Primers and methods for detecting structural variations in cotton mon531 transgenic sequences

Abstract

This invention relates to the field of biotechnology, specifically to primers and methods for detecting transgenic sequence structural variations in cotton MON531. Primer combinations for screening transgenic sequence structural variations in cotton MON531 are provided, including primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8, and P0 / P9, whose nucleotide sequences are shown in SEQ ID NO:7 to SEQ ID NO:16. A method for screening transgenic sequence structural variations in cotton MON531 using the above primer pairs is also provided. The primer combinations and methods of this invention enable continuous monitoring of transgenic sequence structural variations during the cultivation of cotton MON531, laying the foundation for assessing the safety of its long-term application.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to primers and methods for detecting structural variations in the transgenic sequence of cotton MON531. Background Technology

[0002] Genetically modified cotton is one of the major genetically modified crops grown globally. Continuously tracking and monitoring transgenic sequence variations in cotton MON531 is an important aspect of assessing the safety of its long-term application.

[0003] Structural variation (SV) refers to large-scale sequence changes in the genome, including insertions or deletions, tandem repeats, and inversions of long sequences longer than 50 bp. While there are numerous reports on genomic structural variations in studies of genetic trait variation in plants and animals, there are currently no verifiable reports on them in the transgenic sequences of commercially grown genetically modified crops. Summary of the Invention

[0004] To track and monitor the variation of the MON531 transgenic cotton sequence, the inventors developed a rapid and sensitive screening method for structural variations in the MON531 transgenic sequence based on the structural characteristics of the MON531 transgenic insertion sequence. This method screened over 100 transgenic cotton materials containing structural variations of the MON531 transgenic sequence from more than 100,000 market cotton samples. Detailed studies were conducted on five of these structural variants, and specific detection methods for these five variants were established.

[0005] Based on the above research findings, this invention provides primer combinations for screening transgenic sequence structural variations in cotton MON531, comprising primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8, and P0 / P9, with the following nucleotide sequences:

[0006] P0: 5'-AAATTGTCACGGTCTACCAG-3';

[0007] P1: 5'-GAAAGAACTAAACAATTAGTACCC-3';

[0008] P2: 5'-CCTCGTGCTTTACGGTATCG-3';

[0009] P3: 5'-ATGGACAACAACCCAAACATCAACG-3';

[0010] P4: 5'-CCTCTCTATGGAACTATGGG-3';

[0011] P5: 5'-GTTAGACTCAACAGCAGTGG-3';

[0012] P6: 5'-GACGAGAAGGGTGAACTCTC-3';

[0013] P7: 5'-GATGTACTGACCTGAATGAG-3';

[0014] P8: 5'-GATCGAGAACAACACTGACG-3';

[0015] P9: 5'-AGGGAACCTTCATCGTGG-3'.

[0016] The present invention also provides a kit comprising the primer combination described above for screening transgenic sequence structural variations in cotton MON531.

[0017] Preferably, the kit further comprises universal reagents for polymerase chain reaction.

[0018] This invention also provides a method for screening transgenic sequence structural variations in cotton MON531, comprising:

[0019] (1) Extract genomic DNA from the cotton material to be tested;

[0020] (2) Using the genomic DNA as a template, PCR was performed using the primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9 respectively to obtain amplification products;

[0021] (3) Sequencing the amplified product and comparing the determined sequence with the original MON531 transgenic sequence to screen for MON531 transgenic sequences with structural variations.

[0022] Preferably, the PCR reaction system contains 0.5 μmol / L of forward primer and 0.5 μmol / L of reverse primer; the annealing temperature of the PCR is 56 °C and the extension time is 90 s.

[0023] This invention also provides primer combinations for detecting transgenic sequence structural variants of cotton MON531, comprising primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R, and R8877-F / R8877-R, with the following nucleotide sequences:

[0024] R1164-F: 5'-CTCATGCAGGAGCTGTCAGA-3';

[0025] R1164-R: 5'-CTATCAGTGTTTCTGGTTAT-3';

[0026] R7282-F: 5'-CAAGCGTCTCAGCGACGAGA-3';

[0027] R7282-R: 5'-TAAGAGGTATCATGTAGCCT-3';

[0028] R7569-F: 5'-CGTGAATGTCCCAGGTACTG-3';

[0029] R7569-R: 5'-CCGCCTTGGGTCTTGAAGAT-3';

[0030] R8442-F: 5'-AAATCTATCCCAACAACACC-3';

[0031] R8442-R: 5'-GTGTACACTGATAACATAGC-3';

[0032] R8877-F: 5'-ACTGAATAAGTATGTAGTACTA-3';

[0033] R8877-R: 5'-GGATCAGATTGTATATAATAC-3';

[0034] Specifically, R1164-F / R1164-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:1; R7282-F / R7282-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:2; R7569-F / R7569-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:3; R8442-F / R8442-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:4; and R8877-F / R8877-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:5.

[0035] The present invention also provides a kit comprising the primer combination described above for detecting transgenic sequence structural variants of cotton MON531.

[0036] The present invention also provides transgenic sequence structural variants of cotton MON531, the nucleotide sequences of which are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0037] This invention also provides a method for detecting transgenic sequence structural variants of cotton MON531, comprising:

[0038] (1) Extract genomic DNA from the cotton material to be tested;

[0039] (2) Using the genomic DNA as a template, PCR was performed using the primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R and R8877-F / R8877-R respectively to obtain amplification products;

[0040] (3) Detect the amplification product. If the product size is 233 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:1; if the product size is 254 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:2; if the product size is 240 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:3; if the product size is 239 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:4; if the product size is 259 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:5.

[0041] Preferably, the PCR reaction system contains 0.5 μmol / L of forward primer and 0.5 μmol / L of reverse primer; the annealing temperature of the PCR is 60 °C and the extension time is 30 s.

[0042] Experiments have shown that primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8, and P0 / P9 can rapidly screen transgenic cotton materials containing MON531 transgenic sequence structural variants (Example 1). Primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R, and R8877-F / R8877-R can specifically detect cotton MON531 transgenic sequence structural variants R1164, R7282, R7569, R8442, and R8877 (Example 2). Therefore, the primer combination and method of the present invention can realize continuous monitoring of transgenic sequence structural variations during the planting process of cotton MON531, laying the foundation for assessing the safety of its long-term application. Attached Figure Description

[0043] Figure 1 The positions of the screening primers for structural variations in the MON531 transgenic sequence are shown on the MON531 transgenic sequence; P0 is used as one-end primer, and P1–P9 are used as the other-end primers, respectively; P 35S Indicates the promoter (hollow arrow), T NOS and T 7S The empty square indicates the terminator, nptII indicates the marker gene nptII (solid arrow), aad indicates the marker gene aad (solid arrow), cry1Ac indicates the insect-resistant gene cry1Ac (solid arrow), and cry1Ac(t) indicates the truncated insect-resistant gene cry1Ac (solid arrow); the diagonal squares at both ends indicate the cotton genome.

[0044] Figure 2 The amplification results of the screening primers for structural variations of the MON531 transgenic sequence are shown in some cotton materials; P5 to P9 in the figure represent the amplification results of primer pairs P0 / P5, P0 / P6, P0 / P7, P0 / P8, and P0 / P9, respectively; R7569, R8442, and R8877 are the cotton material numbers.

[0045] Figure 3 The structural variations of five MON531 transgenic sequence structural variants relative to the original MON531 transgenic sequence are shown; R1164, R7282, R7569, R8442, and R8877 are structural variants of the cotton MON531 transgenic sequence; P 35S Indicates the promoter (hollow arrow), T NOS and T 7SThe empty square indicates the terminator; nptII indicates the marker gene nptII (solid arrow); aad indicates the marker gene aad (solid arrow); cry1Ac indicates the insect-resistant gene cry1Ac (solid arrow); cry1Ac(t) indicates the truncated insect-resistant gene cry1Ac (solid arrow); the diagonal squares at both ends indicate the cotton genome; the dashed line indicates a deletion.

[0046] Figure 4 The amplification results of five specific detection primers for MON531 transgenic sequence structural variants in cotton material are shown. Five agarose gel electrophoresis images are displayed from top to bottom, representing the amplification results of primer sets R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R, and R8877-F / R8877-R. Each agarose gel electrophoresis image is shown from left to right as lane M and lanes 1-24, where lanes 1-2 are... Variant material R1164, 5-6 are variant material R7282, 7-8 are variant material R7569, 11-12 are variant material R8442, 13-14 are variant material R8877, 3-4 and 9-10 are other variant materials, 15-16 are transgenic cotton MON531, 17-20 are non-transgenic cotton, 21-24 are blank control, M is DL2000 (molecular weight from top to bottom is 2000, 1500, 750, 500, 250, 100bp).

[0047] Sequence Description

[0048] SEQ ID NO:1 Nucleotide sequence of R1164, a transgenic structural variant of cotton MON531;

[0049] The nucleotide sequence of SEQ ID NO:2, the transgenic structural variant R7282 of cotton MON531;

[0050] The nucleotide sequence of SEQ ID NO:3, the transgenic structural variant R7569 of cotton MON531;

[0051] The nucleotide sequence of SEQ ID NO:4, the transgenic structural variant R8442 of cotton MON531;

[0052] SEQ ID NO:5 Nucleotide sequence of R8877, a transgenic structural variant of cotton MON531;

[0053] SEQ ID NO:6 Original transgenic sequence of cotton MON531;

[0054] The nucleotide sequences of primers P0 to P9, SEQ ID NO:7 to SEQ ID NO:16, used to screen for structural variations in the transgenic cotton MON531 sequence;

[0055] The nucleotide sequences of primers R1164-F, R1164-R, R7282-F, R7282-R, R7569-F, R7569-R, R8442-F, R8442-R, R8877-F, and R8877-R used to detect structural variants of the cotton MON531 transgenic sequence are SEQ ID NO:17 to SEQ ID NO:26. Detailed Implementation

[0056] The following implementation plan is provided:

[0057] 1. Primer combinations for screening transgenic sequence structural variations in cotton MON531, comprising primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8, and P0 / P9, with the following nucleotide sequences:

[0058] P0: 5'-AAATTGTCACGGTCTACCAG-3';

[0059] P1: 5'-GAAAGAACTAAACAATTAGTACCC-3';

[0060] P2: 5'-CCTCGTGCTTTACGGTATCG-3';

[0061] P3: 5'-ATGGACAACAACCCAAACATCAACG-3';

[0062] P4: 5'-CCTCTCTATGGAACTATGGG-3';

[0063] P5: 5'-GTTAGACTCAACAGCAGTGG-3';

[0064] P6: 5'-GACGAGAAGGGTGAACTCTC-3';

[0065] P7: 5'-GATGTACTGACCTGAATGAG-3';

[0066] P8: 5'-GATCGAGAACAACACTGACG-3';

[0067] P9: 5'-AGGGAACCTTCATCGTGG-3'.

[0068] 2. A kit comprising the primer combination described in embodiment 1 for screening transgenic sequence structural variations in cotton MON531.

[0069] 3. The kit described in Implementation Scheme 2 further comprises universal reagents for polymerase chain reaction.

[0070] 4. A method for screening transgenic sequence structural variations in cotton MON531, comprising:

[0071] (1) Extract genomic DNA from the cotton material to be tested;

[0072] (2) Using the genomic DNA as a template, PCR was performed on the primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8 and P0 / P9 described in Implementation Scheme 1 to obtain amplification products;

[0073] (3) Sequencing the amplified product and comparing the determined sequence with the original MON531 transgenic sequence to screen for MON531 transgenic sequences with structural variations.

[0074] 5. The method described in Implementation Scheme 4, wherein the PCR reaction system comprises 0.5 μmol / L of forward primer and 0.5 μmol / L of reverse primer; the annealing temperature of the PCR is 56 °C and the extension time is 90 s.

[0075] 6. Primer combinations for detecting transgenic sequence structural variants of cotton MON531, characterized in that they include primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R, and R8877-F / R8877-R, with the following nucleotide sequences:

[0076] R1164-F: 5'-CTCATGCAGGAGCTGTCAGA-3';

[0077] R1164-R: 5'-CTATCAGTGTTTCTGGTTAT-3';

[0078] R7282-F: 5'-CAAGCGTCTCAGCGACGAGA-3';

[0079] R7282-R: 5'-TAAGAGGTATCATGTAGCCT-3';

[0080] R7569-F: 5'-CGTGAATGTCCCAGGTACTG-3';

[0081] R7569-R: 5'-CCGCCTTGGGTCTTGAAGAT-3';

[0082] R8442-F: 5'-AAATCTATCCCAACAACACC-3';

[0083] R8442-R: 5'-GTGTACACTGATAACATAGC-3';

[0084] R8877-F: 5'-ACTGAATAAGTATGTAGTACTA-3';

[0085] R8877-R: 5'-GGATCAGATTGTATATAATAC-3';

[0086] Specifically, R1164-F / R1164-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:1; R7282-F / R7282-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:2; R7569-F / R7569-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:3; R8442-F / R8442-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:4; and R8877-F / R8877-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:5.

[0087] 7. A kit comprising the primer combination described in embodiment 6 for detecting transgenic sequence structural variants of cotton MON531.

[0088] 8. Transgenic structural variants of cotton MON531, the nucleotide sequences of which are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0089] 9. A method for detecting transgenic sequence structural variants of cotton MON531, comprising:

[0090] (1) Extract genomic DNA from the cotton material to be tested;

[0091] (2) Using the genomic DNA as a template, PCR was performed using the primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R and R8877-F / R8877-R as described in Implementation Scheme 6 to obtain amplification products;

[0092] (3) Detect the amplification product. If the product size is 233 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:1; if the product size is 254 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:2; if the product size is 240 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:3; if the product size is 239 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:4; if the product size is 259 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:5.

[0093] 10. The method described in Implementation Scheme 9, wherein the PCR reaction system comprises 0.5 μmol / L of forward primer and 0.5 μmol / L of reverse primer; the annealing temperature of the PCR is 60 °C and the extension time is 30 s.

[0094] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0095] Unless otherwise specified, all reagents used in the following examples are conventional reagents in the art, commercially available, or prepared according to conventional methods in the art. Experimental methods in the following examples that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001), or as recommended by the instrument or reagent manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0096] The main reagents used in the following examples are:

[0097] The plant genomic DNA extraction kit (DP305) was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0098] Go GreenMaster Mix and ddH2O were purchased from Promega Biotechnology Ltd.

[0099] The molecular weight marker DL2000 Marker was purchased from Dalian Takara Bio Engineering Co., Ltd.

[0100] 50×TAE electrophoresis buffer was purchased from Beijing Solarbio Biotechnology Co., Ltd.

[0101] Primer synthesis and Sanger sequencing were performed by Beijing Tsingke Biotech Co., Ltd.

[0102] The main instruments used in the following embodiments:

[0103] Pipettes (Eppendorf)

[0104] Bioprep-24 biological sample homogenizer (Hangzhou Ausen Instruments Co., Ltd.)

[0105] Dry-type thermostat (MK200-4, Hangzhou Aosheng Instrument Co., Ltd.)

[0106] Vacuum pump (Promega, Promega (Beijing) Biotechnology Co., Ltd.)

[0107] Ultra-micro UV spectrophotometer (Thermo Scientific NanoDrop2000, Thermo Fisher Scientific (China) Co., Ltd.)

[0108] High-speed centrifuge (Centrifuge 5425, Eppendorf)

[0109] PCR amplification instrument (Bio-RAD T100™ Thermal Cycler, Bio-Rad)

[0110] Nucleic acid electrophoresis apparatus (DYY-6C model, Beijing Liuyi Instrument Factory)

[0111] Gel imaging system (EC3-Imaging System_UVP, UVP Corporation)

[0112] Electronic balance (PL303-IC, Mettler Toledo Instruments (Shanghai) Co., Ltd.)

[0113] Example 1. Screening for structural variations in the MON531 transgenic sequence

[0114] From 2010 to 2023, commercial cotton seeds from major cotton-producing areas in my country were collected and planted at the experimental base of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Xinxiang City, Henan Province. Seeds were collected from individual cotton plants, resulting in a total of 120,000 cotton seed materials.

[0115] 1. Sample pretreatment

[0116] From 120,000 cottonseed samples, four seeds were randomly selected from each sample, and 100 samples were mixed to form a single sample, which was then ground into powder. To identify samples containing recombinant organisms, four seeds were randomly selected from each sample and ground individually.

[0117] 2. Extraction of cotton genomic DNA

[0118] Genomic DNA was extracted from cotton material using a plant genomic DNA extraction kit (TIANGEN, DP305) following the kit's instruction manual. 5 μl of the extracted DNA solution was subjected to 0.8% agarose gel electrophoresis; the quality of the DNA was initially assessed based on the brightness and pattern of the bands. The concentration and purity of the extracted DNA were determined using a UV spectrophotometer.

[0119] 3. Primer design for screening structural variations in the MON531 transgenic sequence

[0120] Based on the structural characteristics of the cotton MON531 transgenic sequence (SEQ ID NO:6), the focus was on structural variations occurring in the junction regions of its two insert fragments. Primer P0 was designed at the 7S terminator of the truncated insert fragment in the 3' region of the MON531 transgenic sequence, and nine primers P1–P9 were designed in the 5' region of the MON531 transgenic sequence. The corresponding positions of primers P0–P9 in the MON531 transgenic sequence are shown below. Figure 1 Its nucleotide sequence is shown in Table 1.

[0121] Table 1. Primers for screening structural variations of the MON531 transgenic sequence.

[0122]

[0123]

[0124] 4. PCR amplification and screening of structural variations in the MON531 transgenic sequence

[0125] Primer P0 was used as one end primer, and then combined with primers P1–P9 at the other end to obtain primer pairs P0 / P1, P0 / P2, P0 / P3, P0 / P4, P0 / P5, P0 / P6, P0 / P7, P0 / P8, and P0 / P9. Using genomic DNA from cotton material as a template, PCR was performed using these nine primer pairs. PCR reaction system: Go 10 μL GreenMaster Mix, 1 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), 2 μL DNA template, and ultrapure water to a total volume of 20 μL. Reaction program: 95℃ pre-denaturation for 4 min; (95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 90 s) 35 cycles; incubation at 72℃ for 10 min.

[0126] Amplification was performed on 1200 sample pools, and the amplified products were sent for sequencing. The sequenced sequences were analyzed and compared with the original transgenic sequence of MON531 to identify pools with structural variations. Then, the primer combination for identifying these structural variations was used to further identify each cottonseed material in the pools to determine the individual plants with structural variations. Electrophoretic patterns of amplified products from some cottonseed materials are shown below. Figure 2 .

[0127] The identification results showed that a total of 110 structural variant sequences with deletions at different positions on the complete insertion fragment at the 5' end of the original transgenic sequence of MON531 were obtained. Five representative variant materials, R1164, R7282, R7569, R8442 and R8877 (the numbers indicate the sequence deletion start position), were selected for further detailed analysis.

[0128] Example 2. Specific detection of the MON531 variant

[0129] The five representative variant materials R1164, R7282, R7569, R8442 and R8877 selected in Example 1 were planted in the laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Individual plants were identified and seeds were collected through self-pollination.

[0130] 1. Sample pretreatment

[0131] Cotton leaves or seeds should be ground evenly.

[0132] 2. Extraction of cotton genomic DNA

[0133] Genomic DNA was extracted from cotton material using a plant genomic DNA extraction kit (TIANGEN, DP305) following the kit's instruction manual. 5 μl of the extracted DNA solution was subjected to 0.8% agarose gel electrophoresis; the quality of the DNA was initially assessed based on the brightness and pattern of the bands. The concentration and purity of the extracted DNA were determined using a UV spectrophotometer.

[0134] 3. Determination of transgenic sequence structure of variants

[0135] Homozygous materials of the five variants were obtained through single-plant identification, self-pollination, and single-plant identification of progeny materials. Referring to the transgenic sequence of cotton MON531 (SEQ ID NO:6), the transgenic sequences of these five variants were obtained through PCR amplification and sequencing of the amplified fragments.

[0136] Determine sequence structure ( Figure 3 The results showed that the transgenic sequence lengths of the variant materials R1164, R7282, R7569, R8442 and R8877 were 3230bp, 9253bp, 9620bp, 10516bp and 10908bp, respectively, which were deleted by 7935bp, 1912bp, 1545bp, 649bp and 257bp, respectively, compared with the original transgenic sequence of MON531.

[0137] 4. Specific detection of variant materials

[0138] Specific primers were designed at both ends of the deletion regions in the five variant materials. The primer sequences, primer positions, and amplification fragment sizes are shown in Table 2.

[0139] Table 2 Primers for specific detection of the MON531 variant.

[0140]

[0141] Using genomic DNA from cotton materials as templates, PCR was performed using primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R, and R8877-F / R8877-R. The cotton materials included variants R1164, R7282, R7569, R8442, and R8877, other variant materials, transgenic cotton MON531, and non-transgenic cotton. Ultrapure water was used as a blank control instead of the DNA template.

[0142] PCR reaction system: Go 10 μL GreenMaster Mix, 1 μL each of forward primer (10 μmol / L) and reverse primer (10 μmol / L), 2 μL DNA template, and ultrapure water to a total volume of 20 μL. Reaction program: 95℃ pre-denaturation for 4 min; (95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s) 35 cycles; incubation at 72℃ for 10 min.

[0143] Test results ( Figure 4The results showed that primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R, and R8877-F / R8877-R could only amplify characteristic bands from their respective corresponding MON531 variant materials, and did not amplify in other cotton materials. Therefore, these five primer pairs can specifically detect the MON531 transgenic sequence structural variants R1164, R7282, R7569, R8442, and R8877.

Claims

1. A primer set for detecting transgenic sequence structural variants of cotton MON531, characterized in that, The primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R, and R8877-F / R8877-R have the following nucleotide sequences: R1164-F: 5'-CTCATGCAGGAGCTGTCAGA-3'; R1164-R: 5'-CTATCAGTGTTTCTGGTTAT-3'; R7282-F: 5'-CAAGCGTCTCAGCGACGAGA-3'; R7282-R: 5'-TAAGAGGTATCATGTAGCCT-3'; R7569-F: 5'-CGTGAATGTCCCAGGTACTG-3'; R7569-R: 5'-CCGCCTTGGGTCTTGAAGAT-3'; R8442-F: 5'-AAATCTATCCCAACAACACC-3'; R8442-R: 5'-GTGTACACTGATAACATAGC-3'; R8877-F: 5'-ACTGAATAAGTATGTAGTACTA-3'; R8877-R: 5'-GGATCAGATTGTATATAATAC-3'; Specifically, R1164-F / R1164-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:1; R7282-F / R7282-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:2; R7569-F / R7569-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:3; R8442-F / R8442-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:4; and R8877-F / R8877-R is used to detect structural variants of the MON531 transgenic sequence as shown in SEQ ID NO:

5.

2. A reagent kit, characterized in that, The primer combination for detecting transgenic sequence structural variants of cotton MON531 as described in claim 1 is included.

3. A transgenic sequence structural variant of cotton MON531, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:

5.

4. A method for detecting transgenic sequence structural variants of cotton MON531, characterized in that, include: (1) Extract genomic DNA from the cotton material to be tested; (2) Using the genomic DNA as a template, PCR was performed using the primer pairs R1164-F / R1164-R, R7282-F / R7282-R, R7569-F / R7569-R, R8442-F / R8442-R and R8877-F / R8877-R as described in claim 1 to obtain amplification products; (3) Detect the amplification product. If the product size is 233 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:1; if the product size is 254 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:2; if the product size is 240 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:3; if the product size is 239 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:4; if the product size is 259 bp, the cotton material to be tested contains the MON531 transgenic sequence structural variant with the nucleotide sequence shown in SEQ ID NO:

5.

5. The method according to claim 4, characterized in that, The PCR reaction system contains 0.5 μmol / L of forward primer and 0.5 μmol / L of reverse primer; the annealing temperature of the PCR is 60 °C and the extension time is 30 s.

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