Method for cultivating insect-resistant and glyphosate herbicide-resistant cotton variety

Abstract

The invention belongs to the field of biological breeding, and particularly discloses a method for cultivating an insect-resistant and glyphosate herbicide-resistant cotton variety, which comprises the following steps of: hybridizing or backcrossing by taking transgenic cotton GBS8-2 or a derivative line thereof as one of parents and taking a cotton variety with excellent agronomic traits as the other parent for 4-5 generations continuously; a new cotton variety with good insect resistance and high glyphosate herbicide concentration can be bred; wherein the preservation number of the GBS8-2 is CGMCC (China General Microbiological Culture Collection Center) No. 46278. The invention also provides a molecular marker for identifying the GBS8-2 and a filial generation. The insect-resistant gene and the glyphosate-resistant gene in the transgenic cotton GBS8-2 are single insertion sites and single copies, are simple in heredity and are easy to apply in breeding; secondly, the insect-resistant gene provided by the invention is a fusion gene, and compared with a single resistance gene, the insect-resistant gene provided by the invention has the advantage that insects are not easy to generate resistance. In addition, the molecular marker is accurate and reliable and can be used for auxiliary selection in the seedling stage, and the breeding cost is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biological breeding, and in particular relates to a method for cultivating insect-resistant and glyphosate-resistant cotton varieties. Background Art

[0002] Damage caused by cotton bollworms and weeds is a major challenge for cotton production. Transgenic cotton, incorporating Bt insecticide and herbicide-tolerant genes into cotton through transgenic technology, is an effective way to combat these problems. Currently, insect-resistant and herbicide-tolerant transgenic cotton is widely used internationally.

[0003] Monsanto's Cry1Ac transgenic insect-resistant cotton and Cry1Ab transgenic insect-resistant corn were the first commercial insect-resistant crops internationally. In 1992, my country introduced the synthetic GFM Cry1A gene into cotton, successfully producing the first generation of domestically produced single-value insect-resistant cotton and beginning industrial application. However, with the widespread adoption of insect-resistant cotton, lepidopteran pests such as cotton bollworms developed resistance to single-strength insecticidal proteins, rendering crops like transgenic Bt cotton ineffective in killing pests (Tabashnik et al., Nature Biotechnology, 2008). Liao et al. (Journal of Invertebrate Pathology, 2002) found that different Bt proteins have different insecticidal mechanisms against the same pest, making it difficult for pests to develop cross-resistance to two proteins. Therefore, co-expressing two or more Bt proteins can not only enhance the insecticidal activity of transgenic insect-resistant crops but also prevent pests from developing resistance to them.

[0004] Glyphosate is a non-selective, lethal herbicide and one of the most effective weed control agents. However, it can also severely harm crop growth and development. The discovery and application of the glyphosate-tolerant EPSPS gene in crops has addressed this issue. Guo Sandui et al. (Patent ZL2014102047036) simultaneously introduced the glyphosate-tolerant GR79 EPSPS gene and the GAT gene into cotton, resulting in a bivalent transgenic cotton plant resistant to four times the concentration of glyphosate used in production. This plant has now been used in production.

[0005] The construction of co-expression vectors containing multiple genes, including insect-resistant and herbicide-tolerant genes, allows for the simultaneous introduction of both genes into cotton. This approach can address the challenges of bollworm and weed damage in cotton production while also improving transgenic efficiency. However, during the transgenic process, the T-DNA insertion site is random, and the copy number is uncertain. This makes obtaining single-copy transformants that efficiently express both insect-resistant and herbicide-tolerant genes a challenge in achieving transgenic cotton that is both insect-resistant and herbicide-tolerant. Summary of the Invention

[0006] The present invention aims to provide a method for cultivating insect-resistant and glyphosate-resistant cotton varieties.

[0007] Another object of the present invention is to provide a molecular marker for identifying cotton with insect-resistant and glyphosate-resistant genes.

[0008] The third object of the present invention is to provide a primer set for amplifying the above-mentioned molecular markers.

[0009] The fourth object of the present invention is to provide a method for using the above molecular markers to assist in the selection of cotton varieties containing insect-resistant genes and glyphosate-resistant genes.

[0010] To achieve the above object, the technical solution of the present invention is as follows:

[0011] A method for breeding insect-resistant and glyphosate-resistant cotton varieties comprises using GBS8-2 or a derivative of GBS8-2 as one parent and another cotton variety with excellent agronomic and quality traits as the other parent, selecting individual plants with homozygous T-DNA, good insect resistance, and strong glyphosate-resistant properties from offspring through hybridization or backcrossing, and continuously selecting for 4-5 generations to obtain insect-resistant and glyphosate-resistant cotton varieties.

[0012] In the above method, the GBS8-2 described therein is a cotton variety belonging to upland cotton (Gossypium hirsutum), which was deposited in the General Microbiology Center of the China Culture Collection Administration on December 16, 2024, and its deposit number is: CGMCC No.46278.

[0013] In the above method, the GBS8-2 cotton line contains both an insect-resistant gene and a bivalent glyphosate-resistant herbicide gene. The GBS8-2 cotton line was obtained by genetically transferring an expression vector containing both the insect-resistant gene and the bivalent glyphosate-resistant herbicide gene into the Jin668 cotton line (accession number: CCTCC NO: P201519).

[0014] In the above method, the T-DNA is a DNA molecule containing an insect-resistant gene and a glyphosate-resistant gene from GBS8-2; the nucleotide sequence of the T-DNA is shown in SEQ ID NO. 1. The T-DNA is integrated into the cotton D05 chromosome between 12341816 and 12341859 in a single copy.

[0015] In the above method, the insect-resistant gene is an artificially designed fusion gene formed by fusing the coding sequences of the first and second domains of Cry1Ab with the third domain of Cry1Ac. The insect-resistant gene is named GmfCry1A. The nucleotide sequence of the insect-resistant gene is shown in bases 3947 to 432 of SEQ ID NO.1.

[0016] In the above method, the promoter of the insect-resistant gene is the cotton seed globulin gene promoter GhαGLOA (palpha globulin, see patent ZL2015105075070); the nucleotide sequence of the promoter is shown in bases 5637 to 4021 of SEQ ID No. 1.

[0017] In the above method, the glyphosate-resistant herbicide genes are the GR79 EPSPS gene and the GAT gene; the nucleotide sequence of the GR79 EPSPS gene is shown as bases 6428 to 7996 of SEQ ID No. 1; and the nucleotide sequence of the GAT gene is shown as bases 9251 to 9700 of SEQ ID No. 1. Sequences of the GR79 EPSPS and GAT genes and their expression cassettes can also be found in Patent ZL2014102047036.

[0018] The present invention also provides a molecular marker for identifying insect-resistant and glyphosate-resistant transgenic cotton GBS8-2 and its derivatives. The molecular marker is two DNA molecular fragments; one of the DNA molecular fragments is 388 bp in size, and its nucleotide sequence is shown as SEQ ID NO.6. The primers used to amplify the DNA molecular fragment consist of the nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.3; the other DNA molecular fragment is 681 bp in size, and its nucleotide sequence is shown as SEQ ID NO.7. The primers used to amplify the DNA molecular fragment consist of the nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.5.

[0019] The present invention also provides any of the following applications of the above-mentioned molecular markers:

[0020] (1) Application in cotton molecular marker-assisted breeding;

[0021] (2) Application in cotton breeding;

[0022] (3) Application in the preparation of cotton breeding products.

[0023] The present invention also provides a primer set for amplifying the above-mentioned molecular marker, comprising primer pair 1 and primer pair 2; the primer pair 1 consists of the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3, and the nucleotide sequence of the product obtained by PCR amplification thereof is shown in SEQ ID NO.6; the primer pair 2 consists of the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of the product obtained by PCR amplification thereof is shown in SEQ ID NO.7.

[0024] The present invention also provides a kit, which includes the above primer set.

[0025] The present invention also provides any of the following applications of the above primer set or kit:

[0026] (1) Application in cotton molecular marker-assisted breeding;

[0027] (2) Application in cotton breeding;

[0028] (3) Application in the preparation of cotton breeding products.

[0029] The present invention also provides a method for using the above-mentioned molecular markers to assist in the selection of insect-resistant and glyphosate-resistant cotton varieties, comprising using GBS8-2 or a GBS8-2 derivative as one of the parents, identifying offspring at the seedling stage using the above-mentioned molecular markers through hybridization or backcrossing, and selecting offspring with PCR amplification products of 388 bp and 681 bp in size; and continuously selecting 4-5 generations to obtain new insect-resistant and glyphosate-resistant cotton varieties.

[0030] The GBS8-2 derivative line described in the above method refers to a cotton line containing the T-DNA that is bred by hybridization or backcrossing with GBS8-2 as one of the parents.

[0031] PCR amplification primers for identifying homozygous or heterozygous T-DNA plants, wherein the primers consist of the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.5; the nucleotide sequence of the T-DNA is shown in SEQ ID NO.1.

[0032] The method for identifying T-DNA homozygous or heterozygous plants using the above primers comprises using genomic DNA from a hybrid progeny plant of GBS8-2 or a GBS8-2-derived line to be tested as a template and performing PCR amplification using the nucleotide sequences shown in SEQ ID NO. 2 and SEQ ID NO. 5 as primers. If the resulting PCR amplification product is a 517 bp DNA fragment, the plant to be tested is a T-DNA heterozygous plant; if no PCR amplification product is obtained, the plant to be tested is a T-DNA homozygous plant; wherein the nucleotide sequence of the T-DNA is shown in SEQ ID NO. 1.

[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The transgenic cotton GBS8-2 provided by the present invention and the cotton varieties bred with it as a parent have good insect resistance, and the leaf bioassay insecticidal efficiency can reach 99%. At the same time, the transgenic cotton can tolerate more than 4 times the concentration of glyphosate herbicide used in production, and its glyphosate herbicide tolerance is high. (2) The transgenic cotton GBS8-2 provided by the present invention is a transgenic cotton with a single copy and a single insertion site for the insect-resistant gene and the glyphosate herbicide-tolerant gene. Similar to the trait controlled by a single gene, its inheritance is simple, the breeding method is simple, and it is easy to use in breeding. (3) The insect-resistant gene contained in the GBS8-2 provided by the present invention is a fusion gene composed of insect-resistant genes from different sources. Compared with the previous single insect-resistant gene, the fusion insect-resistant gene is not easy to lose insect resistance, and the insect resistance is stable and durable. (4) The molecular markers of the transgenic cotton GBS8-2 provided by the present invention can be used for assisted selection breeding of insect-resistant and glyphosate-resistant genes, allowing selection to be performed directly at the seedling stage, shortening the breeding period and reducing breeding costs. (4) The transgenic cotton GBS8-2 provided by the present invention can simultaneously and efficiently express insect-resistant and herbicide-resistant genes, which is of great significance for reducing the damage caused by lepidopteran pests such as cotton bollworms and weeds during cotton production, reducing pesticide and labor input, lowering production costs, and increasing cotton yield.

[0034] Biological Deposit: GBS8-2 belongs to upland cotton (Gossypium hirsutum). It is a cotton variety bred by the inventors through transgenic methods to transform the new fusion insect-resistant gene and the bivalent glyphosate herbicide-resistant gene created into the cotton receptor Jin668. It has both insect-resistant and glyphosate-resistant characteristics. It was deposited in the General Microbiology Center of the China Microbiological Culture Management Committee on December 16, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and its deposit number is: CGMCC No. 46278. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1Comparative photos of transgenic cotton plants of the present invention identified using herbicide-tolerant protein test strips; 1 is GBS1; 2 is GBS3; 3 is GBS4; 4 is GBS8-2.

[0036] Figure 2 Comparative photos of transgenic cotton plants of the present invention identified using insect-resistant protein test strips; 1 is GBS1; 2 is GBS3; 3 is GBS4; 4 is GBS8-2.

[0037] Figure 3 Comparative photos of the field test of the transgenic cotton GBS8-2 resistant to glyphosate herbicide of the present invention; GBS8-2 is the insect-resistant and glyphosate-resistant transgenic cotton of the present invention; WT is the non-transgenic recipient cotton.

[0038] Figure 4 Comparative photos of leaves of the transgenic cotton GBS8-2 of the present invention in the field insect resistance test; GBS8-2 is the insect-resistant and glyphosate-resistant transgenic cotton of the present invention; WT is the non-transgenic recipient cotton.

[0039] Figure 5 Protein test strip identification results of the transgenic cotton GBS8-2 of the present invention; 1 and 3 are Bt test strips; 2 and 4 are GR79 EPSPS test strips; GBS8-2 is the transgenic cotton of the present invention: WT non-transgenic recipient material.

[0040] Figure 6 . Detection results of T-DNA integration sites in GBS8-2 transgenic cotton; lane 1# is the negative control PCR product amplified using GBS-F1 / GBS-R1 primers; lane 2# is the negative control PCR product amplified using GBS-F2 / GBS-R2 primers; lane 3# is the PCR product of the RB end of the T-DNA integration site in GBS8-2 transgenic plants amplified using GBS-F1 / GBS-R1 primers; lane 4# is the PCR product of the LB end of the T-DNA integration site in GBS8-2 transgenic plants amplified using GBS-F2 / GBS-R2 primers; M: molecular weight standard. DETAILED DESCRIPTION

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0042] Example 1 Cultivation process of transgenic cotton GBS8-2 containing insect-resistant genes and glyphosate-resistant herbicide genes of the present invention

[0043] Proceed as follows:

[0044] (1) Design and artificial synthesis of insect-resistant genes

[0045] Based on the Bacillus thuringiensis Cry1Ab and Cry1Ac protein sequences published in the NCBI database, the coding sequences for the first and second domains of Cry1Ab and the third domain of Cry1Ac were extracted, respectively. The two incomplete coding sequences were fused to form a new 3516bp insect-resistant gene. Based on this, the original insect-resistant gene sequence was optimized based on cotton codon preference. While maintaining the amino acid sequence of the encoded protein, some bacterial-preferred codons were replaced with cotton-preferred codons. Compared to the original nucleotide sequence, the G / C content of the optimized insect-resistant gene sequence increased from 34.22% to 43.46%. The nucleotide sequence of the optimized insect-resistant gene is shown in SEQ ID NO. 1, positions 3947-432. The gene was named Gmf Cry1A.

[0046] (II) Construction of expression vectors containing insect-resistant and glyphosate-resistant herbicide genes

[0047] (1) The cotton seed globulin core promoter GhαGLOA (p_alpha_globulin) (the nucleotide sequence of the promoter is shown in positions 5637 to 4021 of SEQ ID No. 1) was cloned into the PUC19 vector by double digestion with Hind III and BamH I, and the Ω and Cozak enhancer sequences were connected to the 3' end of the above promoter by using BamH I and Pst I; then the Gmf Cry1A gene (synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.) in step (1) was connected to the 3' end of the enhancer sequence by using Pst I and Xho I; finally, the Poly A and T NOS in the bivalent herbicide-resistant gene expression vector pGBIGRGAT were cloned to the 3' end of the Gmf Cry1A gene by double digestion with Pme I and Xho I. Finally, an intermediate expression vector containing the insect-resistant gene Gmf Cry1A of the present invention was constructed and named: PUC19-p alphaglobulin-GMFCry1A-TNOS. The nucleotide sequence of the cotton seed globulin core promoter is shown in positions 5637 to 4021 of SEQ ID NO. 1 in the sequence listing. The nucleotide sequence of the Ω and Cozak enhancer sequences is shown in positions 4022 to 4106 of SEQ ID NO. 1 in the sequence listing. The bivalent herbicide-resistant gene expression vector pGBIGRGAT was constructed according to the method described in the patent "An Expression Vector Containing a Glyphosate-Resistance Gene and Its Application" (Patent No. ZL2014102047036).

[0048] (2) The intermediate expression vector PUC19-p alphaglobulin-GMFCry1A-TNOS obtained in step (1) was double-digested with PmeI and HindIII to obtain a 5643bp Gmf Cry1A gene expression cassette. At the same time, the obtained Gmf Cry1A gene expression cassette was connected to the bivalent herbicide-resistant gene expression vector pGBIGRGAT double-digested with Pme I and Hind III to obtain a plant expression vector containing insect-resistant and glyphosate-resistant herbicide genes. The plant expression vector was named: pGBI-GmfCry1A-GR79EPSPS-GAT. The expression vector pGBIGRGAT was prepared according to the method described in the patent "An expression vector containing a glyphosate-resistant gene and its application" (patent number: ZL2014102047036).

[0049] (3) Using transgenic methods to obtain insect-resistant and herbicide-tolerant transgenic cotton

[0050] (1) Plant expression vector pGBI-Gmf Cry1A-GR79EPSPS-GAT was used to transform Agrobacterium

[0051] The plant expression vector pGBI-Gmf Cry1A-GR79EPSPS-GAT obtained in step (ii) above was transformed into Agrobacterium according to conventional methods. Five resistant clones were randomly selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. An Agrobacterium with the correct expression vector sequence was selected to obtain Agrobacterium containing the plant expression vector pGBI-Gmf Cry1A-GR79EPSPS-GAT.

[0052] (2) Agrobacterium-mediated genetic transformation of cotton and acquisition of transgenic plants

[0053] The upland cotton line Jin668 (its deposit number is: CCTCC NO: P201519, a cotton line bred by Huazhong Agricultural University) was used as a recipient, the Agrobacterium obtained in step (1) was used to infect the cotton hypocotyls, and then transgenic plants were obtained by tissue culture.

[0054] (3) Identification of transgenic plants

[0055] The leaves of the transgenic plants were tested using Cry1Ac test strips (purchased from Beijing Aochuang Jinbiao Biological Company) and GR79 EPSPS test strips (Institute of Biotechnology, Chinese Academy of Agricultural Sciences). Figure 1 and Figure 2 ) found that four plants simultaneously expressed the insecticidal protein and the herbicide-resistant protein. The four transgenic plants were named GBS1, GBS3, GBS4, and GBS8-2.

[0056] (IV) Herbicide tolerance test of transgenic cotton GBS8-2 of the present invention

[0057] (1) The four T0 transgenic plants obtained in step (3) were transplanted into a field and simultaneously sprayed with Roundup glyphosate herbicide at a concentration four times the production concentration (8 mg / L). Finally, a single plant with uninhibited growth and no difference in yield from the control was obtained, designated GBS8-2. Based on this, homozygous transgenic cotton of the T4 generation of GBS8-2 was obtained by screening through multiple generations of continuous glyphosate herbicide spraying.

[0058] (2) GBS8-2 T4 cotton seeds were sown in nutrient pots to cultivate seedlings, yielding a total of 50 seedlings. When the cotton seedlings had three true leaves, they were sprayed with 8 mg / L Roundup glyphosate herbicide. The seedling phenotype was observed 5 days after spraying.

[0059] Results (see Figure 3 ) All 50 T4 transgenic plants derived from GBS8-2 grew normally, while the wild-type recipient WT plants withered and died. This indicates that the transgenic cotton GBS8-2 obtained in the present invention has high tolerance to glyphosate herbicide.

[0060] (V) Insect resistance test of transgenic cotton GBS8-2 of the present invention

[0061] Proceed as follows:

[0062] (1) Cut the T4 homozygous transgenic cotton leaves of GBS8-2 obtained in step (4) into 2 cm squares and place them in a culture dish.

[0063] (2) Five newly hatched cotton bollworm larvae, fed for one day, were inoculated into each culture dish. Leaves from wild-type plants grown at the same time served as negative controls. Mortality and leaf damage were assessed five days after inoculation.

[0064] Results (see Table 1) showed that the corrected mortality of cotton bollworms fed with leaves of transgenic plants reached over 99%. Figure 4 ), the leaves of the non-transgenic plants (WT) were severely damaged and had a large number of insect holes; while the leaves of the transgenic plants (GBS8-2) of the present invention had only a few insect holes and were basically intact, further proving that the transgenic cotton GBS8-2 of the present invention has strong insect resistance.

[0065] Table 1 Insecticidal efficiency test results of transgenic cotton GBS8-2 of the present invention

[0066] Transgenic individual plants 5-day mortality rate (%) Adjusted mortality rate (%) GBS8-2 100 99.72±7.13 WT 1 0.1％

[0067] Example 2 Test strip identification test of insect resistance and herbicide tolerance of transgenic cotton GBS8-2 of the present invention

[0068] Proceed as follows:

[0069] (1) Take 0.1 g of GBS8-2 cotton leaves and place them in a 1.5 mL centrifuge tube. Add a 5 mm steel ball and 600 μL of ddH2O. Use the wild-type strain as a control.

[0070] (2) Place the centrifuge tube in a plant tissue grinder at 1000 rpm and grind for 1 minute.

[0071] (3) After grinding, centrifuge the tube at 12000 rpm for 5 minutes.

[0072] (4) After taking out the centrifuge tube, Bt test strips (purchased from Beijing Aochuang Jinbiao Biological Company) and GR79EPSPS test strips (Institute of Biotechnology, Chinese Academy of Agricultural Sciences) were placed in the centrifuge tube respectively and allowed to stand for 20 minutes.

[0073] (5) Observe the bands displayed on the test strips and qualitatively analyze the expression of insect-resistant and herbicide-resistant proteins in the plants. If the test strips show a band other than the quality control band, it can be proved that the corresponding transgenic plant is a positive plant.

[0074] Results (see Figure 5 ) This indicates that the transgenic cotton GBS8-2 of the present invention can simultaneously express insect-resistant and herbicide-tolerant proteins.

[0075] Example 3 Identification test of T-DNA copy number and integration site in transgenic cotton GBS8-2 of the present invention

[0076] Proceed as follows:

[0077] (1) Genomic DNA was extracted from leaves of T4 plants of GBS8-2 using a plant genome extraction kit. High-quality DNA samples (main band > 30 kb) that passed the test were selected. The DNA fragments were randomly fragmented using an ultrasonic disruptor. 15-18 kb large DNA fragments were enriched and purified using magnetic beads. Damage and end-repair were performed on the fragmented DNA. Stem-loop sequencing adapters were ligated to both ends of the DNA fragments, and fragments that failed to ligate were removed using an exonuclease. The constructed library was sequenced using the Pacbio Sequel II / Pacbio Sequel IIe platform.

[0078] (2) The raw sequencing reads after the machine are dumbbell-shaped structure sequences containing adapters at both ends, called Polymerase reads. The raw data are interrupted at the adapters and the adapter sequences are filtered out to obtain subreads. The subreads filtering standard is Filtering subreads by minimum length = 50. The subreads are further filtered using ccs software (https: / / github.com / PacificBiosciences / ccs), and finally high-precision HiFi reads are generated as valid data for downstream analysis. The ccs parameters are set to min-passes = 3, min-rq = 0.99, that is, all read quality values are above Q20. Finally, 90G high-quality third-generation sequencing reads are obtained, with an average length of 16,156.26bp and a maximum length of 45,965.6bp.

[0079] (3) The three-generation data were assembled using CANU (v2.2) software to obtain the complete contig sequence of the transgenic cotton genome. The assembled cotton genome sequence was indexed and constructed using the bwa (v0.7.17-r1188) index. The bwamem alignment algorithm aligned all sequences of the plant expression vector back to the assembled sequence to confirm the contig sequence where the T-DNA insertion sequence was located. Sequence fragments of 1 kb before and after the transferred fragment were extracted. The extracted sequence was compared with the known reference genome (HAU-TM-1) to confirm its insertion position into the cotton genome. By comparing with the gene annotation file of the reference genome, it was confirmed whether there was a gene at the insertion position or the possibility of causing gene breakage. At the same time, the three-generation sequencing data was aligned back to the assembly results, and the reads that failed to be aligned were extracted and compared with the vector sequence to confirm whether there were residual vector sequences in the unassembled sequencing data.

[0080] Results revealed that the T-DNA in the genome of the transgenic cotton GBS8-2 of the present invention was integrated as a single copy between 12341816 and 12341859 on chromosome D05. The T-DNA insertion resulted in a 42-bp genomic deletion. The T-DNA, consisting of the GMF Cry1A, GR79 EPSPS, and GAT gene expression cassettes, was 10,816 bp in length (its nucleotide sequence is shown in SEQ ID No: 1). Furthermore, aside from the T-DNA, no other plant expression vector backbone sequences were found in the genome of the transgenic cotton GBS8-2. This suggests that the T-DNA, consisting of the insect-resistant and herbicide-tolerant gene expression cassettes in the transgenic cotton GBS8-2, was integrated as a single copy at a single insertion site on the cotton chromosome D05.

[0081] Example 4 Primers and Molecular Markers for Identifying T-DNA in Transgenic Cotton GBS8-2 and Its Hybrid Progeny

[0082] Proceed as follows:

[0083] (1) Primer design: Based on the integration site genomic and T-DNA sequences given in Example 3, primers and molecular markers were designed for PCR identification of T-DNA in the hybrid progeny of transgenic cotton GBS8-2. The primers included primer pair 1 and primer pair 2, wherein primer pair 1 consisted of GBS-F1 and GBS-R1; primer pair 2 consisted of GBS-F2 and GBS-R2. The identification primer sequences are as follows:

[0084] GBS-F1: 5'-GATCACTGGGACCACATGGCCAG-3' (SEQ ID NO. 2);

[0085] GBS-R1: 5'-GAGCTCCGGCCATGCTAGAGTCCG-3' (SEQ ID NO. 3);

[0086] GBS-F2: 5'-GGTGATGGTTCACGTAGTGGGC-3' (SEQ ID NO. 4);

[0087] GBS-R2: 5'-CGTGATCTATGTGAAGGAAGGC-3' (SEQ ID NO. 5).

[0088] (2) Two primer pairs, GBS-F1 / GBS-R1 and GBS-F2 / GBS-R2, can be used to identify transgenic cotton and T-DNA integrity. The PCR reaction system (50 μL) includes: 2X Tag Mix 25 μL, Primer 1 (10 mM): 1 μL, Primer 2 (10 mM): 1 μL, and ddH2O 23 μL. PCR amplification reaction conditions: 98°C for 2 min; 98°C for 2 min, 58°C for 30 s, 72°C for 30 s, 30 cycles; and 72°C for 5 min.

[0089] Results (see Figure 6) Using GBS-F1 / GBS-R1 as primers, a 388 bp DNA fragment was obtained (the nucleotide sequence of which is shown in SEQ ID NO. 6), which includes 142 bp of genomic sequence (positions 1-142 of SEQ ID NO. 6) and 246 bp of RB-terminal T-DNA sequence (positions 143-388 of SEQ ID NO. 6); using GBS-F2 / GBS-R2 as primers, a 681 bp DNA fragment of the RB end of the T-DNA was obtained (the nucleotide sequence of which is shown in SEQ ID NO. 7), which includes 348 bp (positions 1-348 of SEQ ID NO. 7) of LB-terminal T-DNA sequence and 333 bp (positions 349-681 of SEQ ID NO. 7) of genomic sequence.

[0090] If a single plant with PCR amplification products is obtained using both GBS-F1 / GBS-R1 and GBS-F2 / GBS-R2 primer pairs, it is insect-resistant and herbicide-tolerant cotton containing complete T-DNA.

[0091] Based on the above, PCR amplification was performed using GBS-F1 and GBS-R2 as primers. If the amplified DNA fragment was 517 bp in size (the nucleotide sequence of which is shown in SEQ ID NO. 8), the plant was T-DNA heterozygous; if no amplification product was obtained, the plant was T-DNA homozygous and insect-resistant and herbicide-tolerant.

[0092] Example 5 Method for breeding new cotton varieties by hybridization using the transgenic cotton GBS8-2 of the present invention

[0093] Proceed as follows:

[0094] (1) SU12 (a commercial cotton variety in production) was used as the female parent and GBS8-2 (GBS8-2 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on December 16, 2024, and its deposit number is: CGMCC No. 46278) was used as the male parent to hybridize and obtain the F1 generation; the F1 generation was self-pollinated and the F2 generation seeds were obtained after harvest; the F2 generation seeds were sown in the field and sprayed with glyphosate herbicide at a concentration 4 times that used in production, and plants resistant to glyphosate herbicide were screened.

[0095] (2) Identification of homozygosity or heterozygosity of T-DNA of selected individual plants: Genomic DNA of glyphosate-resistant cotton plants in the F2 generation selected in step (1) was extracted, and PCR amplification was performed using GBS-F1 and GBS-R2 as primers. The PCR amplification products were observed by electrophoresis; wherein the PCR amplification reaction system (50 μL) included: 2X Tag Mix 25 μL, Primer 1 (10 mM): 1 μL, Primer 2 (10 mM): 1 μL, ddH2O 23 μL. PCR amplification reaction conditions were: 98°C for 2 min; 98°C for 2 min, 58°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 5 min.

[0096] If there is no PCR amplification product, then the F2 generation plant is a T-DNA homozygous plant; such plants can be selected through systematic breeding methods for 4-5 generations to cultivate new cotton varieties that are resistant to insects and resistant to glyphosate herbicides.

[0097] If the size of the obtained PCR amplification product is 517 bp, then the F2 generation plant is a T-DNA heterozygous plant. This type of plant can be directly eliminated, or the seeds of the plant with good traits can be collected and the above steps can be repeated to screen for T-DNA homozygous plants. Finally, through systematic breeding methods, new varieties that are resistant to insects and tolerant to glyphosate herbicides can be cultivated.

[0098] Example 6 Method for breeding new cotton varieties by backcrossing transgenic cotton GBS8-2 of the present invention

[0099] Proceed as follows:

[0100] (1) SU12 (a commercial cotton variety) was used as the female parent and GBS8-2 (GBS8-2 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 16, 2024, with the deposit number: CGMCC No. 46278) was used as the male parent to obtain F1 hybrid seeds;

[0101] (2) Sowing the F1 generation, spraying glyphosate herbicide at a concentration four times the production concentration at the seedling stage, and screening for glyphosate-resistant plants; then backcrossing the selected F1 generation plants with SU12 as the female parent to obtain BC1 generation seeds.

[0102] (3) Sowing BC1 generation seeds, spraying glyphosate herbicide at a concentration four times the production concentration at the seedling stage, and screening BC1 glyphosate-resistant individual plants; backcrossing the selected BC1 generation individual plants with SU12 as the female parent to obtain BC2 generation seeds.

[0103] (4) Repeat step (3) until backcrossing to the BC5 or BC6 generation; self-pollinate the BC5 or BC6 generation; and then, after sowing the self-pollinated offspring, spray glyphosate herbicide at a concentration four times that used in production, and screen for plants with glyphosate herbicide resistance consistent with SU12 and positive Bt test strip detection.

[0104] (5) Extract genomic DNA from the BC5 or BC6 self-pollinated individual selected in step (4) and perform PCR amplification using GBS-F1 and GBS-R2 as primers; wherein the PCR amplification reaction system (50uL) includes: 2X Tag Mix 25uL, Primer 1 (10mM): 1uL, Primer 2 (10mM): 1uL, ddH2O 23uL. PCR amplification reaction conditions: 98°C for 2 minutes; 98°C for 2 minutes, 58°C for 30 seconds, 72°C for 30 seconds, 30 cycles; 72°C for 5 minutes. The self-pollinated individual without amplification product is selected as the T-DNA homozygous individual, which is the new insect-resistant and glyphosate-tolerant cotton variety developed.

Claims

1. A method for cultivating insect-resistant and glyphosate-resistant cotton varieties, characterized in that: The invention comprises using GBS8-2 or a derivative of GBS8-2 as one parent and another cotton variety with excellent agronomic and quality traits as the other parent, selecting T-DNA homozygous plants with good insect resistance and glyphosate herbicide resistance from offspring through hybridization or backcrossing, and continuously selecting for 4 to 5 generations to obtain an insect-resistant and glyphosate herbicide-resistant cotton variety. The GBS8-2 is a cotton variety belonging to upland cotton (Gossypium hirsutum) and is deposited in the General Microbiology Center of the China Microorganism Culture Collection Committee with a deposit number of CGMCC No. 46278.

2. The method according to claim 1, characterized in that The T-DNA is a DNA molecule containing an insect-resistant gene and a glyphosate-resistant herbicide gene in GBS8-2; the nucleotide sequence of the T-DNA is shown in SEQ ID NO.

1.

3. A molecular marker for identifying insect-resistant and glyphosate-tolerant transgenic cotton GBS8-2 and its derivatives, characterized in that: The molecular markers are two DNA molecular fragments; one of the DNA molecular fragments is 388 bp in size, and its nucleotide sequence is shown in SEQ ID NO.

6. The primers used to amplify the DNA molecular fragment are composed of the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3; the other DNA molecular fragment is 681 bp in size, and its nucleotide sequence is shown in SEQ ID NO.

7. The primers used to amplify the DNA molecular fragment are composed of the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.

5.

4. Any of the following uses of the molecular marker according to claim 3, characterized in that: (1) Application in cotton molecular marker-assisted breeding; (2) Application in cotton breeding; (3) Application in the preparation of cotton breeding products.

5. A primer set for amplifying the molecular marker according to claim 3, characterized in that: It comprises a primer pair 1 and a primer pair 2; the primer pair 1 consists of the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.3; the primer pair 2 consists of the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.

5.

6. A kit, characterized in that The kit includes the primer set according to claim 5.

7. Any of the following uses of the primer set according to claim 5 or the kit according to claim 6, characterized in that: (1) Application in cotton molecular marker-assisted breeding; (2) Application in cotton breeding; (3) Application in the preparation of cotton breeding products.

8. The method for selecting insect-resistant and glyphosate-resistant cotton varieties using the molecular marker-assisted selection method according to claim 3, characterized in that: The method comprises using GBS8-2 or a GBS8-2 derivative as one of the parents, identifying offspring at the seedling stage using the molecular markers described in claim 3 by hybridization or backcrossing, selecting offspring with PCR amplification products of 388 bp and 681 bp in size, and continuously selecting 4-5 generations to obtain a new cotton variety that is resistant to insects and resistant to glyphosate herbicide.

9. PCR amplification primers for identifying homozygous or heterozygous T-DNA plants, characterized in that: The primers are composed of the nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO.5; the nucleotide sequence of the T-DNA is shown in SEQ ID NO.

1.

10. A method for identifying T-DNA homozygous or heterozygous plants using the primers according to claim 9, characterized in that: The method comprises performing PCR amplification using genomic DNA of a hybrid progeny plant of the GBS8-2 or GBS8-2-derived line to be tested as a template and the nucleotide sequences shown in SEQ ID NO. 2 and SEQ ID NO. 5 as primers; if the obtained PCR amplification product is a DNA fragment of 517 bp in size, the tested plant is a T-DNA heterozygous plant; if no PCR amplification product is obtained, the tested plant is a T-DNA homozygous plant; wherein the nucleotide sequence of the T-DNA is shown in SEQ ID NO. 1.

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