A gene and method for inducing parthenogenic haploids in plants

Abstract

The application discloses a gene and a method for inducing parthenogenesis of plants to produce haploid. The application relates to the fields of biotechnology and plant breeding, and provides application of ZmDAZ1 protein or a coding gene thereof in any of the following (A1) and (A2): (A1) inducing parthenogenesis of plants; (A2) cultivating a parthenogenesis plant variety. The application is specifically expressed in an egg cell of a corn inbred line LH244 ZmDAZ1 The gene can endow the corn with the ability of parthenogenesis. Since the parthenogenesis of the plant body can completely inherit the maternal genome, a haploid seed containing only the maternal genome and not containing the paternal genome is generated. Therefore, ZmDAZ1 The gene has important values in inducing the haploid containing only the maternal genome and promoting the haploid breeding process.

Description

[0001] Cross-referencing of related applications: This application claims priority to Chinese patent application (application number 202510318507.X) filed on March 18, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the fields of biotechnology and plant breeding, specifically to a gene and method for inducing haploid production through parthenogenesis in plants. Background Technology

[0003] Plant haploids are individuals with the same number of chromosomes as their gametes, and they are of great value in plant breeding and genetic research. Haploids not only provide a rapid pathway to obtaining homozygous lines in plant breeding, but also offer unique materials for genome research and gene function analysis. Haploids have a significant advantage in accelerating the creation of pure lines; once doubled, they can form homozygous DH (double haploid, DH) lines. Compared to the 7-8 generations required for genetic homozygosity selection in traditional breeding, haploid breeding can rapidly obtain homozygous varieties within two generations, possessing significant technological advantages and application value, and has become a major strategy for maize breeding line selection.

[0004] There are two main pathways for plant haploid formation: artificial induction and spontaneous formation in natural species. The molecular mechanism of the second type of haploid formation is also a hot topic in biological research. Currently, several major genes involved in paternal-induced haploid formation have been cloned, including... PLA1 , DMP However, very few genes controlling parthenogenesis in the maternal gametophyte or sporophyte have been cloned. Furthermore, the probability of naturally forming haploids is very low. Therefore, the currently discovered genes for inducing haploids have very low induction rates, which are insufficient for breeding work. Breeders typically combine artificial haploid induction with other methods to improve breeding efficiency.

[0005] In 2015, CONNER et al. discovered African foxtail grass. PsASGR-BBML Genes can induce haploid embryos in the sexually reproducing species *Pterocarya stenoptera*. In 2019, *Nature* published research by Venkatesan Sundaresan of the University of California, Davis, who first confirmed the presence of an oocyte-specific promoter. AtDD45 Drive rice BBM1 It can also induce rice to produce haploid seeds, which are then combined with MIME ( osd1 / rec8 / spo11 - 1By combining the systems, offspring of rice apomixis were obtained, in which meiosis of the egg cells was replaced by mitosis, and diploid embryos were spontaneously formed, thus completing the fixation of heterosis.

[0006] While many methods exist for inducing haploidy, they all have limitations. Taking maize as an example, very few genes can induce haploidy through single-gene regulation alone. Currently, the induction rate of various paternal induction lines derived from Stock6 germplasm is around 10%, and the offspring exhibit reduced seed setting rate. Furthermore, the MIME system has not yet been implemented in maize. Therefore, exploring new parthenogenetic induction systems for maternal parents with high induction rates and no impact on seed setting is particularly important. Summary of the Invention

[0007] The purpose of this invention is to provide a gene and method for inducing haploid production through parthenogenesis in plants.

[0008] In a first aspect, the present invention claims protection for the use of the ZmDAZ1 protein or its encoding gene in any of the following: (A1) Inducing parthenogenesis in plants; (A2) Cultivating parthenogenetic plant varieties; The ZmDAZ1 protein is any one of the following: (B1) A protein with the amino acid sequence SEQ ID No. 1; (B2) A maize protein having the same function as the amino acid sequence shown in SEQ ID No. 1, by substitution and / or deletion and / or addition of one or more amino acid residues; (B3) has 99%, 95%, 90%, 85% or 80% or more of the same amino acid sequence as any of (B1)-(B2) and is derived from maize and has the same function. (B4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins defined in (B1)-(B3).

[0009] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0010] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0011] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0012] In the aforementioned proteins, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or more identity can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or more identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0013] Secondly, the present invention claims protection for a method for obtaining parthenogenetic plants.

[0014] The method for obtaining parthenogenetic plants claimed in this invention may include the following steps: specifically expressing the ZmDAZ1 protein in the oocytes of a recipient plant to obtain parthenogenetic plants; wherein the ZmDAZ1 protein is any one of those described in (B1)-(B4) above. Furthermore, the specific expression of the ZmDAZ1 protein in the oocytes of the recipient plant can be achieved as follows: The recombinant plasmid was introduced into the recipient plant; The recombinant plasmid contains an expression cassette; The expression cassette contains the gene encoding the ZmDAZ1 protein, and an oocyte-specific expression promoter that initiates the expression of the gene encoding the protein.

[0015] Furthermore, the oocyte-specific expression promoter can be EC1.2EN-EC1.1P; Specifically, the nucleotide sequence of EC1.2EN-EC1.1P is shown in SEQ ID No. 4.

[0016] In one embodiment of the present invention, the recombinant plasmid specifically comprises the DNA molecule shown in SEQ ID No. 4 (i.e., the oocyte-specific promoter EC1.2EN-EC1.1P) and the DNA molecule shown in SEQ ID No. 3 (i.e., the oocyte-specific promoter EC1.2EN-EC1.1P). ZmDAZ1 The CDS sequence of the gene was sequentially linked and then ligated to the HindIII restriction site of the plant expression vector pCM3300M-GFP.

[0017] Thirdly, the present invention claims protection for the use of the expression cassette or recombinant plasmid described in the second aspect above in any of the following: (A1) Inducing parthenogenesis in plants; (A2) Cultivate parthenogenetic plant varieties.

[0018] Fourthly, the present invention claims a method for obtaining plant haploids.

[0019] The method for obtaining plant haploids claimed in this invention may include the following steps: (C1) The recombinant plasmid described in the second aspect above is introduced into the recipient plant to obtain a transgenic plant; (C2) Obtain plant haploids from offspring by self-pollination of the transgenic plant or by hybridization using the transgenic plant as the female parent.

[0020] For example, in step (C2), the transgenic plant is used as the female parent and a plant carrying a haploid selection marker is used as the male parent to perform hybridization, and plant haploids are obtained from the hybrid offspring by screening using the haploid selection marker.

[0021] In one embodiment of the present invention, the haploid selection marker is the R1-nj marker. Specifically, the paternal parent is the maize inbred line Z58 carrying the R1-nj gene.

[0022] Fifthly, the present invention claims a method for obtaining plant DH lines.

[0023] The present invention claims a method for obtaining plant DH lines, which may include the following steps: (D1) Obtain plant haploids using the method described in the fourth aspect above; (D2) The plant haploids are subjected to chromosome doubling to obtain the plant DH line.

[0024] Sixthly, the present invention claims protection for the application of the methods described in the second, fourth and fifth aspects above in plant breeding.

[0025] In the aforementioned relevant aspects, the gene encoding the ZmDAZ1 protein may be any of the following: (E1) The DNA molecule shown in SEQ ID No. 2 or SEQ ID No. 3; (F2) A DNA molecule that hybridizes under stringent conditions with a DNA molecule defined by (F1) and encodes the ZmDAZ1 protein; A DNA molecule that has 99%, 95%, 90%, 85% or more or more identity with any of the defined DNA sequences (F3) and (F1)-(F2) and encodes the ZmDAZ1 protein.

[0026] The stringent conditions for the above-mentioned genes can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1 ...7% SDS, 0.5M Na3PO4, and 1mM EDTA; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS. Hybridize in a mixed solution of SDS, 0.5 M Na3PO4 and 1 mM EDTA, and wash at 65 °C with 0.1 × SSC and 0.1% SDS; alternatively, hybridize in a solution of 6 × SSC and 0.5% SDS at 65 °C, and then wash once each with 2 × SSC and 0.1% SDS and 1 × SSC and 0.1% SDS.

[0027] For the genes mentioned above, nucleotide sequence identity can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastn as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of nucleotide sequences to calculate the identity value (%), then the identity value can be obtained.

[0028] In the aforementioned genes, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or more identity can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or more identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0029] In the aforementioned relevant aspects, the recipient plant is a polyploid plant (such as a diploid plant).

[0030] In the aforementioned relevant aspects, the plant may be any of the following: (F1) Monocotyledons; (F2) Gramineae plants; (F3) Plants of the genus *Zea*; (F4) Corn.

[0031] In one embodiment of the present invention, the recipient plant is the maize inbred line LH244.

[0032] Experiments have shown that this invention is specifically expressed in the egg cells of the maize inbred line LH244. ZmDAZ1 Genes enable parthenogenesis in maize. ZmDAZ1 Genes play a crucial role in induced haploidy. Because parthenogenesis allows for complete inheritance of the maternal genome, without including the paternal genome, therefore... ZmDAZ1 Genes play an important role in inducing haploids containing only the maternal genome and promoting the haploid breeding process. Attached Figure Description

[0033] Figure 1 This is a structural diagram of the main components of the pCM3300M-ZmDAZ1 plasmid.

[0034] Figure 2 Image of T1 generation seeds.

[0035] Figure 3 Images of T2 generation seedlings (3rd from the left are diploids, 3rd from the right are haploids).

[0036] Figure 4 This is a diagram illustrating the identification of Indel molecular markers in T2 generation plants.

[0037] Figure 5 Images of T2 generation plants during pollen shedding (left: diploid, right: haploid).

[0038] Figure 6 The results of flow cytometry analysis of T2 generation plants are shown (left: diploid, right: haploid). Detailed Implementation

[0039] 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.

[0040] 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.

[0041] The haploid screening system described in the following examples utilizes the R1-nj marker. R1-nj is a gene that regulates anthocyanin synthesis in maize. R1 One allele containing R1-nj The aleurone layer and embryo of the corn kernels from this gene are both purple. If the paternal parent contains a homozygous gene... R1-nj The mother parent's seeds, lacking this gene, exhibit a purple embryo and aleurone layer, resulting in white embryos and aleurone layers. Diploid seeds produced after double fertilization of the parents contain this gene. R1-nj The genes are present, hence the embryo and aleurone layer are purple. If the female gamete of the maternal parent can reproduce parthenogenetically to produce haploid seeds, because the fusion of sperm and egg cells does not occur, the embryo does not contain... R1-nj Genes, therefore the embryo of a haploid seed is white; the endosperm that develops from fertilization by the polar nuclei and sperm cells contains R1-nj It is purple due to its genetic makeup.

[0042] The pCM3300M-GFP vector involved in the following examples is described in "Luo H, Meng D, Liu H, et al. Ectopic Expression of the Transcriptional Regulator". silky3The public can obtain this vector from the applicant and it may only be used to repeat the experiments of this invention and may not be used for other purposes.

[0043] The maize inbred line LH244 has been sequenced and is recorded in the NCBI database: https: / / www.ncbi.nlm.nih.gov / assembly / GCA_905067065.1# / def. This material is available to the public from the applicant and may only be used to replicate the experiments of this invention; it may not be used for any other purpose.

[0044] Maize Z58 marker series: will carry R1-nj The maize haploid inducible line CHOI4 was repeatedly backcrossed with the Z58 inbred line, and the offspring were selected for those with purple seed coats and embryos and without [the gene]. PLA1, DMP The lines that induce haploid alleles are the Z58 marker lines. Among them, the maize haploid-induced line CHOI4 is described in the article "Dong Wang, et al. The RUBY reporter enables efficient haploid identification in maize and tomato. PlantBiotechnology Journal (2023) 21, pp. 1707–1715". This germplasm is available to the public from the applicant and can only be used to replicate the experiments of this invention and may not be used for other purposes.

[0045] The amino acid sequence of the ZmDAZ1 protein involved in the following examples is shown in SEQ ID No. 1, its coding gene sequence and cDNA sequence in the maize genome are both SEQ ID No. 2, and its gene CDS sequence is shown in SEQ ID No. 3.

[0046] Example 1, using ZmDAZ1 Induced haploids This invention utilizes ZmDAZ1 The methods for inducing haploids are summarized as follows: ① Selecting EC1.2EN-EC1.1P The promoter specifically expressed in the oocyte, with the nucleotide sequence shown in SEQ ID No. 4, drives... ZmDAZ1The process involves: ① constructing an ectopic expression vector from the CDS or genome sequence of the gene; ② obtaining transgenic positive materials using Agrobacterium-mediated transformation; ③ investigating the field phenotypic traits of wild-type LH244 and T0 generation transgenic positive plants; ④ calculating the seed setting rate of T0 generation transgenic positive materials and performing germination treatment to obtain T1 generation transgenic plants; ⑤ designing InDel polymorphic molecular markers for haploid detection; ⑥ using the Z58 marker line as the male parent and crossing it with T1 generation transgenic positive plants, and using seed color markers and InDel molecular markers to detect haploidy in T2 generation transgenic positive materials; ⑦ using flow cytometry to confirm the ploidy of the plants validated in step ⑥; ⑧ counting the number of haploids in T2 generation transgenic plants and calculating the haploid induction efficiency.

[0047] 1. Experimental materials In this embodiment, the maize inbred line LH244 was selected as the genetic transformation material, and the Z58 marker line was used as the male parent to pollinate the T1 generation positive plants.

[0048] 2. Constructing ectopic expression vectors Select EC1.2EN-EC1.1P The promoter specifically expressed in the oocyte, with the nucleotide sequence shown in SEQ ID No. 4, drives... ZmDAZ1 The CDS sequence (SEQ ID No. 3) of the gene was used to construct an ectopic expression vector.

[0049] 2.1 Obtain the fragments required for constructing the ectopic expression vector.

[0050] (1) Genomic DNA was extracted from Arabidopsis thaliana leaves and used as templates to amplify the EC1.1 and EC1.2 promoters. The fragments were then recombinated to obtain EC1.2EN-EC1.1P Promoter sequence.

[0051] DNA extraction method: Place approximately 1 cm leaf leaf into a 2 mL centrifuge tube with a steel ball, label it, and add 800 μL of Buffer A extraction buffer. Transfer the centrifuge tube to a cell disruptor for vortexing at 100 Hz / sec for 1.5 min. Place the disrupted sample in a 65°C water bath for 30 min, inverting and shaking for 10 min at a time during this period. After removing the sample, cool it to room temperature, then add 800 μL of chloroform / tris-saturated phenol (1:1) mixture in a fume hood, inverting and mixing thoroughly. Centrifuge again at 12000 rpm for 10 min. After centrifugation, aspirate 500 μL of the supernatant into a 1.5 mL sterile centrifuge tube, add 500 μL of pre-chilled isopropanol, gently shake to mix, and then transfer to... Let the sample stand in a 20℃ refrigerator for 30 minutes; after standing, put it in a centrifuge and centrifuge at 12000 rpm for 10 minutes. Discard the supernatant, wash the precipitate twice with 75% ethanol solution, and air dry at room temperature; add 300 μL of ddH2O to dissolve it, shake gently, and store it in a 4℃ refrigerator for later use.

[0052] (2) RNA was extracted from the tassels of maize inbred line LH244 and then reverse transcribed into cDNA sequences, which were used as gene sequences. ZmDAZ1 The CDS sequence provides a template for amplification.

[0053] Method for RNA extraction: Corn tassels at the pollen shedding stage were placed in a sterilized mortar pre-cooled with liquid nitrogen and ground into a white-green powder. The powder was then quickly transferred to an RNase-free EP tube containing 1 mL of Trizol and vortexed for 1 min to mix thoroughly. The mixture was incubated at room temperature for 5 min, then 200 μL of chloroform was added to the EP tube, vortexed for 15 s, and incubated on ice for 2 min. The mixture was then centrifuged at 4°C and 12000 rpm for 10 min. 400 μL of the supernatant was transferred to a 1.5 mL RNase-free EP tube, and 400 μL of isopropanol was added. The mixture was inverted and mixed thoroughly. The mixture was incubated on ice for 10 min, then centrifuged at 4°C and 12000 rpm for 10 min. The supernatant was discarded. ⑧ 1 mL of... Wash the precipitate with 75% ethanol by vortexing, centrifuge at 12000 rpm for 10 min at 4°C; discard the supernatant and repeat the previous step; retain the RNA precipitate at the bottom of the tube, aspirate the residual liquid, air dry on ice, add 100 μL of RNase-free water, gently tap to dissolve, and then measure the concentration and purity. Store at [insert storage location here]. Keep at 80℃ for later use.

[0054] Methods for reverse transcription of RNA into cDNA: via PrimeScript ™ cDNA was prepared using the RT reagent Kit (Perfect RealTime) (RR037Q). The specific steps were as follows: The reagents and RNA template provided in the kit were dissolved on ice; the reagents were mixed according to the RNA reverse transcription system, and the mixture was briefly centrifuged to allow the liquid to collect at the bottom of the tube; the reaction program was then performed, reacting at 42°C for 15 min followed by reacting at 85°C for 5 min; the cDNA was then cooled on ice after the reaction was completed.

[0055] (3) Design amplification primers. Design amplification primers for the EC1.1 and EC1.2 promoter sequences based on the nucleotide sequences. ZmDAZ1 Primers for amplifying the gene CDS sequence.

[0056] (4) Amplify the promoter sequence and gene CDS sequence using amplification primers.

[0057] (5) After detection by 1% agarose gel electrophoresis, the required fragments are cut and recovered. Store at 20℃ for later use.

[0058] 2.2 Enzyme digestion of the backbone vector.

[0059] The pCM3300M-GFP backbone vector was digested with HindIII restriction enzyme to form a sticky end nick, while retaining all other sequences, including the original terminator.

[0060] 2.3 The recovered amplified fragments were ligated into the backbone vector.

[0061] After mixing the ligation system thoroughly, incubate at 50°C for 15 min. After ligation, place the product on ice until ready for use. The amplification and ligation primers are shown in Table 1. Table 1. Primers for vector construction

[0062] 2.4 Conversion and ligation products.

[0063] Methods for converting ligation products: ① Remove competent cells stored at 80℃ and thaw them on ice; ② When the competent cells reach the molten state, immediately add the ligation product; incubate on ice for 30 min; heat shock in a 42℃ water bath for 45 sec; add 500 μL of liquid LB medium; incubate at 37℃ on a shaker for 1 h; centrifuge slowly at 4000 rpm for 3 min and then spread onto LB solid medium with Kan resistance; incubate overnight at 37℃, and perform colony PCR detection after the colonies have grown; pick single colonies and incubate them in liquid medium with Kan resistance at 37℃ on a shaker at 200 rpm for 14 h; extract plasmids and perform Sanger sequencing.

[0064] The recombinant expression vector, after being verified by sequencing, was named pCM3300M-ZmDAZ1. Its structure is described as follows: a recombinant plasmid containing the DNA fragment shown in “SEQ ID No. 4 + SEQ ID No. 3” inserted at the HindIII restriction site of pCM3300M-GFP. The main structural elements of the pCM3300M-ZmDAZ1 plasmid are shown in the diagram below. Figure 1 As shown.

[0065] 3. Obtaining transgenic plants The expression vector pCM3300M-ZmDAZ1 was transformed into Agrobacterium tumefaciens (…). Ag.rhizogenes In strain EHA105, the vector was transferred into the immature embryo of the recipient material maize inbred line LH244 using Agrobacterium-mediated transformation, and the T0 generation transgenic maize was obtained.

[0066] 4. Planting genetically modified plants The genetically modified material was planted in experimental fields during the summer and in greenhouses during the winter. The average daytime temperature in the greenhouse was 32°C, the average nighttime temperature was 24°C, with a 14-hour light / 10-hour dark cycle and a relative humidity of 65%.

[0067] 5. Detection of transgenic plants Primers designed based on the EC1.2EN-EC1.1P promoter sequence and the gene CDS sequence were used as primers for detecting transgenic positive plants. Specific primer information is shown in Table 2. A total of four positive transformation lines were obtained after identification, namely ZmDAZ1-1 to ZmDAZ1-4.

[0068] Table 2. Primers for identifying transgenic plants

[0069] 6. Use color-coding and indel markers to screen haploids. T1 generation positive transgenic lines ZmDAZ1-1 to ZmDAZ1-4 were sown in the transgenic experimental field of China Agricultural University as the female parent and crossed with the Z58 marker line. After harvest at maturity, haploid grains were screened using color markers. Grains with both purple endosperm aleurone layer and embryo were heterozygous diploid grains, while grains with purple endosperm aleurone layer and white embryo were haploid. Figure 2 ).

[0070] To further confirm the chromosome ploidy of the T2 generation plants, based on the genome-wide sequence differences between the maize inbred lines LH244 and Z58, three pairs of Indel polymorphic markers (Table 3) were designed and extracted from the T2 generation plants. Figure 3 Leaf DNA was amplified by PCR, and the presence of the Z58 genome sequence was determined by banding patterns. Figure 4 As shown, the samples with only the LH244 band pattern are haploid, while the plants with both the LH244 and Z58 band patterns are diploid, thus further confirming the ploidy of the T2 generation plants.

[0071] Table 3. InDel polymorphic primers

[0072] 7. Flow cytometry ploidy detection and T2 generation parthenogenesis efficiency statistics To definitively confirm the ploidy of the T2 generation transgenic plants, fresh seedlings at the 3-leaf stage were taken ( Figure 3Leaf samples were examined for ploidy of cell nuclei using flow cytometry, following the method described by Tian S, Zhang J, Zhao H, et al. Production of double haploid watermelon via maternal haploid induction. Plant Biotechnol J. 2023. Results are as follows: Figure 6 As shown, the scattered light signal intensities of cells in the G1 and G2 phases of the sample in the left image are 100 and 200, respectively, indicating a typical diploid plant. In contrast, the scattered light signal intensities of cells in the G1 and G2 phases of the sample in the right image are only half that of the left image, indicating a typical haploid plant. Haploid plants in the pollen shedding stage exhibit typical haploid morphology, including stunted growth and male sterility. Figure 5 ).

[0073] Finally, combining the results of color markers, molecular markers, and flow cytometry, the proportion of haploid plants in the T2 generation was calculated, and the haploid induction efficiency was calculated. The results are shown in Table 4, that is, the haploid induction efficiency of ZmDAZ1-1 to ZmDAZ1-4 ranged from 11.76% to 40.00%.

[0074] Table 4. Statistics on Haploid Induction Efficiency of T2 Generation

[0075] In summary, it can be seen that the specific expression of [the substance] in the egg cells of the maize inbred line LH244 is [indicating this]. ZmDAZ1 Genes can endow maize with the ability to reproduce parthenogenetically. Because parthenogenesis allows for the complete inheritance of the maternal genome, without the paternal genome, it can induce the production of haploids containing only the maternal genome. Therefore, ZmDAZ1 Genes play an important role in inducing the generation of haploids containing only the maternal genome.

[0076] 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. The application of ZmDAZ1 protein or its encoding gene in any of the following: (A1) Inducing parthenogenesis in plants; (A2) Cultivating parthenogenetic plant varieties; The ZmDAZ1 protein is any one of the following: (B1) A protein with the amino acid sequence SEQ ID No. 1; (B2) A protein derived from maize with the same function, having undergone substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1; (B3) has 99%, 95%, 90%, 85% or 80% or more of the same amino acid sequence as any of (B1)-(B2) and is derived from maize and has the same function. (B4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins defined in (B1)-(B3).

2. A method for obtaining parthenogenetic plants, comprising the following steps: specifically expressing ZmDAZ1 protein in the oocytes of a recipient plant, thereby obtaining parthenogenetic plants; The ZmDAZ1 protein is any one of the following: (A1) A protein with the amino acid sequence SEQ ID No. 1; (A2) A protein derived from maize with the same function, having undergone substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1; (A3) has 99%, 95%, 90%, 85% or 80% or more of the same amino acid sequence as any of (A1)-(A2) and is derived from maize and has the same function. (A4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins defined in (A1)-(A3).

3. The method of claim 2, wherein: The specific expression of the ZmDAZ1 protein in the oocytes of the recipient plant is achieved as follows: The recombinant plasmid was introduced into the recipient plant; The recombinant plasmid contains an expression cassette; The expression cassette contains the gene encoding the ZmDAZ1 protein, and an oocyte-specific expression promoter that initiates the expression of the gene encoding the protein.

4. The method of claim 3, wherein: The oocyte-specific expression promoter is EC1.2EN-EC1.1P; Furthermore, the nucleotide sequence of EC1.2EN-EC1.1P is shown in SEQ ID No.

4.

5. The use of the expression cassette or the recombinant plasmid as described in claim 3 or 4 in any of the following: (A1) Inducing parthenogenesis in plants; (A2) Cultivate parthenogenetic plant varieties.

6. A method for obtaining plant haploids, comprising the following steps: (C1) Introducing the recombinant plasmid as described in claim 3 or 4 into a recipient plant to obtain a transgenic plant; (C2) Obtain plant haploids from offspring by self-pollination of the transgenic plant or by hybridization using the transgenic plant as the female parent.

7. A method for obtaining plant DH lines, comprising the following steps: (D1) Obtaining plant haploids using the method described in claim 6; (D2) The plant haploids are subjected to chromosome doubling to obtain the plant DH line.

8. The application of the method according to any one of claims 2-4, 6 and 7 in plant breeding.

9. The use or method of any one of claims 1-8, wherein: The gene encoding the ZmDAZ1 protein is any one of the following: (E1) The DNA molecule shown in SEQ ID No. 2 or SEQ ID No. 3; (E2) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined by (E1) and encodes the ZmDAZ1 protein; A DNA molecule that has 99%, 95%, 90%, 85% or more or more identity with any of the defined DNA sequences in (E3) and (E1)-(E2) and encodes the ZmDAZ1 protein.

10. The use or method of any one of claims 1-9, wherein: The recipient plant is a polyploid plant; or The plant is any one of the following: (F1) Monocotyledons; (F2) Gramineae plants; (F3) Plants of the genus *Zea*; (F4) Corn.

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