SNP molecular marker linked with anthracnose resistance gene, KASP marker primer group Ct6082 and application of SNP molecular marker and KASP marker primer group Ct6082

Abstract

The invention relates to an SNP molecular marker linked with an anthracnose resistance gene, a KASP marker primer group Ct6082 and application of the SNP molecular marker and the KASP marker primer group Ct6082. The SNP molecular marker is located at the 13169223 site of a chromosome 6 of a Zunla-1v 3.0 pepper reference genome, and the basic group of the SNP molecular marker is T or C. The KASP marker primer group Ct6082 is used for detecting or determining the genetic typing of the SNP molecular marker. T: T or T: C is the genotype of the anthracnose-resistant capsicum material, and C: C is the genotype of the anthracnose-sensitive capsicum material. Through group verification, the accuracy rate of the SNP molecular marker can reach 91%, the SNP molecular marker can be used for breeding anthracnose-resistant pepper materials, the material breeding period is shortened, and a practical molecular marker is provided for pepper anthracnose-resistant molecular breeding.

Description

Technical Field

[0001] The 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 the largest vegetable crop in my country. The annual pepper cultivation area in my country is 35.8 million mu, accounting for 9.28% of the country's vegetables, with a total output of 63.994 million tons and an annual output value of 250 billion yuan. The pepper planting area continues to increase, and the losses caused by pepper diseases are also increasing. Among them, anthracnose has become one of the main diseases that endanger pepper production. Pepper anthracnose is a fungal disease caused by the genus Colletotrichum of the subphylum Ascomycetes. It occurs widely around the world and seriously harms pepper production. It can cause a 30-80% reduction in pepper production, greatly reducing the economic benefits of pepper farmers.

[0003] The prevention and control of pepper anthracnose is mainly based on chemical prevention and control, but chemical agents have a single action site on pathogens, and long-term use can easily lead to drug resistance in pathogens; and the abuse of chemical agents will cause water and soil pollution, which is not in line with the concept of green sustainable development. Cultivating and promoting pepper anthracnose-resistant varieties can reduce the use of chemical agents, reduce environmental pollution, reduce pesticide residues, and promote sustainable agricultural development. It is the most effective, economical and environmentally friendly method to prevent and control diseases.

[0004] At present, there is an extreme shortage of pepper anthracnose-resistant varieties in production, and the genetic law of pepper anthracnose resistance is complex, and the molecular genetic progress is slow, which limits the biological breeding process of pepper anthracnose resistance. The existing pepper anthracnose resistance detection method is complex and time-consuming. In order to enrich and simplify the identification of pepper anthracnose-resistant varieties and shorten the material breeding cycle, the present invention is hereby proposed. Summary of the invention

[0005] In order to overcome the defects of the prior art, one of the purposes of the present invention is to provide a KASP marker primer set Ct6082 for determining or detecting the genotype corresponding to the SNP molecular marker linked to the anthracnose resistance gene, which provides a practical molecular marker for molecular breeding of pepper resistance to anthracnose.

[0006] The 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, the above-mentioned method and the kit.

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

[0010] The 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, wherein 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 genotype corresponding to 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.

[0011] Furthermore, the KASP labeling primer set includes:

[0012] Forward primer primer_X, the nucleotide sequence of which is shown in the sequence listing SEQ ID NO.1;

[0013] Forward primer primer_Y, the nucleotide sequence of which is shown in the sequence listing SEQ ID NO.2;

[0014] The nucleotide sequence of the reverse primer primer_C is shown in the sequence listing SEQ ID NO.3.

[0015] SEQ ID NO.1 in the sequence table is specifically: 5'-CATCAGCAGGATTGGATAATCCCA-3';

[0016] SEQ ID NO.2 in the sequence table is specifically: 5'-ATCAGCAGGATTGGATAATCCCG-3';

[0017] SEQ ID NO.3 in the sequence list is specifically: 5'-CATTTTTGTCGCGGTCAAAACCTTG

[0018] G-3';

[0019] Furthermore, in order to simplify the detection method of PCR products, so as to determine the genotyping of SNP molecular markers, the PCR products can be subjected to fluorescence detection. In order to achieve the purpose of fluorescence detection, the 5' ends of the forward primers primer_X and primer_Y need to be connected with different fluorescent linker sequences respectively. 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 luminescent colors of the fluorescent linkers connected to the 5' ends of the two forward primers are different.

[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 pepper varieties 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 marker primer set of the first aspect;

[0023] Performing fluorescence detection and analysis on the amplification 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 anthracnose-resistant pepper material of the present invention refers to pepper material that has a certain resistance to the infection and reproduction and spread of anthrax bacteria, and 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 pepper material that cannot resist the infection and reproduction and spread of anthrax bacteria, and the anthrax bacteria will rapidly expand after the fruits and leaves come into contact with the anthrax bacteria, and the peppers are seriously damaged.

[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 then the annealing temperature was reduced by 0.6°C in each 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, which includes the KASP labeled primer set of the first aspect and a PCR reaction reagent.

[0029] Specifically, the PCR reaction reagents may include at least one of PCR buffer, DNA polymerase, and dNTP. 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 will not be described one by one 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 breeding pepper germplasms or varieties resistant to anthracnose;

[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 at position 13169223 of 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 pepper germplasm or varieties resistant to anthracnose.

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

[0035] The invention uses two pepper inbred lines with significant difference in anthracnose resistance and a RILs group containing 240 strains constructed by taking the two materials as parents as test materials, constructs a high-density genetic map of pepper by using the SLAF-seq technology, locates the main effect QTL of anthracnose resistance to chromosome 6 by combining the anthracnose resistance phenotypic data of three seasons, obtains a tightly linked SNP site and a KASP marker primer group Ct6082, the marker is located at position 13169223 of chromosome 6, and after verification by group tests, the marker accuracy rate can reach 91%, and the marker can be accurately applied to the molecular marker-assisted breeding of pepper anthracnose resistance, shortens the material breeding cycle, and provides a practical molecular marker for the molecular breeding of pepper anthracnose resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the specific implementation methods of the present invention, the drawings required for use in the description of the specific implementation methods will be briefly introduced below. Obviously, the drawings in the following description do not constitute a limitation to the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 It is a high-density complete linkage genetic map of pepper, in which 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 positioning 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 is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0041] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[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 uses pepper materials B158 (highly resistant to anthracnose) and B161 (susceptible to anthracnose) with significant differences in anthracnose resistance as parents to construct F10 recombinant inbred lines (RILs) containing 240 strains. B158, B161 and 240 RILs strains are all set with 3 biological replicates, and 10 strains are planted in each replicate of each strain, and the experiment is designed according to a randomized block design.

[0046] 1.2 Anthrax inoculation method:

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

[0048] Pick red ripe pepper fruits, rinse the fruit surface with tap water, spray the fruit surface with 75% alcohol for disinfection, then rinse with sterile water and dry. When inoculating, pierce one side of the fruit with a sterilized toothpick and drop 6μl of the above spore suspension on the wound. For each pepper material, inoculate 9 pepper fruits with each strain, and repeat 3 times. The fruit is placed in a fresh-keeping box covered with soaked filter paper with the inoculation point facing up, and placed in a 28℃ incubator for moisturizing culture. After 7 days, investigate the disease condition of the fruit and measure the diameter of the lesion with the cross 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 genomic DNA of each strain of the biparental recombinant inbred line was extracted using the Tiangen DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China). The number of markers generated by different enzymes digesting the pepper reference genome (C. annuum cv. Zunla-1) was predicted using the software independently developed by Biomark. The genomic DNA of each strain of the parent and RILs population was digested with HaeⅢ, and Klenow Fragment and dATP were used to add A to the 3' end of the digested fragments, which were then connected to dual-labeled sequencing adapters (PAGE-purified, Life Technologies, USA). The sequences were PCR amplified 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) Purify the PCR products and mix them, separate the mixed samples by gel electrophoresis, and recover and purify the 314-394 bp fragments (SLAFs). Double-end sequencing was performed using the Illumina HiSeq 2500 system (Illuminia, Inc; San Diego, CA, USA). To evaluate the accuracy of the library construction experiment, Nipponbare rice (Oryza sativa L. japonica) was selected 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 is PE125bp. To ensure the quality of the analysis, the adapter sequences and low-quality reads in the original data were filtered out, and the sequencing quality and data volume of the filtered adapter reads were evaluated. The accuracy and effectiveness of the experimental scheme were analyzed by analyzing the alignment efficiency and enzyme digestion efficiency of the control data.

[0057] According to the positioning results of reads on the pepper reference genome (C. annuum cv. Zunla-1), GATK was used for local realignment and variation detection, and samtools was used for variation detection. The intersection of GATK and samtools was taken 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 insertion / deletion alignment results to correct alignment errors caused by insertion / deletion. GATK and samtools were used for SNP and InDel variation detection, and the intersection of the variation sites obtained after GATK and samtools detection was used as the final variation 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 high-density genetic maps

[0059] To ensure the quality of the genetic map, polymorphic SNP markers were filtered to obtain SNP markers that can be used for mapping. The MLOD values ​​between each pair of tags were calculated, the minimum and maximum group tag numbers were set, the MLOD value interval was preset, and the tags were arranged from small to large according to their MLOD values. The tags with the highest MLOD values ​​between tags were grouped in the same linkage group, and the tags with MLOD values ​​lower than 5 with other tags were filtered out. All tags were divided into 12 linkage groups (LGs). In order to ensure the efficient construction of high-density and high-quality genetic maps, HighMAP software was used to construct high-density genetic maps and correct genotyping errors in LGs.

[0060] Based on the constructed pepper high-density complete linkage genetic map, QTL positioning analysis was performed using Icimapping V4.2 software. The software operating 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, the average amount of clean data obtained from the parents was 9.18Gbp, and the total amount of data from the offspring was 332.58Gbp. The Q30 of the sequencing data reached more than 80%. Based on the Zunla-1_v3.0 pepper reference genome information, SLAF-seq was used to construct a pepper high-density complete linkage genetic map containing 5867 bin markers. The total map distance of the map was 2039.72cM, and the average map distance was 0.35cM (see Figure 1 ).

[0064] 2.2 QTL mapping for pepper anthracnose resistance

[0065] Based on the anthracnose resistance phenotype data of pepper in the RILs population, QTL positioning 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 SNP molecular markers on both sides were Block6078 and Block6082, 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] 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.

[0069] 2.3 Development of molecular markers linked to anthracnose resistance in pepper

[0070] According to the SLAF-seq sequencing data, the marker Block6082 on the right side of the QTL positioning interval was developed into the KASP marker Ct6082. The genotyping of the highly anthracnose-resistant pepper material B158 at this marker site was T:T, and the genotyping of the anthracnose-susceptible pepper material B161 at this marker site was C:C.

[0071] According to the information of SNP site Block6082, the KASP primer sequence was designed, see Table 2.

[0072] Table 2 KASP primer sequences

[0073]

[0074] The underlined part 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.

[0075] Example 2 Verification of the Accuracy of the Molecular Markers Developed in Example 1

[0076] 100 materials 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.

[0077] As proposed in Example 1, the genomic DNA of pepper seedling leaf material was used as a template for PCR.

[0078] PCR reaction was performed on a Hydrocycler water bath PCR instrument, and fluorescence detection was performed on a BMG LABTECH GMbH platform. The system was DNA 2.5μL; 2x KASP Master mix 2.5μL; KBD Assay mix (i.e., a mixture of primers, each primer concentration was 10μmol / μL, and forward primer X, forward primer Y, and reverse primer were mixed at a molar ratio of 2:2:5) 0.07μL. The PCR program was pre-denaturation at 94℃ for 15 minutes; then denaturation at 94℃ for 20s, annealing at 61℃ (-0.6℃ / cycle) for 60s, for a total of 10 cycles; then denaturation at 94℃ for 20s, annealing / extension at 55℃ for 60s, for a total of 26 cycles.

[0079] When the amplified product is subjected to fluorescence detection, if the sample PCR product only detects the fluorescence signal corresponding to the forward primer X connected to the fluorescent linker sequence, such as blue fluorescence, it means that the corresponding genotype is T:T, and the pepper strain is determined to be an anthracnose-resistant material; if the sample PCR product only detects the fluorescence signal corresponding to the forward primer Y connected to the fluorescent linker sequence, such as red fluorescence, it means that the corresponding genotype is C:C, and the pepper strain is determined to be an anthracnose-susceptible material; if the fluorescence signal corresponding to the forward primer X and the fluorescence signal corresponding to the forward primer Y, such as green fluorescence, are detected at the same time, it means that the corresponding genotype is T:C, and the pepper strain is determined to be anthracnose-resistant material.

[0080] 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.

[0081] Further population verification was performed using 100 RILs strains. The anthracnose resistance gene was a dominant gene. The phenotype showed that the lesion diameter was greater than 1.00 cm for anthracnose-susceptible materials, and the lesion diameter was less than or equal to 1.00 cm for anthracnose-resistant materials. The KASP typing of anthracnose-resistant materials was (T:T or C:T), and the KASP typing of susceptible materials was (C:C). The t-test was used to test the lesion diameters of the two groups with different KASP typing, and the P value was 1.45E-13. There was a very significant difference between the two groups, indicating that the KASP marker Ct6082 can be used for molecular marker-assisted breeding (see Table 3, Figure 3 ).

[0082] The genotyping results of each pepper material obtained were compared with the actual lesion phenotype. If the genotyping of the material 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 determined to be accurate, otherwise the identification result was considered inaccurate; if the genotyping of the material was C:C, and the actual lesion diameter was greater than 1.00 cm, the genotyping identification result was determined to be accurate, otherwise the identification result was considered inaccurate. From the actual lesion phenotype and genotyping results given in Table 3, it can be seen that the molecular detection accuracy of the molecular marker of the present invention is 91%, indicating that it can be applied to the detection of pepper varieties resistant to anthracnose or susceptible to anthracnose.

[0083] Table 3 Verification of the accuracy of the marking using 100 materials

[0084]

[0085]

[0086]

Claims

1. A KASP marker primer set, used to detect or determine the genotype corresponding to a SNP molecular marker linked to a pepper anthracnose resistance gene, wherein 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 genotype corresponding to 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.

2. The KASP labeling primer set according to claim 1, characterized in that: The KASP labeling primer set includes: Forward primer primer_X, the nucleotide sequence of which is shown in the sequence listing SEQ ID NO.1; Forward primer primer_Y, the nucleotide sequence of which is shown in the sequence listing SEQ ID NO.2; The nucleotide sequence of the reverse primer primer_C is shown in the sequence listing SEQ ID NO.

3.

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

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

5. A method for identifying whether a pepper variety to be tested is an anthracnose-resistant pepper variety, characterized in that: The steps include: Provide genomic DNA of pepper varieties 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 described in any one of claims 1 to 4; 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.

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

7. The method for identifying whether the pepper variety to be tested is an anthracnose-resistant pepper variety according to claim 5 or 6, 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 of the first cycle, and the annealing temperature decreasing 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.

8. A kit comprising the KASP labeling primer set according to any one of claims 1 to 4 and a PCR reaction reagent.

9. Use of the KASP labeling primer set according to any one of claims 1 to 4, the method according to any one of claims 5 to 7, or the kit according to claim 8 in the following (1) or (2): (1) Assist in the breeding of pepper germplasms or varieties resistant to anthracnose; (2) Identify whether the pepper germplasm or variety to be tested is resistant to anthracnose.

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