Wheat mutant protein with glyphosate resistance, gene and application thereof

Abstract

The invention discloses a wheat mutant protein with glyphosate resistance, a gene and an application thereof, and belongs to the technical field of plant breeding. The invention discloses wheat mutant proteins GR1 and GR19 with glyphosate resistance, 6 and 3 mutation sites respectively exist on the ESPSP protein, and the amino acid sequences of positions 128-308 and 133-308 are shown in SEQ ID NO.4 and SEQ ID NO.5 respectively. The invention uses glyphosate as a screening agent to find two mutants with glyphosate resistance and excellent agronomic traits in a wheat mutant population, named common wheat ZM9-GR1 and common wheat ZM9-GR19, and discloses the application of the above mutants in plant breeding. The invention also discloses a method for cultivating glyphosate-resistant wheat by backcross breeding and hybrid breeding using the above mutants. The invention has great application value in plant breeding and agricultural production.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, and in particular to wheat mutant proteins and genes with glyphosate resistance and applications thereof. Background Art

[0002] Farmland weeds are one of the main reasons for crop yield reduction. The economic losses caused by weed damage in wheat production account for 10%-20% of the total output each year. For wheat, my country's staple food crop, the difficulty of weed control in wheat fields has increased due to the increase in weed resistance levels, frequent herbicide damage, prominent production season contradictions, and extensive farming measures. Weeds are a major challenge in farming, restricting the yield and cost of crop planting. Herbicides, as a powerful weapon to control weeds, are an essential part of modern agriculture. Herbicide-resistant crops are an effective means to improve the efficiency of herbicide application, and changing the sensitive sites of herbicide effector proteins is a common strategy for breeding herbicide-resistant crops.

[0003] The molecular formula of glyphosate is C3H8NO5P. It is a highly effective, broad-spectrum, low-toxic, low-residue herbicide that does not damage the soil environment and is lethal to most plants. It is non-toxic to humans and animals, has a low frequency of weeds and crops developing resistance to it under natural conditions, and has low soil residues. It has huge market potential.

[0004] Glyphosate competitively inhibits the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimic acid pathway, resulting in the blockage of the synthesis of aromatic amino acids (phenylalanine, tyrosine and tryptophan), which ultimately causes plant death. Generally, crops are sensitive to glyphosate, which greatly limits its use time and space. For example, herbicides need to be used some time before crop sowing to avoid crop damage. Therefore, cultivating some herbicide-resistant (tolerant) crop varieties can reduce crop damage and broaden the scope of herbicide use.

[0005] The research and development and commercial application of glyphosate-resistant transgenic non-staple crops (such as cotton, soybeans, corn, etc.) have achieved great success, but the development of transgenic staple crops such as GM wheat and GM rice is highly controversial in almost all countries. Mutation breeding is an alternative method for breeding herbicide-resistant / tolerant crops, and has been well applied in some countries because it does not involve genetic modification of the crops themselves. For example, the commercial production of Immi-resistant rice and wheat varieties in the United States has a history of nearly 20 years. Overseas, non-transgenic herbicide-resistant rice led by BASF and others has developed into two types: (1) Clearfield GM rice and (2) GM rice. TMThis type, known as Clean Field Rice in China, is mainly produced by the mutagenesis of the ALS (acetolactate synthase) gene, and the corresponding herbicide is imidacloprid. Some herbicide-resistant rice varieties used in domestic production, such as Jinjing 818 and Clean Field Rice 001 bred by Shenzhen Xingwang Biological Seed Co., Ltd., belong to this type; however, this technology should be used with caution in my country, because imidacloprid has a long residual effect in the soil and has a great impact on the next crop. (2) Provisia TM Type, mainly produced by the mutation of ACCase (acetyl-CoA carboxylase) gene, and the corresponding herbicide is sethoxydim, etc.

[0006] The motif of the EPSPS enzyme is conserved in all plants and most bacteria and is essential for binding phosphoenolpyruvate (pep) or competitively inhibiting glyphosate.

[0007] 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 (calculated based on the EPSPS amino acid positions of the model plant rice) can produce glyphosate resistance, which has been reported in a variety of crops (including corn, wheat, wheat, rapeseed, sunflower, etc.), model plants Arabidopsis and a variety of weeds.

[0008] Common wheat, which is widely cultivated in production, is an allohexaploid with three genomes, A, B and D, that is, each gene has three copies in the wheat genome, and the three copies of the EPSPS gene are located on chromosomes 7A, 4A and 7D. At present, patent CN201680047851.9 discloses that the known glyphosate-resistant mutation sites of wheat include 168 (T168I), 172 (P172S), 173 (T173I), and 177 (P177S). Summary of the invention

[0009] The purpose of the present invention is to provide a wheat mutant protein, gene and application thereof with glyphosate resistance to solve the problems existing in the above-mentioned prior art. The mutant protein provided by the present invention can effectively improve the ability of wheat to resist glyphosate.

[0010] To achieve the above object, the present invention provides the following solutions:

[0011] The present invention provides a wheat mutant protein GR1 with glyphosate resistance. The amino acid sequence at positions 128-308 of the wheat mutant protein GR1 is shown in SEQ ID NO.4.

[0012] The present invention provides a gene encoding the wheat mutant protein GR1. The sequence of the nucleotides from the second exon to the fourth intron of the encoding gene is shown in SEQ ID NO.6.

[0013] The present invention also provides a wheat mutant protein GR19 with glyphosate resistance, and the amino acid sequence at positions 129-308 of the wheat mutant protein GR19 is shown in SEQ ID NO.5.

[0014] The present invention also provides a gene encoding the wheat mutant protein GR19. The sequence of the nucleotides from the second exon to the fourth intron of the encoding gene is shown in SEQ ID NO.7.

[0015] The present invention also provides an application of common wheat (Triticum aestivum L.) ZM9-GR1 in cultivating glyphosate-resistant wheat, wherein the deposit number of the common wheat ZM9-GR1 is CCTCC No: P202419;

[0016] The sequence of the second exon to the fourth intron of the EPSPS gene in the 7D genome of the common wheat ZM9-GR1 is shown in SEQ ID NO.6.

[0017] The present invention also provides an application of common wheat (Triticum aestivum L.) ZM9-GR19 in cultivating glyphosate-resistant wheat, wherein the deposit number of the common wheat ZM9-GR19 is CCTCC No: P202420;

[0018] The sequence of the second exon to the fourth intron of the EPSPS gene in the 7D genome of the common wheat ZM9-GR19 is shown in SEQ ID NO.7.

[0019] Preferably, the common wheat ZM9-GR1 or the common wheat ZM9-GR19 is a donor plant for the glyphosate resistance gene.

[0020] The present invention also provides a method for cultivating glyphosate-resistant wheat, comprising the following steps:

[0021] The common wheat ZM9-GR1 or the common wheat ZM9-GR19 is used as the male parent and the wheat variety to be bred for glyphosate resistance is used as the female parent, and hybridization is performed to obtain hybrid offspring;

[0022] Screening hybrid offspring resistant to glyphosate and backcrossing with the female parent;

[0023] Screening backcross progeny for resistance to glyphosate, backcrossing with the female parent, and repeating for more than 3 times;

[0024] Collect the final offspring and self-pollinate;

[0025] After two generations of self-pollination, the lines with similar phenotypes to the maternal line and stably expressing the glyphosate resistance trait are screened to obtain the glyphosate-resistant wheat.

[0026] The present invention also provides a method for cultivating glyphosate-resistant wheat, comprising the following steps:

[0027] The common wheat ZM9-GR1 or the common wheat ZM9-GR19 is used as the male parent and the wheat variety to be bred for glyphosate resistance is used as the female parent, and hybridization is performed to obtain hybrid first-generation seeds;

[0028] Planting the hybrid first generation seeds, self-pollinating to form hybrid second generation seeds, and continuing to plant;

[0029] Selecting plants of the second generation hybrid seeds, harvesting the selected plants and threshing them separately;

[0030] The steps of self-crossing and plant selection are repeated until plants with more than three completely homozygous mutation sites in the EPSPS gene and excellent target agronomic traits are obtained, thereby obtaining the glyphosate-resistant wheat.

[0031] Preferably, the method of plant selection comprises marker-assisted selection through observation, measurement and mutation site.

[0032] The present invention discloses the following technical effects:

[0033] The experimental results of the present invention through field spraying of glyphosate show that after 40 mL of Roundup / L water (5 times the recommended concentration) is applied to the wheat 3-leaf 1-heart stage seedlings containing the EPSPS protein with herbicide resistance of the present invention, the plants still grow and develop normally and bear fruit, while after 8 mL of Roundup / L water (recommended concentration) is applied to the wild-type wheat seedlings, the plant growth gradually stops, the leaves lose green and dry up, and the whole plant dies in 10-15 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1The figure is a graph showing the characteristics of the "Zhenmai 9" induced M3 generation resistant wheat mutant and wild-type wheat obtained by glyphosate screening of the present invention; wherein A is a resistant mutant obtained by glyphosate screening of Zhenmai 9 induced M3 generation, which was later cultivated into GR19, and B is a comparison of the effects of spraying glyphosate on the wild-type Zhenmai 9;

[0036] Figure 2 This is the result of PCR amplification of the partial fragment of EPSPS gene in wheat 7D genome; among them, the first row 1-24 and the second row 25-44 lanes are the amplification products of 44 glyphosate-resistant mutant wheat strains, and the second row lane 45 is the amplification product of wild-type ZM9; the target fragment length is 950bp;

[0037] Figure 3 It is a comparison of the amino acid sequences of EPSPS proteins in the wheat 7D genome; OsEPSPS is the amino acid sequence of the rice EPSPS protein, TaEPSPS-7D (Louise) is the amino acid sequence of the wheat Louise EPSPS protein; TaEPSPS-7D (ZM9) is a partial amino acid sequence of the wild-type Zhenmai 9 EPSPS protein, TaEPSPS-7D (GR19) is a partial amino acid sequence of the EPSPS protein of the mutant GR19, and the mutation site of the GR19 mutant is indicated by a red 'character + underline';

[0038] Figure 4 Schematic diagram of marker-assisted backcrossing to transfer the glyphosate resistance trait of GR19 to the genetic background of Yangmai 33;

[0039] Figure 5 Schematic diagram of the pedigree breeding method for the combined improvement of Yangmai 33 (with excellent agronomic traits) and GR19 (resistant to glyphosate). DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1: Process for obtaining wheat glyphosate-resistant mutants

[0046] 1 kg of seeds of the 'Zhenmai No. 9' wheat variety (M0, purchased from Nantong Changjiang Seed Co., Ltd.) were soaked in clean water for 5 hours, then transferred to 0.6-1.0% (v / v) ethyl methanesulfonate (EMS) and soaked at room temperature for 5-10 hours, during which the seeds were shaken every hour; the EMS solution was discarded, and the seeds were soaked in tap water for 5 times, each time for 5 minutes, and then the seeds were rinsed with tap water overnight, and the field was sown the next day (M1), and conventional fertilizer and water management was carried out.

[0047] After the plants mature, the seeds are mixed, dried, and stored in a cold storage for autumn sowing experiments. Take 15kg (M2) of mixed seeds and spread them manually over an area of ​​1.5 mu, obtaining 283,000 M2 plants. When the wheat seedlings grow to the 3-leaf 1-heart stage, spray glyphosate (the dosage is 16mL Roundup / L water, 2 times the recommended dosage), and 11 plants grow normally, showing green leaves, normal jointing, heading and fruiting. These are candidate mutants resistant to glyphosate. After the seeds mature, mix them, dry them, and store them in a cold storage.

[0048] The mixed harvested M3 seeds were continued to be planted and sown manually. Glyphosate resistance was further screened and identified. When the wheat seedlings grew to the 3-5 leaf stage, glyphosate was sprayed (the dosage was 40 mL Roundup / L water, 5 times the recommended dosage). After 20 days, it was found that the growth of 44 wheat plants was not affected by glyphosate at all, while the remaining plants and the wild type Zhenmai No. 9 plants were completely dead (such as Figure 1 In addition, secondary screening can eliminate false positives and remove weakly resistant mutants.

[0049] Plant the 44 M3 seeds obtained, and determine the number of rows or areas to plant based on the number of seeds harvested. 3:4 The mutant strains were identified for related agronomic traits, and two mutants with stable glyphosate resistance and excellent agronomic traits were preliminarily identified and named GR1 and GR19 respectively.

[0050] Example 2: Analysis of mutation sites of glyphosate-resistant wheat mutants

[0051] Among the herbicide-resistant wheat mutant lines obtained in Example 1, the M of mutants such as GR1 and GR19 were selected. 3:5 Fifteen seeds of each of the lines and the wild-type control were placed in an incubator for matrix culture. Leaves were taken at the 3-leaf stage to extract genomic DNA. PCR amplification was performed using TaEPSPS-CL as the upstream primer and TaEPSPS-7AR, TaEPSPS-4AR, and TaEPSPS-7DR as the downstream primers. The primer sequences are shown in Table 1.

[0052] For PCR amplification, Novozyme 2×KeyPo Master Mix (Dye Plus) high-fidelity enzyme (Cat. No. PK511) was selected, and the reaction system was as follows: 2×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, and nuclease-free ddH2O 16 μL.

[0053] The amplification procedure is as follows: ①98℃10sec, ②58℃5sec, ③72℃5sec, 35cycles (①→③). The total reaction volume is 40μL, 3μL of the reaction solution is loaded, and after the agarose gel electrophoresis band is correct and bright, the remaining reaction solution is sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing using the forward primer: TaEPSPS-CL. The sequencing results are compared and analyzed using DNAMAN software. The electrophoresis results are shown in Figure 2 shown.

[0054] Table 1: PCR primers for detecting mutations in wheat EPSPS homologous genes

[0055] Primer name Primer sequence (5'-3') TaEPSPS-CL ACAGTGAGGATGTCCACTACATGCTTGA(SEQ ID NO.8) TaEPSPS-7AR ACTTCTCTGACAGAGAACAGAAGTGTGCAC(SEQ ID NO.9) TaEPSPS-4AR TTTGTGTAAGGTCGCATTGATCGTACTACCA(SEQ ID NO.10) TaEPSPS-7DR GAAAACTAGAATCATGCTTTTGTACTCCACTATC(SEQ ID NO.11)

[0056] The sequencing results showed that compared with the wild-type plants, the above-mentioned herbicide-resistant wheat mutants had single-base mutations at multiple sites in the EPSPS gene sequence of the wheat 7D genome, resulting in changes in the 136th, 249th, 250th, 255th, 262nd, 269th, 270th, 272nd, and 296th amino acids in the corresponding encoded amino acid sequences, among which 6 mutation sites were detected in the mutant GR1 and 3 mutation sites were also detected in the mutant GR19 (as shown in Table 2).

[0057] Table 2: Mutation site information in EPSPS gene of wheat chromosome 7D

[0058]

[0059] The nucleotide sequences of the EPSPS gene PCR products of the 7A genome, 4A genome and 7D genome of wild-type wheat plants are shown in SEQ ID NOs. 1 to 3, respectively. The amino acid sequences of the EPSPS proteins of mutant GR1 and mutant GR19 are shown in SEQ ID NOs. 4 to 5, respectively.

[0060] The mutant wheat plants of the present invention are classified and named as common wheat ZM9-GR1 (Triticum aestivum L. ZM9-GR1) and common wheat ZM9-GR19 (Triticum aestivum L. ZM9-GR19).

[0061] The seeds of common wheat ZM9-GR1 (Triticum aestivum L.ZM9-GR1) were named wheat mutant seeds GR1, and the seeds of common wheat ZM9-GR19 (Triticum aestivum L.ZM9-GR19) were named wheat mutant seeds GR19, and the above materials were preserved. The wheat mutant seeds GR1 were deposited in the China Center for Type Culture Collection (CCTCC) on September 4, 2024, address: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC No: P202419; the wheat mutant seeds GR19 were deposited in the China Center for Type Culture Collection (CCTCC) on September 4, 2024, address: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, with a deposit number of CCTCC No: P202420.

[0062] SEQ ID NO.1:

[0063] 5’-CAGGGTAGCTTGGACTCTCCGTGGAGCAGATAAAGTTGCAAAAAGAGCTGTGGT TGTTGGCTGTGGCGGCAGGTTCCCAGTCGAAAAGGACGCCAAAGAGGAAGTAAAGCTCTTCTTGGGTAATGCTGGAACTGCAATGCGGCCACTGACGGCAGCTGTAGTAGCTGCTGGTGGAAATGCAACGTATGTTTTCTTTATCCTAGTGGAAATAAGTATGAGATCCATGGGTATGTTTGGAGACTGATCGTTTCTTTTATTAAAAAAAAACTTCAGTTATGTGCTTGATGGCGTACCAAGAATGAGGGAGCGACCTATTGGTGACTTAGTTGTAGGTTTGCAACAACTCGGCGCAGATGTCGATTGTTTCCTTGGCACAAACTGCCCACCTGTCCGTATCAACGGCAAAGGAGGTCTACCTGGTGGCAAGGTTAGCTACTCATCAACTTGCATGTTATCTACTTTGTGCACACTTCTGTTCTCTGCATAAAAAAGTAATA-3’。

[0064] SEQ ID NO.2:

[0065] 5'--3'。

[0066] SEQ ID NO.3:

[0067] 5'--3'。

[0068] SEQ ID NO.4:

[0069] LGLSVEADKVAKRAVVVGCGGRFPVEKDAKEEVKLFLGNAGTAMRPLTAAVVAAGGNATYVLDGVPRMRERPIGDLVVGLQQLGADADCFLGTNCPPVRINGKGGLPGGKVKLSGSISSQYPRSLLMAAPLALENVEIEIIEILSSVPYVEMTLKLMERFGVTAEHSDSWDRFYIKGGQKY.

[0070] SEQ ID NO.5:

[0071] EADQVAKRAVVVGCGGRFPVEKDAKEEVKLFLGNAGTAMRPLTAAVVAAGGNATYV LDGVPRMRERPIGDLVVGLQQLGADADCFLGTNCPPVRINGKGGLPGGKVKLSGSUSSQY LSSLLMGAPLALEDVEIEIIDKLISVPYVEMTLKLMERFGVTAEHSDRWDRFYIKGGQKY.

[0072]

[0073] The bold and underlined sequences are CDS sequences.

[0074]

[0075]

[0076] The bold and underlined sequences are CDS sequences.

[0077] The amino acid sequence of the EPSPS protein of wheat 7D genome of mutant GR19 obtained in the present invention was compared with that of EPSPS proteins of rice, wheat Louise and wild type Zhenmai 9. Figure 3 shown.

[0078] Example 3 Transformation of glyphosate resistance traits

[0079] The backcross method is used to carry out the breeding of glyphosate resistance traits. The wheat variety that needs to be bred for glyphosate resistance is used as the female parent (also known as the recurrent parent, RP). The mutant common wheat ZM9-GR1 or common wheat ZM9-GR19 is used as the male parent, and the hybrid F1 is obtained by hybridization.

[0080] If the glyphosate resistance trait is dominant, the recurrent parent is used as the female parent, the above F1 is used as the male parent, and the hybridization is used to obtain the backcross generation BC1. Repeat this process, with the recurrent parent as the female parent each time and the glyphosate-resistant backcross generation as the male parent. According to genetic theory, after six backcrosses, the offspring are more than 99% genetically similar to RP on average. After several backcrosses, the individuals in the last backcross generation are self-pollinated to "fix" the introduced glyphosate resistance gene in a homozygous state. Here, marker-assisted selection is used to select backcross offspring plants with a high recovery rate, thereby accelerating the process of backcross breeding.

[0081] If the glyphosate resistance trait is recessive, the recurrent parent is used as the female parent, and the homozygous glyphosate-resistant individuals obtained through self-pollination and phenotypic screening of the offspring group (spraying glyphosate in the field) in the above-mentioned F1 or backcross generation are used as the male parent for backcrossing; similarly, the heterozygous individuals containing the resistance site in the backcross offspring can be selected with the aid of marker-assisted selection technology, which eliminates the need for self-pollination and speeds up the breeding process.

[0082] The present invention takes Yangmai 33 as the recurrent parent to breed the glyphosate-resistant trait from the mutant GR19 as an example. The breeding scheme is shown in Table 3, and the process generally requires 10 generations. In order to speed up the breeding process, marker-assisted backcrossing technology can be used. This method mainly includes three aspects of selection, foreground (target trait) selection, recombinant selection and background selection. Since the selection of glyphosate resistance can be achieved through simple phenotypic screening, the genetic background of the recurrent parent 'Yangmai 33' can be selected through marker information in the backcross offspring population. The markers used are not linked to the target gene. Except for the target gene, all other markers select the genotype of the recurrent 'Yangmai 33'. The more markers there are, the faster the recurrent parent will recover (see the schematic diagram of marker-assisted backcrossing). Figure 4 ). Through marker-assisted backcrossing, a satisfactory recurrent parent recovery rate (>95%) can be obtained after 3-4 consecutive backcrossing generations.

[0083] The transgenic line of Yangmai 33 after backcrossing with the glyphosate resistance gene from GR19 was named 'Yangmai 33GR'. Field spraying tests showed that the resistance of 'Yangmai 33GR' to glyphosate was similar to that of GR19, and could resist 5 times the recommended dose of glyphosate.

[0084] Table 3: Breeding plan for glyphosate resistance from GR19 using Yangmai 33 as the recurrent parent

[0085]

[0086] Example 4: Aggregation of glyphosate resistance and superior agronomic traits

[0087] Wheat varieties (such as Yangmai 33, Zhenmai 18, etc.) that are widely promoted in the target ecological zone are selected as female parents, and mutants GR1 and GR19 are crossed as male parents to obtain hybrid F1. Any such F1 contains a full set of alleles of three or more non-transgenic mutation sites of the EPSPS gene, and is therefore also included in the present invention. These embodiments also include the use of transgenic or backcrossing of wheat varieties with three or more non-transgenic mutations in the EPSPS gene to produce first-generation F1 plants.

[0088] Plant the above-mentioned F1 generation seed, add generation to form F2 seed.Continue to plant the described F2 seed, this is the first separation generation, through observation, measurement and the marker assisted selection of mutation site, carry out the selection of plant, harvest selected individual plant respectively and thresh separately.The F3 seed from each selected plant is planted into different ear rows or plant rows, self-crossing adds generation, then the selected row or the plant of these rows is harvested separately and threshed.This selection is equally based on the observation or measurement of target proterties, for example, contains mutation site in EPSPS gene, and the agronomic traits of multiple expectations.

[0089] In the selection of glyphosate-resistant wheat, except for glyphosate resistance, which is measured by direct field spraying of glyphosate or by marker-assisted selection to select glyphosate-resistant individual plants or ear rows, other traits, especially agronomic traits, refer to conventional wheat breeding selection techniques or standards, and focus on wheat characteristics such as plant height, plant type, maturity, maturity phase, disease resistance (fusarium head blight, powdery mildew, etc.), tillering, ear size, and fruitfulness. Select plants with a plant height of 75-85cm, a full growth period of 198-203d, a compact or loose plant type, good maturity phase, and resistance or medium resistance to fusarium head blight or powdery mildew; in terms of the three factors of yield, select medium-large ears, medium-large grains, and medium tillers. The specific standards are 37-43 grains per ear, 40-46g of 1,000-grain weight, and 295,000-325,000 effective ears per mu. In addition, when selecting seeds indoors, those with yellow or black seeds, high black embryo rate, small seeds, thin and shrunken seeds, and small seed quantity are eliminated, and those with large seeds, horny seeds, bright colors, full seeds, and large seed quantity are retained. The above planting and selection process is repeated many times until three or more mutation sites in the EPSPS gene are completely homozygous and multiple target agronomic traits are excellent. For the hybridization, generation derivation, identification and screening process, see Figure 5 .

[0090] The breeding process of hybridization, selfing and selection can be repeated to produce another wheat breeding population derived from a wheat variety having three or more mutation sites in the EPSPS gene, wherein one of the parents is derived from a mutant or transgenic material containing three or more mutation sites in the EPSPS gene, or both parents are derived from a mutant or transgenic material containing three or more mutation sites in the EPSPS gene.

[0091] Through the above process, two superior lines with excellent yield traits (slightly better than 'Yangmai 33') and excellent glyphosate resistance were obtained, named Chongmai 101GR and Chongmai 102GR. Field spraying tests showed that their glyphosate resistance was similar to that of the parent GR19, and could withstand 5 times the recommended dose of glyphosate.

[0092] Example 5: Marking of mutation sites

[0093] The results of sequencing the PCR amplification products of the three pairs of primer combinations in Table 1 in the present invention showed that the three pairs of primers were confirmed to specifically amplify the EPSPS genes located at 7A, 4A and 7D, respectively, so that the nucleotide differences between the wild-type and mutant sequences can be identified by the sequencing results of the PCR amplification products, and then the mutation sites of the EPSPS genes of the PCR amplification products can be screened.

[0094] The 3-leaf stage leaves of the tested material, GR1 and GR19 were taken respectively to extract genomic DNA, and PCR amplification was performed using TaEPSPS-CL as the upstream primer and TaEPSPS-7DR as the downstream primer (primer sequences are shown in Table 1).

[0095] For PCR amplification, Novozyme 2×KeyPo Master Mix (Dye Plus) high-fidelity enzyme (Cat. No. PK511) was selected, and the reaction system was as follows: 2×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, and nuclease-free ddH2O 16 μL.

[0096] The amplification procedure was as follows: ①98℃10sec, ②58℃5sec, ③72℃5sec, 35cycles (①→③). The total reaction volume was 40μL, 3μL of the reaction solution was loaded, and after the agarose gel electrophoresis band was correct and bright, the remaining reaction solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing using the forward primer: TaEPSPS-CL, and the sequencing results were compared and analyzed using DNAMAN software.

[0097] If the material to be tested contains the same six single base mutations of GR1 or three single base mutations of GR19 as listed in Table 1, it can be considered that the glyphosate resistance gene of the material to be tested is derived from the mutant GR1 or GR19 claimed in the present invention.

[0098] The present invention relates to mutations in EPSPS genes, all of which are single nucleotide polymorphisms, which can be used as markers in crop breeding. Specifically, one or more mutation sites in Table 2 can be used as markers in plant breeding. The relationship between the mutation in EPSPS gene and the resistance of the corresponding mutant to glyphosate is a causal relationship, and some markers including KASP markers / probes can be used to track their separation.

[0099] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An application of common wheat (Triticum aestivum L.) ZM9-GR1 in cultivating glyphosate-resistant wheat, characterized in that: The seeds of the common wheat ZM9-GR1 are named as wheat mutant seeds GR1, and the deposit number of the wheat mutant seeds GR1 is CCTCC No: P202419.

2. The use according to claim 1, characterized in that The common wheat ZM9-GR1 is a donor plant of the glyphosate resistance gene.

3. An application of common wheat ZM9-GR19 in cultivating glyphosate-resistant wheat, characterized in that: The seeds of the common wheat ZM9-GR19 are named as wheat mutant seeds GR19, and the deposit number of the wheat mutant seeds GR19 is CCTCC No: P202420.

4. The use according to claim 3, characterized in that The common wheat ZM9-GR19 is a donor plant of the glyphosate resistance gene.

5. A method for cultivating glyphosate-resistant wheat, characterized in that: The following steps are involved: The common wheat ZM9-GR1 described in claim 1 or the common wheat ZM9-GR19 described in claim 3 is used as the male parent and the wheat variety to be bred for glyphosate resistance is used as the female parent, and hybridization is performed to obtain hybrid offspring; Screening hybrid offspring resistant to glyphosate and backcrossing with the female parent; Screening backcross progeny for resistance to glyphosate, backcrossing with the female parent, and repeating for more than 3 times; Collect the final offspring and self-pollinate; After two generations of self-pollination, the lines with similar phenotypes to the maternal line and stably expressing the glyphosate resistance trait are screened to obtain the glyphosate-resistant wheat.

6. A method for cultivating glyphosate-resistant wheat, characterized in that: The following steps are involved: The common wheat ZM9-GR19 described in claim 3 is used as the male parent and the wheat variety to be bred for glyphosate resistance is used as the female parent, and hybridization is performed to obtain hybrid first generation seeds; Planting the hybrid first generation seeds, self-pollinating to form hybrid second generation seeds, and continuing to plant; Selecting plants of the second generation hybrid seeds, harvesting the selected plants and threshing them separately; Repeating the steps of self-crossing and plant selection until a plant having three completely homozygous mutation sites in the EPSPS gene and excellent target agronomic traits is obtained, thereby obtaining the glyphosate-resistant wheat; The mutation site is located in the EPSPS gene of wheat chromosome 7D, including the following mutation sites: AAA→CAA at position 1479; GCT→GGT at position 2195; AGT→AGG at position 2319.

7. The method according to claim 6, characterized in that The plant selection method includes observation, measurement and marker-assisted selection of mutation sites.

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