Glyphosate-resistant wheat mutant protein gr23, gene and application thereof

Abstract

The application discloses a wheat mutant protein GR23 with glyphosate resistance, a gene and application thereof, and belongs to the technical field of plant breeding.The amino acid sequence of the wheat mutant protein GR23 with glyphosate resistance is shown as SEQ ID NO.8, and the nucleotide sequence of the gene encoding the wheat mutant protein GR23 is shown as SEQ ID NO.9.The application finds the mutant protein GR23 with glyphosate resistance and the mutation type, greatly improves the glyphosate resistance of wheat, and provides a new genetic resource for screening of glyphosate-resistant wheat plants, cultivation of high-resistance wheat strains, and improvement of excellent traits of wheat.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant breeding, and particularly relates to a wheat mutant protein GR23 with glyphosate resistance, a gene and application thereof. BACKGROUND

[0002] Farmland weeds will compete with crops for resources, occupy space, spread diseases and pests, thereby leading to reduction of crop yield and quality, increase of management cost, and economic loss of 10%-20% of total production caused by grass damage in wheat production every year. Due to the reasons such as rising of weed resistance level, frequent occurrence of herbicide phytotoxicity, prominent contradiction in production season, and rough tillage measures, the difficulty of weed control in wheat field is increased. Weeds are a big challenge to tillage, and restrict the yield and cost of crop planting. Herbicide is a powerful weapon to restrict weeds, and is a necessary link of modern agriculture. Herbicide-resistant crops are an effective means to improve the application efficiency of herbicides, and changing the sensitive site of herbicide effect protein is a common strategy for breeding herbicide-resistant crops.

[0003] Glyphosate is a high-efficiency, broad-spectrum, low-toxicity, low-residue, non-soil environment destroying, and low-frequency resistance to most plants and crops herbicide, has no toxicity to human and livestock, and has low soil residual amount. The market potential of glyphosate is huge. Glyphosate competitively inhibits the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimic acid pathway, causes the synthesis of aromatic amino acids (phenylalanine, tyrosine and tryptophan) to be blocked, and finally causes the death of plants. Glyphosate is rapidly decomposed in soil, is one of the most safe commercial herbicides with the least pollution, and is the herbicide with the largest application area. However, general crops are sensitive to glyphosate, which greatly limits the use time and space of glyphosate, and thus it is required to use glyphosate a period of time before the sowing of crops, so as to avoid the phytotoxicity of crops. Therefore, breeding some herbicide-resistant crop varieties can reduce crop phytotoxicity and expand the use range of herbicides.

[0004] The development and commercial application of glyphosate-resistant transgenic non-main crop (such as cotton, soybean and corn) have achieved great success, but the development of transgenic main crops such as GM wheat and GM rice is controversial in almost all countries. Mutagenic breeding as an alternative method for breeding herbicide-resistant crops does not involve genetic modification of crops themselves, and is well applied in some countries. For example, rice and wheat varieties resistant to Immi have been commercially produced in the United States for nearly 20 years, and non-transgenic herbicide-resistant rice led by BASF Company and other foreign companies has developed into two types: (1) Clearfield TMType, also known as Jie Tian rice in China, is mainly induced by mutagenesis of ALS (acetolactate synthase) gene, and the corresponding herbicide is imidazolinone acetochlor. Some herbicide-resistant rice varieties produced in China, such as Jinqian 818, Jie Tian rice 001 bred by Shenzhen Xingwang Biological Seed Industry Co., Ltd., etc., belong to this type; but the use of this technology in China should be cautious, because the soil residual period of imidazolinone acetochlor is long, and it has a great impact on the following crops. TM Type, mainly induced by mutagenesis of ACCase (acetyl coenzyme A carboxylase) gene, and the corresponding herbicide is clethodim.

[0005] The motif of EPSPS enzyme is conserved in all plants and most bacteria, which is essential for binding phosphoenolpyruvate (PEP) or competitively inhibiting glyphosate. The mature EPSPS protein of wheat is composed of about 511 amino acids, and its sequence is highly conserved among different species. Mutations in the EPSPS protein at amino acid positions 96, 97, 101, 106, 168, 172, 173, 177, etc. (calculated based on the EPSPS amino acid position of the model plant rice) can produce glyphosate resistance, which has been reported in various crops (including corn, wheat, wheat, oilseed rape, sunflower, etc.), model plants Arabidopsis thaliana and various weeds. However, different types of mutations can produce different intensities of glyphosate resistance, and the discovery of more types of mutations not only helps to improve the glyphosate resistance of wheat, but also enables the cultivation of more excellent traits of wheat, providing convenience for wheat production. SUMMARY

[0006] The purpose of the present application is to provide a wheat mutant protein GR23 with glyphosate resistance, a gene and its application, in order to solve the problems existing in the prior art. The present application discovers a mutant protein GR23 with glyphosate resistance and its mutation type, which greatly improves the glyphosate resistance of wheat, and provides new genetic resources for the screening of glyphosate-resistant wheat plants, the cultivation of high-resistance wheat lines, and the improvement of excellent traits of wheat.

[0007] To achieve the above purpose, the present application provides the following scheme:

[0008] The present application provides a wheat mutant protein GR23 with glyphosate resistance, and the amino acid sequence of the wheat mutant protein GR23 is shown in SEQ ID NO. 8.

[0009] The present application also provides a gene encoding the above-mentioned wheat mutant protein GR23, and the nucleotide sequence of the gene is shown in SEQ ID NO. 9.

[0010] The present application also provides the application of a reagent for detecting the above-mentioned gene in screening glyphosate-resistant wheat or cultivating glyphosate-resistant wheat.

[0011] The application also provides application of Triticum aestivum L. ZM9-GR23 in breeding glyphosate-resistant wheat, wherein the Triticum aestivum L. ZM9-GR23 has a preservation number of CCTCC NO: P202421.

[0012] The Triticum aestivum L. ZM9-GR23 contains a nucleotide sequence as shown in SEQ ID NO. 9 on the 4A genome.

[0013] Preferably, in the breeding of the glyphosate-resistant wheat, the Triticum aestivum L. ZM9-GR23 is used as a donor plant of the glyphosate-resistant gene.

[0014] The application also provides a method for breeding glyphosate-resistant wheat, comprising the following steps:

[0015] Crossing the Triticum aestivum L. ZM9-GR23 as a male parent with a wheat variety needing to be bred with a glyphosate-resistant trait as a female parent to obtain hybrid offspring;

[0016] Screening the glyphosate-resistant hybrid offspring from the backcrossing with the female parent;

[0017] Screening the glyphosate-resistant backcrossing offspring from the backcrossing with the female parent, and repeating the backcrossing more than 3 times;

[0018] Collecting the final offspring and self-crossing;

[0019] After the self-crossing for 2 generations, screening a strain with a phenotype similar to that of the female parent and stably expressing the glyphosate-resistant trait, i.e. the glyphosate-resistant wheat is obtained.

[0020] The application also provides a method for breeding glyphosate-resistant wheat, comprising the following steps:

[0021] Crossing the Triticum aestivum L. ZM9-GR23 as a male parent with a wheat variety needing to be bred with a glyphosate-resistant trait as a female parent to obtain hybrid offspring;

[0022] Planting the hybrid offspring seeds, self-crossing and generation, forming hybrid offspring seeds, and continuing to plant;

[0023] Selecting plants from the hybrid offspring seeds, and separately harvesting and threshing the selected single plants;

[0024] Repeating the steps of self-crossing and generation and plant selection until plants with more than 5 completely homozygous mutation sites in the EPSPS gene and excellent target agronomic traits are obtained, i.e. the glyphosate-resistant wheat is obtained.

[0025] Preferably, the method for selecting plants comprises observation, measurement and marker-assisted selection of mutation sites.

[0026] The present application discloses the following technical effects:

[0027] The present application obtains a wheat mutant strain ZM9-GR23 with significant glyphosate resistance by spraying glyphosate in the field, analyzes the mutation type of the strain by using PCR and sequencing technology, finds that the mutant strain has single base mutations at multiple sites on the EPSPS gene sequence of the 4A genome of wheat, which causes the change of the 137th, 189th, 198th, 204th and 226th amino acids of the corresponding coded amino acid sequence. This mutation causes the plant to still grow normally and bear fruit after spraying 40 mL of Nongda / L water (5 times the recommended concentration of glyphosate) at the 3-5 leaf stage, while the wild type wheat stops development and dies after spraying 8 mL of Nongda / L water (recommended concentration). The obtaining of the wheat mutant strain, the discovery of the mutant protein and the mutation type provide new genetic resources for the screening of glyphosate-resistant wheat plants, the cultivation of highly resistant wheat lines and the improvement of excellent traits of wheat. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort.

[0029] Figure 1 PCR amplification of part of the EPSPS gene fragment of the 4A genome of wheat is shown in the figure; M is DL5000Marker, 23 lanes are the amplification products of the mutant strain GR23, and 45 lanes are the amplification products of the wild type ZM9; the length of the target fragment is 540 bp;

[0030] Figure 2 The figure is a comparison of the amino acid sequences of the EPSPS proteins of the 4A genome of wheat; OsEPSPS is the amino acid sequence of the EPSPS protein of rice, TaEPSPS-7D1 (Louise) is the amino acid sequence of the EPSPS protein of wheat Louise, TaEPSPS-4A (ZM9) is part of the amino acid sequence of the EPSPS protein of the wild type Zhenmai 9, and TaEPSPS-4A (GR23) is part of the amino acid sequence of the EPSPS protein of the mutant GR23; the mutation sites of the GR23 mutant are represented by red characters;

[0031] Figure 3 The figure is a schematic diagram of marker-assisted backcrossing to transfer the glyphosate-resistant trait of GR23 to the genetic background of Yangmai 33;

[0032] Figure 4 Figure 1 is a schematic diagram of pedigree breeding method for hybridizing Yangmai 33 (with excellent agronomic traits) and GR23 (glyphosate-resistant) to aggregate improved lines. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. The detailed description is merely intended to teach a person of ordinary skill in the art how to make and use the present application and is not intended to limit the scope of the application. Therefore, the description of the exemplary embodiments is not intended to limit the scope of the application.

[0034] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0035] Unless defined otherwise, 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 application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the present specification will control.

[0036] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0037] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0038] Example 1 Obtaining process of wheat glyphosate-resistant mutant variation sites

[0039] A wheat plant of M3 generation of EMS mutagenesis of 3-5 leaf stage of 'Zhenmai 9' was sprayed with glyphosate (40 mL of Nongda / L water, 5 times of recommended dose), and a normal growing wheat plant was harvested, which was a mutant plant line with significant resistance to glyphosate, temporarily named as GR23. In comparison, the wild type plant line was sprayed with 8 mL of Nongda / L water (recommended concentration), and the plant growth gradually stopped, the leaves turned yellow and withered, and the whole plant died in 10-15 days.

[0040] Seeds of 15 grains of the GR23 mutant and 15 grains of the wild type control were placed in a culture box for substrate culture, and leaves were taken at 3 leaf stage, genomic DNA was extracted, and PCR amplification was carried out with TaEPSPS-CL as the upstream primer and TaEPSPS-7AR, TaEPSPS-4AR and TaEPSPS-7DR as the downstream primers, respectively. The primer sequences are shown in Table 1.

[0041] PCR amplification was selected with Nuoyan 2xKeyPo Master Mix (Dye Plus) high-fidelity enzyme (item number PK511), and the reaction system was as follows: 2xKeyPo Master Mix (Dye Plus) 20 μL, upstream primer (10 μM) 1.5 μL, downstream primer (10 μM) 1.5 μL, wheat leaf DNA 1 μL, nuclease-free ddH2O 16 μL.

[0042] The amplification program was as follows: ① 98℃ 10 sec, ② 58℃ 5 sec, ③ 72℃ 5 sec, 35 cycles (①→③). The total reaction volume was 40 μL, 3 μL of the reaction solution was loaded for agarose gel electrophoresis, and after the band was correct and bright, the remaining reaction solution was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing using the forward primer TaEPSPS-CL, and the sequencing results were analyzed by comparison with the DNAMAN software.

[0043] Table 1 PCR primers for detecting mutations of wheat EPSPS homologous genes

[0044]

[0045] The electrophoresis results are shown in Figure 1 . It can be seen that the amplification results are correct and can be used for subsequent sequencing.

[0046] The sequencing results are as follows:

[0047] The nucleotide sequences of the EPSPS gene PCR products of the wild type wheat plant 7A genome, 4A genome and 7D genome are shown as SEQ ID NO. 5-7 respectively, the amino acid sequence of the EPSPS protein of the GR23 mutant is shown as SEQ ID NO. 8, and the nucleotide sequence encoding the EPSPS protein of the GR23 mutant is shown as SEQ ID NO. 9. The alignment result is shown as Figure 2 Compared with the wild type plant, the GR23 mutant has single base mutations at multiple sites on the EPSPS gene sequence of the wheat 4A genome, which causes changes in the 137th, 189th, 198th, 204th and 226th amino acids of the corresponding encoded amino acid sequence, that is, the mutant GR23 detects 5 mutation sites (as shown in Table 2).

[0048] Table 2 Mutation site information in the EPSPS gene of wheat 4A chromosome

[0049]

[0050] The wheat mutant plant of the present application is classified and named as Triticum aestivum L. ZM9-GR23. The seeds of the Triticum aestivum L. ZM9-GR23 were deposited with the China Center for Type Culture Collection (CCTCC) on September 4, 2024, and were named as wheat mutant seed GR23, with the address of Wuhan University Preservation Center, Wuchang District, Wuhan City, Hubei Province, China, and the preservation number of CCTCC NO: P202421.

[0051] SEQ ID NO. 5 (5'-3'):

[0052] CAGGGTAGCTTGGACTCTCCGTGGAGCAGATAAAGTTGCAAAAAGAGCTGTGGTTGTTGGCTGTGGCGGCAGGTTCCCAGTCGAAAAGGACGCCAAAGAGGAAGTAAAGCTCTTCTTGGGTAATGCTGGAACTGCAATGCGGCCACTGACGGCAGCTGTAGTAGCTGCTGGTGGAAATGCAACGTATGTTTTCTTTATCCTAGTGGAAATAAGTATGAGATCCATGGGTATGTTTGGAGACTGATCGTTTCTTTTATTAAAAAAAAACTTCAGTTATGTGCTTGATGGCGTACCAAGAATGAGGGAGCGACCTATTGGTGACTTAGTTGTAGGTTTGCAACAACTCGGCGCAGATGTCGATTGTTTCCTTGGCACAAACTGCCCACCTGTCCGTATCAACGGCAAAGGAGGTCTACCTGGTGGCAAGGTTAGCTACTCATCAACTTGCATGTTATCTACTTTGTGCACACTTCTGTTCTCTGCATAAAAAAGTAATA.

[0053] SEQ ID NO. 6 (5'-3'):

[0054] GGGTCCGGCATCTCGGACTCTCCGTGGAGCAGATAAAGTTGCAAAAAGAGCTGTGGTTGTTGGCTGTGGCGGCAGGTTCCCGGTCGAAAAGGACGCCAAAGAGGAAGTAAAGCTCTTCTTGGGTAATGCTGGAACTGCAATGCGTCCATTGACGGCAGCTGTAGTAGCTGCTGGTGGAAATGCAACGTATGTTTTCTTTATCCTAGTGGAAATAAGTATGAGATCCATGGGTATGTTTGGAAACTGATCGTGTCTTTTATTAAAAAAAAAACTTCATTTATGTGCTTGATGGAATACCAAAAATGAGGGAGCGACCTATTGGTGACTTATTTGTAGGTTTGCAACAACTCGGCGCATATGCTGATTGTTTCCTTGGCACTAACTGCCCACCTGTTCGTATCAATGGCAAAGGAGGGCTACCTGGTGGCAAGGTTAGCTACTTGTGAACTTGCATGTTATGTACTTTTGTGCACATTCAGTTCTCTGTCAAAGAAAAATAATATTATTATGGGTAGTACGATCAATGACCTTTTCCCAAAAAAA.

[0055] SEQ ID NO. 7 (5'-3'):

[0056] ACATCGGACTCTCCGTGGAGCAGATAAAGTTGCAAAAAGAGCTGTGGTTGTTGGCTGTGGCGGCAGGTTCCCGGTCGAAAAGGACGCCAAAGAGGAAGTAAAGCTCTTCTTGGGTAATGCTGGAACTGCAATGCGTCCATTGACGGCAGCTGTAGTAGCTGCTGGTGGAAATGCAACGTATGTTTTCTTTATCCTAGTTGAAATAAGTATGAGATCCATGGGTATGTTTGGAGACTGATCGTGTCTTTTATTAAAAAAAAAACTTCAGTTATGTGCTTGATGGAGTACCAAGAATGAGGGAGCGACCTATTGGTGACTTAGTTGTAGGTTTGCAACAACTCGGCGCAGATGCTGATTGTTTCCTTGGCACTAACTGCCCACCTGTTCGTATCAATGGCAAAGGAGGGCTACCTGGTGGCAAGGTTAGCTACTTGTGAACTTGCATGTTATGTACTTTTGTGCACATTCAGTTCTCTGTCAAAGAAAAATAATATTATTATGGGTAGTACGATGCAACGCGACCTTACACCGTGCACTAAAGTTTAAGAACCAATAAACATTGAATAAATTCAATACTTGAGTATTGCGTGATCAATCATAGATTGGCGCTTAACATTGAATAATTTCACAGGTTAAGCTCTCTGGTTCCATTAGCAGTCAATACCTGAGTTCCTTGCTGATGGCTGCTCCTTTGGCTCTTGAGGATGTCGAGATTGAAATCATTGATAAACTGATCTCCGTTCCTTACGTTGAAATGACATTGAAATTGATGGAGCGTTTTGGCGTGACTGCGGAGCATTCTGATAGTTGGGACAGATTCTACATTAAGGGAGGACAAAAGTACAAGTAAGTTCTACATTGCTTTACTTCTTCTGATAGTGGAGTACAAAGAACTCCCTCGAATCCCACAAAACAAAGAAGAA.

[0057] SEQ ID NO. 8:

[0058] LGLSVEADKAAKRAVVVGCGGRFPVEKDAKEEVKLFLGNAGTAMRPLTAAVVAAGGNATYVFDGVPRMREPPIGDLCVGLQQLGADADCFLGTNCPPVHINGKGGLPGGK.

[0059] SEQ ID NO. 9 (5'-3'):

[0060] GGGGACTGAGCTCGGACTCTCCGTGGAGCAGATAAAGCTGCAAAAAGAGCTGTGGTTGTTGGCTGTGGCGGCAGGTTCCCGGTCGAAAAGGACGCCAAAGAGGAAGTAAAGCTCTTCTTGGGTAATGCTGGAACTGCAATGCGTCCACTGACGGCAGCTGTAGTTGCTGCTGGTGGAAATGCAACGTATGTTTTCTTTATCCTAGTGGAAATAAGTATGAGATCCTGGGGTAGGTTGGGAAATGGACCGGGTCTTTTTTTAAAAAAAAATCTTCTTATATGTTTTTGATGGTACACCAAAAGAGAGGGAGCCACCTATTGGAGACTTATGTGTAGGTTTGCAACCTCTCGGCGCATATGCTGATTGTTTCCTTGGCACTAACTGCCCACCTGTTCATATCAATGGCAAAGGAGGGCTACCTGGTGGCGGGGTTAGCTACTTGTGAACTTGTGTGATATGTACTTTTGTGCACATTCAGTCTCTCTGTCAAAAAAAAATAATATATATATATGGGTAACACGATCAATGACCCTTACCCAAAAAA.

[0061] Example 2: Transformation of glyphosate resistance trait

[0062] The transformation of glyphosate resistance trait was carried out by backcrossing method. The wheat variety in need of transformation of glyphosate resistance trait was used as female parent (also known as recurrent parent, RP), and mutant wheat ZM9-GR23 was used as male parent, and hybrid F1 was obtained by hybridization.

[0063] If the glyphosate resistance trait is dominant, the recurrent parent is used as the female parent and the F1 described above is used as the male parent to obtain the backcross 1 generation (BC1). This process is repeated, with the recurrent parent as the female parent and the glyphosate resistant backcross generation as the male parent. According to the theory of genetics, after six backcrosses, the progeny is, on average, more than 99% similar in genetics to the RP. After several backcrosses, the individuals in the last backcross generation can be self-pollinated to "fix" the introduced glyphosate resistance gene in a homozygous state. Here, the marker assisted selection can be used to select the backcross progeny plants with high rates of reversion in the backcross progeny, thereby speeding up the process of backcross conversion.

[0064] If the glyphosate resistance trait is recessive, the recurrent parent is used as the female parent and the F1 or backcross generation described above is used as the male parent to obtain the backcross generation. Again, the marker assisted selection can be used to select the heterozygous individuals containing the glyphosate resistance site in the backcross progeny, thereby speeding up the process of conversion without the need for the self-pollination process.

[0065] This example uses Yangmai 33 as the recurrent parent to convert the glyphosate resistance trait from mutant ZM9-GR23. The breeding scheme is shown in Table 3, and this process generally requires 10 generations. To speed up the process of conversion, the marker assisted backcross technique can be used, which mainly includes three aspects of selection, the prospect (target trait) selection, the recombination selection and the background selection. Since the selection of glyphosate resistance can be achieved by simple phenotypic screening, the genetic background of the recurrent parent 'Yangmai 33' can be selected in the backcross progeny population by marker information. The markers used are not linked to the target gene, and all markers other than the target gene select the genotype of the recurrent parent 'Yangmai 33'. The more the number of markers, the faster the reversion of the recurrent parent (see the schematic diagram of marker assisted backcross in Figure 1). Figure 3 Through marker assisted backcross, a relatively satisfactory reversion rate of the recurrent parent (>95%) can be obtained after 3-4 generations of continuous backcross.

[0066] The glyphosate resistance gene from ZM9-GR23 converted by Yangmai 33 is named as the converted line 'Yangmai 33GR2'. Field spraying tests show that the resistance of 'Yangmai 33GR2' to glyphosate is similar to that of ZM9-GR23, and it can resist 5 times the recommended dose of glyphosate.

[0067] Table 3 Breeding scheme for converting the glyphosate resistance trait from GR23 using Yangmai 33 as the recurrent parent

[0068]

[0069] Example 3 Aggregation of glyphosate resistance trait and excellent agronomic traits

[0070] A wheat variety (e.g., Yangmai 33, Zhenmai 18, etc.) selected for wide area promotion in a target ecological zone is used as the female parent, and the mutant ZM9-GR23 is used as the male parent to produce hybrid F1. Any such F1 containing a full complement of alleles for 5 or more non-transgenic mutation sites in the EPSPS gene is thus encompassed by the present application. These embodiments also include the use of transgenic or backcross breeding of a wheat variety having 5 or more non-transgenic mutation sites in the EPSPS gene to produce a first generation F1 plant.

[0071] In this example, Yangmai 33 is used as the female parent, and the mutant ZM9-GR23 is used as the male parent to produce hybrid F1. The F1 seeds are planted, and the F2 seeds are produced by further generations. The F2 seeds are planted, which is the first segregating generation. Selection of plants is carried out by observation, measurement, and marker assisted selection of mutation sites. The selected individual plants are harvested and threshed separately. The F3 seeds from each plant are planted in different ear rows or plant rows, selfed, and further generations are produced. The selected rows or plants from these rows are harvested and threshed separately. The selection is also based on observation or measurement of target traits, such as the presence of mutation sites in the EPSPS gene, and various desirable agronomic traits.

[0072] Selection of glyphosate-resistant wheat. In addition to glyphosate resistance, which is selected by direct field spraying of glyphosate or by marker assisted selection of glyphosate-resistant individual plants or ear rows, other traits, especially agronomic traits, can be selected according to conventional breeding techniques or standards for wheat, with a focus on plant height, plant type, maturity, maturity phase, disease resistance (scab, powdery mildew, etc.), tillering, ear size, and seed setting, etc. If it is an ear row, the selection also focuses on stem flexibility, general yield potential, etc. The selection in this example is a plant height of 75-85 cm, a full growth period of 198-203 d, a relatively compact or loose plant type, a good maturity phase, and resistance or moderate resistance to scab or powdery mildew. In terms of yield three factors, the selection is a medium-sized ear, medium-sized grain, and medium tillering, with specific standards of 37-43 grains per ear, 40-46 g per 1000 grains, and 29.5-32.5 thousand effective ears per mu. In addition, during indoor seed selection, seeds with yellow and black color, high black embryo rate, small grain, thin grain, and low seed weight are eliminated, and large grain, cutin, bright color, fullness, and high seed weight are retained. The above planting and selection process is repeated multiple times until the 3 or more mutation sites in the EPSPS gene are completely homozygous, and the multiple target agronomic traits are excellent. The processes of hybridization, generation derivation, and identification and selection are described in detail in the above examples. Figure 4 .

[0073] The breeding process of repeated cross, selfing and selection can produce another wheat breeding population derived from the wheat variety with 5 or more mutation sites in the EPSPS gene, one of the parents is from the mutant or transgenic material containing 5 or more mutation sites in the EPSPS gene, or both parents are from the mutant or transgenic material containing 5 or more mutation sites in the EPSPS gene.

[0074] Through the above process, one elite line with excellent yield traits (slightly better than 'Yangmai 33') and excellent glyphosate resistance characteristics was obtained, named Chongmai 103GR. Field spraying test showed that its glyphosate resistance was similar to that of the father ZM9-GR23, and it could tolerate 5 times the recommended dose of glyphosate.

[0075] Example 4 Marking of mutation sites

[0076] The results of sequencing the PCR amplification products of the three primer combinations in Table 1 showed that the three primers specifically amplified the EPSPS genes located on 7A, 4A and 7D, respectively, so that the nucleotide differences between the wild type and mutant sequences could be identified by sequencing the PCR amplification products, and then the mutation sites of the EPSPS gene in the PCR amplification products could be screened.

[0077] If you want to detect whether the glyphosate-resistant material to be tested is consistent with the mutation in the RPSPS gene of ZM9-GR23, you can extract genomic DNA from the 3-leaf stage leaves of the material to be tested and GR23, respectively, and perform PCR amplification with TaEPSPS-CL as the upstream primer and TaEPSPS-4AR as the downstream primer (primer sequences are shown in Table 1).

[0078] PCR amplification uses NuCyon 2x KeyPo Master Mix (Dye Plus) high-fidelity enzyme (product number PK511), and the reaction system is as follows: 2x KeyPo Master Mix (Dye Plus) 20 μL, upstream primer (10 μM) 1.5 μL, downstream primer (10 μM) 1.5 μL, wheat leaf DNA 1 μL, nuclease-free ddH2O 16 μL.

[0079] The amplification program is as follows: ① 98℃ 10 sec, ② 58℃ 5 sec, ③ 72℃ 5 sec, 35 cycles (①→③). The total reaction volume is 40 μL, 3 μL of the reaction solution is loaded for electrophoresis on agarose gel, and after the band is correct and bright, the remaining reaction solution is sent to GenScript Biotech (Shanghai) Co., Ltd. for sequencing using the forward primer: TaEPSPS-CL. The sequencing results are analyzed by DNAMAN software.

[0080] If the material to be tested contains the same single base mutation type as ZM9-GR23 listed in Table 1, it is reasonable to believe that the glyphosate-resistant gene of the material to be tested comes from ZM9-GR23 of the present application.

[0081] The present application relates to mutations in the EPSPS gene, all of which are single nucleotide polymorphisms, which can be used as markers in crop breeding, and in particular one or more of the mutation sites in Table 2 can be used as markers in plant breeding. The relationship between the mutations in the EPSPS gene and the corresponding mutants' resistance to glyphosate is a causal relationship, and some markers including KASP markers / probes can be used to track their segregation.

[0082] The above-described embodiments are only to describe the preferred modes of the present application, and not to limit the scope of the present application, and various modifications and improvements to the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope determined by the claims of the present application.

Claims

1. The application of a common wheat variety (Triticum aestivum L.) ZM9-GR23 in the breeding of glyphosate-resistant wheat, characterized in that, The seeds of the common wheat ZM9-GR23 are named wheat mutant seeds GR23, and the preservation number of wheat mutant seeds GR23 is CCTCC NO: P202421; The 4A genome of the common wheat ZM9-GR23 contains the nucleotide sequence shown in SEQ ID NO.

9.

2. The application according to claim 1, characterized in that, In the process of cultivating glyphosate-resistant wheat, the common wheat ZM9-GR23 is used as the donor plant for the glyphosate-resistant gene.

3. A method for breeding glyphosate-resistant wheat, characterized in that, Includes the following steps: Using the common wheat ZM9-GR23 described in claim 1 or 2 as the male parent and a wheat variety that needs to be bred to resist glyphosate as the female parent, hybridization is carried out to obtain hybrid offspring; Screen for glyphosate-resistant hybrid offspring and backcross them with the aforementioned maternal parent; Screen for glyphosate-resistant backcross progeny, backcross with the aforementioned maternal parent, and repeat at least 3 times; Collect the final offspring and self-pollinate; After two generations of self-pollination, lines that are phenotypically similar to the maternal parent and stably express glyphosate resistance are selected to obtain the glyphosate-resistant wheat.

4. A method for breeding glyphosate-resistant wheat, characterized in that, Includes the following steps: Using the common wheat ZM9-GR23 described in claim 1 or 2 as the male parent and a wheat variety that needs to be bred to resist glyphosate as the female parent, hybridization is carried out to obtain F1 hybrid seeds; Plant the first generation hybrid seeds, perform self-pollination to generate second generation hybrid seeds, and continue planting them; Plant selection was performed on the second-generation hybrid seeds, and the selected individual plants were harvested and threshed separately. Repeat the steps of self-pollination and plant selection until plants with more than 5 completely homozygous mutation sites in the EPSPS gene and excellent performance of the target agronomic traits are obtained, which is the glyphosate-resistant wheat. The mutation site is located on the EPSPS gene sequence of the wheat 4A genome, with the following changes occurring at amino acid positions 137, 189, 198, 204, and 226: V137A, L189F, R198P, F204C, and R226H.

5. The method according to claim 4, characterized in that, The plant selection methods include selection assisted by observation, measurement, and markers of mutation sites.

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