A method for identifying lantern-shaped pepper fruits and a special SNP primer combination thereof

Abstract

The application discloses a method for identifying lantern-shaped pepper fruits and a special SNP primer combination thereof. The SNP primer combination is composed of one or two primer groups, each primer group is composed of three primer sequences, and each primer sequence is used for amplifying one SNP site. The nucleotide sequences of the primers in the two primer groups are shown in sequence 1 to sequence 6 in the sequence list in sequence. The SNP primer combination provided by the application can be used for identifying whether the fruits of a to-be-tested pepper variety are lantern-shaped, and the DNA extracted from various development stages and different tissue organs of the pepper can be detected, and the method is not limited by environment and season, has the advantages of simple operation, accurate amplification, high throughput, low cost and the like, and has important application value for agricultural production and pepper variety breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for identifying lantern-shaped pepper fruits and its dedicated SNP primer combination. Background Technology

[0002] Chili peppers are the world's most widely cultivated spice and my country's largest-grown vegetable crop. Rich in vitamin C, capsaicin, and other nutrients, chili peppers have numerous processed products, a long industrial chain, and high added value, playing a vital role in the vegetable industry and people's daily lives. After more than sixty years of development, my country's chili pepper breeding industry has reached the forefront globally, entering a new stage of quality and precision breeding.

[0003] Vegetables and fruits exhibit extensive diversity in morphology and characteristics. Fruit shape is a crucial commercial and sensory trait, as well as a significant quality trait affecting total yield. Chili pepper germplasm resources possess rich genetic diversity, with wide variations in traits such as plant shape, fruit shape, fruit color, and fruit weight. Long-term natural evolution and artificial breeding have resulted in mainstream commercial varieties such as lantern peppers, horn peppers, long peppers, facing-heaven peppers, and screw peppers, adaptable to different production areas, cultivation methods, and consumption scenarios. Among these, lantern peppers, with their thick flesh, plump shape, sweet taste, lack of spiciness, and high nutritional value, are suitable for diverse scenarios including fresh consumption, side dishes, and sightseeing picking, leading to a continuously rising market acceptance. This variety is highly adaptable and can be cultivated on a large scale in all provinces and municipalities across my country, with a very wide distribution. It has become a mainstream specialty variety in the vegetable market, facility agriculture, and urban sightseeing agriculture in recent years, demonstrating significant overall economic benefits and industrial promotion value. Chili variety selection and improvement have long been valued. Traditional breeding methods (pedigree selection, backcrossing, hybridization, etc.) can no longer meet the needs of social development and external market competition. Molecular marker design breeding will become an important approach and development trend in chili breeding.

[0004] Single nucleotide polymorphisms (SNPs) are polymorphisms in nucleic acid sequences at the genomic level caused by changes in a single nucleotide, including single base transitions, insertions, and deletions. As third-generation molecular markers, SNPs are characterized by their large number, wide distribution, ease of rapid large-scale screening, and convenient genotyping, making them a widely recognized and promising molecular marker technology. Fruit shape is one of the main target traits in pepper breeding. Based on the market position of bell peppers, developing molecular markers that can quickly identify whether pepper fruits are bell-shaped provides a new approach for pepper fruit shape screening and variety selection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for identifying lantern-shaped pepper fruits and a specific SNP primer set thereof. This primer set can be used to rapidly identify whether pepper fruits are lantern-shaped.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides an SNP primer set, which may include a first primer set for amplifying the DengLJ3 SNP site of the pepper genome and / or a second primer set for amplifying the DengL4 SNP site of the pepper genome. The DengLJ3 SNP site is nucleotide 193057991 on chromosome 9, and it is either A or C. The DengLJ4 SNP site is nucleotide 193059682 on chromosome 9, which is either T or C. The positions of the DengLJ3 and DengLJ4 SNP sites on the chromosome were determined based on the alignment of the Zunla-1 reference genome sequence of the chili pepper, version number V2.0.

[0007] In the SNP primer set, the first primer set can be used to identify the genotype of the DengLJ3 SNP site, and the second primer set can be used to identify the genotype of the DengLJ4 SNP site. The first and second primer sets can be designed based on the KASP principle.

[0008] Further, the first primer set includes a first forward primer, a second forward primer, and a first reverse primer, wherein the specific nucleotide sequence of the first forward primer is shown as SEQ ID NO: 1 from position 22 to 43 from the 5' end, the nucleotide sequence of the second forward primer is shown as SEQ ID NO: 2 from position 22 to 43 from the 5' end, and the nucleotide sequence of the first reverse primer is shown as SEQ ID NO: 3; The second primer set includes a third forward primer, a fourth forward primer, and a second reverse primer, wherein the nucleotide sequence of the third forward primer is shown as SEQ ID NO: 4 from position 22 to 55 from the 5' end, the nucleotide sequence of the fourth forward primer is shown as SEQ ID NO: 5 from position 22 to 55 from the 5' end, and the nucleotide sequence of the second reverse primer is shown as SEQ ID NO: 6; To facilitate the identification of SNP locus genotypes via fluorescence signals, a fluorescent tag sequence is added to the 5' end of the forward primers. The fluorescent tag sequences of the first and second forward primers exhibit different fluorescence signal colors. The fluorescent tag sequences of the third and fourth forward primers also exhibit different fluorescence signal colors.

[0009] Any of the SNP primer combinations described above can be the first primer set alone, the second primer set alone, or a combination of the first primer set and the second primer set.

[0010] In the above text, the nucleotide sequence shown in SEQ ID NO: 1 from position 1 to 21 starting from the 5' end is a fluorescent tag sequence (i.e., a FAM fluorescent tag sequence), and the fluorescence signal is specifically blue. The nucleotide sequence shown in SEQ ID NO: 2 from position 1 to 21 starting from the 5' end is also a fluorescent tag sequence (i.e., a HEX fluorescent tag sequence), and the fluorescence signal is specifically red.

[0011] In the SNP primer combination, as a preferred embodiment, the first primer set may consist of forward primer 1F1 shown in SEQ ID NO: 1, forward primer 1F2 shown in SEQ ID NO: 2, and reverse primer 1R shown in SEQ ID NO: 3. The second primer set may consist of forward primer 2F1 shown in SEQ ID NO: 4, forward primer 2F2 shown in SEQ ID NO: 5, and reverse primer 2R shown in SEQ ID NO: 6.

[0012] In any of the primer sets described above, the molar ratio of primers containing "F1", primers containing "F2", and primers containing "R" in the PCR reaction system can specifically be 2:2:5.

[0013] Kits containing any of the SNP primer combinations described above are also within the scope of protection of this invention. Therefore, a second aspect of this invention provides a kit comprising the SNP primer combinations described in the first aspect.

[0014] The kit may also include other reagents for competitive allele-specific PCR, such as DNA polymerase and buffer.

[0015] The preparation method of the kit is also within the scope of protection of this invention. The preparation method of the kit includes the step of individually packaging each primer from any of the primer sets described above.

[0016] The third aspect of this invention provides the application of the SNP primer combination described in the first aspect or the kit described in the second aspect, wherein the application is any one of the following two: (A) in identifying whether the fruit of the pepper variety to be tested is lantern-shaped or not; (B) in selecting pepper varieties with lantern-shaped fruit.

[0017] The fourth aspect of this invention provides a method for identifying whether the fruit of a chili pepper variety to be tested is lantern-shaped or not, comprising the following steps: detecting the genotype of the chili pepper variety to be tested based on the DengLJ3 SNP site and / or the DengLJ4 SNP site, and then making the following judgments: if the genotype based on the DengLJ3 SNP site is AA and / or the genotype based on the DengLJ4 SNP site is TT, then the fruit of the chili pepper variety to be tested is identified as or suspected to be lantern-shaped; if the genotype based on the DengLJ3 SNP site is CC or CA, and / or the genotype based on the DengLJ4 SNP site is CC or CT, then the fruit of the chili pepper variety to be tested is identified as or suspected to be not lantern-shaped; The DengLJ3 SNP site is nucleotide 193057991 on chromosome 9, and it is either A or C. The DengLJ4 SNP site is nucleotide 193059682 on chromosome 9, which is either T or C. The positions of the DengLJ3 and DengLJ4 SNP sites on the chromosome were determined based on the alignment of the Zunla-1 reference genome sequence of the chili pepper, version number V2.0.

[0018] In the above method, the step of detecting the genotype of the pepper variety to be tested based on the DengLJ3 SNP site and / or the DengLJ4 SNP site can be as follows: (1) Using the genomic DNA of the pepper variety to be tested as a template, PCR amplification was performed using the first primer set and / or the second primer set of the first aspect described above to obtain PCR amplification products; (2) The fluorescence signal of the PCR amplification product obtained in step (1) is detected by an instrument, and the genotype of the pepper variety to be tested based on the DengLJ3 SNP site and / or DengLJ4 SNP site is obtained according to the color of the fluorescence signal.

[0019] Alternatively, in the above method, the step of detecting the genotype of the pepper variety to be tested based on the DengLJ3 SNP site and / or the DengLJ4 SNP site is as follows: (1) Using the genomic DNA of the pepper variety to be tested as a template, PCR amplification was performed using the first primer set and / or the second primer set in the first aspect of the above-mentioned method to obtain PCR amplification products; (2) Take the PCR amplification product obtained in step (1) and sequence it; (3) Based on the sequencing results obtained in step (2), obtain the genotype of the pepper variety to be tested based on the DengLJ3 SNP site and / or the DengLJ4 SNP site.

[0020] Further, the method for determining the genotype of the tested chili pepper variety based on the color of the fluorescence signal at the DengLJ3 SNP site and / or the DengLJ4 SNP site is as follows: If the fluorescence color of the tested chili pepper variety at the DengLJ3 SNP site is the same as the color of the fluorescent tag sequence connected to the 5' end of the first forward primer, then the genotype of the tested chili pepper variety at the DengLJ3 SNP site is AA; if the fluorescence color of the tested chili pepper variety at the DengLJ4 SNP site is the same as the color of the fluorescent tag sequence connected to the 5' end of the third forward primer, then the genotype of the tested chili pepper variety at the DengLJ4 SNP site is TT; if the fluorescence color of the tested chili pepper variety at the DengLJ3 SNP site is the same as the color of the fluorescent tag sequence connected to the 5' end of the second forward primer, then the genotype of the tested chili pepper variety at the DengLJ4 SNP site is CC; if the fluorescence color of the tested chili pepper variety at the DengLJ4 SNP site is AA, then the genotype of the tested chili pepper variety at the DengLJ4 SNP site is TT; if the fluorescence color of the tested chili pepper variety at the DengLJ4 SNP site is AA, then the genotype of the tested chili pepper variety at the DengLJ4 SNP site is CC; if the fluorescence color of the tested chili pepper variety at the DengLJ4 SNP site is AA, then the genotype of the tested chili pepper variety at the DengLJ4 SNP site is TT; if the fluorescence color of the tested chili pepper variety at the DengLJ3 SNP site is the same as the color of the fluorescent tag sequence connected to the 5' end of the second forward primer, then the genotype of the tested chili pepper variety at the DengLJ3 SNP site is CC; if the fluorescence color of the tested chili pepper variety at the DengLJ4 SNP site is AA, then the genotype of the tested chi If the fluorescence color displayed at the SNP site is the same as the color displayed by the fluorescent tag sequence connected to the 5' end of the fourth forward primer, then the genotype of the tested chili variety based on the DengLJ4 SNP site is CC; if the fluorescence color displayed at the DengLJ3 SNP site is a mixture of the colors displayed by the fluorescent tag sequences connected to the 5' ends of the first and second forward primers, then the genotype of the tested chili variety based on the DengLJ3 SNP site is AC; if the fluorescence color displayed at the DengLJ4 SNP site is a mixture of the colors displayed by the fluorescent tag sequences connected to the 5' ends of the third and fourth forward primers, then the genotype of the tested chili variety based on the DengLJ4 SNP site is TC.

[0021] In any of the methods described above, the reaction procedure for "PCR amplification using the first primer set and / or the second primer set of the first aspect" can be as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touch-down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, continued amplification for 26 cycles. If the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20 s, 55℃ annealing and extension for 1 min, 5 cycles) until satisfactory results are obtained.

[0022] Beneficial effects of this invention: With the development of genomics technology, the chili pepper genome has been published, and whole-genome resequencing of over 500 chili pepper resources has been completed. This rich dataset of variant data provides marker resources for screening population-specific genetic loci in chili peppers. Using resequencing data from germplasm resources of both lantern-shaped and non-lantern-shaped chili peppers, population-specific genetic loci are screened from the whole genome, leading to the development of SNP primer combinations for identifying lantern-shaped and non-lantern-shaped chili peppers. The SNP primer combinations provided in this invention can be used to identify whether the fruit of a chili pepper variety is lantern-shaped. Detection is possible at various developmental stages and in different tissues and organs of the chili pepper, without environmental or seasonal limitations. They offer advantages such as simple operation, accurate amplification, high throughput, and low cost, making them of significant application value for agricultural production and chili pepper breeding. Attached Figure Description

[0023] Figure 1 PCA clustering results for resequencing data of 34 representative chili pepper resources.

[0024] Figure 2 This represents the variation in genome-wide SNP frequencies between bell peppers and other types of peppers.

[0025] Figure 3 To identify some SNP typing results of the tested chili pepper varieties using primer set 1.

[0026] Figure 4 To identify some SNP typing results of the tested chili pepper varieties using primer set 2.

[0027] Figure 5 The results of partial SNP typing of the tested chili pepper variety were obtained using primer set 1.

[0028] Figure 6 The results of partial SNP typing of the tested chili pepper varieties were obtained using primer set 2. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] Example 1: Obtaining SNP primer combinations for identifying whether the fruit of a pepper variety is lantern-shaped. I. Discovery of 8 SNP sites This invention utilizes resequencing data from 34 representative chili pepper resources in a vegetable SNP variation database (http: / / www.vegsnpdb.cn). It screens for genome-specific perfect SNP loci with no other variations within 50 bp flanking each SNP, ultimately obtaining 36 SNP loci. These 34 representative chili pepper resources represent broad genetic diversity in chili peppers, including 24 non-bell peppers and 10 bell peppers. The PCA clustering results of the resequencing data from these 34 representative chili pepper resources are shown below. Figure 1 .

[0032] Specifically, the screening criteria for SNP sites are as follows: First, SNP sites with MAF > 0.3, heterozygosity < 0.1, deletion rate < 0.1, and conserved 50bp sequences on both sides (without other variations) were selected throughout the genome, resulting in 24,957 perfect SNPs for peppers; then, the SNP frequency difference between lantern-shaped peppers and other types of peppers was calculated, revealing a significant peak on chromosome 9 in lantern-shaped peppers. Figure 2 The signals on chromosomes 4 and 8 are close to the threshold and are also important candidate regions. Within the above candidate intervals, two sites with a SNP frequency difference of 1 between the two populations and six sites with a difference close to 1 were screened. The basic information of the eight SNP sites is detailed in Table 1. The location of the SNP sites on the chromosomes was determined based on the alignment of the pepper Zunla-1 reference genome sequence. The version number of the pepper Zunla-1 reference genome sequence is V2.0 (download address: (https: / / solgenomics.net / ftp / genomes / Capsicum_annuum / C.annuum_zunla / annotation / )).

[0033] Table 1. Basic information of 8 SNP sites II. Obtaining SNP primer combinations for identifying whether the fruit of a pepper variety is lantern-shaped Based on the eight SNP loci discovered in step one, the inventors of this invention designed and synthesized eight SNP primer sets and performed SNP genotyping on the aforementioned 34 tested chili pepper varieties. According to the SNP genotype results, two SNP loci with a high proportion of homozygous genotypes and 100% linkage to the lantern pepper variety were obtained, namely DengLJ3 and DengLJ4.

[0034] SNP primer sets consist of primer set 1 and / or primer set 2. Primer set 1 is used to amplify DengLJ3. Primer set 2 is used to amplify DengLJ4. Each primer set consists of 3 primer sequences used to amplify one SNP site. The nucleotide sequences of each primer in primer set 1 and primer set 2 are shown in column 4 of Table 2.

[0035] Table 2. SNP primer combinations used to identify whether the tested chili pepper variety is lantern-shaped. Note: A single underscore indicates a FAM fluorescent tag sequence, and a double underscore indicates a HEX fluorescent tag sequence.

[0036] Example 2: Validation of the SNP primer combinations developed in Example 1 Basic information on the 129 tested chili pepper varieties in this embodiment is shown in columns 1 to 4 of Table 3. All 129 tested chili pepper varieties are common market varieties. Based on phenotype, 47 tested chili pepper varieties are bell peppers, and 82 tested chili pepper varieties are non-bell peppers.

[0037] Table 3.129 Basic Information of the Tested Chili Varieties 1. Obtaining genomic DNA from the tested chili pepper varieties Genomic DNA was extracted from leaves of 129 tested chili pepper varieties using the SDS method. The quality and concentration of the genomic DNA from the tested chili pepper varieties had to meet PCR requirements, with the following criteria: agarose gel electrophoresis showed a single DNA band without significant diffusion; UV spectrophotometer Nanodrop 2000 (Thermo) showed an A260 / A280 ratio of approximately 1.8 and an A260 / A230 ratio greater than 1.8; and the concentration of the genomic DNA from the tested chili pepper varieties was 10-30 ng / μL.

[0038] 2. Using genomic DNA from 129 tested chili pepper varieties as templates, PCR amplification was performed using primer sets 1 and 2 from Table 2 of Example 1, respectively, to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2", and primers containing "R" was 2:2:5.

[0039] The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touch down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, and continued amplification for 26 cycles.

[0040] 3. After completing step 2, when the temperature of each PCR amplification product drops below 40℃, the fluorescence value is read by scanning with the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is read at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is read at an excitation wavelength of 528nm and an emission wavelength of 560nm). The genotype of each SNP locus of the 129 tested pepper varieties is determined based on the fluorescence signal color. The specific judgment principles are as follows: If a tested chili pepper variety shows a blue fluorescent signal based on a certain SNP site, then the genotype of the tested chili pepper variety based on that SNP site is homozygous, meaning it is the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F1"; if a tested chili pepper variety shows a red fluorescent signal based on a certain SNP site, then the genotype of the tested chili pepper variety based on that SNP site is homozygous, meaning it is the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F2"; if a tested chili pepper variety shows a green fluorescent signal based on a certain SNP site, then the genotype of the tested chili pepper variety based on that SNP site is heterozygous, with one base being the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F1", and the other base being the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F2".

[0041] It should be noted that if the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20s, 55℃ annealing and extension for 1min, 5 cycles) until the results are satisfactory.

[0042] Partial SNP genotyping results of primer set 1 are shown below Figure 3 The statistical results are shown in column 5 of Table 3.

[0043] Partial SNP genotyping results of primer set 2 are shown below Figure 4 The statistical results are shown in column 6 of Table 3.

[0044] The results showed that primer set 1 and primer set 2 could both achieve good typing results in 129 tested pepper varieties.

[0045] 4. Efficiency evaluation of using primer set 1 or primer set 2 to identify 129 tested chili pepper varieties as bell peppers. (1) The genotypes of 129 tested chili pepper varieties based on the DengLJ3 locus were statistically analyzed.

[0046] The results showed that 47 tested chili pepper varieties with the genotype AA based on the DengLJ3 locus were identified as lantern-shaped chili peppers, and all 47 chili peppers exhibited lantern-shaped fruits with a phenotype of 100%. 81 tested chili pepper varieties with the genotype CC based on the DengLJ3 locus and 1 tested chili pepper variety with the genotype CA heterozygous were identified as non-lantern-shaped chili peppers, and all 82 tested chili peppers exhibited non-lantern-shaped fruits with a phenotype of 100%.

[0047] (2) The genotypes of 129 tested chili pepper varieties based on the DengLJ4 locus were statistically analyzed.

[0048] The results showed that 47 tested chili pepper varieties with the TT homozygous genotype based on the DengLJ4 locus were identified as lantern-shaped chili peppers, and all 47 chili peppers exhibited lantern-shaped fruits with a 100% uniformity. 81 tested chili pepper varieties with the CC homozygous genotype based on the DengLJ4 locus and 1 tested chili pepper variety with the CT heterozygous genotype were identified as non-lantern-shaped chili peppers, and all 82 of these chili peppers exhibited non-lantern-shaped fruits with a 100% uniformity.

[0049] Therefore, the SNP primer combination developed in Example 1 can be used to identify whether the fruit of the pepper variety to be tested is lantern-shaped.

[0050] Example 3: Using the SNP primer combination developed in Example 1 to identify whether the fruit of the tested chili pepper variety is lantern-shaped. The chili pepper varieties to be tested were chili pepper variety 1 to chili pepper variety 12, totaling 12.

[0051] 1. The SNP primer combination developed in Example 1 was used to identify the lantern-shaped fruit of each tested chili pepper variety. The specific steps are as follows: (1) Plant the seeds of the pepper variety to be tested to obtain pepper seedlings; take the leaves or roots of the pepper seedlings to be tested and extract genomic DNA using the SDS method to obtain the genomic DNA of the pepper variety to be tested.

[0052] (2) Using the genomic DNA of the pepper variety to be tested as a template, PCR amplification was performed using primer set 1 and primer set 2 in Table 2 of Example 1, respectively, to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2", and primers containing "R" in their names was 2:2:5.

[0053] The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touch down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, and continued amplification for 26 cycles.

[0054] (3) After completing step (2), when the temperature of each PCR amplification product drops below 40℃, the fluorescence value is read by scanning with the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is read at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is read at an excitation wavelength of 528nm and an emission wavelength of 560nm). The fluorescence signal color is obtained, and the following judgment is made: If the pepper variety to be tested shows a blue fluorescence signal based on a certain SNP site, then the genotype of the pepper variety to be tested based on that SNP site is "the 3' end of the first base of the primer that amplifies the SNP site and whose name contains "F1" is the first base of the primer". The genotype of the tested chili pepper variety is homozygous if it shows a red fluorescent signal based on a certain SNP site. If the tested chili pepper variety shows a red fluorescent signal based on a certain SNP site, then the genotype of the tested chili pepper variety based on that SNP site is homozygous, with one base being the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F2". If the tested chili pepper variety shows a green fluorescent signal based on a certain SNP site, then the genotype of the tested chili pepper variety based on that SNP site is heterozygous, with one base being the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F1" and the other base being the first base at the 3' end of the primer that amplifies the SNP site and whose name contains "F2".

[0055] It should be noted that if the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20s, 55℃ annealing and extension for 1min, 5 cycles) until the results are satisfactory.

[0056] Partial SNP genotyping results of primer set 1 are shown below Figure 5 And column 2 in Table 4.

[0057] Partial SNP genotyping results of primer set 2 are shown below Figure 6 And column 3 in Table 4.

[0058] (4) After completing step (3), make the following judgments: if the genotype based on the DengLJ3 locus is AA and / or the genotype based on the DengLJ4 locus is TT, then the fruit of the pepper variety to be tested is identified as or suspected to be lantern-shaped; if the genotype based on the DengLJ3 locus is CC or CA and / or the genotype based on the DengLJ4 locus is CC or CT, then the fruit of the pepper variety to be tested is identified as or suspected to be non-lantern-shaped. Table 4. Identification results of 12 tested chili pepper varieties 2. Twelve different chili pepper varieties were planted. Based on their phenotypes, the twelve varieties were determined to be either bell peppers or non-bell peppers.

[0059] The phenotypic statistics are shown in column 4 of Table 4.

[0060] The results showed that the identification results of the SNP primer combination developed in Example 1 were completely consistent with the phenotypic identification results.

[0061] Therefore, it can be seen that the SNP primer combination developed in Example 1 can completely identify whether the pepper fruit is lantern-shaped or not.

[0062] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. SNP primer sets, including a first primer set for amplifying the DengLJ3 SNP site in the pepper genome and / or a second primer set for amplifying the DengLJ4 SNP site in the pepper genome; The DengLJ3 SNP site is nucleotide 193057991 on chromosome 9, and it is either A or C. The DengLJ4 SNP site is located at nucleotide 193059682 on chromosome 9, and it is either T or C. The positions of the DengLJ3 and DengLJ4 SNP sites on the chromosome were determined based on the alignment of the Zunla-1 reference genome sequence of the chili pepper, version number V2.

0.

2. The SNP primer combination according to claim 1, characterized in that: The first primer set includes a first forward primer, a second forward primer, and a first reverse primer, wherein the first forward primer contains a specific nucleotide sequence as shown in SEQ ID NO: 1 from position 22 to 43 from the 5' end, the second forward primer contains a specific nucleotide sequence as shown in SEQ ID NO: 2 from position 22 to 43 from the 5' end, and the nucleotide sequence of the first reverse primer is shown in SEQ ID NO: 3; The second primer set includes a third forward primer, a fourth forward primer, and a second reverse primer, wherein the third forward primer contains a specific nucleotide sequence as shown in SEQ ID NO: 4 from position 22 to 55 from the 5' end, the fourth forward primer contains a specific nucleotide sequence as shown in SEQ ID NO: 5 from position 22 to 55 from the 5' end, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO:

6.

3. The SNP primer combination according to claim 2, characterized in that: The 5' ends of the specific nucleotide sequences of the first and second forward primers are respectively linked to fluorescent tag sequences that display different fluorescent signal colors; the 5' ends of the specific nucleotide sequences of the third and fourth forward primers are respectively linked to fluorescent tag sequences that display different fluorescent signal colors.

4. The SNP primer combination according to claim 3, characterized in that: The nucleotide sequence of the first forward primer is shown in SEQ ID NO: 1, the nucleotide sequence of the second forward primer is shown in SEQ ID NO: 2, the nucleotide sequence of the third forward primer is shown in SEQ ID NO: 4, and the nucleotide sequence of the fourth forward primer is shown in SEQ ID NO:

5.

5. A reagent kit, characterized in that: The kit comprises the SNP primer combination as described in any one of claims 1-4.

6. The use of the SNP primer combination according to any one of claims 1 to 4 or the kit according to claim 5 in (A) or (B) below: (A) Application in identifying whether the fruit of a pepper variety to be tested is lantern-shaped or not; (B) Application in the breeding of chili pepper varieties with lantern-shaped fruits.

7. A method for identifying whether the fruit of a chili pepper variety to be tested is lantern-shaped or not, characterized in that: The method includes: detecting the genotype of the pepper variety to be tested based on the DengLJ3 SNP site and / or the DengLJ4 SNP site, and then making the following judgments: if the genotype based on the DengLJ3 SNP site is AA and / or the genotype based on the DengLJ4 SNP site is TT, then the fruit of the pepper variety to be tested is identified as or suspected to be lantern-shaped; if the genotype based on the DengLJ3 SNP site is CC or CA, and / or the genotype based on the DengLJ4 SNP site is CC or CT, then the fruit of the pepper variety to be tested is identified as or suspected to be non-lantern-shaped. The DengLJ3 SNP site is nucleotide 193057991 on chromosome 9, and it is either A or C. The DengLJ4 SNP site is nucleotide 193059682 on chromosome 9, which is either T or C. The positions of the DengLJ3 and DengLJ4 SNP sites on the chromosome were determined based on the alignment of the Zunla-1 reference genome sequence of the chili pepper, version number V2.

0.

8. The method according to claim 7, characterized in that: The steps for detecting the genotype of the chili pepper variety under test based on the DengLJ3 SNP locus and / or the DengLJ4 SNP locus are as follows: (1) Using the genomic DNA of the pepper variety to be tested as a template, PCR amplification was performed using the first primer set and / or the second primer set in claim 3 or 4 respectively to obtain PCR amplification products; (2) The fluorescence signal of the PCR amplification product obtained in step (1) is detected by the instrument, and the genotype of the pepper variety to be tested is determined based on the color of the fluorescence signal based on the DengLJ3 SNP site and / or the DengLJ4 SNP site.

9. The method according to claim 7, characterized in that: The steps for detecting the genotype of the chili pepper variety under test based on the DengLJ3 SNP locus and / or the DengLJ4 SNP locus are as follows: (1) Using the genomic DNA of the pepper variety to be tested as a template, PCR amplification was performed using the first primer set and / or the second primer set in claim 2 to obtain PCR amplification products; (2) Take the PCR amplification product obtained in step (1) and sequence it; (3) Based on the sequencing results obtained in step (2), obtain the genotype of the pepper variety to be tested based on the DengLJ3 SNP site and / or the DengLJ4 SNP site.

10. The method according to claim 8, characterized in that: The method for determining the genotype of the tested chili pepper variety based on the color of the fluorescence signal at the DengLJ3 SNP site and / or the DengLJ4 SNP site is as follows: If the fluorescence color of the tested chili pepper variety at the DengLJ3 SNP site is the same as the color of the fluorescent tag sequence connected to the 5' end of the first forward primer, then the genotype of the tested chili pepper variety at the DengLJ3 SNP site is AA; if the fluorescence color of the tested chili pepper variety at the DengLJ4 SNP site is the same as the color of the fluorescent tag sequence connected to the 5' end of the third forward primer, then the genotype of the tested chili pepper variety at the DengLJ4 SNP site is TT; if the fluorescence color of the tested chili pepper variety at the DengLJ3 SNP site is the same as the color of the fluorescent tag sequence connected to the 5' end of the second forward primer, then the genotype of the tested chili pepper variety at the DengLJ3 SNP site is CC. If the fluorescence color of the chili pepper variety under test based on the DengLJ4 SNP site is the same as the color of the fluorescent tag sequence connected to the 5' end of the fourth forward primer, then the genotype of the chili pepper variety under test based on the DengLJ4 SNP site is CC. If the fluorescence color of the tested chili variety based on the DengLJ3 SNP site is a mixture of the colors displayed by the fluorescent tag sequences connected to the 5' ends of the first and second forward primers, then the genotype of the tested chili variety based on the DengLJ3 SNP site is AC; if the fluorescence color of the tested chili variety based on the DengLJ4 SNP site is a mixture of the colors displayed by the fluorescent tag sequences connected to the 5' ends of the third and fourth forward primers, then the genotype of the tested chili variety based on the DengLJ4 SNP site is TC.

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