence of the homozygous sterile plant (msc-4msc-4) by the co-segregation marker CaMSC4 and the non-cleavability of the matching restriction endonuclease Taq I. The red base represents the key variation site, and the blue base represents the endonuclease recognition sequence.

[0027] Figure 4Genotype analysis results of sterile and fertile plants in the nucleus male sterile dual-purpose line MSC4AB by the cosegregation marker CaMSC4. M represents DNA ladder; MS represents the homozygous sterile plants in the dual-purpose line, all corresponding to A band type; MF represents the heterozygous fertile plants in the dual-purpose line, all corresponding to H band type.

[0028] Figure 5 Genotype analysis results of sterile and fertile plants in the F2 segregation population by the cosegregation marker CaMSC4. M represents DNA ladder; MF represents the fertile plants in the F2 population, all corresponding to H or B band type; MS represents the sterile plants in the F2 population, all corresponding to A band type.

[0029] Figure 6 Genotype analysis results of different genetic background pepper male fertile inbred lines by the cosegregation marker CaMSC4. M represents DNA ladder; MF represents the homozygous fertile plants in the F2 population, MS represents the sterile plants in the dual-purpose line population, and 19 different genetic background male fertile inbred lines all correspond to B band type.

[0030] Figure 7 Application method of the cosegregation marker CaMSC4 in the transformation of the nucleus male sterile dual-purpose line. MSC4A represents the sterile plants in the nucleus male sterile dual-purpose line MSC4AB; Rec represents the recipient parent that needs to be introduced with the nucleus male sterile gene; MAS represents the assisted selection using the cosegregation marker CaMSC4 and its related kit; and breeding year is calculated according to two plantings per year. DETAILED DESCRIPTION

[0031] A. Screening of a locus co-segregated with the GMS gene of pepper, which comprises the following steps:

[0032] 1) DNA sample construction: using the pepper nucleus male sterile dual-purpose line MSC4AB population identified by pollen fertility phenotype, respectively constructing sterile DNA pool (SP) sample, fertile DNA pool (FP) sample, and sterile individual (SI) DNA sample.

[0033] 2) Resequencing and polymorphic site screening: after the above three samples are subjected to high-throughput resequencing, respectively aligning to the Zunla-1 pepper reference genome, and obtaining the variation sites that exist polymorphisms between the SP, FP and SI samples.

[0034] 3) Linkage site screening: According to the genetic characteristics of sterility of MSC4AB, the frequency of sterile alleles of the sites potentially linked to GMS genes in SI, SP and FP should be about equal to 1, 1 and 0.5, respectively. According to this, the above-mentioned sites are filtered, only the sites with polymorphism between SP and FP samples are retained, and a total of 15,767 candidate sites linked to GMS genes are obtained.

[0035] 4) Screening of co-segregation sites: Among the above-mentioned candidate sites linked to GMS genes, if a site co-segregates with GMS genes, the corresponding reads in the resequencing data of SI and SP samples should all be sterile alleles. According to this, 695 sites co-segregating with GMS genes are screened out, of which only one site causes a non-synonymous mutation. The physical coordinates of this site are at 248,789,782 bp of chromosome 3 of Zunla-1 reference genome, and the reference genome at this position is A base. The resequencing results show that in FP samples, this position is a heterozygous type of A base and A base deletion, and in SP samples, this position is a homozygous type of A base deletion.

[0036] B. Development, preparation kit and use method of molecular marker CaMSC4, mainly including the following contents:

[0037] 1) Development of molecular marker CaMSC4: According to the sequence on both sides of the position 248,789,782 bp of chromosome 3 of Zunla-1 reference genome, a dCAPS marker CaMSC4 is designed, which is composed of two single-stranded nucleotide primers CaMSC4-F and CaMSC4-R, and the sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0038] 2) Preparation kit of molecular marker CaMSC4: In addition to containing two primers of co-segregation marker CaMSC4, the kit also contains PCR buffer (containing magnesium ions), dNTP, Taq enzyme and ddH2O. The kit contains all components for PCR amplification of target sequences.

[0039] 3) Instructions for using the molecular marker CaMSC4: Using the kit described above, a small DNA fragment of 81–82 bp in length can be amplified from pepper genomic DNA through a specific PCR reaction procedure. The sequence is shown in SEQ ID NO: 3. When using the genomic DNA of fertile plants in the MSC4AB dual-use line as a template, the resulting PCR fragment, after digestion with restriction endonuclease Taq I and polyacrylamide gel electrophoresis, will show two bands of 81 bp and 58 bp respectively (this band pattern is defined as the H band). When using the genomic DNA of sterile plants as a template, after the same digestion and electrophoresis, only one band of 81 bp will be visible (this band pattern is defined as the A band). When using the genomic DNA of fertile and sterile plants in the F2 population as templates, homozygous fertile plants correspond to a 58 bp band (this band pattern is defined as the B band), heterozygous fertile plants correspond to the H band, and sterile plants correspond to the A band. Furthermore, using this molecular marker to genotype fertile inbred lines will yield the B band pattern. Therefore, the genotype of the target pepper material can be accurately determined based on the electrophoretic banding pattern, thereby achieving the purpose of distinguishing between sterile and fertile plants and being applied to assisted selection breeding.

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0041] The following embodiments are provided to facilitate a better understanding and application of the technical solutions provided by this invention, but the scope of protection of this invention is not limited thereto. Unless otherwise specified, the experimental methods and reagents used in the following embodiments refer to conventional methods and reagents.

[0042] Example 1: Development of co-separated marker CaMSC4

[0043] 1) Pollen fertility phenotype survey of the male-sterile dual-purpose pepper line MSC4AB (isolated and selected from commercial hybrids): The MSC4AB population was planted in a plastic greenhouse using conventional cultivation methods, and fertility phenotype surveys were conducted at the initial flowering stage and the full flowering stage. Individual plants with visible pollen after flower opening and anther dehiscence were considered fertile plants. Figure 1 a) Plants without obvious pollen and unable to set fruit naturally are considered sterile. Figure 1 b) During each survey, at least 3 flowers of each plant should be observed.

[0044] 2) Genetic rule analysis of GMS gene: One sterile individual in MSC4AB was used as the female parent to cross with the fertile inbred line BB3, and F1 seeds were obtained and planted to perform isolated selfing to obtain F2 population seeds. By planting 360 F2 plants in the field, pollen fertility phenotype investigation was performed according to the method of step 1), and the results showed that 283 plants were fertile and 77 plants were sterile. Chi-square goodness-of-fit test found that the ratio of the number of fertile plants to sterile plants was 3:1 (P = 0.11, χ 2 = 0.02), indicating that the inheritance of the GMS gene conforms to the single gene recessive genetic rule.

[0045] 3) DNA acquisition of resequencing samples: According to the pollen fertility phenotype investigation results of step 1), 50 fertile plants were selected from the MSC4AB dual-line population, and then equal amounts of tender leaves were mixed from each plant, ground into powder in a mortar with liquid nitrogen, and genomic DNA was extracted by CTAB method, finally obtaining fertile pool DNA samples (FP). Similarly, sterile pool DNA samples (SP) were obtained. In addition, the DNA sample of one sterile plant (sterile individual, SI) was also extracted for subsequent definition of sterile alleles.

[0046] 4) BSA-seq analysis: The SP, FP and SI samples were resequenced (sequencing depth of about 30x), and after quality control and filtration, they were respectively aligned to the Zunla-1 reference genome for whole genome variation information extraction. According to the genetic characteristics of sterility of MSC4AB, the genotype of the site potentially linked to the GMS gene in SI, SP and FP should be recessive homozygous, recessive homozygous and recessive heterozygous, respectively. Therefore, if the same allele as Zunla-1 is considered as a fertile allele, and the same allele as SI is considered as a sterile allele, then the frequency of the sterile allele of the site linked to the GMS gene should be close to 1 and 0.5 in SP and FP, respectively. Therefore, the screening condition is set as SP·SNP-index≥0.7 and 0.3≤FP·SNP-index≤0.7, and thus 15,767 high-confidence polymorphic sites linked to the sterile gene are obtained. Based on the above markers, BSA-seq analysis of the sterile gene based on Δ(SNP-index) was performed, and 19 intervals associated with the nuclear sterile gene were found (). Figure 2

[0047] ​5) Co-segregation site screening: Since the result of step 2) clearly indicated that the sterile phenotype of MSC4AB was controlled by a single gene, however, the bioinformatics analysis of step 4) co-detected up to 19 intervals on each chromosome linked to the GMS gene, therefore, the BSA-seq analysis method based on Δ(SNP-index) was not suitable for the GMS gene localization in the MSC4AB dual line population. So the analysis strategy was changed, based on the related reads of 15,767 polymorphic sites in step 4), further analysis was carried out: first, screen out the sites with genotype of homozygous sterile (1|1) in SI samples, homozygous sterile (1|1) in SP samples, and heterozygous fertile (0|1) in FP samples, a total of 695; then, further filter (i.e. SNP-index) the above 695 sites, screen out 85 sites with SI·SNP-index = 1 and SP·SNP-index = 1, and these sites are used as candidate co-segregation sites. Based on the Zunla-1 reference genome, annotation analysis of the above 85 candidate co-segregation sites was carried out, and it was found that only one site caused non-synonymous mutation. The physical coordinate of this site is 248,789,782 bp on chromosome 3 of the Zunla-1 reference genome, and the reference genome is A base, the fertile pool is A base and A base deletion heterozygous, and the sterile pool is A base deletion homozygous.

[0048] 6) Co-segregation marker development: Based on the sequence flanking 248,789,782 bp on chromosome 3 of Zunla-1 genome and the variation characteristics of MSC4AB sterile strain at this site Figure 3 a), a pair of dCAPS marker primers CaMSC4 Figure 3 b and c) were designed with the help of Snapgene software, according to the design principle that Taq I restriction endonuclease can cut the target sequence of fertile strains, but cannot cut the target sequence of sterile strains.

[0049] The molecular marker CaMSC4 contains two single-stranded nucleotide primers CaMSC4-F and CaMSC4-R, and the nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the target fragment of the molecular marker CaMSC4 is shown in SEQ ID NO: 3.

[0050] The primer sequences are as follows:

[0051] CaMSC4-F: 5'-TGACTAAGCTTGGCATCATAGAGTCG-3'

[0052] CaMSC4-R: 5'-CTCCAAGGTCTCATACTTTCTCATAAC-3'.

[0053] The target sequence is as follows:

[0054] (underlined is the Taq I restriction enzyme recognition site, and the bold A base indicates that it is deleted in sterile plants).

[0055] Example 2: Application of co-segregation marker CaMSC4 and related kit

[0056] 1) Application of co-segregation marker CaMSC4 in MSC4AB nuclear male sterile line: According to the method in Example 1, 253 MSC4AB nuclear male sterile dual-purpose line populations were identified for fertility phenotype, and the results showed that the population contained 179 fertile plants and 74 sterile plants. The DNA of each single plant was extracted by CTAB method as a template, the kit shown in Table 1 was used, and the target sequence was amplified by PCR according to the reaction program shown in Table 2. Then, the PCR products (target sequence) from each single plant were Taq I enzyme cut according to the Taq I enzyme cut reaction system shown in Table 3 and the Taq I enzyme cut reaction program shown in Table 4. Finally, the products after enzyme cutting were electrophoresed by 6% polyacrylamide gel. The results showed that all sterile plants corresponded to A band type, and all fertile plants corresponded to H band type. Figure 4 ) This is consistent with the characteristics that sterile plants in nuclear male sterile dual-purpose line MSC4AB are recessive homozygous genotype (msc-4msc-4), and fertile plants are heterozygous genotype (Msc-4msc-4). The above results show that the molecular marker CaMSC4 and related kit provided by the present application can achieve 100% discrimination of fertile plants and sterile plants in Capsicum annuum nuclear male sterile line MSC4AB.

[0057] Table 1 Kit composition

[0058]

[0059] Table 2 PCR reaction program

[0060]

[0061]

[0062] Table 3 Taq I enzyme cut reaction system

[0063]

[0064] Table 4 Taq I enzyme cut reaction program

[0065]

[0066] 2) Application of co-segregation marker CaMSC4 in F2 fertility segregation population: one sterile plant MSC4A (msc-4msc-4) in pepper CMS dual line MSC4AB was used as female parent, and a male fertile inbred line BB3 (Msc-4Msc-4) was used as male parent to obtain F1 material, and then F2 fertility segregation population was obtained by selfing. 940 plants of F2 population were planted, and according to the pollen fertility phenotype identification method in example 1, it was found that there were 736 fertile plants and 203 sterile plants in the population. According to the method of step 1) in example 2, genotyping was performed on each single plant of F2 population, and it was found that all fertile plants corresponded to H or B band type, and sterile plants corresponded to A band type. Figure 5 The above results show that using the molecular marker CaMSC4 and the related kit provided by the application to genotype pepper plants at the seedling stage can achieve 100% identification of fertile plants and sterile plants in pepper F2 fertility segregation population.

[0067] 3) Application of co-segregation marker CaMSC4 in natural population: according to the method of step 1) in example 2, 99 male fertile inbred lines with different genetic backgrounds (Table 5) were analyzed by using molecular marker CaMSC4, and the results showed that all materials obtained B band type, and the genotyping results of 19 male fertile inbred lines with different genetic backgrounds were shown in Figure 6 , which shows that the molecular marker CaMSC4 and the related kit provided by the application are also applicable in fertile inbred lines. Accordingly, it is shown that the molecular marker CaMSC4 and the related kit provided by the application have important value in the process of transferring sterile gene msc-4 to other fertile inbred lines to breed new pepper CMS lines.

[0068] Table 5 Genotyping results of marker CaMSC4 on natural population

[0069]

[0070]

[0071] 4) The application of co-segregation marker CaMSC4 and related kit in the process of breeding of nuclear male sterile dual-purpose line: Backcross breeding is the main method to transfer nuclear male sterile gene to target material. When the sterile plant (MSC4A) in MSC4AB is used as the donor parent for backcross breeding, the backcross generation needs to be selfed, and then the sterile single plant is selected from the selfed progeny for backcross breeding; or, the backcross and selfing are simultaneously performed in the backcross generation, and the next generation is planted with both backcross and selfed lines. The lines in the backcross generation whose fertility separates in the selfed progeny can be used for further selection and backcross breeding. The lines in the backcross generation whose fertility does not separate in the selfed progeny can be directly eliminated. However, both methods have the disadvantages of large field workload, high cultivation cost and / or long breeding period. In the process of breeding of nuclear male sterile dual-purpose line, the co-segregation marker CaMSC4 and related kit provided by the present application can quickly identify the genotype before planting, and only the heterozygous backcross progeny carrying the recessive sterile gene is screened for further backcross breeding, thereby the selfing step for field genotype identification and / or unnecessary backcross combination are omitted, so as to shorten the breeding time and save several times of land consumption and cultivation cost. Figure 7

[0072] It should be noted that the above-mentioned embodiments should be understood as illustrative rather than limiting the scope of protection of the present application, and the scope of protection of the present application is subject to the claims. For those skilled in the art, some non-essential improvements and adjustments of the present application without departing from the spirit and scope of the present application still belong to the protection scope of the present application.​

Claims

1. Primer pairs for identifying male-sterile and male-fertile pepper plants, characterized in that: The primer pairs include specific primer pairs CaMSC4-F and CaMSC4-R, whose nucleotide sequences are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

2. A kit comprising the primer pair of claim 1.

3. The reagent kit according to claim 2, characterized in that: The kit also includes a magnesium-containing PCR buffer, dNTPs, Taq enzyme, and ddH2O.

4. The application of the primer pair according to claim 1 or the kit according to any one of claims 2-3 in pepper fertility phenotype identification or assisted selection breeding, wherein the assisted selection breeding refers to the selection of male-sterile lines in pepper.

5. A method for identifying male-sterile and male-fertile pepper plants, characterized in that: Specifically, the following steps are included: Step 1: Extract genomic DNA from the pepper plants to be tested; Step 2: Using the chili genomic DNA extracted in Step 1 as a template, amplify the target sequence using the primer pair described in claim 1; Step 3, adopt Taq I. Restriction endonuclease was used to digest the target sequence amplified in step 2, and then the fertile and sterile plants were identified based on the banding after electrophoresis. In step three, the specific identification method is as follows: When the plant to be tested is from the MSC4AB dual-purpose line, if two bands of 81bp and 58bp appear after electrophoresis, the plant is fertile; if only one band of 81bp appears after electrophoresis, the plant is sterile. When the plant to be tested comes from the F2 population, if only one band of size 58 bp appears after electrophoresis, it is a homozygous fertile plant; if two bands of sizes 81 bp and 58 bp appear after electrophoresis, it is a heterozygous fertile plant; if one band of size 81 bp appears after electrophoresis, it is a sterile plant.

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