Molecular markers, primer sets, kits and applications related to resistance to soybean cyst nematode disease

Abstract

The present invention belongs to the technical fields of molecular biology and genetic breeding, and specifically relates to molecular markers, primer sets, kits and applications related to resistance to soybean cyst nematode disease. The present invention for the first time provides two QTLs related to resistance to soybean cyst nematode disease: qSCN3-15 and qSCN3-18, develops a molecular marker SCN15.2 closely linked to qSCN3-15, and two molecular markers SCN18.1 and SCN18.2 closely linked to qSCN3-18. The molecular markers have a high correlation with resistance to soybean cyst nematode disease. When using the molecular markers to identify soybean varieties resistant to cyst nematode, the identification efficiency of SCN15.2 reaches 66.7%, the identification efficiency of SCN18.1 reaches 80%, and the identification efficiency of SCN18.2 reaches 69.2%.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and genetic breeding, and particularly relates to molecular markers, primer sets, kits and applications related to resistance to soybean cyst nematode disease. Background Art

[0002] Soybean cyst nematode disease is one of the main diseases of soybeans. When the disease occurs severely, the yield reduction can reach more than 80%, and soybeans cannot be planted again in severely diseased soil for 4 to 5 years, which causes great harm to soybean production.

[0003] Selecting new soybean varieties with resistance to soybean cyst nematode disease can fundamentally solve this problem. Conventional phenotypic identification of soybean cyst nematode resistance is time-consuming and expensive, vulnerable to the environment, destructive to the roots of materials, and it is difficult for transplanted plants to survive, resulting in slow progress in the identification of cyst nematode-resistant sources and the cultivation of new disease-resistant germplasms. Compared with phenotypic accurate identification, molecular marker-assisted selection can not only avoid the damage to the variety roots caused by disease resistance identification, but also has the advantages of high efficiency and low cost. Compared with the entire soybean genome, the currently developed functional markers are few and the identification efficiency is low. Satt309, which is only 0.4 cM away from the major locus rhg1, is the most widely used molecular marker in the early molecular marker-assisted selection of soybean cyst nematode resistance. Its disadvantages are that it cannot be detected by high throughput, and it can only select for disease-resistant Peking-type materials, and it cannot distinguish between disease-resistant material PI88788 and susceptible material Lee. Whether it is SSR, InDel or CAPS markers, they all have the problems of small throughput and high cost, and cannot meet the requirements of high-throughput detection, and have limitations in the application of molecular marker-assisted selection.

[0004] KASP (kompetitive allele specific PCR) is a new genotyping technology with the characteristics of low cost and high throughput. It accurately performs dual-allele genotyping on SNP and InDel sites through specific matching of the terminal bases of primers, and has been widely used in molecular marker-assisted selection of crops such as rice, wheat and soybeans. However, currently, the KASP markers developed for soybean cyst nematode resistance traits mainly focus on two loci, rhg1 and Rhg4, and there are still many molecular marker loci related to soybean cyst nematode resistance that have not been mined, which limits the application of molecular marker-assisted breeding technology in breeding soybeans with resistance to soybean cyst nematode disease. Summary of the Invention

[0005] Aiming at the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide molecular markers, primer sets, kits and applications related to soybean cyst nematode disease resistance. The molecular markers SCN15.2, SCN18.1 and SCN18.2, primer sets for amplifying the molecular markers and kits provided by the present invention can be used for the identification, assisted screening and mutant breeding of soybean varieties resistant to cyst nematodes.

[0006] In the first aspect of the present invention, a molecular marker related to soybean cyst nematode disease resistance is provided. The molecular marker is at least one of SCN15.2, SCN18.1 and SCN18.2. The nucleotide sequence of the molecular marker SCN15.2 is shown in SEQ ID NO.1, and has an A / G polymorphism at the 49th bp of this sequence; the nucleotide sequence of the molecular marker SCN18.1 is shown in SEQ ID NO.2, and has a C / T polymorphism at the 50th bp of this sequence; the nucleotide sequence of the molecular marker SCN18.2 is shown in SEQ ID NO.3, and has an A / T polymorphism at the 63rd bp of this sequence.

[0007] Further, the SCN15.2 is closely associated with the QTL locus qSCN3-15, and the SCN18.1 and SCN18.2 are closely associated with the QTL locus qSCN3-18.

[0008] Further, the qSCN3-15 is located in the interval of 117.54 cM to 118.278 cM on the 15th linkage group of soybean, and the qSCN3-18 is located in the interval of 0 cM to 0.495 cM on the 18th linkage group of soybean.

[0009] In the second aspect of the present invention, a primer set for amplifying the molecular marker related to soybean cyst nematode disease resistance is provided. The primer set is composed of two specific primers and one reverse universal primer; the nucleotide sequences of the two specific primers for amplifying SCN15.2 are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.6; the nucleotide sequences of the two specific primers for amplifying SCN18.1 are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.9; the nucleotide sequences of the two specific primers for amplifying SCN18.2 are shown in SEQ ID NO.10 and SEQ ID NO.11 respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.12.

[0010] In the third aspect of the present invention, a kit is provided, which contains any one or several of the primer sets for amplifying SCN15.2, the primer sets for amplifying SCN18.1, and the primer sets for amplifying SCN18.2.

[0011] In the fourth aspect of the present invention, an application of the molecular marker related to soybean cyst nematode disease resistance or the primer set in identifying soybean varieties resistant to cyst nematodes is provided.

[0012] In the fifth aspect of the present invention, an application of the molecular marker related to soybean cyst nematode disease resistance or the primer set in assisting the screening of soybean varieties resistant to cyst nematodes is provided.

[0013] Furthermore, the molecular marker related to soybean cyst nematode disease resistance or the primer set is used for mutation breeding of soybean varieties resistant to cyst nematodes.

[0014] In the sixth aspect of the present invention, a method for identifying soybean varieties resistant to cyst nematodes is provided, including the following steps:

[0015] Extract the DNA of the soybean to be tested;

[0016] Use any one of the primer sets for amplifying SCN15.2, the primer sets for amplifying SCN18.1, or the primer sets for amplifying SCN18.2 to perform PCR amplification on the extracted DNA of the soybean to be tested to obtain a PCR product;

[0017] Detect the gene typing of the PCR product at the polymorphic site, and determine whether the soybean to be tested is a soybean variety resistant to cyst nematodes according to the gene typing result.

[0018] Furthermore, when the genotype at the polymorphic site of SCN15.2 is GG, it is determined that the soybean sample to be tested is a soybean variety resistant to cyst nematodes; or when the genotype at the polymorphic site of SCN18.1 is CC, it is determined that the soybean sample to be tested is a soybean variety resistant to cyst nematodes; or when the genotype at the polymorphic site of SCN18.2 is AA, it is determined that the soybean sample to be tested is a soybean variety resistant to cyst nematodes.

[0019] Furthermore, each 1 μL of the PCR amplification reaction system includes: 4 ng of DNA template, 0.5 μL of 2×KASP Master mix, 0.014 μL of 72×Assay mix, and made up with deionized water; where 72×KASP Assay Mix is composed of two specific primers and one reverse universal primer, the concentrations of the two specific primers are both 0.168 μM, and the concentration of the reverse universal primer is 0.42 μM.

[0020] Furthermore, the reaction procedure for PCR amplification is as follows: (1) Heat activation; (2) Denaturation at 94°C for 20 s; Annealing: from 61°C to 55°C, for 60 s; Decrease by 0.6°C for each cycle, with a total of 10 cycles; (3) Denaturation: 94°C, 20 s; Annealing: 55°C, 60 s; A total of 26 cycles.

[0021] In summary, compared with the prior art, the present invention has the following beneficial advantages and effects:

[0022] (1) The present invention has developed three molecular markers SCN15.2, SCN18.1, and SCN18.2 related to the soybean cyst nematode resistance trait, and provided a primer set and a kit for amplifying the molecular markers. The molecular markers, primer set, and kit provided by the present invention can be used to assist in screening or identifying soybean varieties resistant to cyst nematodes. Among them, the identification efficiency of the molecular marker SCN15.2 is 66.7%, the identification efficiency of the molecular marker SCN18.1 is 80%, and the identification efficiency of the molecular marker SCN18.2 is 69.2%. Moreover, the combined identification efficiency of the two molecular markers SCN15.2 and SCN18.1 is 95.2%. When the three molecular markers SCN15.2, SCN18.1, and SCN18.2 are combined for identification, the identification efficiency is as high as 100%.

[0023] (2) The LOD value ranges of the two QTL loci related to soybean cyst nematode resistance provided by the present invention are greater than 5.730, and the maximum LOD value is 12.033. The contribution rates of qSCN3-15 and qSCN3-18 to the phenotype are 20.731% and 24.476% respectively, which can assist in mining related functional genes. The present invention also provides a method for identifying soybean varieties resistant to cyst nematodes, which can quickly, accurately, and effectively identify soybean materials with the trait of resistance to cyst nematodes. Description of the Drawings

[0024] Figure 1 It is a schematic diagram of a linkage map. The abscissa is the linkage group number, the ordinate is the position of the above-mentioned markers on the chromosome, and the black line represents the above-mentioned markers.

[0025] Figure 2 It is a QTL distribution map related to the soybean cyst nematode resistance trait. The abscissa is the linkage group number, the ordinate is the LOD value, and the gray and white interval background represents different linkage groups; Figure 2 In FIG. A, the black line is the LOD value distribution of the molecular marker and the trait, and the light gray line is the LOD threshold; Figure 2 In FIG. B, the line represents the additive effect at the corresponding position in FIG. A. Detailed Embodiments

[0026] To enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings.

[0027] In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0028] Biological materials involved in the examples:

[0029] Bainong 10, Jiyu 86, and differential hosts for soybean cyst nematode were all obtained from the Soybean Research Institute of Jilin Academy of Agricultural Sciences. Among them, Bainong 10 was labeled as BN10, and Jiyu 86 was labeled as JY86. The differential hosts for soybean cyst nematode were Peking, PI209332, PI437654, PI548316, PI88788, PI89772, and PI90763, respectively.

[0030] Soybean cyst nematode disease causes great harm to soybean production. Breeding new soybean varieties with resistance to soybean cyst nematode can fundamentally solve this problem. Conventional phenotypic identification of resistance to soybean cyst nematode is time-consuming and expensive, vulnerable to the environment, and destructive to the roots of materials, making it difficult for transplanted plants to survive. In the currently reported molecular marker-assisted breeding techniques, whether it is SSR, InDel, or CAPS markers, there are problems of low throughput and high cost, and they cannot meet the requirements of high-throughput detection.

[0031] The present invention provides molecular markers, primer sets, kits, and applications related to resistance to soybean cyst nematode. The present invention uses the disease-resistant soybean variety Bainong 10, the disease-susceptible soybean variety Jiyu 86, and the F 6 and F 7 generation recombinant inbred line population constructed with Bainong 10 and Jiyu 86 as parents as materials to construct a linkage map. By performing trait association analysis on the phenotypic values of soybean resistance to cyst nematode and SNP markers, two QTL loci, qSCN3-15 and qSCN3-18, related to the trait of soybean resistance to cyst nematode were obtained. Then, based on the qSCN3-15 and qSCN3-18 loci, three molecular markers, SCN15.2, SCN18.1, and SCN18.2, were developed, and primer sets for amplifying SCN15.2, SCN18.1, and SCN18.2, respectively, and a kit including amplification of SCN15.2, SCN18.1, and / or SCN18.2 were provided.

[0032] Example 1: Mapping of QTL and determination of molecular markers

[0033] 1. Population construction

[0034] Using the disease-resistant variety BN10 as the female parent and the disease-susceptible variety JY86 as the male parent, a hybrid combination was configured in 2014. Table 1 shows the process of obtaining the families.

[0035] Table 1 Process of Obtaining Families

[0036]

[0037] 2. Construction of Linkage Map

[0038] Using BN10, JY86 and an F population of 150 families and the F recombinant inbred line population as materials, a high-density genetic map and genotyping data of soybean were obtained. Taking each chromosome of soybean as a unit, analyzed by HighMap software, the arrangement order of markers on each chromosome was obtained, and the genetic distance between markers was estimated. Finally, a high-density genetic map with a total map distance of 2755.88 cM as shown in 6 and F 7 was obtained. The average map distance was 0.3 cM, including 9314 SNP markers, distributed on 20 linkage groups. Figure 1 Shown, with an average map distance of 0.3 cM, containing 9314 SNP markers, distributed in 20 linkage groups.

[0039] 3. QTL Mapping Results

[0040] The composite interval mapping method provided by R / qtl was used to conduct trait association analysis and QTL mapping between the phenotypic values of soybean resistance to cyst nematode and SNP markers. Set the threshold by PT testing 1000 times. The results are as shown in Figure 2 Shown. When the LOD value > 5.730, it indicates that the association between the soybean resistance to cyst nematode trait and the SNP marker reaches a highly significant level. Two QTL loci qSCN3-15 and qSCN3-18 related to the soybean resistance to cyst nematode trait were detected on chromosome 15 and chromosome 18 of soybean. The basic information of the associated regions is shown in Table 2.

[0041] Table 2 Basic Information of Associated Regions

[0042]

[0043] qSCN3-15 is located in the interval of 117.54 cM - 118.278 cM on chromosome 15. The maximum LOD value is 11.905, the marker additive effect is -14.720, and the phenotypic contribution rate is 20.731%. It is the major QTL locus for resistance to soybean cyst nematode.

[0044] qSCN3-18 is located in the interval of 0 cM - 0.495 cM on chromosome 18. The maximum LOD value is 12.033, and the additive effect of all markers is -15.995, indicating that its disease-resistant allele comes from BN10. The phenotypic contribution rate is 24.476%. It is the major QTL locus for resistance to soybean cyst nematode.

[0045] 4. Marker Development

[0046] Based on the qSCN3-15 and qSCN3-18 loci, 54 F 6：9 family plants in Table 1, BN10, JY86, Peking, PI209332, PI437654, PI548316, PI88788, PI89772, and PI90763 were selected as test materials, and SNP genotyping was performed using the KASP technology to develop three molecular markers SCN15.2, SCN18.1, and SCN18.2 related to the soybean cyst nematode resistance trait.

[0047] The nucleotide sequence of the molecular marker SCN15.2 is shown in SEQ ID NO.1, and the base at the 49bp of this sequence has A / G polymorphism;

[0048] The nucleotide sequence of the molecular marker SCN18.1 is shown in SEQ ID NO.2, and the base at the 50bp of this sequence has C / T polymorphism;

[0049] The nucleotide sequence of the molecular marker SCN18.2 is shown in SEQ ID NO.3, and the base at the 63bp of this sequence has A / T polymorphism.

[0050] SEQ ID NO.1: GAAGTTCACAAACATAGGTGGAAGCTCTCTGTAGCTTTGGGTTGAATTrTTGGTTGAAAAGAGCAATTGGACGATTTTGTTGCATG, r is A / G.

[0051] SEQ ID NO.2:

[0052] AATTCTAATAGCCAGAGTAAGAAACCAAATTACAGAAAAGACCCGAACAyTAAAGAAACTTTTTAGAATATAATTAAGCTACACTTGTACTCA, y is C / T.

[0053] SEQ ID NO.3:

[0054] TGTACTATACAGATTTCATTTGAACCCTGAATAGCTTCTGCAGTTACTTTAAGTCTTCATTCwTCAACCATGTGTTAGTTCTATCATTTTAAGCGTGCA, w is A / T.

[0055] Example 2: Design of Molecular Marker Primer Sets

[0056] Specific primer combinations were designed for the nucleotide sequences of SCN15.2, SCN18.1, and SCN18.2, respectively.

[0057] The primer set for amplifying SCN15.2 consists of two specific primers and one reverse universal primer. The nucleotide sequences of the two specific primers are shown in SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.6.

[0058] SEQ ID NO.4: 5’-ATCGTCCAATTGCTCTTTTCAACCAAT-3’.

[0059] SEQ ID NO.5: 5’-CGTCCAATTGCTCTTTTCAACCAAC-3’.

[0060] SEQ ID NO.6: 5’-GAAGCTCTCTGTAGCTTTGGGTTGAA-3’.

[0061] The primer set for amplifying SCN18.1 consists of two specific primers and one reverse universal primer. The nucleotide sequences of the two specific primers are shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.9.

[0062] SEQ ID NO.7: 5’-CCAAATTACAGAAAAGACCCGAACAC-3’.

[0063] SEQ ID NO.8: 5’-ACCAAATTACAGAAAAGACCCGAACAT-3’.

[0064] SEQ ID NO.9: 5’-CATAATGCTTTGAGTACAAGTGTAGCTTAA-3’.

[0065] The primer set for amplifying SCN18.2 consists of two specific primers and one reverse universal primer. The nucleotide sequences of the two specific primers are shown in SEQ ID NO.10 and SEQ ID NO.11, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.12.

[0066] SEQ ID NO.10: 5’-TAAAATGATAGAACTAACACATGGTTGAT-3’.

[0067] SEQ ID NO. 11: 5'-CTTAAAATGATAGAACTAACACATGGTTGAA-3'.

[0068] SEQ ID NO. 12: 5'-ACCCTGAATAGCTTCTGCAGTTACTTTAA-3'.

[0069] Example 3: Validation and application of molecular markers

[0070] Select 54 portions of F in Example 1 6：9 Family plants, BN10, JY86, Peking, PI209332, PI437654, PI548316, PI88788, PI89772 and PI90763 are soybean samples to be tested. DNA of the soybean samples to be tested is extracted, and the extracted DNA is PCR amplified using the primer set for amplifying SCN15.2, the primer set for amplifying SCN18.1 and the primer set for amplifying SCN18.2 in Example 2, respectively, to obtain PCR amplification products.

[0071] Each 1 μL PCR amplification reaction system includes: 4 ng DNA template, 0.5 μL 2×KASP Master mix, 0.014 μL 72×Assay mix, and deionized water. The 72×KASP Assay Mix includes two specific primers and one reverse universal primer. The concentrations of the two specific primers are both 0.168 μM, and the concentration of the reverse universal primer is 0.42 μM.

[0072] The PCR amplification reaction procedure was as follows: (1) thermal activation: 94°C, 15 min; (2) denaturation: 94°C, 20 s; annealing: 61°C~55°C, 60 s; the temperature was reduced by 0.6°C each cycle, for a total of 10 cycles; (3) denaturation: 94°C, 20 s; annealing: 55°C, 60 s; for a total of 26 cycles.

[0073] The PCR product is detected in a fluorescent quantitative PCR instrument, the fluorescence intensity signal value is read, and cluster analysis is performed to obtain the genotyping at the polymorphic site. According to the genotyping result, it is determined whether the soybean to be tested is a soybean variety resistant to cyst nematodes.

[0074] The judgment criteria are: when the genotype at the polymorphic site of the molecular marker SCN15.2 is GG, the soybean sample to be tested is judged to be a soybean variety resistant to cyst nematodes; or when the genotype at the polymorphic site of the molecular marker SCN18.1 is CC, the soybean sample to be tested is judged to be a soybean variety resistant to cyst nematodes; or when the genotype at the polymorphic site of the molecular marker SCN18.2 is AA, the soybean sample to be tested is judged to be a soybean variety resistant to cyst nematodes.

[0075] Calculate the identification efficiency of molecular marker disease resistance according to the following formula.

[0076] ;

[0077] After calculation, the identification efficiency of molecular marker SCN15.2 reaches 66.7%, the identification efficiency of molecular marker SCN18.1 reaches 80%, the identification efficiency of molecular marker SCN18.2 reaches 69.2%, and the combined identification efficiency of the two molecular markers SCN15.2 and SCN18.1 is 95.2%. When the three molecular markers SCN15.2, SCN18.1 and SCN18.2 are combined for identification, the identification efficiency is as high as 100%.

Claims

1. Use of a molecular marker associated with soybean cyst nematode resistance or a primer set for amplifying the molecular marker in identifying soybean varieties resistant to cyst nematodes, characterized in that: The molecular marker associated with soybean cyst nematode resistance is at least one of SCN15.2, SCN18.1 and SCN18.2, The nucleotide sequence of the molecular marker SCN15.2 is shown in SEQ ID NO. 1, and there is an A / G polymorphism at 49 bp of this sequence; The nucleotide sequence of the molecular marker SCN18.1 is shown in SEQ ID NO. 2, and there is a C / T polymorphism at 50 bp of this sequence; The nucleotide sequence of the molecular marker SCN18.2 is shown in SEQ ID NO. 3, and there is an A / T polymorphism at 63 bp of this sequence; The primer set is composed of two specific primers and one reverse universal primer; The nucleotide sequences of the two specific primers for amplifying SCN15.2 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.6; The nucleotide sequences of the two specific primers for amplifying SCN18.1 are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.9; The nucleotide sequences of the two specific primers for amplifying SCN18.2 are shown in SEQ ID NO.10 and SEQ ID NO.11 respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.

12.

2. Use of a molecular marker associated with soybean cyst nematode resistance or a primer set for amplifying the molecular marker in assisting the screening of soybean varieties resistant to cyst nematodes, characterized in that: The molecular marker associated with soybean cyst nematode resistance is at least one of SCN15.2, SCN18.1 and SCN18.2, The nucleotide sequence of the molecular marker SCN15.2 is shown in SEQ ID NO. 1, and there is an A / G polymorphism at 49 bp of this sequence; The nucleotide sequence of the molecular marker SCN18.1 is shown in SEQ ID NO. 2, and there is a C / T polymorphism at 50 bp of this sequence; The nucleotide sequence of the molecular marker SCN18.2 is shown in SEQ ID NO. 3, and there is an A / T polymorphism at 63 bp of this sequence; The primer set is composed of two specific primers and one reverse universal primer; The nucleotide sequences of the two specific primers for amplifying SCN15.2 are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.6; The nucleotide sequences of the two specific primers for amplifying SCN18.1 are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.9; The nucleotide sequences of the two specific primers for amplifying SCN18.2 are shown in SEQ ID NO.10 and SEQ ID NO.11 respectively, and the nucleotide sequence of the reverse universal primer is shown in SEQ ID NO.

12.

3. The use according to claim 2, characterized in that: The molecular marker or the primer set is used for mutant breeding of cyst nematode-resistant soybean varieties.

4. A method for identifying soybean varieties resistant to cyst nematodes, characterized in that: The following steps are involved: Extracting DNA from soybeans to be tested; Performing PCR amplification on the extracted soybean DNA to be tested using any one of the primer set for amplifying SCN15.2, the primer set for amplifying SCN18.1, or the primer set for amplifying SCN18.2 in claim 1 to obtain a PCR product; Detect the genotyping of PCR products at polymorphic sites, and determine whether the soybean to be tested is a soybean variety resistant to cyst nematodes based on the genotyping results: When the genotype at the polymorphic site of SCN15.2 is GG, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes; or When the genotype at the polymorphic site of SCN18.1 is CC, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes; or When the genotype at the polymorphic site of SCN18.2 is AA, the soybean sample to be tested is determined to be a soybean variety resistant to cyst nematodes.

5. The method for identifying cyst nematode-resistant soybean varieties according to claim 4, characterized in that: Each 1 μL PCR amplification reaction system includes: 4 ng DNA template, 0.5 μL 2×KASP Master mix, 0.014 μL 72×Assay mix, and deionized water. The 72×KASP Assay Mix is ​​composed of two specific primers and one reverse universal primer. The concentrations of the two specific primers are both 0.168 μM, and the concentration of the reverse universal primer is 0.42 μM.

6. The method for identifying cyst nematode-resistant soybean varieties according to claim 4, characterized in that: The reaction procedure of the PCR amplification is as follows: (1) heat activation; (2) denaturation at 94°C for 20 s; annealing at 61°C to 55°C for 60 s; decreasing by 0.6°C in each cycle, for a total of 10 cycles; (3) denaturation at 94°C for 20 s; annealing at 55°C for 60 s; for a total of 26 cycles.

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