Molecular marker related to soybean cyst nematode disease resistance and application thereof

Abstract

The invention belongs to the technical field of plant molecular breeding, and discloses a molecular marker related to soybean cyst nematode disease resistance and application thereof. The CAPS molecular marker provided by the invention is simple to match and operate, and can quickly distinguish three genotypes of susceptibility, disease resistance and excellent phenotype and disease resistance and scabs. The molecular marker does not need expensive detection equipment, is simple and convenient to operate, low in cost and accurate in typing, is suitable for screening soybean cyst nematode-resistant filial generations, identifying transgenic lines and detecting the purity of disease-resistant varieties, can remarkably improve the breeding efficiency and shorten the breeding period, and provides a practical technical tool for soybean cyst nematode resistance breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a molecular marker related to soybean cyst nematode resistance and its application, which is particularly suitable for low-cost molecular marker-assisted breeding, germplasm resource screening and transgenic line identification for soybean resistance to cyst nematode. Background Technology

[0002] Soybean cyst nematode disease is one of the most devastating diseases affecting soybean-producing regions worldwide, causing economic losses exceeding US$120 million annually. Developing and promoting resistant varieties is the most economical and effective means of controlling this disease. SNAP18 The gene is a key gene regulating soybean resistance to cyst nematode disease, and the c.797G>A functional SNP mutation in exon 9 of its coding region is the core site for resistance formation. This mutation changes the codon encoding tryptophan to a stop codon, leading to premature termination of protein translation and conferring resistance to soybeans through a dual mechanism of interfering with protein-protein interactions and autophagy detoxification. The genotype and phenotype of this SNP site are clearly correlated: A / A homozygous mutant soybeans are nematode resistant but show lesion-like spots on leaves and have poor agronomic traits; G / A heterozygous soybeans have both stable resistance and excellent agronomic phenotypes, making them the core breeding target; G / G wild-type homozygous soybeans are completely susceptible to nematodes. Traditional cyst nematode inoculation and identification methods have a long cycle of 40-45 days and are easily affected by environmental factors, resulting in low identification efficiency. CAPS (amplified polymorphic sequence) markers are based on PCR amplification and restriction endonuclease digestion, requiring only conventional PCR instruments and electrophoresis equipment for detection, and have advantages such as low cost, simple operation, and intuitive results. SNAP18 The application of the c.797G>A gene site has not been reported. Therefore, developing CAPS molecular markers targeting this functional site can fill the gap in low-cost genotyping technology and meet the needs of different breeding scenarios. Summary of the Invention

[0003] This invention aims to overcome the shortcomings of current innovative KASP marker technology, such as high cost, reliance on special equipment, long identification cycle and low efficiency of traditional inoculation. It provides a CAPS molecular marker targeting the c.797G>A site of the SNAP18 gene and its application method, so as to realize low-cost, rapid and accurate identification of soybean cyst nematode resistance genotypes and help the breeding of superior nematode-resistant soybean varieties.

[0004] To address the shortcomings of existing technologies, this invention is achieved through the following solution:

[0005] This invention provides a CAPS molecular marker for identifying resistance to soybean cyst nematode disease, wherein the molecular marker targets the c.797G>A functional SNP site in exon 9 of the soybean SNAP18 gene.

[0006] In this invention, the nucleotide sequence of the coding region of the soybean wild-type SNAP18 gene is shown in SEQ ID NO.1, and the protein sequence is shown in SEQ ID NO.3;

[0007] The nucleotide sequence of the disease-resistant mutant SNAP18 gene is shown in SEQ ID NO.2, and the encoded protein sequence is shown in SEQ ID NO.4.

[0008] The wild-type base at this site is G (corresponding to SEQ ID NO.1 in the gene coding region), and the mutant base is A (corresponding to SEQ ID NO.2 in the gene coding region). Its genotype strictly corresponds to the soybean cyst nematode resistance phenotype and agronomic traits, making it a core indicator site for breeding screening.

[0009] SEQ ID NO.1:

[0010] 。

[0011] SEQ ID NO.2:

[0012] 。

[0013] SEQ ID NO.3:

[0014] MADQLSKGEEFEKKAEKKLSGWGLFGSKYEDAADLFDKAANCFKLAKSWDKAGATYLKLASCHLKLESKHEAAQAHVDAAHCYKKTNINESVSCLDRAVNLFCDIGRLSMAARYLKEIAELYEGEQNIEQALVYYEKSADFFQNE EVTTSANQCKQKVAQFAAQLEQYQKSIDIYEEIARQSLNNNLLKYGVKGHLLNAGICQLCKEDVVAITNALERYQELDPTFSGTREYRLLADIAAAIDEEDVAKFTDVVKEFDSMTPLDSWKTTLLLRVKEKLKAKELEEDDLT.

[0015] SEQ ID NO.4:

[0016] MADQLSKGEEFEKKAEKKLSGWGLFGSKYEDAADLFDKAANCFKLAKSWDKAGATYLKLASCHLKLESKHEAAQAHVDAAHCYKKTNINESVSCLDRAVNLFCDIGRLSMAARYLKEIAELYEGEQNIEQALV YYEKSADFFQNEEVTTSANQCKQKVAQFAAQLEQYQKSIDIYEEIARQSLNNNLLKYGVKGHLLNAGICQLCKEDVVAITNALERYQELDPTFSGTREYRLLADIAAAIDEEDVAKFTDVVKEFDSMTPLDS.

[0017] In practical applications of this invention, the genotype of the SNP locus and the soybean phenotype have the following correspondence:

[0018] (1) A / A homozygous mutant: Soybeans showed resistance to soybean cyst nematode and leaf spots appeared;

[0019] (2) G / A heterozygous type: Soybeans exhibit resistance to soybean cyst nematode, and the plants show an excellent phenotype that combines resistance and agronomic traits;

[0020] (3) G / G wild type homozygous: soybeans showed susceptibility to soybean cyst nematode.

[0021] The present invention also provides a CAPS primer pair for detecting the above-mentioned SNP sites, the nucleotide sequences of which are shown in SEQ ID NO.5 and SEQ ID NO.6; the upstream primer CAPS-SNAP18-F (SEQ ID NO.5) binds to the conserved region flanking the 5' end of the SNP site, and the downstream primer CAPS-SNAP18-R (SEQ ID NO.6) binds to the conserved region flanking the 3' end of the SNP site.

[0022] SEQ ID NO.5:ATTGTGCCTCCCTTGTATGC;

[0023] SEQ ID NO. 6: AGACTCCAGCAACCTCATT.

[0024] In the actual operation of this invention, the PCR product amplified by the primer set is 456 bp in length and contains only a single SNP site c.797G>A. The mutant amplified fragment contains the DdeI restriction site (CTTAG), while the wild-type amplified fragment has the corresponding sequence (CTTGG) and does not contain this restriction site. Therefore, the genotype can be distinguished by the difference in restriction digestion.

[0025] This invention provides a method for detecting the SNAP18 genotype in soybeans, the method comprising the following steps:

[0026] (1) Sample processing: Soybean seedling leaves were collected, and genomic DNA was extracted using the conventional CTAB method or a kit. The DNA concentration was adjusted to 50 ng / μL~200 ng / μL, which met the detection requirements without the need for high-purity purification.

[0027] (2) PCR amplification: Using the extracted DNA as a template, PCR amplification was performed using the above CAPS primer set. The optimized reaction system and procedure were adopted to ensure amplification specificity.

[0028] (3) Enzyme digestion reaction: Mix the PCR amplification product with DdeI enzyme and enzyme digestion buffer, and incubate at 37°C for 2 hours to allow the enzyme digestion reaction to proceed fully;

[0029] (4) Electrophoresis detection: The enzyme digestion products were subjected to 4% agarose gel electrophoresis with added nucleic acid dye (120V, 20~40min), and the bands were observed under a gel imaging system:

[0030] 1) Single band (456bp): G / G wild-type homozygous (susceptible to nematodes);

[0031] 2) Two bands (255bp+201bp): A / A homozygous mutant (nematode resistant + lesion-like);

[0032] 3) Three bands (456bp+255bp+201bp): G / A heterozygous type (nematode resistant + superior phenotype).

[0033] In the actual operation of this invention, it was found that the restriction endonuclease DdeI, as a specific enzyme digestion tool, recognizes the double-stranded DNA sequence 5'-C^TNAG-3' and the complementary strand 3'-GAN^TC-5'. The digestion occurs between C and T of the first strand of the recognized sequence and between N and T of the complementary strand.

[0034] The enzyme digestion products are preferably subjected to electrophoresis on a 4% agarose gel for 20-40 minutes to ensure clear separation of each electrophoretic band.

[0035] In this invention, the PCR amplification reaction system described in step (2) comprises, in 20 μL increments: 10 μL of 2×PCR Mix enzyme, 0.8 μL each of upstream and downstream primers at a concentration of 10 μmol / L, 1 μL of DNA template, and 7.4 μL of ddH2O;

[0036] The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 3-5 min; 94℃ denaturation for 30 s, 54-58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 31-35 cycles; 72℃ final extension for 10 min.

[0037] In this invention, the enzyme digestion system in step (3) comprises, in 20 μL increments: 10 μL of the PCR amplification product, 1 μL of DdeI enzyme at a concentration of 10 U / μL, 2 μL of 10× enzyme digestion buffer, and 7 μL of ddH2O, incubated at 37°C for 2 h.

[0038] This invention also provides the application of the above-mentioned molecular markers, primer pairs, or detection methods in molecular marker-assisted breeding for resistance to soybean cyst nematode disease.

[0039] This invention also provides the application of the above-mentioned molecular markers, primer pairs, or detection methods in the screening of superior allelic hybrid offspring of SNAP18.

[0040] This invention also provides the application of the above-mentioned molecular markers, primer pairs, or detection methods in the identification of transgenic lines.

[0041] This invention also provides the application of the above-mentioned molecular markers, primer pairs, or detection methods in the detection of purity of disease-resistant varieties.

[0042] The innovation of this invention lies in the first design of a specific primer pair for the c.797G>A functional SNP site of the SNAP18 gene, which is highly targeted and has differentiated enzyme digestion. The amplification product contains only a single target site, and three genotypes can be directly distinguished by DdeI enzyme digestion. This solves the problem of the high threshold of current KASP primer equipment and builds a standardized typing tool that is low-cost, highly accurate and easy to use.

[0043] The core is applicable to soybean plants (germplasm resources, hybrid offspring, and transgenic lines) containing the c.797G>A site of the SNAP18 gene; it can be used as a reference for leguminous plants after verifying the functionally conserved SNP. It is not applicable to non-leguminous plants because they lack this functional site.

[0044] Compared with existing technologies, the present invention has the following advantages:

[0045] (1) Accurate typing: It can achieve precise correspondence between genotype and nematode-resistant phenotype, avoid the interference of environmental factors on phenotype identification, and the typing accuracy rate reaches 100%;

[0046] (2) Low cost: No expensive equipment such as real-time PCR instrument is required, only conventional PCR instrument and electrophoresis equipment are needed, and the detection cost is reduced by more than 60% compared with KASP labeling;

[0047] (3) Simple operation: The detection process is simple, and it only takes 1 to 3 days from DNA extraction to genotype determination. It can be completed without professional technicians.

[0048] (4) Wide range of applications: It covers the entire breeding chain, including screening of hybrid offspring, identification of transgenic lines, and detection of variety purity, meeting the needs of grassroots breeding units and large-scale breeding.

[0049] (5) Outstanding breeding value: It can quickly screen out G / A heterozygotes with "nematode resistance + excellent phenotype", solve the breeding pain point of "disease resistance but poor agronomic traits", and accelerate the breeding process of nematode-resistant and excellent soybean varieties.

[0050] In summary, the CAPS molecular marker and its dedicated primers and application methods provided by this invention can directly solve the core pain points of "difficult typing, long cycle, and low efficiency" and "high threshold for typing equipment, difficulty in grassroots promotion, and high cost" in SNAP18 gene breeding. Compared with existing technologies, it has the advantages of low cost, easy operation, and high accuracy, and has clear innovation, strong practicality, and wide promotion value. Attached Figure Description

[0051] Figure 1 Schematic diagram of the c.797G>A site of the SNAP18 gene targeted by the CAPS molecular marker;

[0052] The image shows that the site is located in exon 9, and the mutation causes the codon to change to a stop codon;

[0053] Figure 2 This is a schematic diagram of the CAPS primer binding sites and restriction enzyme sites.

[0054] The figure shows the relative positions of the upstream and downstream primers to the SNP sites and their corresponding restriction enzyme sites;

[0055] Figure 3 Electrophoresis results before and after CAPS-labeled enzyme digestion;

[0056] Figure 4 This is a diagram showing the DdeI restriction enzyme digestion electrophoresis typing results of the CAPS marker (CAPS-SNAP18-F / R) at the c.797G>A site of the soybean SNAP18 gene in Example 1.

[0057] Figure 5 The image shows the DdeI restriction enzyme digestion electrophoresis typing results of the CAPS marker (CAPS-SNAP18-F / R) at the c.797G>A site of the soybean SNAP18 gene in Example 2. Detailed Implementation

[0058] This invention aims to provide a CAPS molecular marker targeting the c.797G>A functional SNP site of the SNAP18 gene and its application method. The core application is in the screening of hybrid offspring, identification of transgenic lines, and detection of the purity of disease-resistant varieties in breeding for resistance to soybean cyst nematode (SCN). The following details the specific technical aspects, primer descriptions, and operating procedures. The CAPS molecular marker of this invention targets the c.797G>A functional SNP site in exon 9 of the soybean SNAP18 gene (…). Figure 1 The wild-type base at this locus is G (corresponding to SEQ ID NO.1 in the gene coding region), and the mutant base is A (corresponding to SEQ ID NO.2 in the gene coding region). Its genotype strictly corresponds to the soybean SCN resistance phenotype and agronomic traits: the A / A homozygous type is resistant to SCN but has lesion-like spots on the leaves, the G / A heterozygous type is resistant to SCN and has excellent agronomic traits, and the G / G wild-type is susceptible to SCN. It is a core indicator locus for breeding screening.

[0059] Based on the aforementioned functional SNP sites, this invention constructs a complete technical system of "label-primer-detection-application," and the specific technical solution is as follows:

[0060] 1. Design and nomenclature of CAPS primer sets targeting SNP sites

[0061] A specific PCR primer set was designed around the conserved sequence flanking the c.797G>A site to ensure that the amplification product contains only this single SNP site, and that this site is the only recognition site for restriction endonucleases (see CAPS primer binding mode and restriction site). Figure 2 ):

[0062] The upstream primer CAPS-SNAP18-F (SEQ ID NO.5): the 5'→3' sequence is ATTGTGCTCCCTTGTATGC, SEQ ID NO.5, which binds to the conserved region of the 5' flanking side of the SNP site;

[0063] The downstream primer CAPS-SNAP18-R (SEQ ID NO.6): 5'→3' sequence is AGACTCCAGCAACCTCATT, SEQ ID NO.6, which binds to the conserved region of the 3' flanking side of the SNP site;

[0064] The PCR product amplified by this primer set is 456 bp in length. The mutant amplified fragment contains the DdeI restriction enzyme site (CTTAG), while the wild-type amplified fragment does not. The three genotypes can be directly distinguished by the difference in enzyme digestion. It should be noted that the upstream primer CAPS-SNAP18-F (sequence shown in SEQ ID NO.5) and the downstream primer CAPS-SNAP18-R (sequence shown in SEQ ID NO.6) used in this invention correspond to primers OL15055 and OL15202 in previous laboratory research papers. The nucleotide sequences of the two primers are completely identical. The primer names have been adjusted only due to differences in technical documentation writing standards, and the binding specificity and function of the primers remain unchanged.

[0065] 2. Selection of restriction endonucleases

[0066] The restriction endonuclease DdeI was selected as a specific digestion tool, with a recognition sequence of C^TTAG (the cleavage site is located between C and T). The digestion reaction conditions are mild and highly specific.

[0067] (1) Wild type (G / G): The sequence corresponding to the c.797G site in the amplified fragment is “CTTGG”, which cannot be digested by DdeI enzyme and is still a complete 456bp fragment;

[0068] (2) Mutant (A / A): The c.797A site in the amplified fragment corresponds to the sequence “CTTAG”, which can be digested by DdeI into two fragments of 255bp and 201bp;

[0069] (3) Heterozygous (G / A): Contains both wild-type and mutant amplified fragments, and after enzyme digestion, it produces three fragments of 456bp, 255bp and 201bp.

[0070] 3. CAPS marker detection method steps

[0071] (1) Sample processing

[0072] 1) Collect fresh leaves of soybean seedlings (2-3 true leaves stage), take 0.1g of each sample, place them in a mortar and grind them into powder with liquid nitrogen;

[0073] 2) Genomic DNA was extracted using the conventional CTAB method or a plant genomic DNA extraction kit. The DNA purity was detected using Nanodrop (OD260 / OD280 was 1.8~2.0), and the DNA integrity was verified by 1% agarose gel electrophoresis.

[0074] 3) Adjust the DNA concentration to 50ng / μL~200ng / μL using ddH2O, store at -20℃ for later use. High-purity purification is not required to meet the detection requirements.

[0075] (2) PCR amplification

[0076] 1) Reaction system (20μL): 10μL 2×PCR Mix enzyme, 0.8μL each of upstream and downstream primers (10μmol / L), 1μL DNA template, and 7.4μL ddH2O.

[0077] 2) Reaction program: 94℃ pre-denaturation for 3~5 min; 94℃ denaturation for 30 s, 54~58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 31~35 cycles; 72℃ final extension for 10 min;

[0078] 3) After amplification, take 5 μL of PCR product for 1% agarose gel electrophoresis (120V, 20min) to verify amplification specificity and ensure no interference from impurities.

[0079] (3) Enzyme digestion reaction

[0080] 1) Enzyme digestion system (20 μL): 10 μL PCR amplification product, 1 μL DdeI enzyme (10 U / μL), 2 μL 10× enzyme digestion buffer, 7 μL ddH2O;

[0081] 2) After mixing the reaction system, incubate it in a 37°C incubator for 2 hours to ensure that the enzyme digestion reaction proceeds fully;

[0082] 3) After enzyme digestion, add 2 μL of 6× loading buffer to terminate the reaction, vortex to mix and set aside.

[0083] (4) Electrophoresis detection and genotyping

[0084] 1) Prepare a 4% agarose gel (containing nucleic acid dye), place the gel in an electrophoresis tank, and add 1×TAE electrophoresis buffer until the gel surface is submerged;

[0085] 2) Take 15 μL of the enzyme digestion product after termination, and simultaneously spot it with the DNA Marker. Perform electrophoresis at 120V for 20-40 min.

[0086] 3) After electrophoresis, place the gel in a gel imaging system for observation and photographing. Determine the genotype based on the number and length of bands. Figure 3 ).

[0087] a. Single band (456bp): G / G wild-type homozygous;

[0088] b. Two bands (255bp + 201bp): A / A homozygous mutant;

[0089] c. Three bands (456bp+255bp+201bp): G / A hybrid.

[0090] 4. Explanation of the advantages of the technical solution

[0091] The CAPS marker detection method provided in this embodiment requires no expensive equipment such as a quantitative real-time PCR instrument, and can be completed using only conventional molecular biology experimental equipment. From DNA extraction to genotype determination, the entire process takes only 1-3 days, and the operation steps are simple and highly reproducible, allowing those skilled in the art to master it without special training. Furthermore, this method achieves 100% genotyping accuracy, precisely distinguishing between the three genotypes and effectively avoiding interference from environmental factors in phenotypic identification. It provides a low-cost, efficient, and easily applicable standardized technical solution for SCN-resistant soybean breeding.

[0092] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions.

[0093] Example 1

[0094] F2 / backcross population CAPS screening

[0095] The lmm3 mutant (A / A, nematode resistant + lesion-like) was backcrossed with the superior variety Williams 82 (G / G, susceptible) to obtain the BC1F2 population, consisting of 56 seedlings. Leaves from the seedling stage were collected, and genomic DNA was extracted using the CTAB method, adjusting the concentration to 50–200 ng / μL. PCR amplification was performed using the CAPS primer set of this invention (CAPS-SNAP18-F / SEQ ID NO.5, CAPS-SNAP18-R / SEQ ID NO.6). After digestion with DdeI, the amplified products were analyzed by 4% agarose gel electrophoresis to determine the band type and genotype. G / A heterozygotes containing three bands (456bp + 255bp + 201bp) were screened.

[0096] The results are as follows Figure 4 As shown in Table 1, 27 G / A heterozygous plants, 16 G / G wild-type plants, and 13 A / A homozygous mutant plants were detected in the 56-plant population; the segregation ratio of the three genotypes conformed to Mendelian inheritance law of 1:2:1. The 27 selected heterozygous plants were cultivated to adulthood, and all heterozygous plants showed no lesion-like spots and excellent agronomic traits; after inoculation with soybean cyst nematodes, the number of cysts per plant was significantly lower than that of the wild type. In conclusion, the CAPS marker of this invention can efficiently and accurately screen G / A heterozygous plants with "nematode resistance + excellent phenotype" in F2 / backcross populations, meeting the needs of early-stage breeding material screening.

[0097] Table 1. Correspondence between CASP phenotypes and soybean cyst nematode resistance phenotypes in each plant of the backcross BC1F2 population.

[0098] Plant number Number of CAPS restriction bands genotype lesion-like manifestations Number of cysts per plant 1 3 strips G / A none 123 2 1 strip G / G none 345 3 1 strip G / G none 378 4 1 strip G / G none 402 5 1 strip G / G none 360 6 1 strip G / G none 356 7 2 strips A / A lesion-like spots 105 8 1 strip G / G none 371 9 3 strips G / A none 118 10 3 strips G / A none 120 11 2 strips A / A lesion-like spots 113 12 1 strip G / G none 396 13 1 strip G / G none 408 14 3 strips G / A none 110 15 2 strips A / A lesion-like spots 99 16 3 strips G / A none 109 17 3 strips G / A none 140 18 3 strips G / A none 117 19 1 strip G / G none 330 20 3 strips G / A none 132 21 3 strips G / A none 151 22 3 strips G / A none 129 23 1 strip G / G none 374 24 3 strips G / A none 136 25 3 strips G / A none 126 26 1 strip G / G none 376 27 1 strip G / G none 334 28 2 strips A / A lesion-like spots 88 29 2 strips A / A lesion-like spots 102 30 3 strips G / A none 106 31 3 strips G / A none 98 32 2 strips A / A lesion-like spots 131 33 2 strips A / A lesion-like spots 109 34 1 strip G / G none 402 35 3 strips G / A none 126 36 3 strips G / A none 112 37 3 strips G / A none 143 38 3 strips G / A none 127 39 2 strips A / A lesion-like spots 103 40 3 strips G / A none 107 41 2 strips A / A lesion-like spots 131 42 1 strip A / A lesion-like spots 376 43 3 strips G / A none 117 44 3 strips G / A none 131 45 3 strips G / A none 126 46 3 strips G / A none 132 47 2 strips A / A lesion-like spots 125 48 3 strips G / A none 96 49 1 strip G / G none 385 50 3 strips G / A none 142 51 1 strip G / G none 377 52 3 strips G / A none 104 53 2 strips A / A lesion-like spots 106 54 2 strips A / A lesion-like spots 114 55 2 strips A / A lesion-like spots 135 56 3 strips G / A none 127

[0099] Example 2

[0100] CAPS identification of superior allelic transgenic lines of SNAP18

[0101] A binary expression vector containing the homozygous mutant SNAP18 genome sequence (A / A) was constructed and transformed into the susceptible variety Williams 82 (G / G) using Agrobacterium-mediated genetic transformation technology to obtain T0 generation heterozygous transgenic lines (genotype G / A). After self-pollination of the T0 generation plants, a T1 generation segregating population was obtained. Genomic DNA was extracted from 22 seedlings in this population, and PCR amplification, DdeI restriction enzyme digestion, and electrophoresis were performed using the CAPS marker of this invention. The reliability of the genotyping was analyzed in conjunction with the lesion-like phenotype identification results. The results showed ( Figure 5(Table 2) Of the 22 T1 generation plants, 12 showed three bands (456bp + 255bp + 201bp), corresponding to genotype G / A (heterozygous), and exhibited no leaf lesions; 4 lines showed two bands (255bp + 201bp), corresponding to genotype A / A (homozygous), and showed obvious leaf lesions; 5 lines showed a single band (456bp), corresponding to genotype G / G (wild-type), and showed no leaf lesions; 1 plant failed to amplify the target band due to DNA quality issues. The segregation ratio of the three genotypes was approximately 2:1:1, consistent with Mendelian segregation patterns observed in self-pollination of T0 generation heterozygous lines.

[0102] This embodiment illustrates the accuracy and reliability of the CAPS marker typing results of the present invention, which can be used for rapid genotyping of transgenic lines containing homozygous mutant SNAP18 genome sequences.

[0103] Table 2. Correspondence between CASP phenotypes and lesion-like phenotypes of each plant in the T1 generation transgenic lines.

[0104] Plant number Number of CAPS restriction bands genotype lesion-like manifestations 1 1 strip G / G none 2 3 strips G / A none 3 3 strips G / A none 4 1 strip G / G none 5 1 strip G / G none 6 3 strips G / A none 7 3 strips G / A none 8 3 strips G / A none 9 3 strips G / A none 10 2 strips A / A lesion-like spots 11 2 strips A / A lesion-like spots 12 3 strips G / A none 13 2 strips A / A lesion-like spots 14 1 strip G / G none 15 1 strip G / G none 16 3 strips G / A none 17 lesion-like spots 18 2 strips A / A lesion-like spots 19 3 strips G / A none 20 3 strips G / A none 21 3 strips G / A none 22 3 strips G / A none

[0105] Example 3

[0106] Comparative verification of the CASP marker of this invention with the traditional SCN disease resistance identification method / KASP marker

[0107] Thirty BC1F2 strains with known genotypes (G / G, G / A, A / A) were selected and compared and validated using the CAPS marker of this invention, the traditional cyst seeding method, and the KASP marker. The detection indicators included time consumption, cost, equipment dependence, and genotyping accuracy. The results are as follows:

[0108] 1. The CAPS marker of this invention has low cost of consumables per sample, takes 1 day to detect (DNA extraction → PCR → enzyme digestion → electrophoresis), has a genotyping accuracy of 100%, and only requires conventional PCR instruments and electrophoresis equipment, without any special equipment dependence;

[0109] 2. Traditional cyst inoculation method: The cost of single sample testing is high, and the whole process takes 45 days (seedling cultivation → inoculation → culture → cyst counting). It is affected by environmental factors such as temperature, light, water and fertilizer, and the typing accuracy is about 96%. The operation is cumbersome and labor-intensive.

[0110] 3. KASP marker: The cost of single-sample detection is 60% higher than that of CAPS marker, the detection time is 1 day, the genotyping accuracy is 100%, but it depends on the real-time PCR instrument (equipment purchase cost is hundreds of thousands of yuan), which is difficult for grassroots breeding units to popularize.

[0111] In summary, the CAPS marker of this invention, under moderate throughput (e.g., detecting 10-200 samples per test), maintains the same detection efficiency and 100% genotyping accuracy as the KASP marker, while significantly reducing detection costs and equipment requirements. It also overcomes the shortcomings of traditional inoculation methods, such as long cycles and susceptibility to environmental interference. These results demonstrate that the CAPS marker of this invention not only achieves accurate genotyping but also possesses unique advantages in terms of low cost, no reliance on high-end equipment, and ease of grassroots promotion. It can significantly reduce the technical threshold and labor costs of SCN-resistant soybean breeding, making it a preferred method for small-scale screening in grassroots units and early-stage material screening in large-scale breeding.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A CAPS primer pair for detecting SNP sites, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO.5 and SEQ ID NO.6; The SNP site is located in soybean. SNAP18 c.797G>A in the 9th exon of the gene; The wild-type soybean SNAP18 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1, and the SNP site at position 797 is G; The disease-resistant mutant soybean SNAP18 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.2, and the SNP site at position 797 is A.

2. A type of soybean SNAP18 The genotype detection method is characterized by, The detection method includes the following steps: (1) Extract soybean genomic DNA. Extract genomic DNA from soybean samples and adjust the concentration to 50 ng / μL~200 ng / μL; (2) Using the DNA as a template, PCR amplification is performed using the primer pair described in claim 1 to obtain PCR amplification primers; (3) Take the PCR amplification product and use restriction endonuclease. Dde I undergoes an enzymatic digestion reaction to obtain the digested product; (4) The enzyme digestion products were detected by 4% agarose gel electrophoresis, and the genotype was determined based on the number and length of the bands.

3. The detection method according to claim 2, characterized in that, The PCR amplification reaction system described in step (2) includes, in 20 μL increments: 10 μL of 2×PCR Mix enzyme, 0.8 μL each of upstream and downstream primers at a concentration of 10 μmol / L, 1 μL of DNA template, and 7.4 μL of ddH2O; The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 3-5 min; 94℃ denaturation for 30 s, 54-58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 31-35 cycles; 72℃ final extension for 10 min.

4. The detection method according to claim 2, characterized in that, The enzyme digestion reaction system described in step (3), in 20 μL increments, includes: 10 μL of the PCR amplification product and a concentration of 10 U / μL of... Dde Incubate 1 μL of enzyme I, 2 μL of 10× enzyme digestion buffer, and 7 μL of ddH2O at 37°C for 2 h.

5. The detection method according to claim 2, characterized in that, The genotype determination criteria mentioned in step (4) are as follows: When a single band of 456bp in length is obtained, it is determined to be G / G wild-type homozygous; When two bands with lengths of 255bp and 201bp are obtained, it is determined to be an A / A homozygous mutant. When three bands with lengths of 456bp, 255bp, and 201bp are obtained, it is determined to be a G / A hybrid type.

6. The application of the primer pair of claim 1 or the detection method of any one of claims 2 to 5 in molecular marker-assisted breeding for resistance to soybean cyst nematode disease.

7. The primer pair according to claim 1 or the detection method according to any one of claims 2 to 5 in... SNAP18 Application in screening of superior allele hybrid offspring.

8. The application of the primer pair according to claim 1 or the detection method according to any one of claims 2 to 5 in the identification of transgenic soybean lines, characterized in that, The genetically modified organism includes SNAP18 The homologous SNP site of gene c.797G>A.

9. The application of the primer pair according to claim 1 or the detection method according to any one of claims 2 to 5 in the purity detection of disease-resistant soybean varieties, characterized in that, The disease-resistant varieties include those resistant to soybean cyst nematode disease.

Images