An Indel marker co-isolated from a trait associated with an antiviral disease in chili peppers and its application.

Abstract

This invention relates to the fields of molecular biology and plant molecular marker breeding technology, and discloses an Indel marker co-segregating with the disease resistance trait in pepper and its application. The Indel marker is located at 29,449,971-29,449,991 bp on chromosome 5 of the pepper cv.CM334 genome; compared with the disease-susceptible pepper, the disease-resistant pepper exhibits a deletion of Indel 73. The marker provided by this invention is tightly linked to the disease resistance gene in pepper, exhibits stable localization, and is suitable for molecular breeding; it allows for the detection of the disease resistance genotype in pepper under laboratory conditions, without environmental limitations. Using this marker, rapid disease resistance testing of individual pepper plants can be performed at any growth stage without affecting their normal growth; the molecular marker of this invention can lay the foundation for cloning the disease resistance gene in pepper and studying the molecular mechanism of disease resistance in pepper, and has application value in the breeding of new varieties.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and plant molecular marker breeding technology, and more specifically, to an Indel marker co-segregated with the disease-resistant trait of pepper and its application. Background Technology

[0002] Chili peppers, belonging to the genus *Capsicum* in the family Solanaceae, originated in Central and South America. Today, chili peppers are widely cultivated globally, used both as a fresh food and in processed foods. They hold an important position in the vegetable industry, boasting abundant germplasm resources, relatively simple cultivation techniques, and significant economic benefits.

[0003] During the growth and development of chili peppers, they are subject to various biotic and abiotic stresses. Among these, blight, also known as "plant death disease," is a serious disease caused by the fungus *Phytophthora capsici*. Once this disease occurs, it can lead to reduced yields or even complete crop failure, causing extremely severe economic losses to chili pepper production. Currently, *Phytophthora capsici* is widely distributed and has become a significant factor affecting chili pepper production. Therefore, the prevention and control of this disease is crucial.

[0004] Phytophthora blight is widespread in peppers, with diverse physiological races, and the pathogen is prone to mutation. Vertical and horizontal resistance coexist in pepper resistance to Phytophthora blight, exhibiting a phased strengthening trend; that is, resistance gradually increases with the progression of the growth stage. Resistance to Phytophthora blight in peppers displays genetic diversity, with different resistant varieties and even different strains exhibiting varying modes of resistance inheritance, primarily encompassing three types: single-gene inheritance, oligogenic inheritance, and polygenic inheritance.

[0005] However, due to the lack of widely applicable chili pepper resources for disease resistance, and the complexity of the genetic patterns of disease resistance in chili peppers and the unclear mechanisms of resistance, current breeding work is progressing relatively slowly. Therefore, marking, locating, and cloning genes related to disease resistance traits has become a core aspect of marker-assisted breeding of disease-resistant varieties. Molecular marker-assisted breeding is a novel approach that combines molecular genetics with traditional phenotypic selection. By directly utilizing molecular markers closely linked to or co-segregating with genes of the target trait, it screens individuals for target regions or even the entire genome, aiming to improve the selection efficiency of the target trait and shorten the breeding cycle.

[0006] Domestic and international scholars have conducted in-depth research on molecular markers of blight resistance genes in peppers and successfully identified several quantitative trait loci (QTLs) associated with blight resistance. These research findings provide strong support for marker-assisted selection in pepper resistance breeding. Current research shows that chromosome 5 of pepper is the main location region for blight resistance genes, and several QTLs closely related to blight resistance have been discovered within this region, along with corresponding molecular markers. However, these publicly available molecular markers have certain limitations in practical applications, particularly in matching phenotypes and genotypes among multiple germplasms, which to some extent restricts the application scope of marker-assisted breeding.

[0007] For the prevention and control of Phytophthora blight in chili peppers, breeding disease-resistant varieties is a key measure to improve yield and economic benefits. However, the gene for disease resistance in chili peppers has not yet been cloned, which to some extent restricts the development of molecular breeding. Therefore, the development of molecular markers closely linked to disease resistance in chili peppers is of great significance for promoting molecular marker-assisted selection breeding of disease-resistant varieties. Summary of the Invention

[0008] The key to marker-assisted selection (MAG) breeding technology lies in identifying DNA molecular markers closely related to important agronomic traits. Among these, Indel polymorphism markers are PCR amplification markers based on specific primers designed for insertion / deletion sites. Essentially, they achieve rapid genotyping by comparing nucleotide insertions or deletions at specific sites in the genomes of different parents. These markers offer advantages such as ease of operation and intuitive results, providing new ideas and methods for marker-assisted selection breeding of disease-resistant pepper varieties. Therefore, this invention proposes an Indel marker co-segregating with disease-resistant traits in peppers and its application, aiming to solve problems in current technologies.

[0009] This invention proposes an Indel marker that co-segregates with the disease resistance trait of pepper. The Indel marker is located at 29,449,971-29,449,991 bp on chromosome 5 of the pepper cv.CM334 genome; the Indel is named Indel 73.

[0010] Compared to chili peppers infected with Phytophthora blight, chili peppers infected with Phytophthora blight exhibit a deficiency of the aforementioned Indel 73.

[0011] This invention also proposes an amplification primer for the Indel marker co-separated from the disease-resistant trait of pepper, wherein the amplification primer comprises:

[0012] Indel 73-F: 5'-GTGGTGGCAGTGGTACTGTG-3';

[0013] Indel 73-R: 5'-TTTGATGGAATGACACCCCT-3'.

[0014] The present invention also proposes a product for identifying disease-resistant traits in chili peppers, the product comprising at least one of the following:

[0015] A) The Indel tag described above;

[0016] The amplification primers described in B).

[0017] Preferably, the product includes:

[0018] Detection reagents, kits, genome chips, or liquid probes.

[0019] The present invention also proposes an application of the Indel marker or the primers used for amplification, the application comprising at least one of the following:

[0020] A) Application in identifying disease-resistant traits in chili peppers;

[0021] B) Application in chili pepper genetic breeding;

[0022] C) Application in the analysis of genetic diversity in chili peppers;

[0023] D) Application in constructing a genetic linkage map of chili peppers.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) The Indel molecular marker co-segregated with the anti-disease trait of pepper and its application in this invention are developed by discovering markers closely linked to the anti-disease gene of pepper. The genetic effect of the anti-disease gene of pepper located by the Indel molecular marker of this invention is obvious and stable, which can meet the needs of molecular breeding.

[0026] (2) This invention allows for laboratory testing of pepper disease resistance genotypes, unaffected by environmental conditions. It enables the testing of single-plant disease resistance at any growth stage in the segregating generations of pepper disease-resistant breeding strains without affecting normal plant growth. Furthermore, this invention provides rapid and cost-effective pepper disease resistance testing.

[0027] (3) Since molecular markers have the advantages of being simple, fast and high-throughput in assisted breeding systems, the molecular markers of this invention can lay the foundation for the final cloning of the pepper anti-disease gene CaphytoX, and thus lay the foundation for the study of the molecular mechanism of pepper anti-disease, and have good application value in the breeding of new varieties. Attached Figure Description

[0028] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0029] Figure 1 This is a phenotypic diagram of the two parents of the present invention on day 6 after inoculation with Phytophthora capsici race 3;

[0030] Figure 2 Root morphology diagrams of the immune material 'ZCM334' (left) and the susceptible material 'Early Calwonder' (right) at different time points after inoculation with Phytophthora capsici in this invention;

[0031] Figure 3 This is the preliminary localization map of the gene for identifying the anti-disease gene in chili peppers using the BSA-Seq method in this invention. In the map, the horizontal axis represents the name and length of each chromosome, the vertical axis represents the SNP-index value, and the point is the SNP-index value corresponding to each SNP. a is the mean SNP-index value within a certain window, b is the 99% confidence line, and c is the 95% confidence line.

[0032] Figure 4 This is a genetic distance map of the fine-mapping region of the CaphytoX gene, a chili pepper disease-resistant gene, according to the present invention.

[0033] Figure 5 This is a gel electrophoresis image of Indel 73, the marker co-segregated with the antiviral properties of pepper in this invention, in the parental line, F2 generation, and BC1F1 generation populations. M represents the DL2000 Marker, showing 195bp and 215bp bands. P1 represents the antiviral immunization material 'ZCM334', and P2 represents the susceptible material 'Early Calwonder'. Individual plants with the same band as the immunization parent 'ZCM334' have genotype AA and exhibit an antiviral phenotype. Individual plants with the same band as the susceptible parent 'Early Calwonder' have genotype aa and exhibit a susceptible phenotype. Individual plants with two bands have genotype Aa and exhibit a resistant phenotype. Detailed Implementation

[0034] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the disclosure to those skilled in the art. It should be noted that, unless otherwise specified, embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0035] Example 1: Establishment of a technical system for inoculation and identification of Phytophthora capsici.

[0036] ZY14 and W18 strains of Phytophthora capsici, physiological race 3, were selected as candidate strains for this study. To optimize culture conditions, two culture medium treatments, V8 and PDA, were designed, and 2cm sections were cut into the culture medium during the sporulation stage. 3 Treatments included both small-piece and uncut samples. Additionally, low-temperature treatments at 10℃ and room-temperature treatments at 28℃ were implemented to investigate the effects of different temperatures on spore release. Specific sporulation capacity data are shown in Table 1.

[0037] After comprehensive comparison and analysis, strain ZY14 of Phytophthora capsici, physiological race 3, was ultimately selected for the inoculation experiment on pepper plants. To ensure the experimental results, the following culture conditions were determined: First, strain ZY14 was rejuvenated and propagated, then transferred to V8 medium and cultured at 28°C for 7 days. After the mycelium had fully colonized the culture dish, the medium was cut into 2cm pieces. 3 Small pieces of culture medium were prepared and soaked in sterile water for 3 days. After sporulation, the culture medium was placed in a 10°C incubator for 1 hour to promote spore release. Subsequently, it was incubated at 24°C for 30 minutes, and the spore concentration was calculated using a hemocytometer. Finally, the *Phytophthora capsici* spore concentration was diluted to 2000 spores / mL as the inoculation concentration (inoculation concentration reference: Sy et al., 2008). This method ensures the production of a large number of zoospores, meeting the requirements for *Phytophthora capsici* inoculation experiments.

[0038] To assess the disease resistance of pepper plants, a root drenching inoculation method was used. First, pepper plants with 4-6 true leaves were removed from their seed trays and transferred to an artificial climate chamber. Next, 5 mL of zoospore suspension was injected into the soil around the roots of each plant using a syringe. After inoculation, the ambient temperature was maintained at 25-28℃, and the relative humidity of the soil was ensured to be greater than 90%. To accurately assess disease resistance, all infected control plants were used as the standard. Symptoms included blackening of the rootstock, approximately 1-2 cm in length, or irreversible wilting of the leaves. This experimental method can accurately evaluate the disease resistance of pepper plants.

[0039] Table 1. Effects of different treatments on sporulation capacity of Phytophthora capsici (sporulations / mL)

[0040]

[0041]

[0042] Example 2: Phenotypic identification of the immune parent 'ZCM334' and the susceptible parent 'Early Calwonder'

[0043] Following preliminary inoculation and identification, using the anti-disease material 'CM334' provided by the Asia Vegetable Research and Development Center (ACN-World Vegetable Research and Development Center), a strain 'ZCM334' resistant to Phytophthora capsici physiological race 3 was successfully bred through multiple generations of self-pollination. Based on this, in-depth hybridization studies were conducted using 'ZCM334' as the immune parent and 'Early Calwonder' as the susceptible parent. During the experiment, the experimental materials were sown in a greenhouse and inoculated with Phytophthora capsici physiological race 3 at a concentration of 2000 zoospores / ml at the four-leaf stage, with each plant receiving 5ml of inoculation. Through hybridization, the F1 generation was obtained, and self-pollination of the F1 generation produced the F2 segregating population. Simultaneously, backcrossing was performed between the F1 generation and the susceptible parent 'Early Calwonder', resulting in the BC1F1 population. To further screen for disease-resistant plants, the obtained disease-resistant plants were backcrossed with the susceptible parent 'Early Calwonder' for multiple generations, ultimately obtaining the BC4F1 population.

[0044] Phenotypic observations of two chili varieties, 'ZCM334' and 'Early Calwonder', after inoculation with *Phytophthora capsici* showed that, from 0 to 96 hours post-inoculation, the roots of 'ZCM334' showed no signs of infection, indicating complete immunity to physiological race 3 of *Phytophthora capsici*. In contrast, the rhizomes of 'Early Calwonder' gradually turned black after inoculation, and this blackening spread upwards with increasing infection time. Six days post-inoculation, 'ZCM334' plants remained normal, while all 'Early Calwonder' plants showed signs of infection, with blackened rhizomes and wilting. These findings provide important evidence for further understanding the differences in resistance to *Phytophthora capsici* between these two chili varieties.

[0045] Example 3: Genetic analysis of the antiviral properties of chili peppers:

[0046] To further investigate the genetic characteristics of pepper resistance to Phytophthora blight, a systematic disease resistance assessment was conducted on multiple generations of pepper populations, including the 'ZCM334', 'Early Calwonder' F1 hybrid, and the backcross BC1 F1 and F2 generations. 'Early Calwonder' was used as a susceptible control, with its complete disease development serving as the standard. Based on this standard, plants without symptoms in the rootstock were classified as resistant, while those showing obvious symptoms such as blackening of the rootstock were considered susceptible. It is noteworthy that, since the appearance of symptoms ultimately leads to death, this study did not differentiate between the severity of disease in infected plants.

[0047] Systematic observation of plant populations across generations after inoculation revealed that all susceptible controls developed symptoms on day 5 post-inoculation. Therefore, this day was selected as the critical time point for assessing plant resistance. At this time, detailed statistical analysis of disease incidence across generations was conducted. Results showed that 'ZCM334' exhibited complete immunity to physiological race 3 of *Phytophthora capsici*, while all 'Early Calwonder' plants developed symptoms and died. Furthermore, all F1 generation plants demonstrated resistance, further confirming that the resistance of 'ZCM334' to *Phytophthora capsici* is controlled by a dominant gene.

[0048] In the BC1 population, 1036 resistant plants and 998 susceptible plants were identified. In the F2 population, 3164 resistant plants and 1086 susceptible plants were identified. To verify whether these results met the expected genetic segregation ratio, a chi-square test was performed. The results showed that the segregation ratio of the BC1F1 population met the expected 1:1 (χ² = 0.67 < χ²<0.05 = 3.84), while the segregation ratio of the F2 population also met the expected 3:1 (χ² = 0.66 < χ²<0.05 = 3.84). These results further confirm the hypothesis that the resistance of 'ZCM334' to pepper blight is controlled by a pair of dominant genes. Specific data are shown in Table 2 (Table 2 shows the number of resistant and susceptible plants investigated on day 5 after inoculation, when all susceptible controls developed symptoms).

[0049] Table 2. Genetic analysis of pepper parents resistant to and susceptible to blight and across generations.

[0050]

[0051] Example 4: Preliminary localization of the anti-disease gene in chili peppers

[0052] This invention uses 'ZCM334' and 'Early Calwonder' as parents to construct a BC4F1 backcross population, aiming to preliminarily locate the disease-resistant gene in peppers. To ensure successful inoculation of all plants and accurately screen resistant and susceptible plants for constructing extreme trait pools, a two-stage inoculation strategy was adopted. After the first inoculation, when all susceptible controls developed symptoms, susceptible individuals from the BC4F1 backcross population were sampled, and 50 individual plants were selected to construct an extreme susceptible pool. Subsequently, the remaining uninfected plants were inoculated a second time, and resistant and susceptible controls were set up. Ten days after the second inoculation, individuals still showing resistance were sampled, and 50 individual plants were selected to construct an extreme resistant pool. DNA was extracted from each individual plant in both pools using the CTAB method, and the DNA concentration was measured using Nanodrop2000 to ensure equal mixing. Subsequently, DNA samples from the immune parent 'ZCM334', the susceptible parent 'Early Calwonder', the resistant pool, and the susceptible pool were sent to Shanghai Paisenno Biotechnology Co., Ltd. for BSA-Seq sequencing.

[0053] Sequencing libraries were meticulously prepared following the standard library construction protocol using the TruSeq DNA PCR-free prep kit provided by Illumina. These libraries contained precise 400 bp inserts. Paired-end (PE) sequencing of these libraries was performed on the Illumina NovaSeq sequencing platform using cutting-edge next-generation sequencing (NGS) technology. Next, the large volume of raw data generated by high-throughput sequencing underwent preliminary quality assessment and screening to ensure high-quality data. Subsequently, this high-quality data was accurately aligned to a reference genome, and the alignment results were statistically analyzed in detail, providing a solid data foundation for subsequent analyses.

[0054] To visually represent the distribution of SNP-index on chromosomes in offspring, a graph was plotted showing the distribution of SNP-index on chromosomes. Figure 3 The red line in the figure shows the SNP-index distribution represented by a window. SNP-index within each window is analyzed using a sliding window approach. The difference in SNP-index between the two progeny pools is calculated as Δ(SNP-index) = SNP-index(extreme trait A) - SNP-index(extreme trait B). After 10,000 permutation tests, 95% ( Figure 3 c (green), 99% ( Figure 3 b) Orange confidence level as the screening threshold. Figure 3The red line represents the distribution of △(SNP-index) in the window format, where windows with a confidence level higher than 99% are identified as candidate regions. SNP-index association analysis determined that the candidate region of the target gene is located within 26.8 Mb of chromosome 5 of pepper (Chr05:17 200 001-44000 000).

[0055] Example 5: Development and Application of Molecular Markers

[0056] Based on the whole-genome resequencing information of the resistant parent and data provided by the reference genome (https: / / solgenomics.net / organism / Capsicum_annuum / genome), primer sequences were carefully designed using Primer5 software. Subsequently, DNA was extracted from the resistant parent and F2, BC1F1, and BC4F1 generation single plants using the CTAB method. To amplify specific DNA fragments, PCR amplification was performed using 2×Accurate Taq Master Mix. After amplification, the PCR products were detected by non-denaturing polyacrylamide gel electrophoresis to ensure the accuracy of the results.

[0057] Using the BC4F1 generation segregating population as the experimental subject, the whole-genome resequencing information of the resistant parents was fully utilized. Within the mapping region, carefully designed Indel primers were employed, and chromosome walking was successfully performed using recombinant individuals as the mapping population by detecting the genotypes of the parents and individual plants in the BC4F1 population. After rigorous screening, Indel markers closely linked to the pepper's disease-resistant gene CaPhytoX were obtained. These markers are of significant value for subsequent genetic linkage map construction.

[0058] Specifically, within the initial mapping interval, 132 pairs of Indel markers were successfully developed (Table 3). Among them, 22 pairs showed polymorphism between resistant and susceptible parents. Using the BC4 mapping population, recombinant single plants were screened, and the pepper disease resistance gene CaPhytoX was precisely mapped to a 1.24Mb interval on chromosome 5. To verify this result, single plants were randomly selected from the F2 and BC1F1 generations for further analysis. The results showed that the Indel 73 molecular marker co-segregated with the pepper disease resistance trait. This discovery provides new molecular markers and mapping information for pepper disease resistance breeding.

[0059] Precise analysis determined the exact location of Indel 73 on chromosome 5 of pepper to be between 29,449,971 and 29,449,991 bp. This 20 bp deletion was present in resistant materials compared to susceptible materials. To further investigate the impact of this genetic variation on resistance, we designed specific primers for the upstream and downstream of Indel 73 and performed PCR amplification on the DNA of segregating populations from resistant parents, F2 to F5 generations, and BC1 to BC6 generations. Subsequently, we performed polyacrylamide gel electrophoresis to more accurately analyze the genetic segregation of Indel 73 across different generations. The results were determined based on the electrophoretic bands, as shown below. Figure 5 As shown.

[0060] Table 3

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] The PCR reaction system contained the following components: 5 μL of 2×Accurate Taq Master Mix, 0.2 μL of forward primer Indel73-F, 0.2 μL of reverse primer Indel 73-R, 0.25 μL of template DNA, and 4.35 μL of double-distilled water, for a total volume of 10 μL. The PCR reaction program was set as follows: first, pre-denaturation at 95℃ for 3 min; then 35 cycles, each cycle consisting of denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s; and finally, extension at 72℃ for 5 min, followed by storage at 4℃.

[0068] For the detection of PCR products, polyacrylamide gel electrophoresis was used. During electrophoresis, 10×TBE was used as the buffer, and electrophoresis was performed at a constant voltage of 200V for 1-1.5 hours. After electrophoresis, silver staining was performed for observation and detection. The silver staining steps included: placing the glass plate with the gel in a plastic container containing the staining solution, and gently shaking the gel on a shaker for 8-10 minutes. The staining solution consisted of 2g silver nitrate and 1L purified water. Then, the silver ions were washed away with purified water, and the gel was gently shaken on a shaker in the dark for 1-2 minutes. Next, the gel was placed in a plastic container containing the developing solution, and gently shaken on a shaker for 8-10 minutes until the bands were clearly visible. The developing solution consisted of 12g sodium hydroxide, 1L purified water, and 1ml formaldehyde. Finally, the gel was rinsed in clean water and observed and photographed under light.

[0069] The Indel 73 primers were used to test pepper samples (including resistant and susceptible parents, as well as F2, BC1F1, and BC4F1 generation individual plants). The disease resistance and genotype of the plants could be determined based on the banding of the PCR amplification products. If the PCR amplification product was a 195 bp DNA band, the tested pepper plant was homozygous resistant; if the PCR amplification product was a 215 bp DNA band, the tested pepper plant was homozygous susceptible; and if the PCR amplification product consisted of two DNA bands, 195 bp and 215 bp in length, the tested pepper plant was heterozygous resistant. This result demonstrates that the molecular marker Indel 73 of this invention can effectively distinguish the disease resistance of pepper plants. The discovery of this molecular marker laid an important foundation for the eventual cloning of the pepper disease resistance gene CaphytoX and the establishment of a molecular marker-assisted breeding system.

[0070] In summary, it can be understood that 'ZCM334' is a disease-resistant material completely immune to physiological races 1, 2, and 3 of Phytophthora capsici, while 'Early Calwonder' is the host for susceptibility identification. This invention, through genetic analysis, discovered that the resistance of 'ZCM334' to Phytophthora capsici is controlled by a pair of dominant genes. Previously, the Phytophthora capsici immunity gene CaPhytoX was preliminarily located in the 1.24Mb region of chromosome 5 of pepper, a region that differs from previously reported resistance regions. This Indel marker was developed based on the whole-genome resequencing results of the immune parent 'ZCM334' and the susceptible parent 'Early Calwonder', combined with BSA-seq mapping results. Through repeated validation in parents, self-cross populations, and backcross populations, the Indel 73 marker of this invention can accurately identify the disease resistance and genotype of pepper plants.

[0071] Through meticulous design and optimization, the Indel 73-labeled primers developed in this invention exhibit superior performance. The primers are designed based on sequence information flanking the insertion / deletion site, ensuring precise binding to the target DNA fragment and avoiding non-specific binding, thereby improving detection accuracy and specificity. The PCR bands of the Indel 73-labeled primers are single and bright, ensuring high stability. Furthermore, the PCR product fragments are small, at 195 bp and 215 bp respectively, significantly shortening identification time. Notably, there is a 20 bp difference between the resistant and susceptible material fragments; this significant distinction simplifies the identification process. In summary, the primers of this invention are highly efficient, stable, and easily distinguishable, providing strong support for research and applications in related fields.

[0072] Through in-depth research, this invention has discovered that the Indel 73 marker exhibits excellent genetic stability during breeding. The variation in the Indel 73 marker is minimal in offspring, a characteristic that is significant for maintaining the stable inheritance of breeding objectives. Furthermore, the Indel 73 marker-assisted breeding technology disclosed in this invention largely overcomes the limitations of traditional molecular marker technologies, providing a new and powerful tool for breeding work.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. The application of primers for amplifying Indel-labeled substances co-isolated with the antiviral trait of chili peppers, characterized in that, The application includes at least one of the following: A) Application in identifying disease-resistant traits in chili peppers; B) Application in chili pepper genetic breeding; The breeding trait is a disease resistance trait, and the primers used for amplification are Indel 73-F: 5'-GTGGTGGCAGTGGTACTGTG-3' and Indel 73-R: 5'-TTTGATGGAATGACACCCCT-3'; The peppers in question are Early Calwonder or ZCM334, and the disease is caused by Phytophthora race 3. The plant's resistance and genotype are determined based on the banding of the PCR amplification products. If the PCR amplification product is a 195bp DNA band, the tested pepper plant is a homozygous resistant material; if the PCR amplification product is a 215bp DNA band, the tested pepper plant is a homozygous susceptible material; if the PCR amplification product consists of two DNA bands, 195bp and 215bp in length respectively, the tested pepper plant is a heterozygous resistant material.

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