SNP molecular marker linked to anthracnose resistance gene, KASP marker primer set Ct6082 and its application

Abstract

The present invention relates to a SNP molecular marker linked to an anthracnose resistance gene, a KASP marker primer set Ct6082, and applications thereof. The SNP molecular marker is located at position 13169223 of chromosome 6 of the Zunla‑1_v3.0 pepper reference genome, and its base is T or C. The KASP marker primer set Ct6082 is used to detect or determine the genotype of the SNP molecular marker. T:T or T:C is the genotype of anthracnose-resistant pepper material, and C:C is the genotype of anthracnose-susceptible pepper material. After population verification, the accuracy of the SNP molecular marker can reach 91%, which can be used to breed anthracnose-resistant pepper materials, shorten the material breeding cycle, and provide a practical molecular marker for the molecular breeding of anthracnose-resistant peppers.

Description

Technical Field

[0001] The present invention belongs to the field of molecular markers, and in particular relates to a SNP molecular marker linked to an anthrax resistance gene, a KASP marker primer set Ct6082 and applications thereof. Background Art

[0002] Pepper is my country's largest vegetable crop, with an annual pepper cultivation area of 35.8 million mu (approximately 1.6 million hectares), accounting for 9.28% of the nation's total vegetable production. The total output is 63.994 million tons, with an annual output value of 250 billion yuan. As pepper cultivation continues to expand, so too are the losses caused by pepper diseases. Anthracnose, a fungal disease caused by the genus Colletotrichum in the subdivision Deuteromycetes, has become one of the major pests endangering pepper production. It is a common fungal disease worldwide, severely impacting pepper production and can lead to a 30-80% yield reduction, significantly reducing the economic benefits for pepper farmers.

[0003] Pepper anthracnose is primarily controlled through chemical control. However, chemical agents target only a single pathogen, and long-term use can easily lead to drug resistance. Furthermore, overuse of chemical agents can cause soil and water pollution, which is inconsistent with the concept of green and sustainable development. Cultivating and promoting anthracnose-resistant pepper varieties can reduce the use of chemical agents, alleviate environmental pollution, reduce pesticide residues, and promote sustainable agricultural development. It is the most effective, economical, and environmentally friendly method for disease control.

[0004] Currently, there is a severe shortage of anthracnose-resistant pepper varieties. Furthermore, the genetics of anthracnose resistance in peppers are complex, and progress in molecular genetics is slow, limiting the progress of anthracnose-resistant pepper breeding. Existing methods for detecting anthracnose resistance in peppers are complex and time-consuming. To enrich and simplify the identification of anthracnose-resistant pepper varieties and shorten the breeding cycle, this invention was developed. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a KASP marker primer set Ct6082 for determining or detecting the genotype corresponding to a SNP molecular marker linked to an anthracnose resistance gene. This provides a practical molecular marker for molecular breeding of pepper varieties for anthracnose resistance.

[0006] A second object of the present invention is to provide a method for identifying whether a pepper variety to be tested is an anthracnose-resistant pepper variety.

[0007] A third object of the present invention is to provide a test kit.

[0008] A fourth object of the present invention is to provide applications of the above-mentioned KASP labeling primer set, method and kit.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A first aspect of the present invention provides a KASP marker primer set for detecting or determining the genotype corresponding to a SNP molecular marker linked to a pepper anthracnose resistance gene, the KASP marker primer set comprising:

[0011] Forward primer primer_X, the nucleotide sequence of which is shown in SEQ ID NO.1 in the sequence listing;

[0012] Forward primer primer_Y, the nucleotide sequence of which is shown in SEQ ID NO.2 in the sequence listing;

[0013] The nucleotide sequence of the reverse primer primer_C is shown in SEQ ID NO.3 in the sequence listing.

[0014] SEQ ID NO. 1 in the sequence listing is specifically: 5'-CATCAGCAGGATTGGATAATCCCA-3';

[0015] SEQ ID NO. 2 in the sequence listing is specifically: 5'-ATCAGCAGGATTGGATAATCCCG-3';

[0016] SEQ ID NO.3 in the sequence table is specifically: 5'-CATTTTTGTCGCGGTCAAAACCTTG

[0017] G-3';

[0018] The genotypes corresponding to the SNP molecular markers are: T:T or T:C, which are genotypes of pepper materials resistant to anthracnose; and C:C, which are genotypes of pepper materials susceptible to anthracnose.

[0019] Furthermore, to simplify the detection method of PCR products and facilitate the determination of SNP molecular marker genotyping, fluorescence detection can be performed on the PCR products. To achieve the purpose of fluorescence detection, the 5' ends of the forward primers primer_X and primer_Y need to be connected to different fluorescent linker sequences. The fluorescent linker sequence can be selected from one of FAM, HEX, FITC, RED, TET, JOE, and R110. It is sufficient to ensure that the fluorescent linkers connected to the 5' ends of the two forward primers emit different colors.

[0020] A second aspect of the present invention provides a method for identifying whether a pepper variety to be tested is an anthracnose-resistant pepper variety, comprising the following steps:

[0021] Provide genomic DNA of the pepper variety to be tested;

[0022] Using the genomic DNA of the pepper variety to be tested as a template, PCR amplification is performed using the KASP labeled primer set of the first aspect;

[0023] Performing fluorescence detection and analysis on the amplified product obtained by the PCR amplification to obtain the genotype of the pepper variety to be tested, thereby determining whether the pepper variety to be tested is an anthracnose-resistant pepper variety;

[0024] When the genotype is T:T or T:C, the pepper variety to be tested is an anthracnose-resistant pepper material; when the genotype is C:C, the pepper variety to be tested is an anthracnose-susceptible pepper material.

[0025] The anthrax-resistant pepper material described in the present invention refers to a pepper material that has a certain resistance to the infection and reproduction and spread of anthrax fungi, and the pepper fruits and leaves can hardly form anthrax lesions or the lesions are small and cannot expand rapidly; while the anthrax-susceptible pepper material refers to a pepper material that cannot resist the infection and reproduction and spread of anthrax fungi, and the anthrax fungi will rapidly expand after the fruits and leaves come into contact with the anthrax fungi, causing serious damage to the peppers.

[0026] Furthermore, the PCR is Touch-down PCR.

[0027] Furthermore, the PCR was performed according to the following procedure: pre-denaturation at 94°C for 15 minutes; then denaturation at 94°C for 20 seconds, annealing at 61°C-55°C for 60 seconds, for a total of 10 cycles, 61°C being the annealing temperature for the first cycle, and the annealing temperature being reduced by 0.6°C in each subsequent cycle; then denaturation at 94°C for 20 seconds, annealing / extension at 55°C for 60 seconds, for a total of 26 cycles.

[0028] The third aspect of the present invention provides a kit comprising the KASP labeled primer set of the first aspect and PCR reaction reagents.

[0029] Specifically, the PCR reaction reagents may include at least one of a PCR buffer, a DNA polymerase, and dNTPs. Of course, for the convenience of the entire operation process, the kit may also include other reagents or experimental equipment required for determining the genotype of the SNP molecular marker of the present invention, which are not detailed here.

[0030] The fourth aspect of the present invention provides the use of the KASP labeling primer set of the first aspect, the method of the second aspect, or the kit of the third aspect in the following (1) or (2):

[0031] (1) Assist in the breeding of anthracnose-resistant pepper germplasm or varieties;

[0032] (2) Identify whether the pepper germplasm or variety to be tested is resistant to anthracnose.

[0033] The KASP marker primer set is used to determine the genotype of the SNP molecular marker located on chromosome 6 of the Zunla-1_v3.0 pepper reference genome, thereby knowing whether the pepper germplasm or variety to be tested is resistant to anthracnose, providing a convenient and accurate breeding method for breeding anthracnose-resistant pepper germplasm or variety.

[0034] The beneficial effects of the present invention are as follows:

[0035] The present invention utilizes two pepper inbred lines with significant differences in anthracnose resistance and a RILs population comprising 240 strains constructed using these two materials as parents as experimental materials, constructs a high-density genetic map of pepper using the SLAF-seq technology, and combines anthracnose resistance phenotypic data from three seasons to locate the major anthracnose resistance QTL to chromosome 6, and obtains tightly linked SNP sites and a KASP marker primer set Ct6082. Population experiments have verified that the marker accuracy rate can reach 91%, and the marker can be accurately applied to molecular marker-assisted breeding of pepper anthracnose resistance, shortening the material breeding cycle, and providing a practical molecular marker for molecular breeding of pepper anthracnose resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the specific embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the specific embodiments. Obviously, the drawings described below do not limit the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 This is a high-density complete linkage genetic map of pepper, where the black horizontal line represents the SLAF marker; the x-axis and y-axis represent the linkage group number and genetic distance (unit: centimorgan), respectively.

[0038] Figure 2 This is the QTL mapping result for pepper anthracnose resistance, where the horizontal axis is the position in the entire genome and the vertical axis is the LOD value.

[0039] Figure 3 The genotyping results of 100 pepper materials were obtained in the present invention. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0041] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0042] Example 1 Acquisition of SNP sites and KASP marker primer set Ct6082

[0043] 1. Materials and Methods

[0044] 1.1 Test materials:

[0045] The present invention constructed F10 recombinant inbred lines (RILs) comprising 240 strains using pepper varieties B158 (highly resistant to anthracnose) and B161 (susceptible to anthracnose), both of which exhibit significant differences in anthracnose resistance. Each of B158, B161, and the 240 RILs was subjected to three biological replicates, with 10 plants per line planted per replicate. The experiment was conducted using a randomized block design.

[0046] 1.2 Anthrax inoculation method:

[0047] Before inoculation, pick up anthrax pathogens with a sterilized inoculation needle, inoculate them on PDA culture medium, and culture them in a 28°C incubator for 7-10 days. After a large number of white spores are produced, prepare a spore suspension with an OD600 of 0.3.

[0048] Harvest red-ripe pepper fruits, rinse the surface with tap water, spray the surface with 75% alcohol for disinfection, then rinse with sterile water and air-dry. For inoculation, pierce one side of the fruit with a sterilized toothpick and apply 6 μl of the spore suspension to the wound. For each pepper material, inoculate nine pepper fruits with each strain three times. Place the fruits, with the inoculation point facing upward, in a fresh-keeping box lined with moistened filter paper and incubate in a 28°C incubator to maintain moisture. After seven days, investigate the fruit for lesion development and measure the diameter of the lesions using the cross-hatch method.

[0049] 1.3 Construction of a high-density genetic map of pepper:

[0050] 1.3.1 SLAF library construction and high-throughput sequencing

[0051] (1) The Tiangen DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China) was used to extract genomic DNA from the parental and recombinant inbred lines. Biomarker's independently developed software was used to predict the number of markers generated by different enzymes in the pepper reference genome (C. annuum cv. Zunla-1). Genomic DNA from the parental and RIL populations was digested with Hae III. Klenow Fragment and dATP were used to add A to the 3' end of the digested fragments. The fragments were then ligated with dual-tag sequencing adapters (PAGE-purified, Life Technologies, USA). The sequences were amplified by PCR using the following PCR primers:

[0052] Forward primer: 5′-ATGATACGGCGACCACCGA-3′ (SEQ ID NO. 6);

[0053] Reverse primer: 5'-CAAGCAGAGAGAGGCATACG-3' (SEQ ID NO. 7).

[0054] (2) PCR products were purified and pooled. The pooled samples were separated by gel electrophoresis, and fragments (SLAFs) ranging from 314 to 394 bp were recovered and purified. Paired-end sequencing was performed using the Illumina HiSeq 2500 system (Illuminia, Inc; San Diego, CA, USA). To evaluate the accuracy of the library construction, Nipponbare rice (Oryza sativa L. japonica) was used as a control and subjected to the same treatment for library construction and sequencing.

[0055] 1.3.2 SLAF-seq Data Analysis and SNP Marker Development

[0056] The original sequencing read length of the SLAF-seq library was 125 bp. To ensure analysis quality, adapter sequences and low-quality reads were filtered out from the raw data. The sequencing quality and data volume of the filtered adapter reads were evaluated. The accuracy and effectiveness of the experimental plan were analyzed by analyzing the alignment efficiency and enzyme digestion efficiency of the control data.

[0057] Based on the reads' localization results on the pepper reference genome (C. annuum cv. Zunla-1), GATK was used for local realignment and variant detection, and samtools was used for variant detection. The intersection of the two methods was used as the final SNP site set to ensure the accuracy of the detected SNPs. First, GATK was used to perform local realignment of sites near the alignment results with insertions / deletions to correct alignment errors caused by insertions / deletions. SNP and indel variant detection was performed using GATK and samtools. The intersection of the variant sites detected by GATK and samtools was used as the final variant site for subsequent analysis. To facilitate subsequent genetic analysis, the polymorphic tags were genotyped according to the common two-allele coding rules in genetics, including 8 segregation types (ab×cd, ef×eg, hk×hk, lm×ll, nn×np, aa×bb, ab×cc, cc×ab). Since the RILs population used in this experiment was derived from two homozygous parents, only polymorphic SNP markers of the aa×bb segregation type were selected for genetic map drawing.

[0058] 1.3.3 Construction of a high-density genetic map

[0059] To ensure the quality of the genetic map, polymorphic SNP markers were filtered to obtain SNP markers suitable for mapping. The MLOD values between each tag were calculated, and the minimum and maximum number of tags in each group were set. The MLOD value intervals were preset, and the tags were sorted from smallest to largest. Tags with the highest MLOD values were grouped together in the same linkage group. Tags with MLOD values less than 5 relative to other tags were filtered out, and all tags were grouped into 12 linkage groups (LGs). To ensure efficient construction of a high-density and high-quality genetic map, HighMAP software was used to construct the high-density genetic map and correct genotyping errors in the LGs.

[0060] Based on the constructed high-density complete linkage genetic map of pepper, QTL mapping analysis was performed using Icimapping V4.2 software. The software parameters were set as follows: PIN = 0.001, step size = 1.0 cM, and LOD = 2.5 as the threshold.

[0061] 2. Research Results

[0062] 2.1 Construction of a high-density genetic map based on the RILs population

[0063] Using SLAF-seq sequencing, an average of 9.18 Gbp of clean data was obtained for the parents, and a total of 332.58 Gbp of data for the progeny. The Q30 of the sequencing data reached over 80%. Based on the Zunla-1_v3.0 pepper reference genome information, SLAF-seq was used to construct a high-density complete linkage genetic map of pepper containing 5867 bin markers. The total distance of the map was 2039.72 cM, with an average distance of 0.35 cM (see Figure 1 ).

[0064] 2.2 QTL mapping for anthracnose resistance in pepper

[0065] Based on the anthracnose resistance phenotypic data of pepper RILs population, QTL mapping analysis was performed using Icimapping V3.3 software. The major QTL for anthracnose resistance in pepper was located on chromosome 6 of the Zunla-1_v3.0 pepper reference genome. The flanking SNP markers were Block 6078 and Block 6082, with an interval size of 0.66 Mb and a contribution rate of 13.2672% (see Figure 2 , Table 1).

[0066] Table 1 QTL mapping results for pepper anthracnose resistance

[0067]

[0068]

[0069] In Table 1, LOD refers to the maximum LOD value of the association of the trait; PVE refers to the phenotypic contribution rate; ADD refers to the additive effect value.

[0070] 2.3 Development of molecular markers linked to anthracnose resistance in pepper

[0071] Based on the SLAF-seq sequencing data, the marker Block6082 on the right side of the QTL mapping interval was developed as the KASP marker Ct6082. The genotype of the highly anthracnose-resistant pepper material B158 at this marker site was T:T, and the genotype of the anthracnose-susceptible pepper material B161 at this marker site was C:C.

[0072] According to the information of SNP site Block6082, KASP primer sequences were designed, see Table 2.

[0073] Table 2 KASP primer sequences

[0074]

[0075] The underlined portion in Table 2 is the fluorescent linker sequence connected to the 5' end of the corresponding forward primer, wherein the underlined sequence in SEQ ID NO.4 is the linker FAM added to the 5' end of the forward primer primer_X, and the underlined sequence in SEQ ID NO.5 is the linker HEX added to the 5' end of the forward primer primer_Y.

[0076] Example 2 Verification of the Accuracy of the Molecular Markers Developed in Example 1

[0077] 100 samples were randomly selected from the RILs population constructed in Example 1 as verification materials, and the molecular markers and KASP marker primers in Example 1 were verified.

[0078] As proposed in Example 1, genomic DNA of pepper seedling leaf material was used as a template for PCR.

[0079] PCR reactions were performed on a Hydrocycler water bath PCR instrument, with fluorescence detection performed on a BMG LABTECH GMbH platform. The system consisted of 2.5 μL of DNA, 2.5 μL of 2x KASP Master mix, and 0.07 μL of KBD Assay mix (a primer mixture with forward primer X, forward primer Y, and reverse primer at a molar ratio of 2:2:5, each at a concentration of 10 μmol / μL). The PCR program was initial denaturation at 94°C for 15 minutes, followed by 10 cycles of denaturation at 94°C for 20 seconds and annealing / extension at 61°C (-0.6°C / cycle) for 60 seconds, followed by 26 cycles of denaturation at 94°C for 20 seconds and annealing / extension at 55°C for 60 seconds.

[0080] When the amplified product is subjected to fluorescence detection, if the sample PCR product only detects the fluorescent signal corresponding to the forward primer X connected to the fluorescent linker sequence, such as blue fluorescence, it indicates that the corresponding genotype is T:T, and the pepper strain is determined to be anthracnose-resistant material; if the sample PCR product only detects the fluorescent signal corresponding to the forward primer Y connected to the fluorescent linker sequence, such as red fluorescence, it indicates that the corresponding genotype is C:C, and the pepper strain is determined to be anthracnose-susceptible material; if the fluorescent signal corresponding to the forward primer X and the fluorescent signal corresponding to the forward primer Y are simultaneously detected, such as green fluorescence, it indicates that the corresponding genotype is T:C, and the pepper strain is determined to be anthracnose-resistant material.

[0081] The phenotype was used to determine whether each material was susceptible to or resistant to anthracnose. For specific detection methods, see Section 1.2 of Example 1. After 7 days, the disease condition of the fruit was investigated, and the diameter of the lesions was measured using the cross method.

[0082] Further population verification using 100 RILs revealed that the anthracnose resistance gene is dominant. Phenotypic lesion diameters greater than 1.00 cm indicate anthracnose-susceptible materials, while lesion diameters less than or equal to 1.00 cm indicate anthracnose-resistant materials. KASP typing for anthracnose-resistant materials was (T:T or C:T), while KASP typing for susceptible materials was (C:C). A t-test of the lesion diameters between the two groups with different KASP typing yielded a P value of 1.45E-13, indicating a highly significant difference between the two groups. This suggests that the KASP marker Ct6082 can be used for marker-assisted breeding (see Table 3, Figure 3 ).

[0083] The genotyping results obtained for each pepper material were compared with the actual lesion phenotype. If the material's genotyping was T:T or C:T and the actual lesion diameter was less than or equal to 1.00 cm, the genotyping identification result was considered accurate; otherwise, the identification result was considered inaccurate. If the material's genotyping was C:C and the actual lesion diameter was greater than 1.00 cm, the genotyping identification result was considered accurate; otherwise, the identification result was considered inaccurate. The actual lesion phenotypes and genotyping results shown in Table 3 indicate that the molecular detection accuracy of the molecular markers of the present invention was 91%, demonstrating its applicability to detecting anthracnose resistance or susceptibility in pepper varieties.

[0084] Table 3: Verification of the accuracy of the labeling using 100 samples

[0085]

[0086]

Claims

1. A KASP marker primer set for detecting or determining the genotype corresponding to a SNP molecular marker linked to a pepper anthracnose resistance gene, the KASP marker primer set comprising: Forward primer primer_X, the nucleotide sequence of which is shown in SEQ ID NO.1 in the sequence listing; Forward primer primer_Y, the nucleotide sequence of which is shown in SEQ ID NO.2 in the sequence listing; The nucleotide sequence of the reverse primer primer_C is shown in SEQ ID NO.3 in the sequence listing.

2. The KASP labeling primer set according to claim 1, characterized in that The 5' ends of the forward primer primer_X and the forward primer primer_Y are respectively connected to different fluorescent linker sequences.

3. The KASP labeling primer set according to claim 2, characterized in that The fluorescent linker sequence is selected from one of FAM, HEX, FITC, RED, TET, JOE, and R110.

4. A method for identifying whether a pepper variety to be tested is anthracnose-resistant pepper variety, characterized in that: The steps include: Provide genomic DNA of the pepper variety to be tested; Using the genomic DNA of the pepper variety to be tested as a template, PCR amplification is performed using the KASP marker primer set according to any one of claims 1 to 3; Performing fluorescence detection and analysis on the amplified product obtained by the PCR amplification to obtain the genotype of the pepper variety to be tested, thereby determining whether the pepper variety to be tested is an anthracnose-resistant pepper variety; When the genotype is T:T or T:C, the pepper variety to be tested is an anthracnose-resistant pepper material; when the genotype is C:C, the pepper variety to be tested is an anthracnose-susceptible pepper material.

5. The method for identifying whether a pepper variety to be tested is an anthracnose-resistant pepper variety according to claim 4, characterized in that: The PCR is Touch-down PCR.

6. The method for identifying whether a pepper variety to be tested is an anthracnose-resistant pepper variety according to claim 4 or 5, characterized in that: The PCR was performed according to the following procedure: pre-denaturation at 94°C for 15 minutes; then denaturation at 94°C for 20 seconds, annealing at 61°C-55°C for 60 seconds, for a total of 10 cycles, 61°C being the annealing temperature for the first cycle, and the annealing temperature being decreased by 0.6°C in each subsequent cycle; then denaturation at 94°C for 20 seconds, annealing / extension at 55°C for 60 seconds, for a total of 26 cycles.

7. A kit comprising the KASP labeling primer set according to any one of claims 1 to 3 and PCR reaction reagents.

8. Use of the KASP labeling primer set according to any one of claims 1 to 3, the method according to any one of claims 4 to 6, or the kit according to claim 7 in the following (1) or (2): (1) Assist in the breeding of anthracnose-resistant pepper germplasm or varieties; (2) Identify whether the pepper germplasm or variety to be tested is resistant to anthracnose.

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