Method for creating a millet haploid induction line using a crisper / cas12i.3 system and application thereof

Abstract

The application discloses a method for creating a millet haploid induction line by using a CRISPR / Cas12i.3 system and application thereof, and belongs to the technical field of genetic engineering. The technical problem to be solved by the application is how to prepare a haploid millet by using a simpler method, such as a gene editing tool. To this end, the application provides the application of a genome editing system and related biological materials in the preparation of a millet haploid induction line, a method for preparing a millet haploid induction line based on a genome editing system, and a gene edited millet obtained as a haploid induction line. The application successfully constructs a double-mutation homozygous plant by targeting editing two key MTL (MILKED EAR1-LIKE) homologous genes in the millet, and verifies that the double-mutation homozygous plant can stably induce a female parent to produce a haploid offspring as a male parent, fills the blank of the millet haploid induction technology, and provides a new path for rapid pure line breeding of the crop.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to a method for creating millet haploid inducible lines using the CRISPR / Cas12i.3 system and its application. Background Technology

[0002] Millet, as an important specialty grain crop, suffers from long and inefficient conventional breeding cycles. Currently, an effective haploid breeding system has not been established for millet, primarily due to the lack of efficient haploid inducible lines. Although studies have successfully created mtl mutant inducible lines in other gramineous crops using the CRISPR / Cas9 system, the Cas9 system faces challenges such as large protein volume, difficulties in vector loading, and potential risks associated with foreign patents.

[0003] In existing technologies, the main method for creating haploid inducible lines is to use the CRISPR / Cas9 gene editing system to target and knock out the MTL (MATRILINEAL) gene in gramineous crops (such as maize and rice). This approach involves designing specific sgRNAs targeting key exons of the MTL gene, constructing a Cas9 / sgRNA expression vector to achieve functional knockout of the MTL gene, and then using the resulting mutants as male parents for hybridization to induce the production of haploid offspring.

[0004] This technology has been successfully applied in major crops such as maize and rice, confirming that the MTL gene is a key target for regulating haploid induction, and providing a theoretical basis and technical path for haploid breeding in other species.

[0005] The CRISPR / Cas12i.3 system is a novel miniaturized gene editing tool independently developed by Chinese researchers. It boasts advantages such as short recognition sites (TTN PAM), high editing efficiency, strong specificity, and complete independent intellectual property rights. Its application to the targeted knockout of the millet MTL gene holds promise for the efficient creation of localized haploid inducible lines, breaking through the bottlenecks in millet breeding technology.

[0006] In theory, systems like Cas9 and Cas12a can also be used to knock out key haploid induction genes (such as MTL homologs) in millet, thereby creating inducible lines. These systems are technologically mature and widely used, and have successfully achieved haploid induction in various crops. However, the core patents for Cas9 and Cas12a are mainly held by foreign institutions (such as the Broad Institute and the CVC team), and there is a lack of independent underlying patents in China. When applying them for commercial breeding in China, there may be patent licensing barriers or infringement risks, especially given the increasing emphasis on intellectual property compliance in the field of agricultural biotechnology.

[0007] Anther / microspore culture combined with chromosome doubling: This method does not rely on gene editing. Haploid plants are obtained directly from male gametophytes through in vitro culture, and then homozygous diploids (DH lines) are obtained through colchicine treatment. This approach completely avoids gene editing patent issues. However, it is highly dependent on species-specific tissue culture systems, and millet flowers are small and difficult to regenerate. Currently, there is no efficient and stable anther culture protocol, making it technically challenging, time-consuming, and highly genotype-dependent, thus hindering large-scale application. Summary of the Invention

[0008] The technical problem this application aims to solve is: how to prepare haploid millet using a simpler method, such as gene editing tools. To solve this technical problem, this application provides the following technical solution: The first aspect: The application of genome editing tools in the preparation of haploid millet induction lines This application provides for the use of genome editing systems and related biomaterials in at least one of the following: A1) Application in the preparation of millet haploid induction systems; A2) Its application as a product for preparing millet haploid induction lines; A3) Application in millet breeding or assisted breeding; A4) Application in the preparation of products for millet breeding or assisted breeding; The genome editing system includes: (i) Protein components, which are proteins or conjugates thereof whose amino acid sequence is shown in SEQ ID NO:9, and (ii) A nucleic acid component containing the RNA sequence shown in SEQ ID NO:10 from the 5' to 3' direction. The protein component and the nucleic acid component combine to form a complex.

[0009] The biomaterial is at least one of B1) to B4) below: B1) Nucleic acid molecules, wherein the nucleic acid molecules express the aforementioned protein components and / or nucleic acid components; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0010] Wherein, the nucleic acid molecule expressing the above-mentioned protein components is a DNA molecule as described in at least one of g1) and g2) below: g1) DNA molecules with nucleotide sequences such as positions 3143 to 6286 of SEQ ID NO:7; g2) A DNA molecule that has more than 70% identity with the DNA molecule at positions 3143 to 6286 of SEQ ID NO:7 and encodes the above-mentioned protein components; The nucleic acid molecule expressing the above-mentioned nucleic acid components is a DNA molecule as described in at least one of g3) and g4) below: g3), DNA molecules with nucleotide sequences such as positions 427 to 474 of SEQ ID NO:7; g4) DNA molecules that have more than 70% identity with the DNA molecule at positions 427 to 474 of SEQ ID NO:7 and that transcribe the above-mentioned nucleic acid components.

[0011] In this application, the genome editing system is a CRISPR / Cas12i.3 system, in which the protein component is Cas12i.3. Cas12i.3 protein is smaller in size, making it easier to construct plant expression vectors and significantly improving transformation efficiency.

[0012] In this application, the expression cassette of the protein component contains a DNA fragment with a nucleotide sequence as shown in positions 3443 to 6286 of SEQ ID NO:7; the transcribed DNA molecule (expression cassette) of the nucleic acid component contains a DNA fragment with a nucleotide sequence as shown in positions 427 to 474 of SEQ ID NO:7.

[0013] In this application, "identity" refers to the similarity of amino acid or nucleotide sequences. The similarity of amino acid sequences (or nucleotide sequences) can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of amino acid sequences, the similarity value (%) can be obtained.

[0014] Specifically, the consistency of 70% or more can be 75% or more. Specifically, the consistency of 75% or more can be 80% or more. Specifically, the consistency of 80% or more can be 85% or more. Specifically, the consistency of 85% or more can be 90% or more. Specifically, the consistency of 90% or more can be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. More specifically, the consistency of 70% or more can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% consistency.

[0015] In this application, the expression box further includes a promoter and a terminator.

[0016] The expression cassette of the protein component is promoted by the Ubi promoter.

[0017] The promoter of the transcribed DNA molecule of the nucleic acid component is the U3 promoter, and the terminator is the U3t terminator.

[0018] In this application, the genome editing system is introduced into the immature embryo of the millet grain to be edited in the form of a plasmid.

[0019] In this application, the plasmid is transformed into the immature embryo of millet grains to be edited via Agrobacterium-mediated transformation.

[0020] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to polymers of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, proteins, peptides, or polypeptides are at least 3 amino acids in length. Proteins, peptides, or polypeptides can refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications. Proteins, peptides, or polypeptides can also be single molecules or can be multi-molecular complexes. Proteins, peptides, or polypeptides can simply be fragments of naturally occurring proteins or peptides. Proteins, peptides, or polypeptides can be naturally occurring, recombinant, or synthetic, or any combination thereof. Any protein provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers.

[0021] The terms "nuclear localization signal," "nuclear localization sequence," or "NLS," used interchangeably in this article, refer to the amino acid sequence that "tags" a protein for transport into the nucleus via nuclear transport. Typically, this signal consists of a short sequence of one or more positively charged lysine or arginine residues exposed on the protein surface. Different nuclear localization proteins may share the same NLS. The NLS functions in the opposite way to the nuclear export signal, aiming to expel the protein from the nucleus.

[0022] As used in this article, the term "fusion protein" refers to a hybrid polypeptide containing protein domains from at least two different proteins. One protein may be located at the N-terminal (N-terminal) portion or the C-terminal (C-terminal) portion of the fusion protein, thus forming an "N-terminal fusion protein" or a "C-terminal fusion protein," respectively.

[0023] The term "biomaterial" refers to any material that carries genetic information and is capable of self-replication or replication within a biological system, such as genes, plasmids, microorganisms, animals, and plants.

[0024] As is commonly understood in the art, the term "promoter" generally refers to a DNA containing an RNA polymerase binding site, a transcription start site, and / or a TATA box that assists or promotes the transcription of transcribed DNA. Promoters can be artificially synthesized, modified, or derived from known or naturally occurring promoters. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this application may include variants of promoter sequences that are compositionally similar but not identical to other promoter sequences provided herein.

[0025] Promoters can be classified according to various criteria related to the expression patterns of the associated coding or transcribed sequences or genes (including transgenes) operably linked to them, such as constitutive, developmental, tissue-specific, and inducible promoters. A promoter that drives expression in all or most tissues of a plant is called a "constitutive" promoter. A promoter that drives expression at certain times or stages of development is called a "developmental" promoter. A promoter that drives enhanced expression in certain tissues of a plant relative to other tissues is called a "tissue-enhancing" or "tissue-preferred" promoter. Therefore, a "tissue-preferred" promoter elicits relatively high or preferential expression in a specific tissue of the plant, but lower expression levels in other tissues. A promoter that is expressed in a specific tissue of the plant but rarely or not expressed in other tissues is called a "tissue-specific" promoter. An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli (e.g., cold, drought, or light) or other stimuli (e.g., injury or chemical application). Promoters can also be classified according to their origin, such as heterologous, homologous, chimeric, synthetic, etc.

[0026] The term "transcribed DNA" refers to DNA that can be transcribed into RNA molecules.

[0027] In this application, the genetically modified plant organs may be the roots, stems, leaves, flowers, fruits, and seeds of the genetically modified plant.

[0028] In this application, the transgenic plant cells, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0029] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes).

[0030] The recombinant microorganisms described in this application can be yeast, bacteria, algae, or fungi. The bacteria can be Gram-positive or Gram-negative bacteria.

[0031] The term "operationally ligated" can refer to a functional connection between a promoter and transcribed DNA, enabling the promoter to function and initiate transcription of the transcribed DNA. The term "operationally ligated" can also refer to a functional connection between other regulatory elements and a target gene to regulate the transcription and / or expression of the target gene.

[0032] As used herein, an "expression cassette" refers to a cassette containing at least transcribed DNA operatively linked to one or more regulatory elements, typically at least a promoter and a 3' UTR (such as a terminator).

[0033] As used herein, the term "vector" refers to any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell. Examples include plasmids, granules, viruses, bacteriophages, or linear or circular DNA.

[0034] In this application, "editing" or "genome editing" means using targeted genome editing technology to produce a targeted mutation, deletion, inversion, or substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, or at least 10,000 nucleotides of endogenous plant genome nucleic acid sequence.

[0035] In this application, “editing” or “genome editing” also covers the use of targeted genome editing technology to target and insert or site-specifically integrate at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides into the endogenous genome of a plant.

[0036] In this application, the “target sequence” or “target site” for genome editing refers to the location of a polynucleotide sequence within the plant genome that is targeted and cleaved by a site-specific nuclease, thereby introducing a double-strand break (or single-strand nick) into the nucleic acid backbone and / or its complementary DNA strand. The site-specific nuclease may bind to the target site, for example, via a non-coding guide RNA (e.g., but not limited to CRISPR RNA (crRNA) or single-strand guide RNA (sgRNA)). The non-coding guide RNA provided herein may be complementary to the target site (e.g., complementary to the strand of a double-stranded nucleic acid molecule or the chromosome of the target site). “Target site” also refers to the location of a polynucleotide sequence within the plant genome that is bound and cleaved by another site-specific nuclease, which may not be guided by a non-coding RNA molecule, such as a broad-spectrum nuclease, zinc finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN), to introduce a double-strand break (or single-strand nick) into the polynucleotide sequence and / or its complementary DNA strand.

[0037] In this application, the term "guide RNA" or "gRNA" is a short RNA sequence comprising (1) a structural or scaffold RNA sequence required to bind or interact with RNA-guided nucleases and / or other RNA molecules (e.g., tracrRNA), and (2) an RNA sequence that is identical or complementary to a target sequence or target site (referred to herein as the "guide sequence"). A "single-stranded guide RNA" (or "sgRNA") is an RNA molecule comprising tracrRNA and crRNA covalently linked by a linker sequence, which may be expressed as a single RNA transcript or molecule. The guide RNA comprises a guide or target sequence ("guide sequence") identical or complementary to a target site within the plant genome, for example, at or near a GA oxidase gene. An interphase sequence adjacent motif (PAM) may be present immediately adjacent to the 5' end of a genomic target site sequence complementary to the guide RNA's target sequence and upstream of it in the genome, i.e., downstream (3') of the sense (+) strand immediately adjacent to the genomic target site (relative to the guide RNA's target sequence), as is known in the art. The genomic PAM sequence (relative to the target sequence of the guide RNA) on the sense (+) strand adjacent to the target site may contain 5'-NGG-3'. However, the corresponding sequence of the guide RNA (i.e., immediately downstream (3') of the target sequence of the guide RNA) is typically not complementary to the genomic PAM sequence. The guide RNA can usually be a non-coding RNA molecule that does not encode a protein. In some embodiments of this application, the composition for genome editing may be co-delivered with a DNA molecule containing a selection or screening marker gene.

[0038] The second aspect: Methods for preparing millet haploid induction lines This application provides a method for preparing a millet haploid induction line, the method comprising at least one of the following C1) and C2): C1) Knock out the gene encoding pollen-specific phospholipase in the millet seed to be edited using the genome editing system described above; C2) The genome of the millet seed to be edited is mutated as follows: the DNA fragment with nucleotide sequence as SEQ ID NO:1 at positions 5017 to 5058 and the DNA fragment with nucleotide sequence as SEQ ID NO:4 at positions 524 to 531 are deleted from the millet seed to be edited; The millet seed to be edited contains the encoding gene of the pollen-specific phospholipase, which includes PmMTLa and PmMTLb. The amino acid sequence of PmMTLa is shown in SEQ ID NO:3, and the amino acid sequence of PmMTLb is shown in SEQ ID NO:6.

[0039] In this application, the gene encoding PmMTLa is shown below: (1) The encoded sequence is shown in SEQ ID NO:2; (2) The nucleotide sequence is shown in SEQ ID NO:1.

[0040] In this application, the gene encoding PmMTLb is as follows: (1) The encoded sequence is shown in SEQ ID NO:5; (2) The nucleotide sequence is shown in SEQ ID NO:4.

[0041] The third aspect: gene-edited millet This application also provides gene-edited millet, wherein the gene-edited millet contains a DNA fragment with a nucleotide sequence of SEQ ID NO:1, such as the DNA fragment at positions 5017 to 5058 of SEQ ID NO:1, and a DNA fragment with a nucleotide sequence of SEQ ID NO:4, such as the DNA fragment at positions 524 to 531 of SEQ ID NO:4.

[0042] The gene-edited millet in this application is a millet haploid inducible line.

[0043] The gene-edited millet seed is obtained by gene editing of the millet seed to be edited, and the millet seed to be edited contains the gene encoding the pollen-specific phospholipase mentioned above.

[0044] The gene-edited millet was prepared using the method described above for preparing millet haploid inducible lines.

[0045] The fourth aspect: Application of gene-edited millet This application also provides the application of the above-mentioned gene-edited millet in at least one of the following: E1) Application in the preparation of haploid millet; E2), its application in the preparation of haploid millet products; E3), its application in millet haploid breeding; E4) Application in the preparation of millet haploid breeding products.

[0046] In this application, the evaluation indicators for millet breeding include the genomic haplotype of millet.

[0047] In this application, the purpose of the millet breeding is to obtain millet with a haplotype genome.

[0048] Fifth aspect: Methods for preparing haploid millet. This application provides a method for preparing haploid millet seeds, the method comprising at least one of the following: F1) The above-mentioned gene-edited millet (millet haploid inducible line) was used as the male parent for hybridization and millet was used as the female parent to prepare the haploid millet; F2) The above-mentioned gene-edited millet (millet haploid inducible line) was self-crossed to prepare the haploid millet; The process also includes a step of emasculating the millet seedlings used as the female parent. Maestrosculation involves immersing the entire ear of millet in a constant temperature water bath at 46-49°C for 7-10 minutes before the female parent begins to head but before pollen is released.

[0049] The emasculated female parent and the gene-edited millet seedlings were placed together in the same pollination bag. On the 4th day after pollination, the female parent ear was replaced separately with a clean white sulfuric acid paper isolation bag. After another 3 days (about the 6th day after pollination), when pollination was completed and the surface of the ear was dry and free of pollen residue, the isolation bag was replaced with a breathable nylon mesh bag with a small pore size (about 0.5–1.0 mm).

[0050] The sixth aspect is haploid millet and diploid millet obtained by chromosome doubling. This application also provides millet as described in at least one of the following: G1), haploid millet, wherein the haploid millet is obtained by hybridization or self-pollination using the above-mentioned gene-edited millet as a hybrid parent (father); Homozygous diploid millet obtained by chromosome doubling of haploid millet described in G2) and F1).

[0051] As used in this article, “gene-edited millet” and / or “haploid millet” includes plant cells, explants, plant parts, seedlings, plantlets, or whole plants at any stage of regeneration or development.

[0052] As used herein, "plant part" can refer to any organ or intact tissue of a plant, such as meristematic tissue, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryo, endosperm, and seed coat), fruits (e.g., mature ovaries), propagules, or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof. The plant part in this application can be viable, non-viable, renewable, and / or non-renewable. "Propagule" can include any plant part that can grow into a whole plant.

[0053] Plant cells are biological cells of plants, which are taken from plants or derived from cultures obtained by culturing cells taken from plants. Cells can include original transformed plant cells, transgenic plant cells regenerated or developed from R0 generation transgenic plant cells, transgenic plant cells cultured from another transgenic plant cell, or transgenic plant cells from any progeny or offspring of a transformed R0 generation plant, including cells of plant seeds or embryos, or cultured plant cells, callus cells, etc.

[0054] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0055] The term "comprising" is not intended to be restrictive, but rather inclusive and implies the presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially consisting of". In this document, the terms "including" and "comprise" are used interchangeably.

[0056] The beneficial technical effects achieved by this application are as follows: (1) First-ever efficient and controllable haploid induction in millet: As a typical self-pollinating, densely floreted, and small-flowered grass, effective haploid breeding methods have long been lacking. This invention successfully constructed double-mutant homozygous plants by targeting and editing two key MTL (MILKED EAR1-LIKE) homologous genes (longmi014834 and longmi037254) in millet, and verified that they can stably induce haploid offspring in the maternal parent when used as the male parent, filling the gap in millet haploid induction technology and providing a new path for rapid pureline breeding of this crop.

[0057] (2) The invention employs a self-developed miniaturized Cas12i.3 system: Unlike the commonly used Cas9 or Cas12a systems, this invention uses a miniaturized CRISPR / Cas12i nuclease with independent intellectual property rights. This system has advantages such as small protein size, loose PAM sequence (e.g., TTN), low off-target rate, and easy delivery, making it particularly suitable for genetic manipulation of difficult-to-transform crops such as millet. Furthermore, Cas12i.3 has a low off-target rate, lower biosafety risks, and is more in line with future trends in agricultural biotechnology regulation.

[0058] (3) Optimizing the hybridization pollination procedure significantly improves the recovery rate of haploid grains: Addressing the problems of short flowering period, difficulty in emasculation, and susceptibility to contamination in millet, this invention optimizes the hot water emasculation parameters (46-49℃ treatment for 7-10 minutes) and adopts a phased isolation strategy of "changing the sulfuric acid paper bag on the 3rd day after pollination and changing it to a small-pore nylon mesh bag on the 7th day." This scheme effectively prevents contamination by foreign pollen, reduces ear mold, and avoids bird and rain damage. While ensuring pollination success rate, it greatly improves the grain setting rate and harvest integrity of haploid grains.

[0059] (4) Constructing a dual identification system of molecular markers and flow cytometry to ensure accurate and reliable haploid screening: This invention employs a set of highly specific co-dominant molecular markers combined with flow cytometry to verify the parental origin and ploidy of candidate plants. This method can effectively eliminate false-positive hybrid seeds and interference from spontaneous polyploids, increasing the accuracy of haploid identification to over 95%, which is significantly better than traditional morphological or single-method judgment, providing high-quality starting materials for subsequent chromosome doubling and DH line construction.

[0060] (5) Significantly shortens the breeding cycle and accelerates the genetic improvement process of millet: Traditional pure-line breeding of millet requires 6-8 generations of self-pollination to achieve genetic stability, taking as long as 4-6 years. However, this invention, through haploid induction and chromosome doubling, can obtain completely homozygous double haploid (DH) lines within 1-2 growing seasons, shortening the breeding cycle by more than 60%. At the same time, the DH population has a uniform genetic background, which greatly improves the efficiency and accuracy of QTL mapping, gene function verification, and hybrid parent selection.

[0061] (6) The inducible line can be used for gene editing breeding without exogenous T-DNA: By using the inducible line of this invention as the male (or female) parent and crossing it with the target millet variety, gene-edited plants without transgenic elements (i.e., without T-DNA integration) can be directly obtained in the offspring. This strategy not only effectively circumvents the strict approval and supervision of transgenic crops in my country, but also solves the technical bottleneck that prevents direct gene editing of some millet varieties due to difficulties in genetic transformation, providing a feasible path for precision breeding without transgenic attributes.

[0062] (7) Promoting the development of functional genomics research and molecular design breeding of millet: This invention is not only a breakthrough in breeding technology, but also establishes the first heritable haploid platform for millet, an important and distinctive miscellaneous grain crop. This platform can be used for basic research such as large-scale screening of recessive mutants, gene interaction analysis, and comparison of allele functions, providing a powerful tool for elucidating the molecular mechanisms of millet's excellent traits such as drought resistance, tolerance to poor soil, and high nutrition.

[0063] In summary, this invention integrates gene editing, hybridization, precise identification, and breeding applications. It has a complete technical system, strong operability, and high scalability. It not only solves the haploid breeding problem of millet, but also provides a reference paradigm for the development of haploid technology for other similar small-flowered gramineous crops (such as foxtail millet, sorghum, and sorghum spp.), and has important scientific value and broad application prospects. Attached Figure Description

[0064] Figure 1 This is a structural diagram of a gene editing vector.

[0065] Figure 2 The results are for MTL gene editing.

[0066] Figure 3 The results are from flow cytometry of diploid and haploid (induced) cells.

[0067] Figure 4 The plants in question are Longmi No. 4 and haploid (induced) plants.

[0068] Figure 5 The grains are those of Longmi No. 4 and haploid (induced). Detailed Implementation

[0069] This invention is the first to identify and validate endogenous genes related to haploid induction (such as the MTL homologs longmi014834 and / or longmi037254) in millet, and successfully create a stable haploid induction line by precisely knocking out these genes using the CRISPR / Cas12i.3 gene editing system with independent Chinese intellectual property rights. This induction line can efficiently produce haploid grains through conventional hybridization. Combined with optimized pollination management and a dual identification system of "molecular markers + flow cytometry," the haploid recovery rate and screening accuracy are significantly improved, thus achieving rapid pure-line breeding of millet. Crucially, this induction line can be used as a universal paternal (or maternal) parent for hybridization with other non-transgenic millet varieties, directly obtaining gene-edited progeny plants without transgenic elements without introducing exogenous T-DNA. This strategy effectively circumvents my country's strict regulatory approval process for transgenic crops, while simultaneously solving the technical bottleneck of some millet varieties being difficult to genetically transform and directly gene-edit.

[0070] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are 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 present application in any way.

[0071] 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.

[0072] The Longmi No. 4 in the following embodiments is owned by the applicant and is described in the following published literature: "Shi J, Ma X, Zhang J, Zhou Y, Liu M, Huang L, Sun S, Zhang X, Gao X, Zhan W, Li P, Wang L, Lu P, Zhao H, Song W, Lai J. Chromosome conformation capture resolved nearcomplete genome assembly of broomcorn millet. Nat Commun. 2019 Jan 25;10(1):464. doi: 10.1038 / s41467-018-07876-6. PMID: 30683940; PMCID: PMC6347627." The public can apply to obtain this biological material from the applicant. The obtained biological material can only be used to verify the technical solution of this application and cannot be used for other purposes.

[0073] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0074] Example 1. Preparation and phenotypic detection of gene-edited millet. 1.1 Target Design and Vector Construction: Based on the millet genome database, key genes PmMTLa ( longmi014834 ), PmMTLb ( longmi037254 The encoded sequence of ).

[0075] The genomic sequence of the PmMTLa gene is SEQ ID NO:1 (including introns), its coding sequence is shown in SEQ ID NO:2, and the amino acid sequence encoding the protein is shown in SEQ ID NO:3.

[0076] The genomic sequence of the PmMTLb gene is SEQ ID NO:4 (including introns), its coding sequence is shown in SEQ ID NO:5, and the amino acid sequence encoding the protein is shown in SEQ ID NO:6.

[0077] Based on the PAM recognition characteristics (such as TTN) of the Cas12i.3 system, target sequences were designed for screening the exon regions of the PmMTLa and PmMTLb genes, respectively. The target sequences for the PmMTLa and PmMTLb genes are 5'-GTCAGCGTGTCGGACACCCTCG-3', located at positions 5028 to 5049 of SEQ ID NO:1 or positions 524 to 525 of SEQ ID NO:4.

[0078] DNA fragments targeting the PmMTLa and PmMTLb genes were synthesized based on the target sequences and constructed into a binary plant expression vector containing the proprietary Cas12i.3 protein expression cassette. Figure 1 The vector is named Cas12i.3-MTL. The Cas12i.3-MTL vector is a circular DNA, with one strand of the circular DNA consisting of segment 1 (shown in SEQ ID NO:7) and segment 2 (shown in SEQ ID NO:8) linked together from the 5' end to the 3' end. Specifically, positions 27 to 426 of SEQ ID NO:7 represent the nucleotide sequence of the promoter U6p, positions 427 to 474 represent the nucleotide sequence of crRNA, and positions 475 to 811 represent the nucleotide sequence of the terminator TaU3t. Positions 831 to 3014 of SEQ ID NO:7 represent the nucleotide sequence of the promoter UBi, positions 3143 to 6286 represent the coding sequence of Cas12i.3, and positions 6373 to 6275 represent the nucleotide sequence of the terminator NOS.

[0079] The Cas12i.3-MTL vector can express gene editing systems targeting the PmMTLa and PmMTLb genes, including: (i) Cas12i.3, whose amino acid sequence is shown in SEQ ID NO:9, is an effector protein of the gene editing system; (ii) crRNA with a nucleotide sequence as shown in SEQ ID NO:10, wherein positions 1 to 26 of the crRNA are repeat sequences and positions 27 to 48 hybridize with the target sequences of the PmMTLa and PmMTLb genes; Cas12i.3 binds to crRNA to form a complex.

[0080] After being introduced into the receptor, the transcribed crRNA can target the target sequences near the PAM of the PmMTLa and PmMTLb genes through base complementarity pairing. The Cas12i.3 protein forms a genome editing effect complex with the crRNA, causing double-strand breaks in the DNA near the target sites of the PmMTLa and PmMTLb genes. Through the organism's own DNA damage repair response mechanism, gene mutations occur in the cleaved regions during the repair process, thereby achieving the knockout of the PmMTLa and PmMTLb genes.

[0081] Agrobacterium EHA105 was transformed with the Cas12i.3-MTL vector, positive clones were screened and cultured to obtain Agrobacterium culture for later use.

[0082] 1.2 Seed sterilization and explant preparation: Mature millet seeds (Longmi 4) with lemma and husk removed were used to induce embryogenic callus. The dehulled seeds were first surface sterilized with 75% (v / v) ethanol aqueous solution for 2 minutes, then transferred to sterile filter paper to absorb excess moisture. Ten seeds were placed in each dish on callus induction medium (CIM, formulation: 4.33 g / L MS salt, 30 g / L sucrose, 1 mg / L D-biotin, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 50 mg / L inositol, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L kinetin, 2 mg / L 2,4-dichlorophenoxyacetic acid, 0.3 g / L casein hydrolysate, 0.6 mg / L copper sulfate, and 2 mg / L AgNO3, solvent: water, pH 5.8) and cultured in the dark at 28 ℃. Eighteen days later, the callus tissue was transferred to fresh CIM medium and subcultured for two weeks under the same conditions. Embryogenic callus tissue was then selected for genetic transformation.

[0083] 1.3 Agrobacterium tumefaciens-mediated genetic transformation: Callus tissue was cut into small pieces of about 5 mm and placed in liquid co-culture medium (CoIM, formulation: 4.33 g / L MS salt, 30 g / L sucrose, 1 mg / L D-biotin, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 50 mg / L inositol, 0.1 mg / L thiamine hydrochloride, 0.5 mg / L kinetin, 2 mg / L 2,4-dichlorophenoxyacetic acid and 200 µM acetylsylgenone, solvent: water, pH 5.4). The medium was pre-cooled on ice for 20 minutes, and then fresh Agrobacterium tumefaciens culture (OD600 = 0.8-1.0) was added. The medium was then co-cultured at 26 ℃ and 100 rpm on a shaker for 30 minutes. The callus tissue was then placed on sterile filter paper to remove excess Agrobacterium tumefaciens culture, and transferred to solid CoIM medium supplemented with 3 g / L Phytagel. The culture was then carried out in the dark at 22°C for 3 days.

[0084] After co-culture, the callus tissue was transferred to CIM medium containing 150 mg / L Timentin and cultured in the dark at 28°C for 2 weeks. Then it was transferred to screening medium (SM, formula: CIM + 100 mg / L Timentin + 50 mg / L Hygromycin B, solvent: water, pH 5.8, 3 g / L Phytagel) and screened for 10 days under the same conditions to select resistant callus tissue.

[0085] 1.4 Plant regeneration: Resistant callus was transferred to pre-regeneration medium (PRM, formulation: 4.43 g / L MS salt, 15 g / L sucrose, 15 g / L glucose, 3 mg / L 6-benzylaminopurine, 0.2 mg / L 2,4-dichlorophenoxyacetic acid, 0.3 g / L casein hydrolysate, 0.6 mg / L copper sulfate, 1 mg / L D-biotin, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 50 mg / L inositol, 0.1 mg / L thiamine hydrochloride, 3 g / L Gelzan and 100 mg / L Timentin, solvent: water, pH 5.8) and cultured for 10 days at 28°C under a 16-hour light / 8-hour dark light cycle. The regenerated shoots were then transferred to regeneration medium (RM, formulated as follows: 4.43 g / L MS salt, 15 g / L sucrose, 15 g / L glucose, 0.3 g / L casein hydrolysate, 0.6 mg / L copper sulfate, 1 mg / L D-biotin, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L nicotinic acid, 50 mg / L inositol, 0.1 mg / L thiamine hydrochloride, 3 g / L Gelzan and 50 mg / L Timentin, in water, pH 5.8) and cultured for 20-30 days under the same light and temperature conditions.

[0086] When the regenerated shoots reach 3-5 cm in length, they are transferred to a seedling culture medium (PM, formulated as follows: 2.21 g / L MS salt, 15 g / L sucrose, 100 mg / L inositol, 2.6 g / L Gelzan, and 50 mg / L Timentin, with water as the solvent, pH 5.8), and cultured at 28 ℃ under 16 hours of light / 8 hours of darkness for 10-14 days. Complete T0 generation candidate regenerated plants are obtained after approximately 2-3 weeks. The obtained T0 generation candidate regenerated plants can be directly transplanted into pots or in the field.

[0087] 1.5 Mutant Identification and Screening: For molecular identification of T0 generation candidate regenerated plants, genomic DNA was first extracted from fresh leaves using the CTAB method or commercially available plant genomic DNA extraction kits (such as TIANGEN, Omega, etc.). The integrity, concentration, and purity of the DNA were then detected by agarose gel electrophoresis and spectrophotometry (such as NanoDrop) (A260 / A280 ratio controlled between 1.8 and 2.0). Subsequently, based on the millet... MTL Gene sequence was obtained, and specific primers were designed approximately 150-200 bp upstream and downstream of the Cas12i.3 target site. Using extracted DNA as a template, PCR amplification was performed using a high-fidelity DNA polymerase (such as PhantaMax or Q5). The PCR products were verified by 1.5% agarose gel electrophoresis, then excised and recovered for Sanger bidirectional sequencing. Sequencing results were compared with wild-type sequences using software such as SnapGene, Chromas, or DSDecodeM to analyze the presence of insertions, deletions, or base substitutions at the target site and to determine the mutation type (homozygous, heterozygous, or chimeric). All sequences containing... MTL Plants that produce frameshift mutations or premature stop codons in the coding region are considered successfully edited T0 generation regenerated plants, which are used for subsequent phenotypic verification and genetic analysis.

[0088] Successfully edited T0 generation regenerated plants were self-pollinated to obtain edited line T1 plants, from which Casfree and homozygous edited lines were selected. Figure 2 T1 generation gene-edited millet was used to measure phenotype.

[0089] The detection method for Casfree T1 generation gene-edited millet, in which the Cas12i.3 system was removed, included the following: the primers used were primer pairs specifically designed for the coding region of the hygromycin phosphotransferase gene (hpt) (upstream primer: 5'-GCCTGAACTCACCGCGACGT-3', downstream primer: 5'-TCGGACGAGTGCTGGGGCGT-3'); the detection method involved PCR amplification after genomic DNA extraction using the CTAB method, and the results were observed by agarose gel electrophoresis; the result judgment criteria were that the presence of a specific band (approximately 950 bp) in the electrophoresis pattern consistent with the positive control position was considered positive (i.e., still carrying the T-DNA backbone), while the absence of a band was considered a Casfree negative plant; the positive control was plasmid DNA containing the hpt gene or genomic DNA of transgenic millet that had been confirmed positive, the negative control was wild-type millet genomic DNA, and the blank control was a PCR reaction system using sterile water instead of template.

[0090] Genomic DNA was extracted from leaves of Casfree T1 generation gene-edited millet at the three-leaf stage, and the gene editing type was detected. The results are as follows: Figure 2.

[0091] The results showed that, compared with the wild-type Longmi 4, the mtl double mutant genome had nucleotide sequences at positions 5017 to 5058 of SEQ ID NO:1 and positions 524 to 531 of SEQ ID NO:4, which resulted in the knockout of the coding genes for pollen-specific phospholipases (PmMTLa and PmMTLb) in PmMTLa / b-1.

[0092] 1.6 Millet emasculation and hybridization using warm water To ensure hybridization efficiency and accurate identification and harvesting of haploid grains, this invention simultaneously optimizes and standardizes the artificial hybridization process for millet, employing a hot water demasking method to achieve complete demasking of the female parent. The specific steps are as follows: Before the female parent ear emerges but before pollen shedding (usually 1-2 days before pollen shedding), select uniformly developed and robust main ears, manually prune the ear branches, remove spikelets that have shed pollen or are obviously developing too early / too late, retaining the middle spikelets that are in the synchronous flowering stage to improve the seed setting rate. Then, immerse the entire ear in a constant temperature water bath at 46–49℃ for 7–10 minutes. This temperature and time combination effectively kills pollen viability while maximally protecting pistil activity. Immediately after the hot water treatment, gently absorb the moisture from the ear surface with absorbent paper. Quickly place the female parent ear and the prepared male parent ear (i.e., the MTL double mutant) together into the same pollination bag, and carefully secure them to the ear neck with paper clips or thin wire to prevent mechanical breakage of the ear branches due to wind or handling. During pollination, keep the two parent ears in close contact to promote natural pollen dispersal.

[0093] Among them, millet pollen is dispersed from top to bottom, so pollen can be seen at the top of the ear while pollen has not yet dispersed in the middle of the ear, which is 1-2 days before pollen dispersal.

[0094] Four days after pollination, the female parent ear is individually replaced with a clean white tracing paper isolation bag to prevent subsequent contamination by foreign pollen. Three days later (approximately six days after pollination), once pollination is complete and the ear surface is dry and free of pollen residue, the isolation bag is replaced with a breathable nylon mesh bag with small pores (approximately 0.5–1.0 mm). This effectively prevents birds from pecking at the ears and rainwater from washing them away, while also allowing for ventilation and light penetration, reducing the risk of ear mold, and ensuring normal grain filling and development. The ear development is then regularly observed. Once the grains are fully mature, each ear is harvested individually and named hybridization-induced seeds for subsequent haploid identification and doubling treatment.

[0095] 1.7 Preparation of self-induced seeds Seeds obtained by self-pollination of the MTL double mutant are named self-induced seeds. The preparation method of self-induced seeds is to not emasculate the MTL double mutant, and to bag each ear of the plant individually before pollination to prevent pollination from neighboring plants.

[0096] 1.8 Molecular marker-assisted parental origin identification (1) Material preparation: Self-induced seeds and hybrid induced seeds were sown in the greenhouse, and the seedlings were named self-induced population and hybrid induced population, respectively. When the seedlings grew to the three-leaf-one-heart stage (about 7-10 days), fresh leaf tissue (about 50-100 mg) was collected, and immediately placed in liquid nitrogen for quick freezing and stored at -80°C for later use.

[0097] (2) DNA extraction and labeling development: Total DNA was extracted using the CTAB method, and its concentration and purity (A) were determined. 260 / A 280 ≈1.8–2.0). Based on previous resequencing or simplified genome sequencing (such as GBS) data, in the maternal and paternal (i.e., mtl Screening for highly polymorphic codominant molecular marker sites among mutants, with preference given to InDel or SNP sites located on different chromosomes (usually 5–10 evenly distributed sites are selected to improve reliability).

[0098] The InDel loci obtained through screening were located on chromosome 9, between 2971337 and 2971873. These loci were present in the father but not in the mother. Primers were designed before and after the InDel locus. The chromosome location was based on the genome version number GCA_002895445.2 (Panicum miliaceum genome assembly ASM289544v2 - NCBI - NLM) on NCBI.

[0099] Primer F, chr9-F: 5'-ATCGACTAGGGTTCATAGCT-3'; Primer R, chr9-R: 5'-CCGTATGCGACCTGAGCGTC-3'.

[0100] (3) PCR amplification and electrophoresis analysis: Specific primers were designed to amplify the target polymorphic sites by PCR. The PCR products were subjected to agarose gel electrophoresis, and the size and number of bands were used to determine whether the millet seedlings being tested were normal fertilized diploid hybrid offspring or haploid candidates.

[0101] The PCR reaction system is as follows:

[0102] PCR reaction conditions are as follows

[0103] The judgment criteria are as follows: The PCR product showed the target band between 1379 and 1419 bp, indicating that the tested millet seed only had the paternal genome. The PCR product showed the target band between 1018 and 1058 bp, indicating that the tested millet seed had only the maternal genome. The presence of target bands in the PCR product between 1018 and 1058 bp and between 1379 and 1419 bp indicates that the tested millet is a hybrid, meaning it possesses the genomes of both the father and mother.

[0104] (4) Result interpretation and screening: A hybridization-induced population: If the offspring simultaneously exhibit maternal and paternal alleles at multiple loci (e.g., double banding), they are determined to be diploid hybrid offspring produced by normal fertilization and are eliminated; only individuals that show a pure maternal single banding pattern at all detection loci (i.e., no paternal allele infiltration) are retained as haploid candidate plants.

[0105] B self-induced population: Since the paternal parent is itself a mutant, there is theoretically no difference in the genomic structure of the parents. Therefore, the observation is mainly based on seedling morphology. Haploid plants are obviously shorter and the grains are shriveled and aborted.

[0106] 1.9 Flow cytometry ploidy verification (1) Sample preparation: From the haploid candidate plants screened by the above molecular markers and the normal diploid control plants (such as the maternal original material Longmi 4), take about 0.5 cm² of young leaves and place them in a grinding dish containing 1 mL of pre-cooled nuclear extraction buffer (LB01: 15 mM Tris, 2 mM Na2EDTA, 80 mM KCl, 20 mM NaCl, 30 mM sodium citrate, 0.1% Triton X-100, pH 7.0). Quickly mince the tissue on ice with a sharp blade (about 30–50 times), and let it stand for 10–15 minutes to allow the cell nuclei to be fully released to obtain a homogenate.

[0107] (2) Filtration and staining: Filter the homogenate through a 30–50 μm nylon filter to remove cell debris and collect the filtrate. Add 50 μg / mL of propidium iodide (PI) dye and 50 μg / mL of RNase A to a final concentration, and incubate at 4°C in the dark for at least 30 minutes (or at room temperature in the dark for 15 minutes) to allow PI to intercalate into the DNA double strand and emit red fluorescence.

[0108] (3) Flow cytometry detection and data analysis: Flow cytometry was used for detection. Forward scattering (FSC) and side scattering (SSC) gating were set to exclude impurities, and PI fluorescence intensity was recorded. At least 5,000 nuclear events were obtained for each sample. The nuclear fluorescence peak of the G0 / G1 phase cells of the normal diploid control was set as the 2C standard (relative DNA content = 100%). If the main peak of the candidate plant appeared at about 50% relative fluorescence intensity (i.e., 1C peak), and the peak shape was sharp without obvious tailing or multi-peak phenomenon, it was judged as haploid. Figure 3 ).

[0109] (4) Result confirmation and follow-up treatment: Candidate plants are only confirmed as true haploids when they meet the following two conditions: A. Molecular marker analysis shows that their genome is entirely derived from the maternal parent with no paternal genetic material infiltration; B. Flow cytometry shows that their nuclear DNA content is about half that of the diploid control, and their ploidy is n (i.e., 1C).

[0110] Once confirmed, haploid plants will be used for subsequent chromosome doubling treatment (e.g., soaking seedlings in 0.1–0.2% colchicine solution for 24–48 hours) to obtain homozygous double haploid (DH) lines for genetic research or breeding applications.

[0111] After dual verification: the average induction efficiency of the induction line created by this invention is 3.28%, and the haploids exhibit a phenotype of shorter plants and complete grain abortion. Figure 4 , Figure 5 ).

[0112] In summary, the beneficial technical effects achieved by this application include the following steps: (1) First efficient and controllable haploid induction in millet: As a typical self-pollinating, densely floreted, and small-flowered grass, effective haploid breeding methods have long been lacking. This invention successfully constructed a double mutant homozygous plant by targeting and editing two key MTL (MILKED EAR1-LIKE) homologous genes (longmi014834 and longmi037254) in millet, and verified that it can stably induce haploid offspring in the maternal parent as the male parent, filling the gap in haploid induction technology in millet and providing a new path for rapid pureline breeding of this crop.

[0113] (2) The invention employs a self-developed miniaturized Cas12i.3 system: Unlike the commonly used Cas9 or Cas12a systems, this invention uses a miniaturized CRISPR / Cas12i nuclease with independent intellectual property rights. This system has advantages such as small protein size, loose PAM sequence (e.g., TTN), low off-target rate, and easy delivery, making it particularly suitable for genetic manipulation of difficult-to-transform crops such as millet. At the same time, Cas12i.3 has a low off-target rate and lower biosafety risk, which is more in line with the future trend of agricultural biotechnology regulation.

[0114] (3) Optimizing the hybridization pollination procedure to significantly improve the recovery rate of haploid grains: In response to the problems of short flowering period, difficulty in emasculation, and susceptibility to contamination in millet, this invention optimizes the parameters for hot water emasculation (treatment at 46–49℃ for 7–10 minutes) and adopts a phased isolation strategy of "changing sulfuric acid paper bags on the 3rd day after pollination and changing to small-pore nylon net bags on the 7th day". This scheme effectively prevents contamination by foreign pollen, reduces ear mold, and avoids bird and rain damage. While ensuring the pollination success rate, it greatly improves the grain setting rate and harvest integrity of haploid grains.

[0115] (4) Constructing a dual identification system of molecular markers and flow cytometry to ensure accurate and reliable haploid screening: This invention employs a set of highly specific co-dominant molecular markers combined with flow cytometry to verify the parental origin and ploidy of candidate plants. This method can effectively eliminate false-positive hybrid seeds and spontaneous polyploid interference, increasing the accuracy of haploid identification to over 95%, which is significantly better than traditional morphological or single-method judgment, providing high-quality starting materials for subsequent chromosome doubling and DH line construction.

[0116] (5) Significantly shortens the breeding cycle and accelerates the genetic improvement process of millet: Traditional pure line breeding of millet requires 6-8 generations of self-pollination to achieve genetic stability, taking as long as 4-6 years. However, this invention, through haploid induction and chromosome doubling, can obtain completely homozygous double haploid (DH) lines within 1-2 growing seasons, shortening the breeding cycle by more than 60%. At the same time, the DH population has a uniform genetic background, which greatly improves the efficiency and accuracy of QTL mapping, gene function verification, and hybrid parent selection.

[0117] (6) The inducible line can be used for gene editing breeding without exogenous T-DNA: By using the inducible line of this invention as the male (or female) parent and crossing it with the target millet variety, gene-edited plants without transgenic elements (i.e., without T-DNA integration) can be directly obtained in the offspring. This strategy not only effectively circumvents the strict approval and supervision of transgenic crops in my country, but also solves the technical bottleneck that prevents direct gene editing of some millet varieties due to difficulties in genetic transformation, providing a feasible path for precision breeding without transgenic attributes.

[0118] (7) Promoting the development of functional genomics research and molecular design breeding of millet: This invention is not only a breakthrough in breeding technology, but also establishes the first heritable haploid platform for millet, an important and distinctive miscellaneous grain crop. This platform can be used for basic research such as large-scale screening of recessive mutants, gene interaction analysis, and comparison of allele functions, providing a powerful tool for elucidating the molecular mechanisms of millet's excellent traits such as drought resistance, tolerance to poor soil, and high nutrition.

[0119] In summary, this invention integrates gene editing, hybridization, precise identification, and breeding applications. It has a complete technical system, strong operability, and high scalability. It not only solves the haploid breeding problem of millet, but also provides a reference paradigm for the development of haploid technology for other similar small-flowered gramineous crops (such as foxtail millet, sorghum, and sorghum spp.), and has important scientific value and broad application prospects.

[0120] The sequences in this application are as follows: SEQ ID NO:1, Genomic sequence of the PmMTLa gene (including introns):

[0121] SEQ ID NO:2, coding sequence of the PmMTLa gene:

[0122] SEQ ID NO:3, Amino acid sequence of the protein encoded by the PmMTLa gene: MASYSSRRPCSVCRTKAMAGSVVSEPVVPGQRVTVLTIDGGGIRGLIPGTILAFLEARLQELDGPHYRKQLADYFDCIAGTSTGGLITSMITTPGEDKRLLFAARDIN RFYFDNCPRIFRQSRSSLTAAMSALRKPRYSGKYLRSTIRSMLGETRVSDTLTNVVIPTFDIKLLQPIIFSTYDARSTPLKNALLSDVCISTSAAPTYLPAHYFKTQD AGGKAREYNLTDGGVAANNPTMVAMTQITKKMLGKDKEELFPVKPADCRKFLVLSIGTGSASDEGLFTARQCSRWGVVRWLRNKGMAPIIDIFMAASSDLVDIHAAVL FQSLHGDRDYLRIQDSSLRGAAATVDAATPENMRTLVGIGERMLAQRVSRVDVETGRNEPVPGEGSNADALAGLARQLSEERRTRLARRAAAGCAAGSRCCPPAET*.

[0123] SEQ ID NO:4, Genomic sequence of the PmMTLb gene (including introns):

[0124] SEQ ID NO:5, coding sequence of the PmMTLb gene:

[0125] SEQ ID NO:6, Amino acid sequence of the protein encoded by the PmMTLb gene: MASYSSRRPCSVCRTKAMAGSVVGEPVVPGQRVTVLTIDGGGIRGLIPGTILAFLEARLQELDGPEARLADYFDCIAGTSTGGLITSMITTPGEDKRPLFAARDINR FYFDNCPRIFPQSRSSLTAAMSALRKPRYSGKYLRSIIRSMLGETRVSDTLTNVVIPTFDIKLLQPIIFSTYDARSTPLKNALLSDVCISTSAAPTYLPAHYFKTQDA GGKAREYNLIDGGVAANNPTMVAMTQITKKMLGKDKEELFPVKPADCRKFLVLSIGTGSASDEGLFTARQCSRWGVVRWLRNKGMAPIIDIFMAASSDLVDIHAAVL FQSLHSDRDYLRIQDSSLRGAAATVDAATPENMRTLVGIGERMLAQRVSRVNVETGRNEPVPGEGSNADALAGLARQLSEERRTRLARRAAAGCAGGSTCCSPVKT*.

[0126] SEQ ID NO:7, nucleotide sequence of the first part of the Cas12i.3-MTL vector:

[0127] SEQ ID NO:8, nucleotide sequence of the second part of the Cas12i.3-MTL vector:

[0128] The amino acid sequence of the Cas12i.3 protein, SEQ ID NO:9:

[0129] SEQ ID NO:10, crRNA: gtgagagaatgtgtgcatagtcacacGTCAGCGTGTCGGACACCCTCG.

[0130] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Applications of genome editing systems and related biomaterials in at least one of the following: A1) Application in the preparation of millet haploid induction systems; A2) Its application as a product for preparing millet haploid induction lines; A3) Application in millet breeding or assisted breeding; A4) Application in the preparation of products for millet breeding or assisted breeding; The genome editing system includes: (i) Protein components, which are proteins or conjugates thereof whose amino acid sequence is shown in SEQ ID NO:9, and (ii) A nucleic acid component containing the RNA sequence shown in SEQ ID NO:10 from the 5' to 3' direction. in, The protein components and nucleic acid components combine to form a complex.

2. The application according to claim 1, characterized in that, The biomaterial is at least one of B1) to B4) below: B1) Nucleic acid molecules, wherein the nucleic acid molecules express the protein components and / or nucleic acid components described in claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

3. The application according to claim 2, characterized in that, The nucleic acid molecule expressing the protein component of claim 1 is a DNA molecule as described in at least one of the following g1) and g2): g1) DNA molecules with nucleotide sequences such as positions 3143 to 6286 of SEQ ID NO:7; g2), which shares more than 70% identity with the DNA molecule at positions 3143 to 6286 of SEQ ID NO:7 and DNA molecules encoding the protein components described in claim 1; The nucleic acid molecule expressing the nucleic acid component of claim 1 is a DNA molecule as described in at least one of the following g3) and g4): g3), DNA molecules with nucleotide sequences such as positions 427 to 474 of SEQ ID NO:7; g4) A DNA molecule that has more than 70% identity with the DNA molecule at positions 427 to 474 of SEQ ID NO:7 and transcribes the nucleic acid components described in claim 1.

4. A method for preparing a millet haploid inducible line, characterized in that, The method includes at least one of the following C1) and C2): C1) Knock out the gene encoding pollen-specific phospholipase in the millet seed to be edited using the genome editing system described in claim 1; C2) The genome of the millet to be edited is mutated as follows: the DNA fragment with nucleotide sequence as SEQ ID NO:1 from position 5017 to 5058 and the DNA fragment with nucleotide sequence as SEQ ID NO:4 from position 524 to 531 are deleted from the millet to be edited. The millet seed to be edited contains the encoding gene of the pollen-specific phospholipase, which includes PmMTLa and PmMTLb. The amino acid sequence of PmMTLa is shown in SEQ ID NO:3, and the amino acid sequence of PmMTLb is shown in SEQ ID NO:

6.

5. Gene-edited millet, characterized in that, In the gene-edited millet, the DNA fragment with nucleotide sequence 5017 to 5058 in the gene shown in SEQ ID NO:1 is deleted, and the DNA fragment with nucleotide sequence 524 to 531 in the gene shown in SEQ ID NO:4 is deleted.

6. The gene-edited millet seed according to claim 5, characterized in that, The gene-edited millet seed is obtained by gene editing of millet seed to be edited, and the millet seed to be edited contains the gene encoding pollen-specific phospholipase as described in claim 4.

7. The gene-edited millet according to claim 5 or 6, characterized in that, The gene-edited millet is prepared by the method described in claim 4.

8. The use of the gene-edited millet according to any one of claims 5 to 7 in at least one of the following: E1) Application in the preparation of haploid millet; E2), its application in the preparation of haploid millet products; E3), its application in millet haploid breeding; E4) Application in the preparation of millet haploid breeding products.

9. A method for preparing haploid millet, characterized in that: The method includes at least one of the following: F1) The haploid millet is prepared by hybridizing the gene-edited millet as described in at least one of claims 5 to 7 with millet as the female parent; F2) The haploid millet is prepared by self-pollination of the gene-edited millet according to at least one of claims 5 to 7; The process also includes a step of emasculating the millet seedlings used as the female parent. Maestrosculation involves immersing the entire ear of millet in a constant temperature water bath at 46-49°C for 7-10 minutes before the female parent begins to head but before pollen is released.

10. Millet as described in at least one of the following: G1), haploid millet, wherein the haploid millet is obtained by hybridization or self-pollination using the gene-edited millet as any one of claims 5 to 7 as a hybrid parent; Homozygous diploid millet obtained by chromosome doubling of haploid millet described in G2) and F1).

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