Application of soybean resistance gene GmCSLA2 excellent haplotype and dCAPS molecular marker for soybean rhizosphere disease

Abstract

The application discloses a soybean Cercospora sojina resistance gene GmCSLA2 The application of excellent haplotype and its dCAPS molecular marker belongs to the technical field of molecular biology detection. GmCSLA2 The application discloses a reagent for detecting a gene haplotype and application of the reagent in identifying soybean Cercospora sojina resistance, preparing a detection tool and cultivating a disease-resistant variety. GmCSLA2 Hap1 (SEQ ID NO. 3) and a susceptible haplotype (SEQ ID NO. 4), wherein, GmCSLA2 Hap2 (SEQ ID NO. 4), wherein, GmCSLA2 GmCSLA2 Hap1 is an excellent haplotype. The reagent and the method can efficiently and accurately identify the soybean Cercospora sojina resistance, and can significantly improve the soybean disease-resistant breeding efficiency.

Description

Technical Field

[0001] This application belongs to the field of molecular biological detection technology, specifically relating to soybean stem rot resistance genes. GmCSLA2 Applications of superior haplotypes and their dCAPS molecular markers. Background Technology

[0002] Soybean pseudostem spot seed rot (PSD) is caused by the fungus *Phomopsis spp.* (Spp. spp.). Phomopsis longicolla Stem spot rot is a fungal disease of soybean caused by infection. It primarily affects soybean seeds during storage and seedling stages. Infected seeds become shriveled and cracked, with a chalky white seed coat accompanied by mold, leading to loss of seed viability, reduced oil content, and altered fatty acid ratios. During germination, infected seeds germinate slowly or not at all. In later stages of plant growth, the disease further causes stem and pod dieback, resulting in severe losses in soybean yield and quality. Therefore, breeding and planting disease-resistant varieties is currently the most economical and effective control method.

[0003] Currently, the identification of resistance to soybean stem rot mainly relies on artificial greenhouse or field planting identification methods. The identification steps are usually as follows: (1) Pathogen culture: Isolate, purify and culture stem rot fungus in large quantities ( Phomopsis longicolla (2) Artificial inoculation: During specific growth stages of soybeans (such as budding, seedling, or maturity stages), artificial inoculation is carried out on soybean plants or seeds by means of spraying with mycelium cakes, spore suspensions, or embedding with toothpicks. (3) Disease investigation and assessment: After inoculation, the disease incidence of the plants is observed and recorded at a certain time (such as 7 days to 14 days). Assessment indicators include, but are not limited to, lesion length, seed rot rate, mycelial coverage rate on seed surface, and seed infection rate. Based on these indicators, soybean varieties are classified into different levels such as highly resistant, moderately resistant, and susceptible according to a set of grading standards (such as levels 1-5).

[0004] However, the above-mentioned identification methods have long identification cycles and low efficiency, and fluctuations in environmental factors can affect the severity of disease occurrence, resulting in poor repeatability. At the same time, inoculation identification requires causing infection damage to seeds or plants, making non-destructive identification impossible, especially for precious or newly created breeding intermediate materials. In addition, traditional inoculation identification must be carried out after the plants have entered a specific growth stage, and can only be judged after the disease symptoms have fully manifested. It is impossible to screen and eliminate the target disease resistance traits in the seedling stage, thus prolonging the breeding cycle.

[0005] In summary, current identification methods are insufficient to meet the urgent needs of modern molecular breeding for high-throughput and precise identification of disease-resistant germplasm. Summary of the Invention

[0006] Therefore, the primary objective of this application is to provide a gene for resistance to soybean stem rot. GmCSLA2 The application of superior haplotypes and their dCAPS molecular markers has led to the discovery of genes for resistance to soybean stem rot. GmCSLA2 Excellent haplotype, and based on GmCSLA2 dCAPS molecular markers were developed from functional SNP variant sites in gene promoter regions, enabling rapid and accurate differentiation at the DNA level. GmCSLA2 Hap1 (Superior disease-resistant haplotype) and GmCSLA2 Hap2 (Susceptible haplotypes) provide an efficient and reliable technical tool for molecular breeding of soybean disease resistance, significantly improving the efficiency of disease-resistant germplasm identification and utilization.

[0007] To achieve the above objectives, this application adopts the following technical solution: One aspect of this application discloses the detection... GmCSLA2 The application of haplotype reagents in any of the following: (i) To identify or assist in the identification of soybean resistance to stem spot disease; (ii) Prepare detection tools for identifying or assisting in the identification of soybean stem rot resistance; (iii) Breed soybeans resistant to stem rot; in, GmCSLA2 The haplotype of the gene is GmCSLA2 Hap1 or GmCSLA2 Hap2 ; The GmCSLA2 Hap1 The nucleotide sequence is SEQ ID NO.3, indicating a disease-resistant haplotype; GmCSLA2 Hap2 The nucleotide sequence is SEQ ID NO.4, which is the disease-susceptible haplotype.

[0008] Another aspect of this application discloses methods for amplification. GmCSLA2 Primer pair for the gene SNP-dCAPS molecular marker, wherein the primer pair consists of an upstream primer with the sequence shown in SEQ ID NO.5 and a downstream primer with the sequence shown in SEQ ID NO.6.

[0009] Another aspect of this application discloses a detection tool containing reagents as defined in this application.

[0010] Another aspect of this application discloses a method for identifying or assisting in the identification of soybean stem rot resistance, comprising at least the following steps: Testing soybeans GmCSLA2 Gene haplotype, haplotype isGmCSLA2 Hap1 The soybeans are disease-resistant haplotypes, and the haplotype is... GmCSLA2 Hap2 The soybeans in question are susceptible haplotypes.

[0011] This application has at least the following beneficial effects: This application identified and characterized genes associated with resistance to soybean stem rot— GmCSLA2 and to GmCSLA2 Variation analysis was performed on the coding region of the gene and the upstream 1500 bp promoter region. Based on the variation sites, all materials were divided into two main haplotypes. GmCSLA2 Hap1 and GmCSLA2 Hap2 Based on its haplotype, it is possible to achieve efficient and accurate identification of soybean resistance to pseudostem rot.

[0012] Specifically, this application directly detects specific functional variant sites at the DNA level, providing objective and stable results unaffected by environmental factors. It exhibits high reproducibility across different batches, with extremely high accuracy and consistency with phenotypic data. The detection requires only a small amount of tissue (such as leaves, seeds, or pods) for DNA extraction, causing no damage to the plant. This facilitates the screening of valuable early-generation breeding materials and significantly shortens the breeding cycle. Furthermore, all reagents used are standard molecular biology reagents, and the PCR amplification and enzyme digestion reactions are mild and easy to operate, requiring no expensive large-scale instruments. This allows for high-throughput, automated screening, making it suitable for widespread use in conventional breeding units.

[0013] The method described in this application, from DNA extraction to genotyping, regardless of whether enzyme digestion or sequencing is used, can be completed within one day, significantly faster than the 7-14 days required for traditional field inoculation and identification, thus greatly improving the efficiency of disease-resistant germplasm identification. Furthermore, utilizing the method provided in this application… GmCSLA2 Molecular markers enable rapid, large-scale identification of a vast amount of soybean germplasm resources; combined with pedigree data, they can efficiently identify markers carrying molecular markers. GmCSLA2 Hap1 Superior haplotype disease-resistant parental materials provide precise parental selection criteria for improving soybean disease-resistant varieties, thus serving soybean variety improvement. Attached Figure Description Figure 1 This is a genome-wide association diagram of the pseudostem spot rot phenotype based on 308 natural germplasms. Figure 1 In diagram A, the Manhattan plot represents the genome-wide association analysis of seed rot rate after inoculation of 308 germplasm accessions with the pathogen. Figure 1 B in the diagram represents the QQ plot of the genome-wide association analysis.

[0014] Figure 2 for GmCSLA2The relative expression levels of genes at different infection time points in susceptible and resistant varieties.

[0015] Figure 3 for GmCSLA2 Gene mutation sites and their haplotypes.

[0016] Figure 4 for GmCSLA2 Comparison of disease resistance phenotypes of the two haplotypes in 308 materials, respectively, seed surface mycelial coverage ( Figure 4 a) Seed rot rate ( Figure 4 (b) and spore density on the seed surface ( Figure 4 (c)

[0017] Figure 5 for GmCSLA2 Results of resistance identification of different haplotypes before and after inoculation at the bud and seedling stages.

[0018] Figure 6 for GmCSLA2 Different haplotype gel images.

[0019] Figure 7 For 20 soybean germplasm accessions GmCSLA2 Phenotypic indices of different haplotype plants inoculated with *Pterygotympanum* at the bud stage for 72 hours were compared, with the following values ​​being the mycelial coverage rate on the seed surface (…). Figure 7 a) Seed rot rate ( Figure 7 (b) and spore density ( Figure 7 (c)

[0020] In the diagram, ns represents P >0.05 (not significant) express P <0.05 (significant) express P <0.01 (highly significant) express P <0.001 (extremely significant). Detailed Implementation

[0021] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0022] The first aspect of this application discloses the identification. GmCSLA2The application of haplotype reagents, particularly in identifying resistance to soybean stem rot. Specifically, through detection... GmCSLA The haplotype of gene 2 can quickly and accurately determine the resistance and susceptibility of soybean to stem spot rot, and thus be applied to the screening of disease-resistant germplasm, molecular marker-assisted breeding, and the preparation of related detection products.

[0023] In this application, genes for resistance to soybean stem spot disease were discovered and identified through previous research. GmCSLA2 (The nucleotide sequence is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2). Furthermore, the haplotype refers to a combination of alleles at multiple loci commonly inherited on the same chromosome; for the purposes of this application, GmCSLA2 Gene haplotypes specifically refer to the two main forms of variation existing in a specific sequence segment of a gene, namely... GmCSLA2 Hap1 and GmCSLA2 Hap2 .in, GmCSLA2 Hap1 The nucleotide sequence is shown in SEQ ID NO.3. This haplotype is associated with resistance to *Sterculia vesicularis* and is a resistant haplotype. GmCSLA2 Hap2 The nucleotide sequence is shown in SEQ ID NO.4. This haplotype is associated with susceptibility to *Sterculia vesicularis*, and is a susceptible haplotype. Through association analysis of a large number of soybean germplasm resources, the carrier was identified. GmCSLA2 Hap1 Soybean plants exhibited resistance to stem rot during the bud stage, while those carrying... GmCSLA2 Hap2 Soybean plants in these areas showed signs of susceptibility to the disease. Therefore, by detecting the pathogens in the soybean materials being tested... GmCSLA2 The specific type of gene haplotype can be used to identify or assist in the identification of disease resistance traits.

[0024] In this application, the detection GmCSLA2 Reagents for haplotype testing refer to reagents that can detect haplotypes in the sample being tested. GmCSLA2 The haplotype of a gene is GmCSLA2 Hap1 still GmCSLA2 Hap2 Any molecular biological assay substance for differentiation. It should be understood that the reagents encompass a variety of forms, including but not limited to oligonucleotide primer pairs, specific probes, chips for allelic typing, and compositions containing the above components. Any substance capable of specifically recognizing or reflecting the nucleotide sequence difference between SEQ ID NO. 3 and SEQ ID NO. 4 is considered a reagent as defined in this application.

[0025] In some specific embodiments, the reagent is a primer pair, which is based on... GmCSLA2 Hap1 and GmCSLA2 Hap2 The sequence difference design enables haplotype differentiation through amplification and subsequent product analysis. As a preferred example, the primer pair consists of one upstream primer and one downstream primer, with nucleotide sequences SEQ ID NO.5 and SEQ ID NO.6, respectively. The example primer pair is targeted at… GmCSLA2 Single nucleotide polymorphism sites on genes, designed based on derivatized enzyme digestion amplification polymorphic sequence technology, constitute... GmCSLA2 Primers for SNP-dCAPS molecular marker amplification of genes. In this application, the SNP-dCAPS molecular marker refers to a single nucleotide polymorphism site that cannot be directly recognized by restriction endonucleases by introducing a mismatched base, transforming it into a restriction enzyme site that can be recognized by a specific restriction endonuclease, thereby utilizing the fragment length polymorphism after enzyme digestion to achieve SNP genotyping. When using this primer pair, the upstream primer introduces a mismatched base that is not fully paired with the template at a specific position near the 3' end. When the haplotype of the template DNA to be tested is... GmCSLA2 Hap2 At that time, the product obtained by PCR amplification can form restriction endonucleases. Nsp The recognition sequence of I; and when the template haplotype is GmCSLA2 Hap1 At this time, the recognition sequence cannot be formed in the amplification product. Therefore, restriction endonucleases are used to detect it. Nsp The amplification products are digested with enzymes and then separated by electrophoresis, which clearly distinguishes the two haplotypes. However, it should be understood that the primer pairs in this application are not limited to the examples shown here. Depending on the selected restriction endonuclease, other dCAPS can be designed to introduce different restriction endonucleases (such as...). Taq I, Hae The restriction enzyme sites (such as III, etc.) are sufficient to distinguish between the two haplotypes.

[0026] The above tests GmCSLA2 The specific applications of haplotype reagents include the following aspects: (i) Identification or auxiliary identification of soybean stem spot disease resistance This reagent was used to analyze the genomic DNA of the soybean sample to be tested, and to obtain... GmCSLA2 Genetic haplotype information can directly determine or assist in determining the soybean's resistance to *Stipa rot*. If the test results show a haplotype of... GmCSLA2 Hap1 If it is identified as a disease-resistant haplotype, the individual possesses disease resistance; if it is... GmCSLA2 Hap2If the result is positive, it is identified as a susceptible haplotype, and the individual will exhibit susceptibility to the disease. This identification method does not require pathogen inoculation or long-term phenotypic observation, and the results are objective and unaffected by the environment.

[0027] (ii) Preparation of detection tools for identifying or assisting in the identification of soybean stem rot resistance. The aforementioned reagents can be used as core detection elements to prepare various easy-to-use and commercially viable detection tools. For example, the primer pair containing SEQ ID NO.5 and SEQ ID NO.6 can be premixed with DNA polymerase, dNTPs, buffer, etc., required for PCR, or further assembled with restriction endonucleases, electrophoresis standards, etc., to form a detection kit. This detection tool can be a single-use sealed reaction system, a microarray chip immobilized on a carrier, or a microfluidic device integrating reaction and interpretation functions; this application does not impose strict limitations on these aspects.

[0028] (iii) Breeding soybeans resistant to stem rot In the process of disease-resistant breeding, large-scale single-plant experiments are conducted on hybrid offspring or mutant populations. GmCSLA2 Gene haplotype testing to screen for carriers GmCSLA2 Hap1 Superior individual plants with disease-resistant haplotypes are used as parents for further hybridization, backcrossing, or self-pollination, thereby directionally introducing the disease-resistant haplotype into the background of superior cultivars and accelerating the breeding of new soybean varieties resistant to stem rot. This method transforms traditional phenotypic selection into genotypic selection, significantly improving selection efficiency and shortening the breeding cycle.

[0029] The second aspect of this application discloses methods for amplification. GmCSLA2 Primer pair for the SNP-dCAPS molecular marker of the gene. As described above, this primer pair consists of an upstream primer (SEQ ID NO. 5) and a downstream primer (SEQ ID NO. 6). Each of the upstream and downstream primers is typically 18–25 nucleotides in length, and their combination allows for specific binding to soybean genomic DNA at a suitable annealing temperature. GmCSLA2 The target region of the gene is identified, and a single product containing the SNP site to be tested is amplified. GmCSLA2 Hap1 or GmCSLA2 Hap2 Using the template, the full-length amplification products are all 433 bp, but due to haplotype sequence differences, they may contain different restriction endonuclease recognition sites.

[0030] It is understood that primer pair design can be carried out in a manner well-known in the art, such as selecting sequences with moderate GC content and good 3' end stability to ensure the specificity and efficiency of PCR amplification. This application does not impose any limitations in this regard.

[0031] A third aspect of this application further discloses a detection tool that includes the detection methods defined above. GmCSLA2 A reagent for gene haplotype detection. This detection tool can be provided as a kit, and in some specific embodiments, the kit contains at least a primer pair consisting of SEQ ID NO. 5 and SEQ ID NO. 6. As a further improvement, the detection tool also contains a restriction endonuclease. Nsp I. Restriction endonucleases Nsp I can specifically recognize a specific double-stranded DNA sequence in PCR amplification products formed by primer mismatch bases and haplotype SNP sites, and cut it within or near the recognition site.

[0032] In addition, the testing tool may also contain other standard components required for the test, such as Taq DNA polymerase premix, dNTPs, PCR reaction buffer, sterile water, and positive control DNA (e.g., GmCSLA2 Hap1 Type and GmCSLA2 Hap2 The system includes genomic DNA or plasmid standards, DNA molecular weight standards, agarose gels, and electrophoresis buffers. These components can be packaged individually or premixed into several working solutions as needed, allowing operators to begin testing with simple mixing after obtaining the DNA sample, thus reducing operational errors.

[0033] The fourth aspect of this application discloses a method for identifying or assisting in the identification of soybean resistance to stem rot, which involves detecting the resistance of the soybean to be tested. GmCSLA2 Gene haplotype, and haplotype as GmCSLA2 Hap1 The soybean was identified as a disease-resistant haplotype, and the haplotype was... GmCSLA2 Hap2 The soybeans were identified as susceptible haplotypes.

[0034] In some specific implementations, the soybeans to be tested are detected. GmCSLA2 Methods for determining gene haplotypes include steps such as genomic DNA extraction, PCR amplification, and analysis of the amplification products. In a typical example, the specific steps include: (1) Extract genomic DNA from the soybean to be tested. Take any tissue part of the soybean to be tested, such as leaves, cotyledons, roots or seeds, and extract it using conventional methods for plant genomic DNA extraction, such as the cetyltrimethylammonium bromide method (CTAB method) or commercial plant DNA extraction kits. The extracted DNA must be kept in a certain purity and integrity. After evaluation by UV spectrophotometer or gel electrophoresis, adjust the DNA concentration to about 100 ng / μL as a template for PCR amplification. It should be understood that other template concentrations within a reasonable range, such as 50~200 ng / μL, can also meet the detection requirements and can be adjusted according to the actual yield.

[0035] (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the primer pair provided in this application (upstream primer SEQ ID NO.5, downstream primer SEQ ID NO.6) to obtain PCR amplification products. In a preferred PCR amplification system, the amounts of each component added were: 1.0 μL of template DNA at a concentration of 100 ng / μL, 1.0 μL each of upstream and downstream primers at a concentration of 10 μmol / L, 7.5 μL of PCR premix, and 4.5 μL of ddH2O, forming a reaction system with a total volume of 15 μL. It should be understood that in practical applications, other brands of DNA polymerase premix or self-prepared reaction solutions can also be selected, and this application does not limit this.

[0036] The PCR reaction conditions were set as follows: Pre-denaturation at 95°C for 5 minutes to fully denature the template DNA; followed by cyclic amplification with parameters of 95°C denaturation for 15 seconds, 62°C annealing for 15 seconds, and 72°C extension for 10 seconds, for a total of 36 cycles; after each cycle, an extension at 72°C for 5 minutes was performed to complete the ends, and finally, the temperature was lowered to 16°C for storage. Specific PCR reaction conditions can be adjusted or optimized depending on the selection of primer pairs, and are not limited to the examples in this application.

[0037] (3) Analyze the obtained PCR amplification products to confirm GmCSLA2 Gene haplotype.

[0038] In some specific examples, the analysis employs enzymatic digestion, specifically, restriction endonucleases. Nsp The PCR amplification products are digested with restriction enzymes, and then the digested products are subjected to agarose gel electrophoresis. Specifically, if the PCR amplification products cannot be digested by restriction endonucleases... Nsp After digestion with enzyme I and electrophoresis, only one band of 433 bp in length was observed, and no 408 bp band was seen. Therefore, the haplotype of the soybean plant being tested was determined to be [missing information]. GmCSLA2 Hap1The type exhibits a phenotype resistant to stem spot rot; if the PCR amplification product can be restricted by restriction endonucleases... Nsp After digestion with enzyme I and electrophoresis, a distinct 408 bp band was observed (possibly accompanied by a smaller band of approximately 25 bp, which may be faint or difficult to observe in conventional electrophoresis due to its lower molecular weight), and the original 433 bp band disappeared. Therefore, the haplotype of the soybean plant being tested was determined to be [unclear - likely a specific haplotype or type]. GmCSLA2 Hap2 The type is characterized by a phenotype similar to that of stem spot rot.

[0039] It should be noted that if two bands of 433 bp and 408 bp appear simultaneously on the electrophoretic pattern, it indicates that the plant is heterozygous and carries both disease-resistant and disease-susceptible haplotypes. In this case, further comprehensive judgment can be made by combining phenotypic identification or other markers. However, when the purpose is to determine whether the haplotype is homozygous disease-resistant or disease-susceptible, the above criteria are sufficient.

[0040] In other specific examples, the type of mutation can be determined by directly sequencing the PCR amplification products, such as Sanger sequencing, or by using labeling techniques such as quantitative real-time PCR or KASP (Kompetitive Allele Specific PCR). GmCSLA2 Haplotype classification.

[0041] Using the above method, the entire process from sample collection to resistance identification can be completed within one day using only standard molecular biology laboratory conditions. Compared with existing phenotypic identification methods that rely on pathogen inoculation, this method is simple to operate, has a short cycle time, is not affected by climate or pathogen inoculation conditions, and provides stable and reproducible results.

[0042] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0044] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0045] Example 1: GmCSLA2 Gene mining and superior haplotype analysis To discover resistance genes for soybean seed rot, this study, referring to the soybean bud-stage seed rot resistance identification method (Miao Long, Yang Lei, Xu Jinghao, Li Na, Wang Feiyu, Qiu Lijuan, Wang Xiaobo. Construction of soybean bud-stage seed rot identification system and screening of resistant germplasm [J]. Chinese Agricultural Science, 2024, 57(11): 2092-2101.), conducted resistance identification and genome-wide association analysis (GWAS) of disease traits on 308 core germplasm accessions with rich genetic background and stable phenotypic variation. Through GWAS, the major QTL (quantitative trait locus) was located in the interval 16294264–16300400 bp on chromosome 11 (…). Figure 1 This region encodes β-mannan synthase. GmCSLA2 The expression of *Pseudomonas stearans* was significantly upregulated 24 h after infection, and the expression level in the resistant variety Qihuang 34 was significantly higher than that in the susceptible variety Williams, suggesting that it may be involved in the disease resistance response. Figure 2 ).in, GmCSLA2 The nucleotide sequence of the gene and the amino acid sequence it encodes are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0046] Furthermore, based on the germplasm resources used in the 308 genome-wide association analyses, [the following was conducted]... GmCSLA2 Variation analysis was performed on the coding region of the gene and the upstream 1500 bp promoter region. Based on the variation sites, all materials were divided into two main haplotypes. GmCSLA2 Hap1 and GmCSLA2 Hap2 ( Figure 3 ).in, GmCSLA2 Hap1 The nucleotide sequence of this type is shown in SEQ ID NO.3. GmCSLA2 Hap2 The nucleotide sequence of the type is shown in SEQ ID NO.4. By comparing the disease resistance phenotype data of 308 materials in the population through association analysis, it was found that... GmCSLA2 Hap1 Type II soybeans have stronger disease resistance than GmCSLA2 Hap2 type( Figure 4 This conclusion was verified in the disease resistance identification before and after inoculation with *Pseudomonas stolonifer* during the bud and seedling stages. Figure 5 ).

[0047] Example 2: Development of dCAPS molecular markers According to Example 1 GmCSLA2A dCAPS molecular marker was developed at the 642 bp upstream site of the gene start codon. Specifically, restriction endonucleases were selected using the online enzyme recognition software dCAPS Finder 2.0. Nsp I, the restriction endonuclease Nsp The enzyme digestion recognition sequence for I is 5'-RCATGY-3', specifically the following four sequences: ACATGC, ACATGT, GCATGC, and GCATGT, for primer pair design.

[0048] The designed primer pair (5'→3') is as follows: Upstream sequence: CTTAATTATTTGATCTTTCGTTACA (SEQ ID NO.5); Downstream sequence: CTCTCACTCACAATTGGCGC (SEQ ID NO.6).

[0049] Example 3: GmCSLA2 Identification of genotypes In this embodiment, PCR and enzyme digestion techniques were used to identify the genotype of SNP loci in order to determine resistance to soybean stem rot. The specific steps are as follows: (1) Twenty natural soybean germplasms shown in Table 1 were selected, and their genomic DNA was extracted using the CTAB method.

[0050] (2) Using the genomic DNA from step (1) as a template, PCR amplification was performed using the primer pair developed in Example 2.

[0051] The PCR amplification system (15 μL) consisted of: 1.0 μL template DNA at a concentration of 100 ng / μL, 1.0 μL each of forward and reverse primers at a concentration of 10 μmol / L, 7.5 μL KOD One™ PCR Master Mix, and 4.5 μL ddH2O.

[0052] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min → 95℃ denaturation for 15 s, 62℃ annealing for 15 s, 72℃ extension for 10 s, 36 cycles → final extension at 72℃ for 5 min → storage at 16℃ to obtain the amplification product.

[0053] (3) Take the amplification product from step (2) and digest it with enzymes.

[0054] The enzyme digestion reaction system (15 μL) consisted of: 10.0 μL of PCR amplification product, 0.5 μL of restriction endonuclease Nsp I (10 U / μL), 2.5 μL of ddH2O, and 2.0 μL of CutSmart Buffer.

[0055] After gently mixing, briefly centrifuge and place in a PCR instrument at 37°C for 8 hours. Finally, store at 16°C to ensure complete enzyme digestion.

[0056] (4) Take the enzyme digestion product from step (3) for agarose gel electrophoresis. Specifically, place the prepared agarose gel into the electrophoresis tank, add 1×TAE electrophoresis buffer until the liquid surface covers the gel by 2-3 mm, use a pipette to draw 5 μL of 2000 DNA Marker into the first well, and then draw 5 μL of enzyme digestion product into the subsequent wells. Turn on the electrophoresis apparatus and set the electrophoresis parameters: 120 V, 120 mA, and electrophoresis time of 40 min.

[0057] The agarose gel was prepared as follows: Weigh 4 g of agarose and add it to 100 mL of 1×TAE electrophoresis buffer. After shaking well, heat it in a microwave oven until the agarose is completely dissolved. Add 4 μL of nucleic acid dye, mix well, pour the dissolved agarose into a gel casting plate, insert a comb, and cool it to a solid state for later use.

[0058] The results are as follows Figure 6 As shown in the image. If the PCR amplification product cannot be digested by restriction endonucleases... Nsp If the soybean plant is digested with enzyme I and the fragment length is 433 bp, then the soybean plant to be tested is... GmCSLA2 Hap1 The type corresponds to the phenotype of resistance to stem spot rot during the bud stage; if the amplified product can be restricted by restriction endonucleases... Nsp If the soybean plant was digested with enzyme I, and the resulting fragment lengths were 408 bp and 25 bp, then the soybean plant being tested was... GmCSLA2 Hap2 Type, corresponding to the phenotype of stem rot during the bud stage.

[0059] Example 4: GmCSLA2 Application of the dCAPS gene molecular marker in screening soybean germplasm resistant to stem rot After extracting genomic DNA from the 20 soybean germplasms in Table 1, PCR amplification and enzyme digestion identification were performed according to the method in Example 3. GmCSLA2 Genetic haplotype. Based on haplotype typing results, it is divided into... GmCSLA2 Hap1 and GmCSLA2 Hap2 Two groups were tested, and the resistance to *S. stalk rot* during the soybean sprouting stage was evaluated under the same environmental conditions using the method described in Example 1. The results are shown in Table 1 and... Figure 7 As shown. GmCSLA2 Hap1 The average mycelial coverage rate of this type of soybean is 5%. GmCSLA2 Hap2The average mycelial coverage of the two types of soybeans was 93.17%, showing a highly significant difference. P <0.001); GmCSLA2 Hap1 The average seed rot rate of this type of soybean is 4%. GmCSLA2 Hap2 The average seed decay rate of soybean of both types was 82.33%, showing a highly significant difference between the two. P <0.001); GmCSLA2 Hap1 The average spore density on the seed surface of this type of soybean is 336.70 × 10⁻⁶. 4 cells / mL GmCSLA2 Hap2 The average spore density on the seed surface of this type of soybean is 2637.72 × 10⁻⁶. 4 The number / mL showed a highly significant difference between the two ( P <0.001)( Figure 7 This indicates that based on GmCSLA2 Haplotype marker-assisted selection can effectively screen for disease-resistant germplasm.

[0060] Table 1 Natural Soybean Germplasm GmCSLA2 Statistical analysis of disease indicators of different haplotypes

[0061] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. Detection GmCSLA2 The application of haplotype reagents in any of the following: (i) To identify or assist in the identification of soybean resistance to stem spot disease; (ii) Prepare detection tools for identifying or assisting in the identification of soybean stem rot resistance; (iii) Breed soybeans resistant to stem rot; in, GmCSLA2 The haplotype of the gene is GmCSLA2 Hap1 or GmCSLA2 Hap2 The GmCSLA2 Hap1 The nucleotide sequence is SEQ ID NO.3, and this haplotype is a disease-resistant haplotype; GmCSLA2 Hap2 The nucleotide sequence is SEQ ID NO.4, and this haplotype is a susceptible haplotype.

2. The application as described in claim 1, characterized in that, The reagents include amplification. GmCSLA2 Primer pair for the gene SNP-dCAPS molecular marker, wherein the primer pair consists of an upstream primer with the sequence shown in SEQ ID NO.5 and a downstream primer with the sequence shown in SEQ ID NO.

6.

3. Used for amplification GmCSLA2 Primer pairs for gene SNP-dCAPS molecular markers, characterized in that, The primer pair consists of an upstream primer with the sequence shown in SEQ ID NO.5 and a downstream primer with the sequence shown in SEQ ID NO.

6.

4. A testing tool, characterized in that, Contains the reagent as defined in claim 1 or 2.

5. The detection tool as described in claim 4, characterized in that, It also contains restriction endonucleases Nsp I.

6. A method for identifying or assisting in the identification of soybean stem rot resistance, characterized in that, At least the following steps are included: Testing soybeans GmCSLA2 Gene haplotype, haplotype is GmCSLA2 Hap1 The soybeans are disease-resistant haplotypes, and the haplotype is... GmCSLA2 Hap2 The soybeans in question are susceptible haplotypes.

7. The method as described in claim 6, characterized in that, The soybean to be tested GmCSLA2 The method for identifying gene haplotypes includes the following steps: Genomic DNA was extracted from the soybeans to be tested; Using the genomic DNA as a template, PCR amplification was performed using the primer pair described in claim 3 to obtain the PCR amplification product; Analyze the PCR amplification products to confirm GmCSLA2 Gene haplotype.

8. The method as described in claim 7, characterized in that, The PCR amplification system consisted of: 1.0 μL of template DNA at a concentration of 100 ng / μL, 1.0 μL each of forward and reverse primers at a concentration of 10 μmol / L, 7.5 μL of PCR premix, and 4.5 μL of ddH2O. The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min → 95℃ denaturation for 15 s, 62℃ annealing for 15 s, 72℃ extension for 10 s, 36 cycles → 72℃ extension for 5 min → storage at 16℃.

9. The method as described in claim 7, characterized in that, The analysis of the PCR amplification products was performed using an enzyme digestion method, which included using restriction endonucleases on the PCR amplification products. Nsp After digestion with enzyme I, the digestion products were subjected to agarose gel electrophoresis.

10. The method as described in claim 9, characterized in that, The haplotype and disease resistance type of the soybean to be tested can be determined based on the gel electrophoresis bands. If the PCR amplification product cannot be digested by restriction endonucleases Nsp If the soybean plant is digested with enzyme I and the fragment length is 433 bp, then the soybean plant being tested is determined to be... GmCSLA2 Hap1 The type is a phenotype that is resistant to stem rot; If the PCR amplification product can be restricted by restriction endonucleases Nsp If the fragment is digested with enzyme I, and the resulting fragment lengths are 408 bp and 25 bp, then it is determined to be... GmCSLA2 Hap2 This type represents the phenotype of stem spot rot.

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