KASP marker for detecting yellow 6106 in transgenic soybean and application of KASP marker

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a KASP marker for detecting yellow 6106 in transgenic soybeans and application of the KASP marker. The specific sequence where the KASP marker is located comprises ZH6106-2R and / or ZH6106-3R, and the specific sequence where the KASP marker is located comprises ZH6106-2R; the nucleotide sequence of the ZH6106-2R is as shown in SEQ ID NO: 1; the nucleotide sequence of the ZH6106-3R is as shown in SEQ ID NO: 2. Through the KASP marker and the primer, whether a sample to be detected contains the yellow 6106 component in the transgenic soybean or not can be effectively detected, and the detection method is rapid, simple, convenient, accurate and reliable.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a KASP marker for detecting transgenic soybean Zhonghuang 6106 and application thereof. BACKGROUND

[0002] Zhonghuang 6106 is a high glyphosate-tolerant transgenic soybean transformant obtained by using Agrobacterium-mediated cotyledon node method to superimpose g2-epsps and gat genes with different glyphosate tolerance mechanisms into Zhonghuang 10, and then screening by glyphosate. Cultivating new glyphosate-tolerant herbicide soybean varieties using Zhonghuang 6106 and promoting its application have important significance for improving the efficiency of weed control in the field, reducing production costs, improving planting benefits, and enhancing the competitiveness of domestic soybeans.

[0003] At present, the commonly used transformant detection methods include DNA-based and protein-based detection methods. The DNA-based detection methods include ordinary PCR and real-time fluorescent PCR, etc. The former has low detection cost but low sensitivity, and the latter has high sensitivity but high detection cost. The common deficiency of the two methods is low throughput, and it is difficult to efficiently detect a large number of samples. The protein-based detection methods include enzyme-linked immunosorbent assay and transgenic rapid detection test strips, which have the advantages of simplicity and rapidness, but the disadvantages are that the development of antibodies and the establishment of experimental systems are relatively complex, and the early development cost is high. It is difficult to realize high-throughput and low-cost specific detection of plant samples by using the above methods.

[0004] Competitive allele PCR (KASP) is a genotyping technology based on fluorescence detection, which detects the possible two genotypes of a locus in a sample through double-color fluorescence (FAM fluorescence and HEX fluorescence) in each reaction well. KASP genotyping technology has high sensitivity, low detection cost, and can be used for high-throughput detection, and has become an important technical means for molecular assisted breeding. However, there is no KASP technology for identifying transgenic soybean Zhonghuang 6106. SUMMARY

[0005] The purpose of the present application is to provide a KASP marker for detecting transgenic soybean Zhonghuang 6106 and application thereof. The KASP marker and primer of the present application can effectively detect whether the sample contains the components of transgenic soybean Zhonghuang 6106, and the detection method is rapid, simple, accurate and reliable.

[0006] The present invention provides a KASP marker for detecting Huang 6106 in transgenic soybeans. The specific sequence of the KASP marker includes ZH6106-2R and / or ZH6106-3R; the nucleotide sequence of the ZH6106-2R is shown in SEQ ID NO: 1; the nucleotide sequence of the ZH6106-3R is shown in SEQ ID NO: 2.

[0007] As a preferred embodiment, an insertion / deletion polymorphism of the exogenous sequence shown in SEQ ID NO:3 exists at 239bp-4977bp of SEQ ID NO:1; an insertion / deletion polymorphism of the exogenous sequence shown in SEQ ID NO:4 exists at 306bp-3961bp of SEQ ID NO:2.

[0008] The present invention also provides a primer set for detecting the above-mentioned KASP marker, including a primer set for specifically detecting ZH6106-2R and / or a primer set for specifically detecting ZH6106-3R. As a preferred embodiment, the primer set for specifically detecting ZH6106-2R includes Primer_AlleleFAM-2R, Primer_AlleleHEX-2R, and Primer_Common-2R; the nucleotide sequence of Primer_AlleleFAM-2R is shown in SEQ ID NO:5; the nucleotides 1-21 at the 5' end of Primer_AlleleFAM-2R are a specific linker sequence for labeling FAM fluorescence; the nucleotide sequence of Primer_AlleleHEX-2R is shown in SEQ ID NO:6; the nucleotides 1-21 at the 5' end of Primer_AlleleHEX-2R are a specific linker sequence for labeling HEX fluorescence; and the nucleotide sequence of Primer_Common-2R is shown in SEQ ID NO:7.

[0009] As a preferred solution, the primer set for specifically detecting ZH6106-3R comprises Primer_AlleleFAM-3R, Primer_AlleleHEX-3R and Primer_Common-3R; the nucleotide sequence of Primer_AlleleFAM-3R is shown as SEQ ID NO: 8; the 1-21 nucleotides at the 5' end of Primer_AlleleFAM-3R are specific linker sequences for labeling FAM fluorescence; the nucleotide sequence of Primer_AlleleHEX-3R is shown as SEQ ID NO: 9; the 1-21 nucleotides at the 5' end of Primer_AlleleHEX-3R are specific linker sequences for labeling HEX fluorescence; and the nucleotide sequence of Primer_Common-3R is shown as SEQ ID NO: 10.

[0010] The application further provides a kit for detecting the KASP marker, comprising the primer set and the PCR amplification reagent.

[0011] The application further provides the application of the KASP marker, the primer set or the kit, which comprises at least one of the following: specifically detecting transgenic soybean Zhonghuang 6106 and / or transgenic soybean Zhonghuang 6106 related materials; the transgenic soybean Zhonghuang 6106 related materials comprise parents and / or offspring; the detection object comprises one or more of plants, tissues, seeds and soybean products; assisting breeding of glyphosate-resistant soybeans; preparing products for assisting breeding of glyphosate-resistant soybeans; identifying or assisting in identifying soybean glyphosate resistance; preparing products for identifying or assisting in identifying soybean glyphosate resistance; breeding or assisting in breeding glyphosate-resistant soybeans; and preparing products for breeding or assisting in breeding glyphosate-resistant soybeans.

[0012] The application further provides a method for specifically detecting transgenic soybean Zhonghuang 6106 and / or transgenic soybean Zhonghuang 6106 related materials, comprising the following steps: using the DNA of the sample to be detected as a template, and using the primer set or the kit to perform PCR amplification; and judging whether the sample to be detected contains the components of transgenic soybean Zhonghuang 6106 according to fluorescence.

[0013] As a preferred solution, the system of the PCR amplification is 10.14 μL, including 5.0 μL of DNA template, 5.0 μL of 2xMaster Mix and 0.14 μL of primer mixture; the PCR amplification program is as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61-55℃ annealing for 60 s, 10 cycles of decreasing 0.6℃ each time; 94℃ denaturation for 20 s, 55℃ annealing for 60 s, 26 cycles.

[0014] The present invention also provides a method for breeding glyphosate-tolerant soybeans, comprising the following steps: hybridizing a soybean plant containing the transgenic soybean Zhonghuang 6106 component with another soybean plant, identifying the resulting progeny plants by detecting the KASP marker using the method described above, and selecting soybeans containing specific sequences for breeding to obtain soybeans with significantly improved tolerance to glyphosate herbicides; the specific sequences include ZH6106-2R and / or ZH6106-3R; the nucleotide sequence of ZH6106-2R is shown in SEQ ID NO: 1; and the nucleotide sequence of ZH6106-3R is shown in SEQ ID NO: 2.

[0015] Beneficial Effects: The present invention provides a competitive allele specific PCR (KASP) marker for detecting Huang 6106 in transgenic soybeans. The specific sequence of the KASP marker includes ZH6106-2R and / or ZH6106-3R; the nucleotide sequence of ZH6106-2R is shown in SEQ ID NO:1; the nucleotide sequence of ZH6106-3R is shown in SEQ ID NO:2; an insertion / deletion polymorphism of an exogenous sequence as shown in SEQ ID NO:3 exists at 239bp-4977bp of SEQ ID NO:1; and an insertion / deletion polymorphism of an exogenous sequence as shown in SEQ ID NO:4 exists at 306bp-3961bp of SEQ ID NO:2. The KASP marker and primers of the present invention can effectively detect whether a sample contains Huang 6106 in transgenic soybeans. The detection method is rapid, simple, accurate, and reliable.

[0016] The test results from the examples of the present invention demonstrate that the KASP markers and detection methods of the present invention not only possess high accuracy, a 100% detection rate, and clear typing, but also enhance the reliability of the results through dual-labeling verification, while also offering practical benefits such as rapidity, simplicity, and ease of scalability. The KASP markers and detection methods of the present invention enable high-throughput sample testing, and the soybeans identified as containing the transgenic soybean Zhonghuang 6106 component exhibit high tolerance to the herbicide glyphosate. This method fully meets the needs for accurate identification, breeding assistance, and regulatory testing of transgenic soybean Zhonghuang 6106, providing an efficient, stable, and reliable molecular detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0018] Figure 1Figure 2 shows the different sample genotyping results detected by ZH6106-2R specific fragment in Example 2, wherein the blue dot is the genotype without the insertion fragment "-"; the red dot is the genotype with the insertion fragment "GGTGT…"; and the black dot is the blank control.

[0019] Figure 2 Figure 3 shows the different sample genotyping results detected by ZH6106-3R specific fragment in Example 2, wherein the red dot is the genotype without the insertion fragment "-"; the green dot is the genotype with the insertion fragment "- / AACAC"; and the black dot is the blank control. DETAILED DESCRIPTION

[0020] The present application provides a KASP marker for detecting ZH6106 in transgenic soybean, wherein the specific sequence of the KASP marker comprises ZH6106-2R and / or ZH6106-3R. As a specific embodiment, the nucleotide sequence of ZH6106-2R is shown in SEQ ID NO: 1. As a specific embodiment, the nucleotide sequence of ZH6106-3R is shown in SEQ ID NO: 2. As a specific embodiment, there is an insertion / deletion polymorphism of the exogenous sequence shown in SEQ ID NO: 3 at 239bp-4977bp of the sequence of SEQ ID NO: 1; the nucleotide sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 3. As a specific embodiment, there is an insertion / deletion polymorphism of the exogenous sequence shown in SEQ ID NO: 4 at 306bp-3961bp of the sequence of SEQ ID NO: 2; the nucleotide sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 4.

[0021] The present invention also provides a primer set for detecting the above-mentioned KASP marker, including a primer set for specifically detecting ZH6106-2R and / or a primer set for specifically detecting ZH6106-3R. As a specific embodiment, the primer set for specific detection of ZH6106-2R includes Primer_AlleleFAM-2R, Primer_AlleleHEX-2R and Primer_Common-2R; Primer_AlleleFAM-2R (SEQ ID NO: 5): 5'-GAAGGTGACCAAGTTCATGCTGTTTGAGACTGTAAATCACCCAC-3'; the 1-21 nucleotides at the 5' end of the Primer_AlleleFAM-2R are a specific linker sequence for labeling FAM fluorescence; Primer_AlleleHE X-2R (SEQ ID NO: 6): 5'-GAAGGTCGGAGTCAACGGATTTGCGTGTGTAGTATGATTCATTGTT-3'; the 1-21 nucleotides at the 5' end of the Primer_AlleleHEX-2R are a specific linker sequence for labeling HEX fluorescence; Primer_Common-2R (SEQ ID NO:7): 5'-AGAGAAAGGTGTGAAATGTGAACG-3'. As a specific embodiment, the primer set for specific detection of ZH6106-3R includes Primer_AlleleFAM-3R, Primer_AlleleHEX-3R and Primer_Common-3R; Primer_AlleleFAM-3R (SEQ ID NO: 8): 5'-GAAGGTGACCAAGTTCATGCTAGCTTGA ACACCTTGAGTGGAGT-3'; nucleotides 1-21 at the 5' end of the Primer_AlleleFAM-3R are a specific linker sequence for labeling FAM fluorescence; Primer_AlleleHEX-3R (SEQ ID NO: 9): 5'-GAAGGTCGGAGTCAACGGATTGATTGTCGTTTCCCGCCTTCAGT-3'; nucleotides 1-21 at the 5' end of the Primer_AlleleHEX-3R are a specific linker sequence for labeling HEX fluorescence; Primer_Common-3R (SEQ ID NO: 10): 5'-TCAAATCCTATGGGCATTCT TCC-3'.As a specific embodiment, at least one primer set of the present invention can be used to specifically identify transgenic soybean Zhonghuang 6106 and / or transgenic soybean Zhonghuang 6106-related materials. Using two primer sets together for detection results in more accurate detection. Information on the transgenic soybean Zhonghuang 6106 described in the present invention can be found in patent application number 202510453111.6, publication number CN120210227A, and titled "Specific Sequence of Transgenic Soybean Zhonghuang 6106 and Its Applications."

[0022] The present invention also provides a kit for detecting the aforementioned KASP marker, comprising the aforementioned primer set and a PCR amplification reagent. In a specific embodiment of the present invention, the PCR amplification reagent is 2×MasterMix.

[0023] The present invention also provides applications of the above-mentioned KASP marker, the above-mentioned primer set or the above-mentioned kit, wherein the applications include at least one of the following: specific detection of transgenic soybean Zhonghuang 6106 and / or transgenic soybean Zhonghuang 6106-related materials; the transgenic soybean Zhonghuang 6106-related materials include parents and / or offspring; detection objects include one or more plants, tissues, seeds and soybean products; assisted breeding of glyphosate-resistant soybeans; preparation of products for assisted breeding of glyphosate-resistant soybeans; identification or assisted identification of soybean glyphosate resistance; preparation of products for identifying or assisted identification of soybean glyphosate resistance; breeding or assisted breeding of glyphosate-resistant soybeans; preparation of products for breeding or assisted breeding of glyphosate-resistant soybeans.

[0024] The present invention also provides a method for specifically detecting transgenic soybean Huang 6106 and / or transgenic soybean Huang 6106-related materials, comprising the following steps: using DNA from a test sample as a template, performing PCR amplification using the aforementioned primer set or kit, and determining whether the test sample contains transgenic soybean Huang 6106 components based on fluorescence. In one embodiment, the PCR amplification system, calculated as 10.14 μL, comprises: 5.0 μL DNA template, 5.0 μL 2× MasterMix, and 0.14 μL primer mixture. In one embodiment, the PCR amplification procedure is as follows: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing at 61-55°C for 60 seconds, decreasing the temperature by 0.7°C each cycle, for 10 cycles; denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, decreasing the temperature by 0.7°C each cycle, for 26 cycles.

[0025] In the embodiment of the present application, when the KASP marker with the specific sequence of ZH6106-2R is used for detection, if the FAM fluorescence signal is detected, it indicates that the exogenous fragment is inserted, and the sample to be tested contains the composition of transgenic soybean Zhonghuang 6106; if the HEX fluorescence signal is detected, it indicates that the exogenous fragment is not inserted, and the sample to be tested does not contain the composition of transgenic soybean Zhonghuang 6106. When the KASP marker with the specific sequence of ZH6106-3R is used for detection, if the FAM fluorescence signal is detected, it indicates that the exogenous fragment is not inserted, and the sample to be tested does not contain the composition of transgenic soybean Zhonghuang 6106; if the HEX fluorescence signal is detected, it indicates that the exogenous fragment is inserted, and the sample to be tested contains the composition of transgenic soybean Zhonghuang 6106.

[0026] In the embodiment of the present application, 382 samples are detected, and the detection results are 100% consistent with the expected results, without false positives or false negatives. The detection rate of the two KASP markers is more than 99%, whether for samples containing exogenous fragments or for samples not containing exogenous fragments. It is proved that the sensitivity and specificity of the KASP marker of the present application are sufficient to cover samples with different genetic backgrounds (including derived offspring and non-derived offspring), and even in the case of increasing sample quantity or more complex genetic diversity, stable detection can still be achieved, and the applicability is strong. At the same time, the detection period of the present application is short, the operation steps are simplified, and it is suitable for high-throughput screening (such as large-scale seedling detection in breeding, seed purity identification, processing product traceability and the like), which significantly improves the detection efficiency.

[0027] The present application also provides a breeding method of glyphosate-resistant soybean, comprising the following steps: crossing a soybean plant containing the composition of transgenic soybean Zhonghuang 6106 with another soybean plant, using the above-mentioned method to detect the KASP marker to identify the offspring plants, selecting soybean containing specific sequences for breeding, and obtaining soybean with significantly improved tolerance to glyphosate herbicide; the specific sequence comprises ZH6106-2R and / or ZH6106-3R; the nucleotide sequence of ZH6106-2R is shown as SEQ ID NO: 1; the nucleotide sequence of ZH6106-3R is shown as SEQ ID NO: 2. In order to further illustrate the present application, the KASP marker for detecting transgenic soybean Zhonghuang 6106 and its application provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0028] Unless otherwise specified, the raw materials used in the present application have no special requirements and commercially available products known to those skilled in the art can be used.

[0029] The experimental reagents used in the present application are shown in Table 1, and the instruments used are shown in Table 2.

[0030] Table 1 Reagents and sources

[0031] Reagent name Reagent source Catalogue number KASPTM Indirect LGC Corporation KBS-2100-100-OLI Primer LGC Corporation KBD Assay RNase-free water Tiangen RT121-02

[0032] Table 2 Instruments and Models

[0033] Instrument name Instrument source Model number PCR machine ABI Corporation 9700 Centrifuge Eppendorf 5418 NanoDrop Thermo 2000 Vortex mixer IKA QL-901 Centrifuge Eppendorf 5810R Quantitative PCR machine ABI Corporation 7900

[0034] In the present application, "GGTGT..." means the sequence as shown in SEQ ID NO: 3, and "AACAC..." means the sequence as shown in SEQ ID NO: 4.

[0035] Example 1

[0036] 1. Primer design: Primers were designed for specific sequences ZH6106-2R and ZH6106-3R using Primer Premier 6 software, see Table 3 for specific sequences and primer design. Primer_Common-2R (SEQ ID NO: 7) is a common primer designed for the common sequence part of both variants of ZH6106-2R, Primer_AlleleFAM-2R (SEQ ID NO: 5) is a specific primer designed for the insertion variant sequence and contains a specific adaptor sequence for labelling FAM fluorescence at the 5' end, Primer_AlleleHEX-2R (SEQ ID NO: 6) is a specific primer designed for the deletion variant sequence and contains a specific adaptor sequence for labelling HEX fluorescence at the 5' end, where the specific adaptor sequence is indicated by lower case letters. Primer_Common-3R (SEQ ID NO: 10) is a common primer designed for the common sequence part of both variants of ZH6106-3R, Primer_AlleleFAM-3R (SEQ ID NO: 8) is a specific primer designed for the deletion variant sequence and contains a specific adaptor sequence for labelling FAM fluorescence at the 5' end, Primer_AlleleHEX-3R (SEQ ID NO: 9) is a specific primer designed for the insertion variant sequence and contains a specific adaptor sequence for labelling HEX fluorescence at the 5' end, where the specific adaptor sequence is indicated by lower case letters.

[0037] Table 3 Sequences and primers

[0038]

[0039]

[0040] 2. DNA sample processing: Extract DNA from 382 samples to be tested, and use NanoDrop spectrophotometer to determine the concentration of the samples to be tested. Dilute the DNA samples to about 50 ng / μL based on the concentration of the tested DNA samples and dispense them into 384-well plates for subsequent experiments.

[0041] 3. KASP labeling detection: (1) Preparation of working solution: Prepare the working solution according to Table 4. Add the corresponding volume of 2× Master reaction mixture and primer mixture according to the number of detection reactions required. Carefully check the name and amount of the added reagents during preparation. After preparation, gently pipette 5 times to mix, and centrifuge at 3000 rpm.

[0042] Table 4 Preparation of working solution

[0043] Component Per reaction volume (μL) n reaction volumes (μL) 2x Master mix 5 5×n Primer mix (100 μM concentration of each primer) 0.14 0.14×n

[0044] (2) Working solution aliquoting: aliquot 5.14 μL of the prepared working solution into each well of a 384-well plate according to the order of the wells in the plate.

[0045] (3) DNA sample packaging: Take 5.0 μL of the DNA with adjusted concentration and add it to the corresponding position of the 384-well plate according to the well plate layout.

[0046] (4) Blank control setting: Add two blank control reagents at the corresponding positions according to the well plate arrangement table, including 5.14 μL working solution and 5.0 μL sterile water, which does not contain DNA components.

[0047] (5) Sealing the 384-well plate: Seal the plate with a special sealing film and scrape the plate tightly around the edges. Place the 384-well plate in a 4°C centrifuge and centrifuge briefly at 1200 rpm to collect the sample at the bottom of the well. If there are bubbles at the bottom of the well, flick it twice and centrifuge again at 1200 rpm.

[0048] (6) Perform PCR amplification according to the PCR program in Table 5.

[0049] Table 5 PCR program

[0050]

[0051] 5. Data scanning: (1) After the PCR is completed, remove the plate and perform data scanning. For details, refer to the "ABI7900 HTFast RealTime PCR System Experimental Procedure"

[0052] Example 2

[0053] 1. Using the method of example 1, 382 samples were detected, and the sample detection rate results are shown in Table 6. Using the KASP marker ZH6106-2R, 379 samples can be detected, and the sample detection rate is 99.2%; using the KASP marker ZH6106-3R, 382 samples can be detected, and the sample detection rate is 100%.

[0054] Table 6 Sample detection rate

[0055] ID Total number of samples Number of detections Detection rate ZH6106-2R 382 379 9. ZH6106-3R 382 382 1.

[0056] 2. The statistics of different sample typing results are shown in Table 7 and Figure 1 . Using the ZH6106-2R marker detection, 379 of the 382 samples can be detected, of which 202 samples do not contain foreign fragments, and 177 samples contain foreign fragments, and the 177 samples contain the components of transgenic soybean Zhonghuang 6106; using the ZH6106-3R marker detection, 382 samples (all detection sites are homozygous), 204 samples do not contain foreign fragments, and 178 samples contain foreign fragments, and the 178 samples contain the components of transgenic soybean Zhonghuang 6106.

[0057] Table 7 Statistics of different sample typing results

[0058]

[0059]

[0060] 3. Sample detection result analysis: The sample detection results are shown in Table 8. As expected, the samples containing inserted foreign fragments and containing the components of transgenic soybean Zhonghuang 6106 can tolerate glyphosate herbicide after spraying glyphosate herbicide, and the samples not containing inserted fragments and not containing the components of transgenic soybean Zhonghuang 6106 are not resistant to glyphosate herbicide after spraying glyphosate herbicide. The detection results are consistent with the expectation, indicating that the detection method of the present application is fast, simple, accurate and reliable.

[0061] Table 8 Analysis of sample detection results

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071] Therefore, the KASP marker specific sequence of the application includes ZH6106-2R and / or ZH6106-3R; the nucleotide sequence of the ZH6106-2R is shown as SEQ ID NO:1; and the nucleotide sequence of the ZH6106-3R is shown as SEQ ID NO:2. The KASP marker and primer of the application can effectively detect whether the sample to be detected contains the component of transgenic soybean Zhonghuang 6106, and the detection method is rapid, simple, accurate and reliable.

[0072] Although the above embodiment has made a detailed description of the application, it is only a part of the embodiment of the application, not all the embodiments, and people can also obtain other embodiments according to the embodiment without creativity, which all belong to the protection scope of the application.

Claims

1. A KASP marker for detecting Huang 6106 in transgenic soybeans, characterized in that: The specific sequence of the KASP marker includes ZH6106-2R and / or ZH6106-3R; The nucleotide sequence of ZH6106-2R is shown in SEQ ID NO: 1; The nucleotide sequence of ZH6106-3R is shown in SEQ ID NO: 2; There is an insertion / deletion polymorphism of the exogenous sequence shown in SEQ ID NO: 3 at 239 bp-4977 bp of the sequence of SEQ ID NO: 1; At 306 bp to 3961 bp of SEQ ID NO: 2, there is an insertion / deletion polymorphism of the exogenous sequence shown in SEQ ID NO:

4.

2. A primer set for detecting the KASP marker according to claim 1, characterized in that: It includes a primer set for specifically detecting ZH6106-2R and / or a primer set for specifically detecting ZH6106-3R.

3. The primer set according to claim 2, characterized in that The primer set for specific detection of ZH6106-2R includes Primer_AlleleFAM-2R, Primer_AlleleHEX-2R and Primer_Common-2R; The nucleotide sequence of the Primer_AlleleFAM-2R is shown in SEQ ID NO: 5; the nucleotides 1-21 at the 5' end of the Primer_AlleleFAM-2R are specific linker sequences for labeling FAM fluorescence; The nucleotide sequence of Primer_AlleleHEX-2R is shown in SEQ ID NO: 6; nucleotides 1-21 at the 5' end of Primer_AlleleHEX-2R are specific linker sequences for labeling HEX fluorescence; The nucleotide sequence of Primer_Common-2R is shown in SEQ ID NO:

7.

4. The primer set according to claim 2, characterized in that The primer set for specific detection of ZH6106-3R includes Primer_AlleleFAM-3R, Primer_AlleleHEX-3R and Primer_Common-3R; The nucleotide sequence of the Primer_AlleleFAM-3R is shown in SEQ ID NO: 8; the nucleotides 1-21 at the 5' end of the Primer_AlleleFAM-3R are a specific linker sequence for labeling FAM fluorescence; The nucleotide sequence of Primer_AlleleHEX-3R is shown in SEQ ID NO: 9; nucleotides 1-21 at the 5' end of Primer_AlleleHEX-3R are specific linker sequences for labeling HEX fluorescence; The nucleotide sequence of Primer_Common-3R is shown in SEQ ID NO:

10.

5. A kit for detecting the KASP marker according to claim 1, characterized in that: The method comprises the primer set according to any one of claims 2 to 4 and a PCR amplification reagent.

6. Use of the KASP marker according to claim 1, the primer set according to any one of claims 2 to 4, or the kit according to claim 5, characterized in that: The application includes at least one of the following: Specific detection of transgenic soybean Zhonghuang 6106 and / or transgenic soybean Zhonghuang 6106-related materials; the transgenic soybean Zhonghuang 6106-related materials include parents and / or offspring; detection objects include one or more plants, tissues, seeds and soybean products; assisted breeding of glyphosate-tolerant soybeans; Preparation of products to assist in the breeding of glyphosate-tolerant soybeans; Identify or assist in identifying soybean glyphosate tolerance characteristics; Preparation of products for identifying or assisting in identifying soybean glyphosate resistance; Breeding or assisting in the breeding of glyphosate-tolerant soybeans; Prepare products for breeding or assisting in breeding glyphosate-tolerant soybeans.

7. A method for specifically detecting Huang 6106 in transgenic soybeans and / or Huang 6106-related materials in transgenic soybeans, characterized in that: The following steps are involved: The DNA of the test sample is used as a template, and PCR amplification is performed using the primer set described in any one of claims 2 to 4 or the kit described in claim 5. Whether the test sample contains the component of Huang 6106 in the genetically modified soybean is determined based on the fluorescent signal.

8. The method according to claim 7, characterized in that The PCR amplification system, calculated as 10.14 μL, comprises: 5.0 μL of DNA template, 5.0 μL of 2×MasterMix, and 0.14 μL of primer mixture.

9. The method according to claim 7, characterized in that The PCR amplification program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing at 61-55°C for 60 s, decreasing the temperature by 0.6°C each cycle, for 10 cycles; denaturation at 94°C for 20 s, annealing at 55°C for 60 s, for 26 cycles.

10. A method for breeding glyphosate-tolerant soybeans, characterized in that: The following steps are involved: Crossing a soybean plant containing the transgenic soybean Zhonghuang 6106 component with another soybean plant, identifying the resulting progeny plants by detecting the KASP marker using the method described in any one of claims 7 to 9, and selecting soybeans containing the specific sequence for breeding to obtain soybeans with significantly improved tolerance to glyphosate herbicide; The specific sequence includes ZH6106-2R and / or ZH6106-3R; The nucleotide sequence of ZH6106-2R is shown in SEQ ID NO: 1; The nucleotide sequence of ZH6106-3R is shown in SEQ ID NO: 2.

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