A dCAPS marker for the dark green trait of Hainan yellow lantern pepper in the early stage of fruit development and its application

Abstract

The invention provides a new SNP site associated with the early dark green trait of Hainan yellow lantern pepper fruit development, which is located at position 22921311 on chromosome 1 (reference genome version: ASM227189v2); for the above SNP site, the invention also provides a corresponding dCAPS molecular marker, a specific primer and a method for identifying the presence of the Hainan yellow lantern pepper fruit development early dark green trait (including homozygotes and heterozygotes) in the seedling stage by using the dCAPS molecular marker. The method is based on the characteristics of the SNP site and the application of the mismatch base enzyme cleavage site, combined with PCR amplification and enzyme cleavage methods, and can identify whether a Chinese pepper material to be tested contains the Hainan yellow lantern pepper fruit development early dark green trait (including homozygotes and heterozygotes) in the seedling stage. The development of the marker is of great significance to the Hainan yellow lantern pepper fruit development early dark green trait in the efficient genetic improvement of Chinese pepper fruit color.

Description

Technical Field

[0001] The invention relates to the fields of pepper genetic breeding and molecular biology, and in particular to a dCAPS marker for the dark green trait of Hainan yellow lantern pepper fruit in the early development stage and its application. Background Art

[0002] Hainan Yellow Lantern Pepper is a local pepper germplasm distributed in Hainan. It belongs to Chinese pepper (Capsicum chinense) in taxonomy. Chinese pepper is one of the five cultivated species of the genus Capsicum. Hainan Yellow Lantern Pepper has a prominent aroma and a strong spicy taste. The fruit is dark green in the early stages of development. The color of pepper fruit is an important breeding goal. Genetic studies have shown that when dark green pepper fruits are hybridized with light green pepper fruits, the F1 representative is dark green, indicating that dark green is a dominant trait and light green is a recessive trait. Therefore, through traditional phenotypic identification methods, plants with dark green pepper fruits may have dark green and light green pepper fruits in their offspring, making the color of pepper fruits unstable in the early stages of development, which profoundly affects breeding efficiency.

[0003] Molecular marker breeding is an important means of breeding. By developing molecular markers that are closely linked to the target traits, stable, and easy to detect, the genotype can be directly selected, and the target traits can be selected in the early stages of plant growth and development. At the same time, co-dominant molecular markers can also distinguish between homozygous and heterozygous genotypes, which can greatly improve the accuracy and efficiency of selection in breeding and significantly shorten the breeding cycle. Therefore, in the process of breeding new varieties with Hainan yellow lantern pepper as one of the parents, it is very urgent to identify whether the plant to be tested contains the dark green fruit gene in Hainan yellow lantern pepper in the early stages of plant growth and development, and at the same time determine whether its genotype is homozygous. It is urgent to carry out molecular marker breeding. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide a SNP site related to the early dark green trait of Hainan yellow lantern pepper fruit development, wherein the SNP site is at position 22921311 on chromosome 1 of Hainan yellow lantern pepper (reference genome version: ASM227189v2), and the base is G.

[0005] The second aspect of the present invention aims to provide an application of the SNP site in identifying or screening the early green trait of fruit development in hybrid offspring with Hainan yellow lantern pepper as a parent. Specifically, it can be determined whether the fruit is dark green in the early stage of development in the hybrid offspring seedling stage.

[0006] The third aspect of the present invention aims to provide a specific dCAPS molecular marker associated with the early dark green trait of Hainan yellow lantern fruit development, the nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1, and the polymorphic site of the sequence is at position 22921311 (reference genome version: ASM227189v2).

[0007] The fourth aspect of the present invention aims to provide a primer pair for detecting the specific dCAPS molecular marker of the present invention, wherein the primer pair consists of a primer F and a primer R;

[0008] The primer F is as follows a1) or a2):

[0009] a1) the sequence shown in SEQ ID NO: 2;

[0010] a2) a single-stranded DNA molecule having the same function as the sequence shown in SEQ ID NO: 2 after one or more nucleotides are substituted and / or deleted and / or added;

[0011] The primer R is as follows b1) or b2):

[0012] b1) the sequence shown in SEQ ID NO: 3;

[0013] b2) A single-stranded DNA molecule having the same function as the sequence shown in SEQ ID NO: 3 after one or more nucleotides are substituted and / or deleted and / or added.

[0014] In one or more embodiments, the primer pair consists of a primer F and a primer R, wherein the primer F is a1), and the primer R is b1).

[0015] In one or more embodiments, the primer pair consists of a primer F and a primer R, wherein the primer F is a1), and the primer R is b2).

[0016] In one or more embodiments, the primer pair consists of a primer F and a primer R, wherein the primer F is a2) and the primer R is b1).

[0017] In one or more embodiments, the primer pair consists of primer F and primer R, wherein primer F is a2) and primer R is b2).

[0018] It is well known to those skilled in the art that the design of primers must comply with certain principles, such as controlling the length of the primers, the GC content of the primers, the Tm value of the primers, the number of mismatched bases, etc., and can be designed using conventional software in the art, or can be designed directly on the relevant website. As long as the primers comply with the above design principles, they may be used for the detection of the present application. Preferably, the primer pair described in the present application includes the sequence shown in SEQ ID NO:2 and the sequence shown in SEQ ID NO:3.

[0019] The fifth aspect of the present invention aims to provide the application of the specific dCAPS molecular marker in hybrid offspring with Hainan yellow lantern pepper as parent to identify whether the color of the early fruit development is dark green at the seedling stage and determine whether it is homozygous or heterozygous.

[0020] The sixth aspect of the present invention aims to provide a method for identifying whether the color of the early fruit development is dark green in the seedling stage, and determining whether it is homozygous or heterozygous in the hybrid offspring with Hainan yellow lantern pepper as the parent, comprising the following steps:

[0021] S1. Extract genomic DNA of seedlings from offspring produced by Hainan yellow lantern pepper and other Chinese peppers as parents;

[0022] S2. performing PCR amplification on the extracted genomic DNA using the primer pair of the present invention;

[0023] S3. The amplified PCR product was digested with BamHI and then electrophoresed;

[0024] S4. Result judgment: If the enzyme cleavage product electrophoresis obtains an electrophoresis band of 107bp, the genotype of the SNP site of the pepper sample is HH (H is one of the bases T, A, and C), and the color of the fruit of the sample in the early stage of development is non-dark green; if the enzyme cleavage product electrophoresis obtains an electrophoresis band of 86bp, the genotype of the pepper sample is GG, and the color of the fruit of the sample in the early stage of development is dark green; if the enzyme cleavage product electrophoresis obtains electrophoresis bands of 107bp and 86bp, the genotype of the SNP site of the pepper sample is GH (H is one of the bases T, A, and C), and the color of the fruit of the sample in the early stage of development is dark green.

[0025] In one or more embodiments, the PCR amplification system (25 μl) in step S2 is: 5 μl genomic DNA, 12.5 μl 2x Rapid Taq Master Mix, 2 μl upstream primer at a concentration of 10 μM / L, 2 μl downstream primer at a concentration of 10 μM / L, and ddH2O to make up to 25 μl.

[0026] In one or more embodiments, the conditions for PCR amplification in step S2 are:

[0027] Step 1: pre-denaturation at 94°C for 5 min; Step 2: denaturation at 94°C for 30 s;

[0028] Step 3: anneal at 56℃ for 30s; Step 4: extend at 72℃ for 60s;

[0029] Step 5: Repeat Step 2-4 for 35 cycles; Step 6: Extend at 72°C for 7 min.

[0030] In one or more embodiments, the enzyme digestion system (25 μl) in step S3 is: 1 μl of restriction endonuclease, 5 μl of restriction endonuclease buffer, 10 μl of PCR product, and ddH2O to make up to 25 μl.

[0031] In one or more embodiments, the conditions for the enzyme cleavage in step S3 are: Step 1: incubate at 37° C. for 15 min; Step 2: add 5 μl of purple termination reaction staining solution.

[0032] The seventh aspect of the present invention aims to provide a product for a method of identifying whether the color of the early fruit development is dark green in the seedling stage in hybrid offspring with Hainan yellow lantern pepper as the parent, and determining whether it is homozygous or heterozygous, the product comprising any of the above-mentioned primer pairs and BamHI enzyme, preferably, the product also comprises a DNA extraction reagent.

[0033] In one or more embodiments, the method for using the product of the present invention for identifying or screening whether a Chinese pepper sample to be tested contains the dark green trait of Hainan yellow lantern pepper in the early stage of fruit development is as follows: extracting the genome of the pepper to be tested by using the DNA extraction reagent provided by the product, then performing PCR amplification using the primer pair provided by the product, and performing electrophoresis on the amplified product after enzyme digestion with the BamHI enzyme provided by the product, and judging the color of the fruit of the Chinese pepper sample to be tested in the early stage of fruit development according to the electrophoresis band; if the enzyme digestion product electrophoresis obtains an electrophoresis band of 107bp, the genotype of the SNP site of the Chinese pepper sample is HH (H is one of the bases T, A, and C), and the color of the fruit of the sample in the early stage of fruit development is non-dark green; if the enzyme digestion product electrophoresis obtains an electrophoresis band of 86bp, the genotype of the Chinese pepper sample is GG, and the color of the fruit of the sample in the early stage of fruit development is dark green; if the enzyme digestion product electrophoresis obtains electrophoresis bands of 107bp and 86bp, the genotype of the SNP site of the Chinese pepper sample is GH (H is one of the bases T, A, and C), and the color of the fruit of the sample in the early stage of fruit development is dark green.

[0034] The present invention provides a new SNP site related to the early color of Hainan yellow lantern pepper fruit development, which is located at position 22921311 on chromosome 1 of Hainan yellow lantern pepper; by identifying this site, it is possible to accurately identify whether the fruit of an unknown Chinese pepper sample is dark green in the early stage of development, and batch identification can be performed, greatly improving the identification efficiency and accuracy.

[0035] For the above-mentioned SNP sites, the present invention also provides corresponding dCAPS molecular markers, specific primers, and a method for quickly identifying the early color of Hainan yellow lantern pepper fruit development at the seedling stage using dCAPS molecular markers. The method is based on the characteristics of the SNP site and the application of the mismatch base enzyme cleavage site, combined with PCR amplification and enzyme cleavage methods, which greatly improves the identification efficiency of the early color of the fruit development of the Chinese pepper to be tested. The development of this marker is of great significance to the breeding of different fruit colors of Chinese peppers. The dCAPS marker of the early color trait of the fruit development of Hainan yellow lantern pepper described in the present invention can also identify whether the genotype of the Chinese pepper sample is homozygous, can be used for molecular marker-assisted breeding, greatly shortens the breeding cycle, and has important theoretical and practical significance for the cultivation of new varieties of early color of Chinese pepper fruit development. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Illustration of the design of 100bp sequences upstream and downstream of the SNP and ISKL_dCAPS primers.

[0037] Figure 2 Validation diagram of the developed dCAPS molecular markers in natural populations and progeny.

[0038] M is D2000 Marker; double bands represent heterozygous genotypes, and single band represents homozygous genotypes. DETAILED DESCRIPTION

[0039] The following is a detailed description of the present invention. The following description of the technical features is based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted that:

[0040] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0041] In the present specification, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0042] In this specification, the word "may" includes both performing a certain process and not performing a certain process.

[0043] In this specification, the use of "optional" or "optional" indicates that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0044] In this specification, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used indicates weight or mass percentage.

[0045] In this specification, unless otherwise stated, "plurality" means that there are two or more.

[0046] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0048] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0049] Example 1 The acquisition of SNP loci related to the early color of Hainan yellow lantern pepper fruit development was based on the simplified genome sequencing (GBS) technology, and GATK and other software were used to detect population SNPs:

[0050] (1) Select reads that map to unique positions on the genome for subsequent analysis;

[0051] (2) GATK (-type UnifiedGenotyper) was used to detect population SNPs in the filtered bam files;

[0052] (3) To reduce false positive SNPs caused by sequencing errors, the SNP base support number for parents and offspring is required to be no less than 4.

[0053] (4) Statistical SNP-related information: number of heterozygous SNPs, number of homozygous SNPs, and heterozygous SNP ratio.

[0054] By analyzing the test results of the two parental genotypes (Chinese peppers with light green fruit in the early stage of fruit development and Hainan yellow lantern peppers with dark green fruit in the early stage of fruit development), polymorphic markers between parents were developed. The sites where both parents were homozygous and polymorphic between parents were screened. The early color of fruit development was associated with SNP through the F2 segregation population, and a SNP site significantly associated with the color trait of the early fruit development of Hainan yellow lantern pepper was obtained. The SNP site in Hainan yellow lantern pepper is at position 22921311 on chromosome 1 (reference genome version: ASM227189v2), the base is G, and the other parent is T at this site.

[0055] Example 2 Development of dCAPS molecular markers related to early fruit color of Hainan yellow lantern pepper (named ISKL_dCAPS in the present invention)

[0056] 1. Primer design:

[0057] The dCAPS primers were designed according to the SNP sites obtained in Example 1, and the specific steps were as follows:

[0058] Using the online software dCAPS Finder 2.0, the SNP mutation site was selected, the mismatch base (TT→GG) was introduced, and the appropriate restriction endonuclease (BamHI) was selected to design one of the primers; the other primer was designed using Snapgene viewer, the primer was designed to be 20-25bp, and the GC content was 35-60%; the annealing temperature was 55-60°C, and the annealing temperature and length of the upstream and downstream primers were kept consistent as much as possible.

[0059] The final primer sequences are as follows:

[0060]

[0061] like Figure 1 The figure shows the upstream and downstream 100 bp sequences of the SNP of the present invention and the design diagram of ISKL_dCAPS primers, wherein [G / T] is the SNP mutation site, the arrows underline the upstream and downstream primer positions, and the arrows are the restriction sites.

[0062] Using the two parental genomic DNAs in Example 1 as templates, PCR amplification was performed using the above primers to obtain a product of 107 bp, the sequence of which is shown in SEQ ID NO: 1 in the sequence listing. The 101st base of the sequence is G / T, which is a polymorphic site.

[0063] 2. Extraction of sample genomic DNA

[0064] Select the young leaves of the Chinese pepper sample at the seedling stage of the parent or F2 segregating population to be tested in Example 1

[0065] Extraction of genomic DNA:

[0066] DNA extraction kit for Plant DNA Isolation Mini Kit DC104 (Novizan Biotech Co., Ltd., Nanjing).

[0067] (1) Take 100 mg of fresh plant tissue and grind it into a homogenate using a grinder;

[0068] (2) Immediately add 400 μl buffer A1 and 4 μl RNase A to the ground sample and vortex to fully lyse.

[0069] (3) 65°C water bath for 10 min. During the water bath, invert the centrifuge tube 2-3 times to mix the sample.

[0070] (4) Add 130 μl of Buffer A2 to the mixture, mix thoroughly, place on ice for 5 min, and then centrifuge at 14,000 rpm (18,400 × g) for 10 min. Pipette the supernatant into a new centrifuge tube.

[0071] (5) Take 1.5 times the volume of the supernatant, Buffer A3, and mix immediately by pipetting;

[0072] (6) The mixture in (5) was transferred to an adsorption column, centrifuged at 12000 rpm (13400 × g) for 1 min, and the filtrate was discarded;

[0073] (7) Add 600 μl of Buffer AW, centrifuge at 12000 rpm (13400 × g) for 1 min, discard the filtrate, and repeat the process;

[0074] (8) Place the adsorption column in a new 1.5 ml centrifuge tube, add 50 μl of preheated Elution Buffer to the center of the adsorption column, leave at room temperature for 5 min, centrifuge at 12000 rpm (13400 × g) for 1 min, discard the adsorption column, and store at -20°C for future use.

[0075] 3. PCR amplification

[0076] Using the genomic DNA obtained above as a template, the following amplification system was constructed:

[0077] (1) The PCR amplification system is (25 μl):

[0078] 2x Rapid Taq Master Mix: 12.5μl

[0079] Upstream primer: 2 μl (concentration 10 μM)

[0080] Upstream primer: 2 μl (concentration 10 μM)

[0081] Genomic DNA: 5 μl

[0082] ddH2O: make up to 25μl.

[0083] (2) The PCR amplification procedure is as follows:

[0084] Step 1: Pre-denaturation at 94°C for 5 min;

[0085] Step 2: denaturation at 94°C for 30 seconds;

[0086] Step 3: anneal at 56℃ for 30s;

[0087] Step 4: 72℃ extension for 60s;

[0088] Step 5: Repeat Step 2-4 for 35 cycles;

[0089] Step 6: Extend at 72℃ for 7 minutes.

[0090] 4. Enzyme Digestion

[0091] The PCR product obtained in the above step was digested with restriction endonuclease BamHI (NEB)

[0092] (1) Enzyme digestion system (25 μl) is as follows:

[0093] Restriction enzyme: 1 μl

[0094] Restriction enzyme buffer: 5 μl

[0095] PCR product: 10 μl

[0096] ddH2O: make up to 25 μl

[0097] (2) Enzyme digestion reaction conditions are as follows:

[0098] Step 1: Incubate at 37°C for 15 min;

[0099] Step 2: Add 5 μl of purple termination reaction staining solution.

[0100] 5. Electrophoresis

[0101] The above enzyme-digested products were subjected to agarose gel electrophoresis, stained with green fluorescent dye and photographed using a gel imager. The electrophoresis conditions were: 3.5% agarose gel, voltage: 150 V, time: 35 min.

[0102] 6. Result judgment: If the electrophoresis of the enzyme cleavage product obtains an electrophoresis band of 107bp, the genotype of the SNP site of the pepper sample is TT, and the color of the fruit of the sample in the early stage of development is light green; if the electrophoresis of the enzyme cleavage product obtains an electrophoresis band of 86bp, the genotype of the pepper sample is GG, and the color of the fruit of the sample in the early stage of development is dark green; if the electrophoresis of the enzyme cleavage product obtains electrophoresis bands of 107bp and 86bp, the genotype of the SNP site of the pepper sample is GT, and the color of the fruit of the sample in the early stage of development is dark green.

[0103] Example 3 Application of dCAPS molecular marker (named ISKL_dCAPS in the present invention)

[0104] Hainan yellow lantern pepper (Capsicum chinense) is used as the male parent, collected from Hainan Lingshui local varieties, numbered as 'HNUCC16', and the fruit is dark green in the early stage of development; the female parent is another Chinese pepper (Capsicum chinense) germplasm, collected and numbered as 'HNUCC22' by the College of Horticulture of Hainan University, and the fruit is light green in the early stage of development. The F1 generation population is obtained by hybridization, and then the F2 generation population is obtained by selfing. The present invention randomly selects 40 Chinese pepper seedlings from 200 F2 generation populations as samples to be tested, and uses the method described in the present invention to identify or predict the color of the early stage of fruit development of each sample; transplant the F2 generation seedlings of each Chinese pepper to be tested to the test field, water and fertilize during the period to ensure the normal growth of each sample, and fruiting around 60 days; the early stage color of the fruit development of each sample identified by the method described in the present invention is compared with the actual early stage color of the fruit development of pepper, and the accuracy of the detection method described in the present invention is judged.

[0105] 1. Select young leaves of each sample at the seedling stage for genomic DNA extraction

[0106] 2. Using the genomic DNA obtained above as a template, perform PCR amplification respectively:

[0107] The PCR amplification system was (25 μl): 2x Rapid Taq Master Mix 12.5 μl, upstream primer (SEQ ID NO: 2) 2 μl (concentration 10 μM), downstream primer (SEQ ID NO: 3) 2 μl (concentration 10 μM), genomic DNA 5 μl, and ddH2O was added to make up to 25 μl.

[0108] The PCR amplification procedure is:

[0109] Step 1: pre-denaturation at 94°C for 5 min; Step 2: denaturation at 94°C for 30 s;

[0110] Step 3: anneal at 56℃ for 30s; Step 4: extend at 72℃ for 60s;

[0111] Step 5: Repeat Step 2-4 for 35 cycles; Step 6: Extend at 72°C for 7 min.

[0112] 3. The amplified products obtained above were digested with restriction endonuclease BamHI (NEB):

[0113] The enzyme digestion system (25 μl) is: 1 μl of restriction endonuclease, 5 μl of restriction endonuclease buffer, 10 μl of PCR product, and ddH2O to make up to 25 μl.

[0114] The enzyme digestion reaction conditions are as follows: Step 1: incubate at 37°C for 15 min; Step 2: add 5 μl of purple termination reaction staining solution.

[0115] 4. The digested product was subjected to agarose gel electrophoresis and photographed with a gel imager after being stained with green fluorescent dye. The electrophoresis conditions were: 3.5% agarose gel, voltage: 150 V, time: 35 min.

[0116] 5. Identification or prediction is performed according to the following standards: If the enzyme cleavage product electrophoresis obtains an electrophoresis band of 107bp, the genotype of the SNP site of the Chinese pepper sample is TT, and the color of the fruit of the sample in the early stage of development is light green; if the enzyme cleavage product electrophoresis obtains an electrophoresis band of 86bp, the genotype of the Chinese pepper sample is GG, and the color of the fruit of the sample in the early stage of development is dark green; if the enzyme cleavage product electrophoresis obtains electrophoresis bands of 107bp and 86bp, the genotype of the SNP site of the Chinese pepper sample is GT, and the color of the fruit of the sample in the early stage of development is dark green.

[0117] 6. The early fruit development colors of each sample identified or predicted by the above method were compared with the actual early fruit development colors of each sample grown in the field. The results are as follows:

[0118]

[0119]

[0120] The above test shows that the results of identifying or predicting the early fruit development colors of 40 randomly selected Chinese pepper samples to be tested by the method of the present invention are completely consistent with their actual early fruit development colors, with an accuracy rate of 100%. Therefore, the molecular markers of the present invention have strong applicability, and can be used in practical breeding applications to quickly identify and screen whether the Chinese pepper samples to be tested contain the early fruit development color traits of Hainan yellow lantern pepper in the seedling stage.

[0121] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. An application of a SNP locus associated with the dark green trait of Hainan yellow lantern pepper in the early development stage to identify or screen the green trait of fruit development in the early development stage in the hybrid offspring of Hainan yellow lantern pepper as a parent, characterized in that: The SNP site is located at position 22921311 on chromosome 1 of the reference genome of Hainan yellow lantern pepper, the base is G / T, and the reference genome version is: ASM227189v2. It can be judged whether the fruit is dark green in the early stage of development during the seedling stage of the hybrid offspring of Hainan yellow lantern pepper.

2. A primer pair of dCAPS molecular markers for the dark green trait of Hainan yellow lantern fruit development in the early stage developed based on the SNP locus according to claim 1, characterized in that: The primer pair consists of primer F and primer R; The primer F is the sequence shown in SEQ ID NO: 2; The primer R is the sequence shown in SEQ ID NO:

3.

3. Use of the primer pair of the specific dCAPS molecular marker according to claim 2 in identifying the green trait in the early stage of fruit development and whether the fruit is homozygous in the hybrid offspring with Hainan yellow lantern pepper as the parent.

4. A method for identifying the green trait of early fruit development and whether it is homozygous in the hybrid offspring of Hainan yellow lantern pepper as the parent, characterized in that: The following steps are involved: S1. Extracting genomic DNA from leaves of pepper parents and hybrid offspring seedlings; S2. performing PCR amplification on the extracted genomic DNA using the primer pair according to claim 2; S3. The amplified PCR product was digested with BamHI and then electrophoresed; S4. Result judgment: If the enzyme cleavage product electrophoresis obtains an electrophoresis band of 107bp, the genotype of the SNP site of the pepper sample is HH, H is one of the bases T, A, and C, and the color of the fruit of the sample in the early stage of development is non-dark green; if the enzyme cleavage product electrophoresis obtains an electrophoresis band of 86bp, the genotype of the pepper sample is GG, and the color of the fruit of the sample in the early stage of development is dark green; if the enzyme cleavage product electrophoresis obtains electrophoresis bands of 107bp and 86bp, the genotype of the SNP site of the pepper sample is GH, H is one of the bases T, A, and C, and the color of the fruit of the sample in the early stage of development is dark green.

5. A product for identifying the green trait of early fruit development and whether it is homozygous in the hybrid offspring of Hainan yellow lantern pepper as the parent, characterized in that: The product comprises the primer pair according to claim 2 and BamHI enzyme.

6. The product according to claim 5, characterized in that Also contains DNA extraction reagents.

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