A method for preparing maize haploids

Abstract

The application discloses a method for preparing maize haploid. The method comprises the following steps: (1) reducing the activity and / or expression amount of protein ZmGEX1 in diploid maize to obtain transgenic maize; the protein ZmGEX1 is a protein with an amino acid sequence as shown in SEQ ID NO: 3; (2) selfing the transgenic maize or crossing the transgenic maize with diploid maize as a female parent to obtain offspring; and separating maize haploid from the offspring. The application firstly utilizes the protein ZmGEX1 to prepare maize haploid, avoids the phenotype of weak growth and poor fertility of an induction system, and can prepare haploid by selfing, so that excellent crop traits are completely reserved. Meanwhile, the method has a high haploid induction rate for preparing maize haploid. The application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing maize haploids. Background Technology

[0002] The basic procedure for conventional maize haploid breeding is to cross a haploid inducing line (male parent) with ordinary female parent material to obtain haploids, and then double the line to form a double haploid pure line; this inducing line is called a maize female parent haploid inducing line. Stock6 was the first maize female parent haploid inducing line discovered. Using inducing lines derived from Stock6, a certain proportion of haploids can be obtained in almost all female parent materials. Breeders in various countries have used this as a basis to select and breed a large number of excellent inducing lines, and the induction rate has been continuously improved.

[0003] Currently, although some genes regulating maize maize haploid induction lines have been identified, the following problems still need to be addressed: First, gene mutations often lead to weakened plant growth, abnormal pollen development, germination, or fertilization, affecting seed setting rate and thus reducing haploid induction efficiency; Second, detecting male sterility is an important method for rapid haploid identification, but the male sterility phenotype caused by gene mutations can interfere with haploid identification. Therefore, establishing other methods for preparing maize haploids has significant application potential. Summary of the Invention

[0004] The purpose of this invention is to prepare maize haploids.

[0005] This invention first protects a method for preparing maize haploids, which may include the following steps:

[0006] (1) Reduce the activity and / or expression level of protein ZmGEX1 in diploid maize to obtain transgenic maize;

[0007] The protein ZmGEX1 is (a1), (a2), or (a3):

[0008] (a1) A protein with the amino acid sequence shown in SEQ ID NO: 3;

[0009] (a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1);

[0010] (a3) A maize ploidy-related protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in (a1) or (a2).

[0011] (2) After completing step (1), the transgenic corn is self-pollinated or used as the female parent to cross with diploid corn B to obtain offspring;

[0012] (3) After completing step (2), isolate maize haploids from the offspring.

[0013] The labels in (a2) above are shown in Table 1.

[0014] Table 1. Sequence of Labels

[0015] Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tagII 8 WSHPQFEK c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

[0016] In the above method, the reduction of the activity and / or expression level of protein ZmGEX1 in diploid maize A is achieved by mutating the gene encoding protein ZmGEX1 on one homologous chromosome of diploid maize A, while leaving the gene encoding protein ZmGEX1 on the other homologous chromosome unchanged.

[0017] In the above method, the phrase "mutating the gene encoding the protein ZmGEX1 on one homologous chromosome of diploid maize A while keeping the gene encoding the protein ZmGEX1 on the other homologous chromosome unchanged" can be interpreted as mutating the ZmGEX1 gene shown in SEQ ID No: 2 on one homologous chromosome of diploid maize A to the ZmGEX1 / GA gene, while keeping the ZmGEX1 gene shown in SEQ ID No: 2 on the other homologous chromosome unchanged; the ZmGEX1 / GA gene is a DNA molecule obtained by changing the G at position 18 from the 5' end of SEQ ID No: 2 to A, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged.

[0018] In the above method, the gene encoding the protein ZmGEX1 on a homologous chromosome of the mutant diploid maize A is obtained by EMS mutagenesis, T-DNA insertion, RNA interference, homologous recombination, gene site editing, zinc finger nuclease or transcription activator-like effector nuclease, to induce missense mutations in the exons of the gene encoding the protein ZmGEX1.

[0019] The gene encoding any of the aforementioned proteins ZmGEX1 can be a DNA molecule of the following type: (b1) or (b2) or (b3) or (b4) or (b5):

[0020] (b1) A DNA molecule with a coding region as shown in SEQ ID NO:2;

[0021] (b2) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO:2;

[0022] (b3) A DNA molecule with a nucleotide sequence as shown in SEQ ID NO:1;

[0023] (b4) A DNA molecule that hybridizes with the DNA molecule defined by (b1) or (b2) or (b3) and encodes the protein ZmGEX1;

[0024] (b5) A DNA molecule derived from corn that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the DNA molecule defined in (b1), (b2), or (b3) and that encodes the protein ZmGEX1.

[0025] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein ZmGEX1 of this invention using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides that have 70% or higher identity with the nucleotide sequence of the protein ZmGEX1 isolated according to this invention, as long as they encode the protein ZmGEX1, are derived from and equivalent to the nucleotide sequence of this invention.

[0026] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 70% or higher, or 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence of the protein ZmGEX1, which encodes the amino acid sequence shown in SEQ ID NO:3 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0027] The diploid maize A mentioned above can be maize inbred line B73.

[0028] The diploid maize B mentioned above can be maize inbred line B73.

[0029] In any of the methods described above, the genetically modified corn may be zmgex1. + / - Heterozygous mutant; compared with the genome of maize inbred line B73, zmgex1 + / - The only difference between the heterozygous mutants is that the ZmGEX1 gene shown in SEQ ID No: 2 remains unchanged on one homologous chromosome, while the ZmGEX1 gene on the other homologous chromosome is mutated into the ZmGEX1 / GA gene; the ZmGEX1 / GA gene is a DNA molecule obtained by changing the G at position 18 from the 5' end of SEQ ID No: 2 to A, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged.

[0030] In an embodiment of the present invention, zmgex1 + / -The methods for preparing heterozygous mutants can be as follows:

[0031] ① The corn seed was purchased from the EMS mutagenesis mutant library, specifically the zmgex1 strain. + / - Seeds of the mutant (Mutant ID: EMS4-0999b1) were cultured using standard methods to obtain maize plants. Genomic DNA was extracted from the leaves of the maize plants and used as a template. PCR amplification was performed using primers consisting of primer check-F: 5'-TGATGCCCTTCAATTCGCTGTT-3' and primer check-R: 5'-GGGAGGAGAAGATGTTCCACGAGA-3', yielding the PCR amplification product.

[0032] ② Sequencing the PCR amplification product obtained in step ①. The sequencing results are compared with the ZmGEX1 gene (DNA fragment shown at positions 805-1046 from the 5' end of SEQ ID NO: 1) to select heterozygous mutants; a heterozygous mutant is defined as a plant in which the ZmGEX1 gene (nucleotide sequence shown in SEQ ID No: 1) of one of its two homologous chromosomes is mutated, while the ZmGEX1 gene of the other homologous chromosome is not mutated;

[0033] The mutation in the ZmGEX1 gene specifically involves a one-nucleotide mutation in the coding region of the ZmGEX1 gene. Specifically, in SEQ ID No: 1, the G at position 967 from the 5' end is mutated to A, thereby changing the codon TGG encoding tryptophan (Trp) to the stop codon TGA, leading to premature termination of the translation of the protein ZmGEX1.

[0034] ③ The heterozygous mutant obtained in step ② (as the female parent) was backcrossed with maize B73 (as the male parent) to obtain F1 generation seeds; the F1 generation seeds were sown and cultured conventionally to obtain maize plants. Genomic DNA was extracted from the leaves of the maize plants and used as a template. PCR amplification was performed using primers check-F: 5'-TGATGCCCTTCAATTCGCTGTT-3' and check-R: 5'-GGGAGGAGAAGATGTTCCACGAGA-3' to obtain PCR amplification products. The PCR amplification products were sequenced. The sequencing results were compared with the ZmGEX1 gene (DNA fragment shown at positions 805-1046 from the 5' end of SEQ ID NO: 1), and F1-heterozygous mutants were screened based on the comparison results.

[0035] The ZmGEX1 gene on one homologous chromosome of the F1 heterozygous mutant was not mutated, while a 1-nucleotide mutation occurred in the coding region of the ZmGEX1 gene on the other homologous chromosome. Specifically, in SEQ ID No: 1, the G at position 967 from the 5' end was mutated to A.

[0036] ④ The F1 heterozygous mutant obtained in step ③ (as the female parent) was backcrossed with maize B73 (as the male parent) to obtain F2 generation seeds; the F2 generation seeds were sown and cultured conventionally to obtain maize plants. Genomic DNA was extracted from the leaves of the maize plants and used as a template. PCR amplification was performed using primers check-F: 5'-TGATGCCCTTCAATTCGCTGTT-3' and check-R: 5'-GGGAGGAGAAGATGTTCCACGAGA-3' to obtain PCR amplification products. The PCR amplification products were sequenced. The sequencing results were compared with the ZmGEX1 gene (DNA fragment shown in SEQ ID NO: 1 from the 5' end, positions 805-1046), and F2 heterozygous mutants were screened based on the comparison results.

[0037] The F2 heterozygous mutant has no mutation in the ZmGEX1 gene on one homologous chromosome, but a one-nucleotide mutation occurs in the coding region of the ZmGEX1 gene on the other homologous chromosome. Specifically, in SEQ ID No: 1, the G at position 967 from the 5' end is mutated to A.

[0038] ⑤ The F2-heterozygous mutant obtained in step ④ was self-pollinated to obtain F3 generation seeds; F3 generation seeds were sown and cultured conventionally to obtain maize plants. Genomic DNA was extracted from the leaves of the maize plants and used as a template. PCR amplification was performed using primer pair consisting of primer check-F: 5'-TGATGCCCTTCAATTCGCTGTT-3' and primer check-R: 5'-GGGAGGAGAAGATGTTCCACGAGA-3', and the PCR amplification product was obtained. The PCR amplification product was sequenced. The sequencing results were compared with the ZmGEX1 gene (DNA fragment shown at positions 805-1046 from the 5' end of SEQ ID NO: 1), and zmgex1 was obtained from the comparison results. + / - Heterozygous mutant;

[0039] Compared to the genome of maize inbred line B73, zmgex1 + / - The only difference between the heterozygous mutants is that the ZmGEX1 gene on one homologous chromosome is not mutated, while a one-nucleotide mutation occurs in the coding region of the ZmGEX1 gene on the other homologous chromosome. Specifically, in SEQ ID No: 1, the G at position 967 from the 5' end is mutated to A.

[0040] The application of any of the methods described above in improving the efficiency of maize haploid breeding also falls within the scope of this invention.

[0041] The application of maize haploids prepared by any of the above methods in improving maize haploid breeding efficiency also falls within the scope of this invention.

[0042] The application of any of the above-described transgenic maize or any of the above-described proteins ZmGEX1 in the preparation of maize haploids also falls within the scope of this invention.

[0043] The application of any of the above-described transgenic maize or any of the above-described proteins ZmGEX1 in improving maize haploid breeding efficiency also falls within the scope of this invention.

[0044] Experiments have shown that transgenic maize obtained by reducing the activity and / or expression level of the protein ZmGEX1 in diploid maize, through self-pollination or by crossing it with diploid maize as a female parent, can produce maize haploid offspring. This invention is the first to utilize the protein ZmGEX1 to prepare maize haploids, avoiding the weak growth and poor fertility phenotypes of the inducing line, and allowing haploids to be prepared through self-pollination, fully preserving the crop's superior traits. The method of this invention can prepare maize haploids with a high haploid induction rate. This invention has significant application value. Attached Figure Description

[0045] Figure 1 zmgex1 + / - Sequencing results of heterozygous mutants and maize B73.

[0046] Figure 2 This is a schematic diagram of the ZmGEX1 gene structure and the location of the mutated nucleotides in the ZmGEX1 gene.

[0047] Figure 3 The results of haplotype identification in Example 2 are shown.

[0048] Figure 4 The results of flow cytometry identification of haploids in Example 2 are shown.

[0049] Figure 5 This is the result of haploid identification by chromosome counting in Example 2. Detailed Implementation

[0050] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0052] The maize inbred line B73 was obtained from the National Germplasm Bank (website: http: / / www.cgris.net / ) and can be obtained by the public from Henan Agricultural University (the applicant) to replicate this experiment. In the following text, maize inbred line B73 will be referred to as Maize B73.

[0053] zmgex1 + / - The mutant was purchased from the EMS Mutagenesis Library, Mutant ID: EMS4-0999b1. The URL of the EMS Mutagenesis Library is: http: / / www.elabcaas.cn / memd / public / index.html# / . zmgex1 + / - The mutant was obtained by mutating the ZmGEX1 gene against the background of maize B73.

[0054] The nucleotide sequence of the ZmGEX1 gene in the genomic DNA of maize B73 is shown in SEQ ID NO: 1. The nucleotide sequence of the ZmGEX1 gene in the cDNA of maize B73 is shown in SEQ ID NO: 2. The ZmGEX1 gene encodes the protein ZmGEX1, and the amino acid sequence of the protein ZmGEX1 is shown in SEQ ID NO: 3.

[0055] Example 1, zmgex1 + / - Identification of mutants and zmgex1 + / - Obtaining heterozygous mutants

[0056] 1. Sow corn in the field (zmgex1) + / - Seeds of mutant maize (or maize B73) were cultured conventionally for approximately 20 days to obtain maize plants. Genomic DNA was extracted from the leaves of the maize plants and used as a template. PCR amplification was performed using primers consisting of primer check-F: 5'-TGATGCCCTTCAATTCGCTGTT-3' and primer check-R: 5'-GGGAGGAGAAGATGTTCCACGAGA-3', yielding the PCR amplification product.

[0057] 2. Sequencing of the PCR amplification product obtained in step 1. The sequencing results are compared with the ZmGEX1 gene (focusing on the DNA fragment shown in SEQ ID NO: 1 from position 805 to 1046 from the 5' end) to determine the mutation type.

[0058] The genomic DNA of maize B73 has not been mutated. The nucleotide sequence of its ZmGEX1 gene is shown in SEQ ID No: 1. The nucleotide at position 967 from the 5' end of SEQ ID No: 1 (equivalent to position 18 from the 5' end of SEQ ID No: 2) is G. The sequencing chromatogram shows a single peak characteristic.

[0059] zmgex1 + / - Theoretically, mutants can include heterozygous mutants, homozygous mutants, wild-type mutants, and haploid mutants, with the following specific characteristics:

[0060] A homozygous mutant is a plant in which the ZmGEX1 gene on two homologous chromosomes has the same mutation; for example, the nucleotide at position 967 from the 5' end of SEQ ID No: 1 (equivalent to position 18 from the 5' end of SEQ ID No: 2) is non-G, and the sequencing chromatogram shows a single peak feature;

[0061] A heterozygous mutant is a plant in which the ZmGEX1 gene on one of its two homologous chromosomes is mutated, while the ZmGEX1 gene on the other homologous chromosome is not mutated; the sequencing chromatogram shows a bimodal characteristic.

[0062] Wild-type plants refer to plants in which the ZmGEX1 gene on both homologous chromosomes has not been mutated; the sequencing chromatogram shows a single-peak characteristic.

[0063] A haploid plant refers to a plant that has only one set of chromosomes, and its sequencing data shows a single peak.

[0064] Heterozygous mutants exhibiting a bimodal pattern were selected and further identified. One heterozygous mutant showed no mutation in the ZmGEX1 gene on one homologous chromosome, while a single nucleotide mutation occurred in the coding region of the ZmGEX1 gene on the other homologous chromosome. Specifically, at position 967 from the 5' end of SEQ ID No. 1 (equivalent to position 18 from the 5' end of SEQ ID No. 2 or position 163 from the 5' end of the PCR amplification product), the G nucleotide was mutated to A, resulting in a change from the tryptophan (Trp) codon TGG to the stop codon TGA, leading to premature termination of translation of the ZmGEX1 protein. In this heterozygous mutant, the nucleotides at position 967 from the 5' end of SEQ ID No. 1 (equivalent to position 18 from the 5' end of SEQ ID No. 2 or position 163 from the 5' end of the PCR amplification product) were G and A, and the sequencing chromatogram showed a bimodal pattern. Since this heterozygous mutant was generated by EMS mutagenesis, in order to eliminate interference from other gene mutations, the inventors used multiple generations of backcrossing to remove the background of EMS mutagenesis, obtaining zmgex1. + / - Heterozygous mutant. The specific steps are as follows:

[0065] (1) The above heterozygous mutant (as the female parent) was backcrossed with maize B73 (as the male parent) to obtain F1 generation seeds.

[0066] (2) F1 generation seeds were sown in the field and cultured for about 20 days to obtain maize plants. Genomic DNA was extracted from the leaves of the maize plants and used as a template. PCR amplification was performed using primers check-F: 5'-TGATGCCCTTCAATTCGCTGTT-3' and check-R: 5'-GGGAGGAGAAGATGTTCCACGAGA-3' to obtain PCR amplification products. The PCR amplification products were sequenced. The sequencing results were compared with the ZmGEX1 gene (focusing on the DNA fragment shown in SEQ ID NO: 1 from position 805-1046 from the 5' end). Based on the comparison results, F1-heterozygous mutants were screened.

[0067] The F1 heterozygous mutant has no mutation in the ZmGEX1 gene on one homologous chromosome, but a one-nucleotide mutation occurs in the coding region of the ZmGEX1 gene on the other homologous chromosome. Specifically, in SEQ ID No: 1, the G at position 967 from the 5' end is mutated to A.

[0068] (3) Backcross the F1-heterozygous mutant (as the female parent) with maize B73 (as the male parent) to obtain F2 generation seeds.

[0069] (4) Following the method in step (2), replace the F1 generation seed with the F2 generation seed, and keep all other steps unchanged to obtain the F2-heterozygous mutant.

[0070] The F2 heterozygous mutant has no mutation in the ZmGEX1 gene on one homologous chromosome, but a one-nucleotide mutation occurs in the coding region of the ZmGEX1 gene on the other homologous chromosome. Specifically, in SEQ ID No: 1, the G at position 967 from the 5' end is mutated to A.

[0071] (5) Self-pollinate the F2-heterozygous mutant to obtain F3 generation seeds.

[0072] (6) Following the method in step (2), replace the F1 generation seed with the F3 generation seed, keeping all other steps unchanged, to obtain zmgex1. + / - Heterozygous mutant.

[0073] Compared to the genome of maize inbred line B73, zmgex1 + / -The only difference between the heterozygous mutants is that the ZmGEX1 gene on one homologous chromosome is not mutated, while a one-nucleotide mutation occurs in the coding region of the ZmGEX1 gene on the other homologous chromosome. Specifically, in SEQ ID No: 1, the G at position 967 from the 5' end is mutated to A.

[0074] zmgex1 + / - Partial sequencing results of heterozygous mutants and maize B73 are shown below. Figure 1 (zmgex1 + / - zmgex1 + / - Heterozygous mutant, ZmGEX1 is maize B73).

[0075] A schematic diagram of the ZmGEX1 gene structure and the location of the mutated nucleotide (SEQ ID No: 1, position 967 from the 5' end (equivalent to position 18 from the 5' end of SEQ ID No: 2)) in the ZmGEX1 gene are shown below. Figure 2 (Mutant is zmgex1) + / - Heterozygous mutant (Wild Type: maize B73).

[0076] Example 2, zmgex1 + / - Application of heterozygous mutants in the preparation of maize haploids

[0077] 1. zmgex1 + / - Maize haploids are prepared by self-pollination or hybridization of heterozygous mutants.

[0078] 1. Sowing zmgex1 + / - Seeds of the heterozygous mutant were self-pollinated after maturation, yielding 207 F4 generation offspring. These were then sown with zmgex1 seeds. + / - Seeds of heterozygous mutants and maize B73 were then used with zmgex1 + / - A heterozygous mutant was used as the female parent and maize B73 was used as the male parent for hybridization, resulting in 178 F4 generation hybrids.

[0079] 2. 207 F4 self-pollinated progeny, 178 F4 hybrid progeny, and maize B73 were sown in the field and cultured conventionally for approximately 20 days to obtain maize plants. Genomic DNA was extracted from the leaves of the maize plants and used as templates. PCR amplification was performed using primers check-F: 5'-TGATGCCCTTCAATTCGCTGTT-3' and check-R: 5'-GGGAGGAGAAGATGTTCCACGAGA-3', yielding the PCR amplification products.

[0080] 3. Sequencing the PCR amplification product obtained in step 2. The sequencing results were compared with the DNA fragment shown in SEQ ID NO: 1 from position 805 to 1046 starting from the 5' end, and the mutation status was counted.

[0081] The results showed that only one plantlet among the 207 F4 self-pollinated progeny met the criteria for maize haploid: the nucleotide at position 163 of the PCR amplification product (equivalent to position 18 from the 5' end of SEQ ID No: 2) had a single-peak characteristic and a G mutation to A; this plantlet was preliminarily identified as a haploid plantlet and named Haploid 1. Only four plants among the 178 F4 hybrid progeny met the criteria for maize haploid: the nucleotide at position 163 of the PCR amplification product (equivalent to position 18 from the 5' end of SEQ ID No: 2) had a single-peak characteristic and a G mutation to A; these four plants were preliminarily identified as haploid plants and named Haploid 2, Haploid 3, Haploid 4, and Haploid 5, respectively.

[0082] II. Phenotypic Identification of Haploids

[0083] Observe the phenotypes of Haploid 1, Haploid 2, Haploid 3, Haploid 4, and Haploid 5 obtained in step one, as well as maize B73. Maize B73 is diploid.

[0084] Some results can be found Figure 3 (Haploid refers to Haploid 1, Diploid refers to maize B73, a is a comparison of Haploid 1 and maize B73, b is the plant height measurement). The results showed that Haploid 1, Haploid 2, Haploid 3, Haploid 4 and Haploid 5 all had characteristics such as short plants (plant height 100±20cm), narrow leaves, compact plant type, and male sterility; while the diploid maize B73 showed characteristics such as tall plants (plant height 200±20cm), wide leaves, spreading plant type, and normal fertility.

[0085] III. Flow Cytometry for Ploidy Identification

[0086] 1. Extract cell nuclei from young leaves of corn B73, then detect the cell nucleus signal using flow cytometry, and set the signal peak of corn B73 cell nuclei to 100.

[0087] Since corn B73 is diploid, the genetic material in diploid cells is twice that in haploid cells. Therefore, the signal peak in the haploid cell nucleus appears around 50.

[0088] 2. After completing step 1, extract the cell nuclei from the young leaves of the plants to be tested (Haploid 1, Haploid 2, Haploid 3, Haploid 4, or Haploid 5), then detect the cell nucleus signals using flow cytometry, and make the following judgments:

[0089] If the signal peak of the cell nucleus of the plant under test appears around 100 (the location of the signal intensity enrichment in the cell nucleus of the plant under test is basically the same as that of the corn B73 cell nucleus), then the plant under test is determined to be diploid; if the signal peak of the cell nucleus of the plant under test appears around 50, then the plant under test is determined to be haploid.

[0090] Some test results can be found Figure 4 (Diploid is maize B73). The results showed that Haploid1, Haploid2, Haploid3, Haploid4 and Haploid5 were all haploids, as detected by flow cytometry.

[0091] IV. Chromosome Display for Haploid Identification

[0092] 1. Pretreatment

[0093] Between 9:00 and 11:00 AM, root tips (1-1.5 cm in length, representing the root tip meristem) were taken from the plants to be tested (Haploid 1, Haploid 2, Haploid 3, Haploid 4, Haploid 5, or corn B73), submerged in distilled water, and then placed in an ice-water mixture at 4°C for 24 hours.

[0094] 2. Fixed

[0095] After completing step 1, rinse 2-3 times with distilled water, absorb the water, and then fix in a fixative (acetic acid and ethanol mixed in a volume ratio of 1:3) at room temperature for 24 hours.

[0096] 3. Dissociation

[0097] After completing step 2, remove the root tip from the fixative, rinse it with distilled water, then add 1N HCl solution to dissociate it for 20 minutes. When the root tip is transparent and milky white, remove it and rinse it with distilled water 2-3 times, 3-5 minutes each time.

[0098] 4. Staining

[0099] After completing step 3, remove excess water, place the root tip on a glass slide, cut off 2-3 mm of the root tip, add modified phenol-fuchsin staining solution to submerge the root tip, and stain for 10 minutes.

[0100] 5. Tableting

[0101] After completing step 4, crush the root tip with tweezers, cover it with a coverslip, cover it with double layers of filter paper, and press vertically with your thumb until the root tip appears cloudy (0.5-1cm).

[0102] 6. Observation

[0103] After completing step 5, use a 10x scope to find the field of view, 40x to observe, and 100x to take a picture.

[0104] Some test results can be found Figure 5 (Diploid is maize B73, Haploid is Haploid 1). The results showed that Haploid 1, Haploid 2, Haploid 3, Haploid 4 and Haploid 5 have 10 chromosomes, while maize B73 has 20 chromosomes.

[0105] Based on the results of steps two through four above, Haploid 1, Haploid 2, Haploid 3, Haploid 4, and Haploid 5 obtained in step one are all haploids.

[0106] V. Statistics zmgex1 + / - Haploid induction rate of maize haploids prepared from heterozygous mutants

[0107] Maize haploids were prepared twice according to steps one through four. The haploid induction rate was calculated, and the average of the three results was taken.

[0108] zmgex1 + / - The haploid induction rate of maize haploids prepared by hybridization of heterozygous mutants is shown in Table 2.

[0109] Table 2

[0110]

[0111]

[0112] The results showed that zmgex1 + / - The haploid induction rate of maize haploids prepared by self-pollination of heterozygous mutants is relatively stable, around 0.5%; zmgex1 + / - The haploid induction rate of maize haploids prepared by hybridization of heterozygous mutants was relatively high, about 2.02% ((2.25%+3.07%+0.75%) / 3=2.02%).

[0113] The above results indicate that using zmgex1 + / - Heterozygous mutants can be used to prepare maize haploids, zmgex1 + / - The heterozygous mutant is diploid, avoiding the phenotypes of weak growth and poor fertility found in the induced line; meanwhile, zmgex1+ / - Heterozygous mutants can be self-pollinated to produce maize haploids, which can completely retain the crop's desirable traits. Therefore, mutations in the ZmGEX1 gene (specifically, the mutation of G to A at position 18 from the 5' end of SEQ ID No: 2) can lead to the production of maize haploids, providing a new perspective on the biological role of the ZmGEX1 gene in the production of maize haploids.

[0114] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for preparing maize haploids, comprising the following steps: (1) Reduce the expression level of protein ZmGEX1 in diploid maize A to obtain transgenic maize; The protein ZmGEX1 is (a1) or (a2): (a1) A protein with the amino acid sequence shown in SEQ ID NO: 3; (a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1); (2) After completing step (1), the transgenic corn is self-pollinated or used as the female parent to cross with diploid corn B to obtain offspring; (3) After completing step (2), isolate maize haploids from the offspring.

2. The method according to claim 1, characterized in that: The reduction in the expression level of protein ZmGEX1 in diploid maize A is achieved by mutating the gene encoding protein ZmGEX1 on one homologous chromosome of diploid maize A, while leaving the gene encoding protein ZmGEX1 on the other homologous chromosome unchanged.

3. The method according to claim 2, characterized in that: The phrase "the gene encoding the protein ZmGEX1 on one homologous chromosome of the diploid maize α1 mutant, while the gene encoding the protein ZmGEX1 on the other homologous chromosome remains unchanged" refers to the gene encoded by the protein ZmGEX1 on one homologous chromosome of the diploid maize α1 mutant, as shown in SEQ ID No:

2. ZmGEX1 Gene mutation ZmGEX1 / GA The gene, shown in SEQ ID No: 2 on another homologous chromosome. ZmGEX1 The genes remain unchanged; ZmGEX1 / G- A A gene is a DNA molecule obtained by changing the G at position 18 from the 5' end of SEQ ID No: 2 to A, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged.

4. The method according to claim 2, characterized in that: The protein ZmGEX1 encoding gene on a homologous chromosome of the mutant diploid maize A is obtained by causing missense mutations in the exons of the protein ZmGEX1 encoding gene through T-DNA insertion, RNA interference, homologous recombination, gene site editing, zinc finger nucleases or transcription activator-like effector nucleases.

5. The method according to claim 1, characterized in that: The diploid maize A or diploid maize B is maize inbred line B73.

6. The method according to claim 1, characterized in that: The genetically modified corn is zmgex1 + / - Heterozygous mutant; compared to the genome of maize inbred line B73. zmgex1 + / - The only distinguishing feature of heterozygous mutants is the presence of the chromosome shown in SEQ ID No: 2 on a single homologous chromosome. ZmGEX1 The genes remain unchanged, but on another homologous chromosome ZmGEX1 Gene mutation ZmGEX1 / GA Genes; the stated ZmGEX1 / GA A gene is a DNA molecule obtained by changing the G at position 18 from the 5' end of SEQ ID No: 2 to A, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged.

7. The application of the method according to any one of claims 1 to 6 in improving the efficiency of maize haploid breeding.

8. The application of maize haploids prepared by the method according to any one of claims 1 to 6 in improving maize haploid breeding efficiency.

9. The use of the transgenic maize as described in claims 1 to 6 in the preparation of maize haploids.

10. The application of the transgenic maize as described in claims 1 to 6 in improving the haploid breeding efficiency of maize.

11. The application of reducing the expression level of the protein ZmGEX1 described in claim 1 in the preparation of maize haploids or in improving the efficiency of maize haploid breeding; The application is achieved by reducing the expression level of the protein ZmGEX1 described in claim 1 in diploid maize to obtain transgenic maize; and by self-pollinating or hybridizing the transgenic maize to isolate maize haploids from the offspring.

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