Method for propagating maize nuclear male sterile line through seed morphology marking

Abstract

The invention belongs to the field of plant genetic breeding and seed production, and particularly discloses a method for propagating a maize nuclear male sterile line through seed morphology marking. The technical problem to be solved by the invention is how to improve a nuclear male sterility system and / or how to obtain high-purity male sterility offspring. The method comprises the following steps: a male gamete of a maintainer line B containing a restoring gene and a screening gene is fertilized with a female gamete of a sterile line A containing a homozygous recessive male sterility gene, and the screening gene silences the Su1 gene; and distinguishing the obtained seeds into the seeds of the sterile line A and the seeds of the maintainer line B according to the external characters of the screened genes. The method can be used for expanding propagation of male sterile line and corresponding maintainer line plants.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic breeding and seed production, and in particular relates to a method for propagating corn nuclear male sterile lines by seed morphology marking. Background Art

[0002] There are three types of plants in nature: self-pollination, cross-pollination, and cross-pollination. Self-pollination refers to the phenomenon that the pollen of a plant pollinates the pistil of the same individual. In plants with bisexual flowers, it can be divided into same-flower pollination (Phaseolus genus), which is pollination between the stamens and pistils of the same flower, and neighbor-flower pollination, which is pollination between different flowers in an inflorescence (individual), and cross-flower pollination, which is pollination between different flowers on the same plant. Some plants have stamens and pistils that do not grow in the same flower, or even on the same plant, and cannot self-pollinate. Their pistils can only get pollen from other flowers, which is called cross-pollination. Crops with a natural hybridization rate higher than 50% and self-pollination depression are classified as cross-pollination crops, such as corn.

[0003] Corn is monoecious, with male and female flowers located in different parts of the plant. Corn can reproduce through self-pollination as well as cross-pollination. Under natural conditions, natural pollination is completed when the wind blows pollen from the male ear to the filaments of the female ear.

[0004] Due to the existence of hybrid vigor, the biomass, resistance to pests and diseases, and stress tolerance (drought, high temperature, low temperature, salinity, etc.) of hybrids are significantly improved compared to their parents. For example, the yield of hybrid corn and hybrid rice is much higher than that of the homozygous parents. The method commonly used to produce hybrids is to plant the female parent and the male parent together, remove the male tassel of the female parent, and retain the male tassel of the male parent. The seeds harvested from the female parent are hybrids.

[0005] In corn breeding, homozygous corn inbred lines should be developed first, and then two inbred lines should be hybridized. The yield, stress resistance, etc. of the hybrid offspring should be evaluated to determine whether they have commercial potential. Each inbred line may have one or more excellent traits that the other inbred line lacks, or supplement one or more undesirable traits of the other inbred line. The first generation seeds of the hybrid of two inbred lines are F1 generation seeds. After the F1 generation seeds germinate, the F1 generation plants are obtained. The F1 generation plants are stronger than the two inbred line parents (parents) and have more biomass.

[0006] Hybrids can be produced by artificial emasculation of the female parent, that is, the tassels of the female parent that has not shed pollen (which can be sown in the field with the male parent at intervals, such as sowing 5 rows of female parent and one row of male parent) are removed, and the tassels of the male parent are retained. Subsequently, as long as the foreign corn pollen is isolated, the female ears of the female parent can only receive pollen from the male parent, and the resulting seeds are hybrids (F1), which can be used for agricultural production.

[0007] In the process of producing hybrid seeds, environmental changes may cause the plants to become tassels again after emasculation, or incomplete emasculation. The above two situations can lead to self-pollination of the female parent, resulting in the hybrid seeds produced being mixed with seeds of the female parent's inbred line. The yield of the female parent's inbred line is much lower than that of the hybrid seeds. Such seeds are unqualified products, which will affect both farmers' income and the reputation of the seed production company. In serious cases, the seed production company will bear corresponding compensation liability.

[0008] Machine detasseling can also be done on female plants with about the same reliability as hand detasseling, but faster and at a lower cost. However, most machine detasseling methods cause more damage to the plant than hand detasseling, so there is currently no completely satisfactory method of detasseling, and people are still looking for cheaper and more complete alternative methods.

[0009] The stable male sterility system provides a simple and efficient means. By using the nuclear-cytoplasmic male sterility (CMS) inbred line, the heavy work of detasseling can be avoided in some genotypes. This method includes three main materials, namely the sterile line: male sterile material, the maintenance line: can provide pollen for the sterile line so that the offspring of the sterile line is still sterile, and the restorer line: can restore the fertility of the sterile line. The sterile line and the restorer line are hybridized to produce F1, which is a hybrid used for agricultural production. More specifically, the nuclear-cytoplasmic sterility type is manifested as nuclear-cytoplasmic inheritance. Not only does it require the sterility gene S in the cytoplasm, but it also requires the homozygous sterility gene (rfrf) in the nucleus. The two exist at the same time to make the plant show male sterility. If the cytoplasmic gene is fertile N, then regardless of whether the nuclear gene is fertile (RfRf) or sterile (rfrf), it will show male fertility. Similarly, if there is a fertility gene (RfRf) or (Rfrf) in the nucleus, then no matter whether the cytoplasmic gene is fertile N or sterile S, it will also show male fertility. This male sterile line formed by nuclear-cytoplasmic interaction has a genetic composition of S (rfrf) and cannot produce normal pollen, but can be used as a hybrid mother. Since we can find the maintenance line N(rfrf) [when it is hybridized with the sterile line, the resulting F1 can still maintain male sterility, that is: S(rfrf)(♀)×N(rfrf)→S(rfrf)(sterile)] and can accept the pollen of the restorer line S(RfRf) or N(RfRf) [when it is hybridized with the sterile line, the resulting F1 is fertile, that is: S(rfrf)(♀)×S(RfRf)→S(Rfrf)(F1)(fertile), or S(rfrf)(♀)×N(RfRf)→S(Rfrf)(F1)(fertile)], the F1 can be restored to male fertility, and the F1 plants can be self-pollinated to produce F2, so it can be widely used in agricultural production. Male sterile lines can avoid artificial emasculation, save manpower, reduce seed costs, and ensure seed purity. Currently, crops such as rice, corn, sorghum, onion, castor, sugar beet and rapeseed have used nuclear-cytoplasmic male sterility to produce hybrid seeds; extensive research is also being conducted on nuclear-cytoplasmic male sterility lines of other crops.

[0010] CMS also has its drawbacks. First, it has been observed that some CMS materials are susceptible to diseases. Second, it is difficult to find recovery lines. Third, sterile lines are easily affected by the environment, and some individual plants or anthers may be fertile. Fourth, the fertility of the hybrids produced is easily affected by the environment, resulting in large-scale sterility. These problems hinder the widespread application of the CMS system in seed production.

[0011] Brar et al. discloses a type of genetic sterility in U.S. Patents 4,654,465 and 4,727,219. However, this type of genetic sterility requires the maintenance of corresponding genotypes at multiple different sites in the genome, and molecular marker tracking detection of these sites is required for each generation. Patterson also describes a chromosome translocation gene system that may be useful, but the system is more complicated (see U.S. Patents Nos. 3,861,709 and 3,710,511).

[0012] People have been trying to optimize the male sterility system. For example, Fabijanski et al. developed a method for making plants male sterile (EPO 89 / 3010153.8 Publication No. 329308 and PCT application PCT / CA90 / 00037 published as WO 90 / 08828). The male flower fertility of plants is mainly inhibited through the following two ways. One method is to connect the promoter specifically expressed by male tissues with the cytotoxin gene and transfer it into the plant, so that the male flowers cannot shed pollen normally without affecting other traits; the other is to interfere with the cloned gene controlling the male flower fertility of the plant through transgenic means, so that it cannot function normally. There is also a method of inhibiting gene expression through some gene regulatory elements, thereby affecting the fertility of plants (WO90 / 08829).

[0013] In most cases, only plants with recessive homozygous (msms) nuclear genes controlling male sterility will show male sterility. Since male sterile plants cannot self-pollinate, they can only be hybridized with heterozygous plants (Msms) to obtain male sterile plants (msms). In addition, male sterile grains (msms) and fertile heterozygous grains (Msms) coexist on the same ear. It is impossible to distinguish which are sterile grains and which are fertile grains through the grains. They can only be distinguished after sowing when the plants shed pollen, and it is too late to emasculate them at this time.

[0014] In recent years, transgenic methods have also been used to maintain the sterility of male sterile plants (US6743968). This method constructs a pollen lethal gene and a male fertility restoration gene into a vector and introduces it into male sterile plants. The transgenic offspring appear fertile, but can only produce pollen without the restoration gene. When such plants are hybridized with male sterile plants, the homozygous recessive state of the recessive sterile plants is maintained. First, a transgenic vector is constructed, which contains a pollen cell lethal gene and a dominant gene that restores the fertility of the plant. The vector is transferred into male sterile plants, and the vector exists in a heterozygous state in the transgenic plants. Due to the presence of the fertility restoration gene, the plants are fertile. When hybridized with male sterile plants, the pollen containing the restoration gene (Msms) also contains the lethal gene, which makes the pollen aborted. Therefore, only the pollen (ms) without the restoration gene can be hybridized with the female gametes (ms) of the male sterile plants, and the offspring are all recessive homozygous individuals (msms). The inventors previously used the gene mn1 that controls grain size for marking (ZL201210406155.6), but mn1 is different in different genetic backgrounds, and some backgrounds cannot be effectively applied.

[0015] As mentioned above, an important issue in many seed production works using male sterility systems is how to utilize male sterility genes and how to distinguish between male sterile seeds and fertile seeds. At the same time, it is also necessary to consider how to maintain the sterility of sterile individuals. Summary of the invention

[0016] The technical problem to be solved by the present invention is how to utilize male sterility genes and how to distinguish male sterile seeds from fertile seeds. At the same time, it is also necessary to consider how to maintain the sterility of sterile individuals.

[0017] Through careful design and a large number of experiments, the inventors of the present application have obtained a method for producing a maintainer plant that can maintain the sterility of a male sterile plant. When the maintainer plant is hybridized with a male sterile plant, distinguishable sterile and maintainer offspring can be obtained simultaneously, which significantly improves the propagation efficiency and breeding efficiency of the sterile plant.

[0018] Therefore, in the first aspect of the present application, a method for propagating a maize nuclear male sterile line by seed morphology markers is provided, comprising the following steps:

[0019] a) providing a maintenance line B, wherein the maintenance line B is an inbred line comprising the same homozygous recessive male sterility gene as that in the sterile line A and containing a nucleic acid molecule in a heterozygous state; the nucleic acid molecule comprises a first polynucleotide and a second polynucleotide; the first polynucleotide comprises a restoration gene, and the restoration gene can restore the male fertility of a plant that is male sterile due to the recessive male sterility gene; the second polynucleotide comprises a screening gene that can regulate the grain shape and / or endosperm development morphology, wherein the grain shape comprises the size, length, width, or / and thickness of the seed, and the endosperm development morphology comprises whether the endosperm is sunken, wrinkled, or / and powdery; the sterile line A is a sterile line comprising a homozygous recessive male sterility gene;

[0020] The screening gene silences the Su1 gene; the Su1 gene encodes the Su1 protein, and the Su1 protein is a protein of C1, C2 or C3 as follows:

[0021] C1. A protein having an amino acid sequence as shown in SEQ ID No.10;

[0022] C2. A protein having more than 80% identity with the protein shown in C1) and having the same function as the protein obtained by replacing and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No.10;

[0023] C3. A fusion protein obtained by connecting a protein tag at the N-terminus or / and C-terminus of C1) or C2);

[0024] b) Fertilizing the female gametes of the sterile line A with the male gametes of the maintenance line B, and distinguishing the obtained seeds into seeds of the sterile line A and seeds of the maintenance line B according to the external characteristics of the screening gene, thereby completing the propagation of the male sterile line and the corresponding maintenance line.

[0025] In the above-mentioned propagation method, the sterile line is a plant sterile line, and the maintainer line is a plant maintainer line.

[0026] In the above-mentioned propagation method, the plant is a monocot or a dicot. The plant can be corn (Zea mays), rapeseed (Brassica napus), rice (Oryza sativa), Arabidopsis thaliana, barley (Hordeum vulgare), wheat (Triticum aestivum), sorghum (Sorghum bicolor), soybean (Glycinemax), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), cotton (Gossypium hirsutum), sunflower (Helianthus annuus) or sugarcane (Saccharum officinarum). In an embodiment of the present invention, the plant is corn (Zea mays).

[0027] In the above-mentioned propagation method, the recessive male sterile gene causes plant male sterility in a homozygous state. The recessive male sterile gene is selected from at least one of ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, ms10, ms11, ms12, ms13, ms14, ms15, ms16, ms17, ms18, ms19, ms20, ms21, ms22, ms23, ms24, ms25, ms26, ms27, ms28, ms29, ms30, ms31, ms32, ms33, ms34, ms35, ms36, ms37, ms38, ms43, ms45, ms47, ms48, ms49, ms50 and ms52. In an embodiment of the present invention, the male sterile gene is ms45.

[0028] In the above-mentioned propagation method, the restoration gene can restore the male fertility of plants that are male sterile due to the recessive male sterility gene. The restoration gene is selected from at least one of Ms1, Ms2, Ms3, Ms4, Ms5, Ms6, Ms7, Ms8, Ms9, Ms10, Ms11, Ms12, Ms13, Ms14, Ms15, Ms16, Ms17, Ms18, Ms19, Ms20, Ms21, Ms22, Ms23, Ms24, Ms25, Ms26, Ms27, Ms28, Ms29, Ms30, Ms31, Ms32, Ms33, Ms34, Ms35, Ms36, Ms37, Ms38, Ms43, Ms45, Ms47, Ms48, Ms49, Ms50 and Ms52. In an embodiment of the present invention, the restoration gene is Ms45.

[0029] It is easy to understand that in the nucleic acid molecule of the present application, the restoration gene should correspond to the male sterility gene, so that it can rescue the sterility trait caused by the recessive male sterility gene. In the embodiment of the present invention, the recessive male sterility gene is ms45, and the corresponding restoration gene is Ms45.

[0030] Specifically, the restoration gene Ms45 encodes the Ms45 protein, and the Ms45 protein is the following protein A1, A2 or A3:

[0031] A1, a protein having an amino acid sequence as shown in SEQ ID No. 2;

[0032] A2, a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by replacing and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No.2;

[0033] A3. A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

[0034] In the above-mentioned propagation method, the restoration gene Ms45 can specifically be a nucleic acid molecule with a coding sequence of SEQ ID No.1.

[0035] In the above-mentioned propagation method, the first polynucleotide also contains expression control elements, such as promoters and enhancers, which are operably connected to the nucleotide sequence of the restoration gene. In certain embodiments, the expression control element is selected from: a promoter, an enhancer, a regulatory sequence, an inducible element, and any combination thereof. In the above-mentioned nucleic acid molecule, the promoter is selected from: a constitutive promoter, an inducible promoter, a tissue-preferred promoter, a tissue-specific promoter, and a growth phase-preferred promoter. The promoters that can be used in this application are not limited to the promoters listed above. It is easy to understand that in the above-mentioned nucleic acid molecules, any promoter known to those skilled in the art can be used according to actual needs.

[0036] In the above-mentioned enrichment method, the first polynucleotide sequence may be SEQ ID No.3.

[0037] In the above-mentioned propagation method, the Su1 gene may specifically be a nucleic acid molecule whose coding sequence of the coding chain is a nucleotide sequence of SEQ ID No.9.

[0038] In the above-mentioned propagation method, the screening gene comprises a forward interference fragment, a reverse interference fragment, and an intron forming a hairpin structure between the forward interference fragment and the reverse interference fragment; the sequence of the forward interference fragment is SEQ ID No.4; the sequence of the reverse interference fragment is SEQ ID No.5; and the sequence of the intron forming the hairpin structure is SEQ ID No.6.

[0039] In the above-mentioned propagation method, the second polynucleotide also comprises an expression control element, such as a promoter and an enhancer, which is operably connected to the nucleotide sequence of the screening gene. In certain embodiments, the expression control element is selected from: a promoter, an enhancer, a regulatory sequence, an inducible element, and any combination thereof. The promoter is selected from: a constitutive promoter, an inducible promoter, a tissue-preferred promoter, a tissue-specific promoter, and a growth phase-preferred promoter. The promoters that can be used in this application are not limited to the promoters listed above. It is easy to understand that in the second polynucleotide, any promoter known to those skilled in the art can be used according to actual needs.

[0040] In the above-mentioned expansion method, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0041] In the above-mentioned enrichment method, the above-mentioned 80% or more identity may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0042] In the above-mentioned amplification method, the first polynucleotide and the second polynucleotide are genetically linked. For example, the first polynucleotide and the second polynucleotide are covalently linked. The covalent linkage may be through or without a linker nucleotide. The length of the linker nucleotide is no more than 10 kb, no more than 5 kb, no more than 1 kb, no more than 500 bp, no more than 100 bp, no more than 50 bp, no more than 10 bp, no more than 5 bp, or shorter.

[0043] In the second aspect of the present application, a method for constructing a maintenance line is provided, the method comprising introducing the nucleic acid molecule described above into a sterile line comprising a homozygous recessive male sterility gene, to obtain a maintenance line in which the nucleic acid molecule exists in a heterozygous state and the recessive male sterility gene is homozygous, and the maintenance line is the maintenance line B used for the above-mentioned propagation method.

[0044] In the third aspect of the present application, an isolated nucleic acid molecule is provided, which comprises a first polynucleotide and a second polynucleotide; the first polynucleotide comprises a restoration gene, which can restore the male fertility (male fertility) of a plant that is male sterile due to a recessive male sterility gene; the second polynucleotide comprises a screening gene that can regulate the grain shape and / or endosperm development morphology, the grain shape comprises the size, length, width and / or thickness of the seed, and the endosperm development morphology comprises whether the endosperm is sunken, wrinkled and / or powdery.

[0045] In the fourth aspect of the present application, a biological material related to the above-mentioned nucleic acid molecule is provided, which is any one of the following B1) to B8):

[0046] B1) an expression cassette containing the nucleic acid molecule;

[0047] B2) a recombinant vector containing the nucleic acid molecule, or a recombinant vector containing the expression cassette described in B1);

[0048] B3) a recombinant microorganism containing the nucleic acid molecule, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2);

[0049] B4) a transgenic plant cell line containing the nucleic acid molecule, or a transgenic plant cell line containing the expression cassette described in B1);

[0050] B5) transgenic plant tissue containing the nucleic acid molecule, or transgenic plant tissue containing the expression cassette described in B1);

[0051] B6) A transgenic plant organ containing the nucleic acid molecule, or a transgenic plant organ containing the expression cassette described in B1).

[0052] The recombinant expression vector described in B2) can be constructed with existing plant expression vectors. The plant expression vector includes binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. Such as pCAMBIA3301, pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company), etc. The plant expression vector can also contain the 3' end non-translated region of the foreign gene, i.e., contain polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as the non-translated region transcribed at the 3' end of the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos) and the plant gene (such as the soybean storage protein gene) all have similar functions. When using the gene of the present invention to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation initiation region can come from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes encoding enzymes or luminescent compounds that can produce color changes and can be expressed in plants (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bialaphos resistance gene (bar gene) that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin, and the β-catenin gene that confers resistance to methatrexate). The selective marker gene may be a dhfr gene, an EPSPS gene that confers resistance to glyphosate) or a chemical resistance marker gene (such as a herbicide resistance gene), a mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose, a neomycin resistance gene (such as a gene encoding neomycin phosphotransferase), a hygromycin resistance gene (such as a gene encoding hygromycin phosphotransferase), a chloramphenicol resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, a bleomycin resistance gene, a sulfonamide resistance gene, a bromoxynil resistance gene, a glyphosate resistance gene, and a glufosinate resistance gene. In an embodiment of the present invention, the selective marker gene is a herbicide resistance gene, and specifically the herbicide resistance gene is a bar gene. The vector can express the nucleic acid molecule as described above in a plant cell (such as corn).

[0053] Among the above biological materials, the recombinant microorganisms described in B3) can specifically be yeast, bacteria, algae and fungi.

[0054] In the above biological materials, the plant cell line in B4) is a cell line of a monocotyledonous plant or a dicotyledonous plant.

[0055] In the above biological materials, the plant cell line B4) is a cell line of the following plants: corn (Zea mays), rapeseed (Brassica napus), rice (Oryza sativa), Arabidopsis thaliana, barley (Hordeum vulgare), wheat (Triticum aestivum), sorghum (Sorghum bicolor), soybean (Glycinemax), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), cotton (Gossypium hirsutum), sunflower (Helianthus annuus) or sugarcane (Saccharum officinarum).

[0056] In the fifth aspect of the present application, there is provided the use of the above-mentioned propagation method, and / or the above-mentioned construction method, and / or the above-mentioned nucleic acid molecule, and / or the above-mentioned biological material in plant hybrid seed production.

[0057] Beneficial effects:

[0058] The present application provides a method for propagating a male sterile line and a corresponding maintainer line and a nucleic acid molecule used therein. After hybridizing the maintainer line plant provided in the present application with the male sterile plant, the offspring of the male sterile plant and the maintainer line plant can be harvested at the same time and effectively distinguished. Thus, the method and maintainer line plant provided in the present application can achieve efficient propagation of male sterile plants and improve breeding efficiency.

[0059] In the present invention, the inventors have invented a new method for efficiently propagating male sterile lines of plants by using genes controlling plant male fertility, seed morphology marker genes and transgenic technology. In one embodiment of the present invention, the seed morphology marker gene is a screening gene that regulates grain shape (size, length, width or / and thickness) and / or endosperm development morphology (whether the endosperm is sunken, wrinkled or / and whether it is powdery). In the present invention, the wild-type nucleotide sequence controlling male fertility and the seed morphology marker gene sequence are linked together and transferred into conventional corn, and then backcrossed to homozygous recessive male sterile line plants. After hybridizing the obtained transgenic plants with the homozygous recessive sterile line, a large number of sterile line and maintenance line seeds can be obtained at the same time. Due to the effect of the nucleotide sequence controlling grain shape or endosperm development morphology, the sterile line and the maintenance line can be distinguished by the grain shape or endosperm development morphology. Among them, seeds with normal shape or normal endosperm development are sterile lines (do not contain transgenic sequences), and seeds with abnormal shape (such as changes in grain size, length, width, thickness, etc.) or abnormal endosperm development (such as concave, wrinkled, powdery endosperm, etc.) are maintainer lines.

[0060] In one embodiment of the present invention, the inventors constructed a plant transformation vector, which contains an expression element of a gene that restores male fertility and an interference sequence that controls grain shape and / or endosperm development morphology, and transferred the vector into HiIIA×HiIIB corn hybrids, and then backcrossed the obtained transgenic plants with male sterile plants, thereby introducing the nucleotide sequence that controls plant male fertility, grain shape (such as size, length, width, thickness, etc.) or endosperm development morphology (such as concave, wrinkled, powdery endosperm, etc.) into the male sterile plants. Due to the presence of the restorer gene, the plant appears fertile. When the transgenic heterozygous plant (Msmsms) is crossed with the male sterile plant (msms), the following two types of offspring will be produced: one is male sterile grains with normal grains (sterile line, genotype is msms), and the fertility of this sterile line can be restored by any wild-type plant; the other is fertile grains with abnormal grains (maintainer line, genotype is Msmsms), and this maintainer line is recessive homozygous at the locus controlling male fertility. Since it contains complementary transgenic sequences, the plant appears fertile. Since it also contains nucleotide sequences that affect grain shape (such as size, length, width, thickness, etc.) or endosperm development morphology (such as concave, wrinkled, powdery endosperm, etc.), the grain shape (such as size, length, width, thickness, etc.) or endosperm development morphology (such as concave, wrinkled, powdery endosperm, etc.) of this seed is different from that of the wild type. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The male flower phenotypes of the male sterile mutant and its wild type. Figure 1The left picture shows the male flower phenotype of the male sterile mutant (containing the homozygous ms45 gene); Figure 1 The right picture shows the male flower phenotype of the wild type (containing the Ms45 gene).

[0062] Figure 2 The phenotypes of the su1 mutant and wild type, which control the grain size gene. 1 is a mutant grain containing the homozygous su1 gene, and 2 is a wild type grain containing the Su1 gene.

[0063] Figure 3 The plant expression vector pMs45-Su1RNAi contains the expression element of the male fertility gene Ms45 and the expression element of the interference fragment of the grain size control gene Su1.

[0064] Figure 4 The male flower and grain phenotypes of plants transformed with plant expression vectors containing the expression element of the male fertility gene Ms45 and the expression element of the interference fragment of the grain size controlling gene Su1. Figure 4 The right picture shows the results of judging fertility by grain size, where B is the hybrid offspring grain containing the transgenic element Ms45-Su1RNAi, and A is the hybrid offspring grain not containing the transgenic element Ms45-Su1RNAi. Figure 4 The left picture is Figure 4 The picture on the right shows the male flower of the plant after the small seed B has grown.

[0065] Figure 5 It is a roadmap for seed production using nuclear male sterility genes, genes that control grain size or endosperm development morphology, and transgenic technology. DETAILED DESCRIPTION

[0066] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0067] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0068] Maize variety B73 was obtained from the National Maize Improvement Center of China Agricultural University. It is available to the public from the applicant to replicate this experiment.

[0069] Maize varieties HiIIA and HiIIB are both described in the following document: "Armstrong CL, Green CE and Phillips RL. Development and availability of germplasm with high Type II culture formation response. Maize Genetics Cooperation News Letter, 1991, 65: 92-93". The public can obtain them from the applicant to repeat the experiment.

[0070] Maize ms45 male sterile material 905I is a product of Maize Genetics Cooperation Stock Center.

[0071] All technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the field to which the invention belongs. Unless otherwise specified, the techniques used or mentioned in this article are standard techniques recognized by ordinary technicians in the field. The materials, methods and examples are for illustrative purposes only and are not limited.

[0072] The following provides a more detailed description by way of illustration and description, which is not intended to limit the scope of the invention.

[0073] Example 1

[0074] Nuclear male sterility is the result of mutation, inhibition or other effects of key genes in the microspore formation process, which are collectively referred to as male sterility genes. Pollen development is controlled by many genes, so mutations in many genes will eventually lead to male sterility. Currently, a large number of male sterility mutants have been identified in maize (as shown in Table 1). Each male sterility gene has its specific restoration gene, that is, each male sterility mutant can only be restored by its wild-type allele.

[0075] Table 1 Male sterile mutants caused by nuclear genes

[0076]

[0077]

[0078]

[0079] The above genes have been cloned one after another, such as ms45 (Albertsen et al. 1993) and ms26 (PTC / US2006 / 024273). At the same time, some male sterility genes have also been cloned in rice, such as dpw (Jing Shi et al. 2011) and some male sterility genes identified in Arabidopsis, such as (Aarts, et al. 1993).

[0080] Integration of these genes with the marker gene of the present invention can achieve effective separation of sterile lines and maintainer lines.

[0081] The present invention takes a male sterile mutant in Table 1 as an example. For example, the male flowers of the mutant containing the homozygous ms45 gene cannot shed pollen normally (e.g. Figure 1 As shown, Figure 1 The left picture shows the male flower phenotype of the male sterile mutant, which contains the homozygous ms45 gene; Figure 1 The right picture shows the male flower phenotype of the wild type, containing the Ms45 gene). The mutant sexuality can be restored by the wild type plants.

[0082] The Su1 (Sugary1) gene encodes a maize isoamylase. When the gene is inactivated, the kernels become wrinkled and translucent, the kernels shrink and become smaller (e.g. Figure 2 As shown, Figure 2 1 is a mutant grain containing the homozygous su1 gene. Figure 2 2 is a wild-type grain containing the Su1 gene). Most of the su1 mutants obtained under natural conditions or artificial mutagenesis are recessive mutants. The inventors interfered with the wild-type Su1 gene at the mRNA level through transgenic technology, so that the grains containing the transformation interfering nucleotide fragments would become smaller. Therefore, when the transformation fragment exists in the grain in a heterozygous state, the inventors can quickly distinguish the grains containing the transformation fragments by the size of the grains. Similarly, the same operation can be performed on other genes that affect the development of grain shape (such as size, length, width, thickness, etc.) by the same method to obtain transgenic grains that are easy to distinguish. The Su1 gene RNAi interference sequence in the present invention is derived from B73, but is not limited to the inbred line B73. It can also be derived from any other wild-type corn inbred line, or homologous genes of other species, or artificially synthesized nucleotide sequences.

[0083] The key to the present invention is to construct the interference fragments of the male restoration gene Ms45 and the grain marker gene Su1 in a vector, which can restore the fertility of the male sterile mutant ms45 and make the grains containing the transgenic sequence smaller, that is, to mark the grains containing the restoration gene so as to distinguish fertile grains (maintainer lines) from sterile grains (sterile lines). The present invention provides an efficient seed marking method, which is not only applicable to corn (Zea mays), but also to rice (Oryza sativa), sorghum (Sorghum bicolor), wheat (Triticum aestivum), soybean (Glycinemax), cotton (Gossypium hirsutum), sunflower (Helianthus annuus) and other crops.

[0084] The method of the present invention is as follows:

[0085] 1. Construction of plant transformation vector pMs45-Su1RNAi containing DNA fragments (DNA constructs) for controlling maize male fertility and maize kernel size

[0086] 1. Synthesis of the Ms45 wild-type allele (Ms45 expression element) that restores male fertility to the maize male sterile mutant ms45

[0087] The male sterility restoration gene Ms45 in the present invention is derived from the inbred line B73. The genomic DNA of B73 is used as a reference to synthesize the Ms45 expression element, as shown in SEQ ID No.3, which includes a promoter, a coding region and a terminator, an EcoRI enzyme recognition sequence is added to the 5' end, and a BstEII enzyme recognition sequence is added to the 3' end. The coding sequence (CDS) of the Ms45 gene is shown in SEQ ID No.1, and the amino acid sequence of the Ms45 protein encoded by it is shown in SEQ ID No.2. After the sequence of SEQ ID No.3 is transferred into the ms45 male sterile mutant, the mutant plant becomes fertile.

[0088] 2. Synthesis of interference fragments regulating genes related to grain shape and endosperm development morphology

[0089] Synthesis of Su1 interference fragment expression element

[0090] The present invention silences the Su1 gene by RNAi technology, and the Su1 interference fragment (as a screening gene) used is named Su1 RNAi. The Su1 gene coding sequence (CDS) is shown in SEQ ID No.9, and the corresponding Su1 protein sequence is shown in SEQ ID No.10. Su1 RNAi comprises a forward interference fragment SEQ ID No.4, a reverse interference fragment SEQ ID No.5, and an intron SEQ ID No.6 forming a hairpin structure between the forward interference fragment and the reverse interference fragment. SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6 are artificially synthesized by an artificial synthesis method, and a promoter SEQ ID No.7 for starting the expression of Su1 RNAi and a terminator SEQ ID No.8 for terminating the expression of Su1 RNAi are also synthesized, which together constitute the Su1 interference fragment expression element, and BstEII restriction sites are added at the 5' end and 3' end thereof, respectively.

[0091] 3. Construction of a plant expression vector containing the male fertility gene Ms45 expression element and the interference fragment expression element of the gene related to grain shape and endosperm development morphology

[0092] Plasmid pCAMBIA3301 (CAMBIA product of the International Center for the Application of Agricultural Molecular Biology, Australia) is used as the backbone DNA, and the vector contains the selection marker gene bar. The bar gene coding sequence (CDS) is shown in SEQ ID No.11.

[0093] The following plant expression vector was constructed using the vector skeleton:

[0094] Plant expression vector pMs45-Su1RNAi: The synthesized Ms45 wild-type allele expression element and the Su1 interference fragment expression element were respectively integrated into the pCAMBIA3301 vector using EcoRI, BstEII and BstEII digestion to obtain pMs45-Su1RNAi, which contains its own selection marker gene bar. Figure 3As shown, the vector contains a DNA fragment (DNA construct) and a selection marker gene for controlling the male fertility of corn and the size of corn kernels. Among them, the DNA fragment for controlling the male fertility of corn and the size of corn kernels is named Ms45-Su1RNAi, which is a DNA fragment between LB and RB of pMs45-Su1RNAi. Ms45-Su1RNAi comprises the Ms45 expression element (first polynucleotide) of SEQ ID No:3 and the expression element (second polynucleotide) for silencing the Su1 gene. The expression element for silencing the Su1 gene is connected by the promoter (Su1promoter) of Su1, the Su1 interference fragment Su1RNAi (as a screening gene) and the terminator. The Ms45 expression element and the expression element for silencing the Su1 gene are closely connected. When pMs45-Su1RNAi is transferred into plants, these two expression elements exist simultaneously in the plants.

[0095] 2. Plant transformation vectors transform corn to obtain transgenic corn

[0096] 1. Plant transformation vector transformation of corn

[0097] The recipients used in the genetic modification process in the laboratory are the F1 generation of the hybrid of the inbred lines HiIIA and HiIIB.

[0098] The plant transformation vector pMs45-Su1RNAi was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pMs45-Su1RNAi containing transgenic elements.

[0099] The invention obtains transgenic plants by a method of infecting maize immature embryos with Agrobacterium. First, maize inbred lines HiIIA and HiIIB are planted in the field, and are bagged when the inbred lines shed pollen; then, pollination is prepared, and there are two pollination methods: HiIIA is used as the female parent and HiIIB is used as the male parent; HiIIA is used as the male parent and HiIIB is used as the female parent. 9-11 days after pollination, immature hybrid embryos on the grains of the pollinated cobs are taken, and then recombinant Agrobacterium EHA105 / pMs45-Su1RNAi is infected indoors, and the immature embryos infected with the recombinant Agrobacterium EHA105 / pMs45-Su1RNAi are placed on a selection medium, and after screening with the herbicide bialaphos, resistant callus tissue is obtained, and the resistant callus tissue is regenerated into seedlings to obtain transgenic T0 generation plants. After obtaining the transgenic T0 generation, the pollen of the T0 generation transgenic plants is used to hybridize some seed production female parents and Ms45 male sterile materials, and the phenotype is observed.

[0100] The specific method is as follows:

[0101] (I) Peeling off the young embryo

[0102] 1) Remove the bracts. Cut off about 1 cm from the top of the F1 hybrid fruit ear of HiIIA and HiIIB, insert the fruit ear from the top with tweezers, so that the tweezers can be used as a handle, which is convenient for operation, and then put the fruit ear into a beaker containing disinfectant. According to actual needs, 4-6 fruit ears can be placed in the same beaker.

[0103] 2) Add about 700 ml of disinfectant (50% bleach or 5.25% sodium hypochlorite, and add a drop of Tween 20) to the beaker to soak the fruit ears. During the 20-minute disinfection process, rotate the fruit ears from time to time and gently tap the beaker to remove bubbles on the surface of the grains to achieve the best disinfection effect. After the disinfection is completed, take out the fruit ears and put them in a beaker filled with sterile water. Wash them in water for 3 times, and then prepare to peel the embryo.

[0104] 3) Place one end of the sterilized ear on a large culture dish and use a large scalpel to cut off the top of the kernel (1.5-1.8mm). During this process, you should frequently sterilize the tools used, such as surgical blades, culture dishes, embryo peeling knives, etc.

[0105] 4) Insert the tip of the embryo peeling knife between the embryo and the endosperm, then gently pry out the young embryo upwards, and gently hold up the young embryo with the tip of a small surgical knife to ensure that the young embryo is not damaged in any way. Place the embryo axis surface close to the N6E culture medium with filter paper. The density of the embryos is about 2×2cm (30 per dish).

[0106] 5) Seal the culture dish with sealing film and culture in the dark at 28 degrees for 2-3 days.

[0107] (ii) Agrobacterium infection

[0108] 1) The recombinant Agrobacterium EHA105 / pMs45-Su1RNAi was cultured on YEP medium (containing 33 mg / L kanamycin and 100 mg / L rifampicin) one week in advance.

[0109] 2) The recombinant Agrobacterium cultured above was transferred to fresh YEP medium (containing 33 mg / L kanamycin and 50 mg / L rifampicin) and cultured at 19°C for 3 days.

[0110] 3) After 3 days, pick up Agrobacterium tumefaciens EHA105 and put it into a 50 ml centrifuge tube containing 5 ml infection medium, add AS (inf + AS) (solute as shown in Table 2, solvent is water), and shake the bacteria at room temperature (25°C) and 75 rpm for 2-4 hours.

[0111] 4) Infecting embryos: Place the freshly peeled embryos into a centrifuge tube containing AS (inf + AS) liquid medium (2 ml), with about 20-100 embryos per tube. Wash twice with this medium, and then add 1-1.5 ml of a specific concentration (OD550 =0.3-0.4) of Agrobacterium, gently invert the centrifuge tube 20 times, and then place it upright in a dark box for 5 minutes to ensure that all the young embryos are immersed in the Agrobacterium liquid. Avoid vortexing during the whole process.

[0112] (III) Co-cultivation

[0113] 1) After infection, the infected immature embryos are transferred to a co-cultivation medium (solutes are shown in Table 2, and the solvent is water), so that the embryonic axis of the immature embryo contacts the surface of the medium, and at the same time, excess Agrobacterium on the surface of the medium is removed.

[0114] 2) Seal the culture dish with sealing film and culture in the dark at 20°C for 3 days.

[0115] (IV) Quiescent cultivation

[0116] After 3 days of co-culture, the young embryos were transferred to a resting culture medium (solutes as shown in Table 2, the solvent was water), and the culture dish was sealed with a sealing film and cultured in the dark at 28°C for 7 days.

[0117] (V) Selection

[0118] 1) After 7 days, all the embryos were transferred to the selection medium (solute as shown in Table 2, solvent is water) (35 embryos / dish), cultured for two weeks, the selection medium contained 1.5 mg / L of bialaphos, and subcultured after two weeks, the concentration of bialaphos could be raised to 3 mg / L.

[0119] 2) About 5 weeks after infection, the cells containing the transformants will grow into visible type II callus.

[0120] 6. Regeneration of transgenic plants

[0121] 1) Grow on regeneration medium I (solutes as shown in Table 2, solvent is water) for 3 weeks, then germinate on regeneration medium II (solutes as shown in Table 2, solvent is water) (in a light culture room) to obtain 100 T0 generation pMs45-Su1RNAi transformed corns.

[0122] 2) When the regenerated seedlings grow 3-4 leaves, they are transferred to a greenhouse and pollinated when they grow to the silk-spinning and pollination stage.

[0123] Table 2 Solutes and contents of culture medium

[0124]

[0125] In Table 2, MS salt was purchased from Phyto Technology Laboratories, with the product number being M524.

[0126] 2. Analysis of transgenic plants obtained

[0127] The T0 generation pMs45-Su1 RNAi transformed maize obtained in step 1 and its progeny were evaluated for overall plant morphology, and analyzed for pollen, plant and grain phenotypes.

[0128] The T0 generation pMs45-Su1RNAi transformed maize was crossed with the ms45 homozygous recessive ms45 male sterile material 905I to obtain hybrid offspring.

[0129] 1) Detect the genotype of hybrid offspring

[0130] The bar gene detection is used to determine whether the transgenic element Ms45-Su1RNAi is present. The bar gene detection method is as follows: the hybrid offspring is amplified by PCR using a primer pair consisting of Bar669F and Bar669R. If the hybrid offspring contains a target fragment of 669 bp in size, the hybrid offspring is a hybrid offspring containing the transgenic element. If the target fragment of 669 bp in size is not present, the hybrid offspring is a hybrid offspring not containing the transgenic element.

[0131] The sequences of the primers Bar669F and Bar669R are as follows:

[0132] Bar669F:5'-TCTCGGGTGACGGGCAGGAC-3';

[0133] Bar669R:5'-TGACGCACAATCCCACTATCCTT-3'.

[0134] The hybrid offspring containing the transgenic element Ms45-Su1RNAi were obtained.

[0135] 2) Detect the phenotype of hybrid offspring

[0136] The hybrid progeny containing the transgenic element Ms45-Su1RNAi were observed in the field. The results are shown in Figure 4 :

[0137] The results of judging fertility by grain size are as follows Figure 4 As shown in the right figure, B is the hybrid offspring grain containing the transgenic element Ms45-Su1RNAi, and the grain has become smaller (tested as fertile); A is the hybrid offspring grain without the transgenic element Ms45-Su1RNAi, and the grain is normal (tested as infertile);

[0138] Figure 4 The male flowers of the plant after the small seeds grow up are as follows Figure 4 The left picture shows the hybrid offspring containing the transgenic element Ms45-Su1RNAi. Since the plant contains Ms45, it appears fertile (fertility test).

[0139] 3. Large-scale propagation of male sterile lines using male sterile maintainer lines

[0140] according to Figure 5 The route is to use the male sterile maintenance line to carry out large-scale propagation of the male sterile line, as follows:

[0141] 1. Convert the Ms45Ms45 wild-type inbred line into the ms45ms45 homozygous recessive inbred line

[0142] A ms45 homozygous recessive mutant (such as 905I) is used as the female parent and hybridized with different inbred lines (such as Zheng 58 (zheng58), a variety of the Grain Crop Research Institute of the Henan Academy of Agricultural Sciences). The obtained F1 is further backcrossed with the corn inbred line Zheng 58, and the genotype analysis is performed on the obtained BC1 population. Plants that are heterozygous for the Ms45 locus are identified and further backcrossed with Zheng 58. After 5-6 backcross generations, molecular markers are used to screen for single plants that are heterozygous for the Ms45 locus and Zheng 58 for other loci, and self-pollination is performed to obtain the ms45 homozygous recessive inbred line Zheng 58 (called: Zheng 58 (ms45ms45)). This inbred line can be used as a sterile line and is called the first plant.

[0143] The above method for screening the genotype of the Ms45 locus is as follows: the plant genome is PCR amplified using the following primers Ms45F1 and Ms45R1, and the amplification results are sequenced. The target fragment size of Ms45 is 859bp, and the target fragment size of ms45 is 811bp. If the amplified product contains both the 859bp and 811bp target fragments, the genotype of the locus is heterozygous Ms45 / ms45; if the amplified product does not contain the 811bp fragment, the genotype of the locus is Ms45 / Ms45 dominant homozygous; if the amplified product does not contain the 859bp target fragment, the genotype of the locus is ms45 / ms45 recessive homozygous.

[0144] Ms45F1:5'-CTTGAGCGACAGCGGGAACT-3';

[0145] Ms45R1:5'-TGTTGTTTCTTGGCAAAGGTCAG-3'.

[0146] 2. Preparation of the second plant

[0147] Preparation of a second plant heterozygous for Ms45-Su1RNAi and homozygous for ms45

[0148] The obtained ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)) (first plant, female parent) is hybridized with the obtained T0 generation pMs45-Su1RNAi transformed corn (male parent), and then multiple generations of backcrossing are carried out with the ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)) (first plant) as the recurrent parent, and the T0 generation pMs45-Su1RNAi transformed corn is transformed into an inbred line containing Ms45-Su1RNAi heterozygosity and homozygous recessive ms45 locus. This inbred line is the second plant that is heterozygous for Ms45-Su1RNAi and homozygous for ms45, also known as Zheng 58 (Ms45-Su1RNAi heterozygosity and homozygous recessive ms45).

[0149] The specific method is as follows:

[0150] The T0 generation of pMs45-Su1RNAi transgenic corn was used as the male parent and hybridized with the obtained ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)) (female parent). Small-grained grains were selected from the hybrid offspring, sown in the field and sprayed with 200mM of bispyribac, and the surviving plants were backcrossed with Zheng 58 (Zheng 58 (ms45ms45)). After 5-6 generations of backcrossing, since small-grained plants were always selected to hybridize with the first plant during the backcrossing process, the small-grained grains or plants contained the transgenic site (Ms45-Su1RNAi), so the small-grained grains or plants in the obtained offspring were all heterozygous for the transgenic site (Ms45-Su1RNAi). When the first plant is pollinated with pollen from a small-grain plant, if the normal grains (large-grain grains) obtained are all sterile, the plant providing the pollen is heterozygous for the transgenic site Ms45-Su1RNAi and is recessive homozygous for the ms45 site, which is the second plant.

[0151] 3. Obtaining the maintenance system

[0152] The second plant Zheng 58 (Ms45-Su1RNAi heterozygous and ms45 homozygous recessive) was used as the male parent and hybridized with the first plant ms45 homozygous recessive self-bred line Zheng 58 (Zheng 58 (ms45ms45)) (female parent). The offspring produced not only Zheng 58 (ms45ms45), but also the male sterile Zheng 58 maintenance line Zheng 58 (Ms45-Su1RNAi heterozygous ms45ms45). In addition, the grains of Zheng 58 (ms45ms45) were normal, while the grains of its maintenance line Zheng 58 (Ms45-Su1RNAi heterozygous ms45ms45) were small (such as Figure 4 (The grain is shown in Figure B on the right).

[0153] 4. Large-scale propagation of the ms45 male sterile inbred line using the male sterile maintainer line

[0154] Taking the Zheng 58 inbred line as an example, the male sterile line Zheng 58 (ms45ms45) and the male sterile maintenance line Zheng 58 (Ms45-Su1RNAi heterozygous ms45ms45) were sown in the field. The two materials were sown alternately, and 5 rows of the sterile line were sown for every 1 row of the maintenance line. It was ensured that no other corn was sown within 300 meters around the breeding seed, so that the sterile line and the maintenance line were naturally pollinated in the field. The maintenance line can only accept its own pollen. Since the seeds containing homozygous transgenic components and heterozygous seeds in the produced offspring cannot be distinguished, these seeds are discarded, and the normal-sized seeds (large seeds) can be used as sterile lines. The sterile line materials accepted the pollen of the maintenance line, and the normal-sized seeds in their offspring were sterile lines without transgenic components, while the small-sized seeds were maintenance lines containing transgenic components. All maintenance lines are used to expand the sterile lines and maintenance lines in the next year, while most of the sterile lines are used to produce varieties, and the remaining small part is used to expand the sterile lines and maintenance lines in the next year. The specific production process is as shown in the attached Figure 5 shown.

[0155] 4. Large-scale production of hybrids using male sterile lines

[0156] As long as a male sterile (ms45ms45) inbred line, such as male sterile Zheng 58 (ms45ms45) with high compatibility, such as Chang 7-2, is selected for hybridization, a hybrid with excellent agronomic traits can be produced. In order to achieve the above purpose, the inventor sows the male sterile inbred line and the wild-type inbred line in alternate rows in the field to ensure that no other corn is sown within 300 meters around the breeding seed. The ears of the sterile line can only accept pollen from the wild-type inbred line, and the wild-type inbred line can only self-pollinate. In this way, the seeds produced on the ears of the sterile line are hybrids.

[0157] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for propagating maize nuclear male sterile lines by seed morphology markers, characterized in that: The method comprises the following steps: a) providing a maintenance line B, wherein the maintenance line B is an inbred line comprising the same homozygous recessive male sterility gene as that in the sterile line A and containing a nucleic acid molecule in a heterozygous state; the nucleic acid molecule comprises a first polynucleotide and a second polynucleotide; the first polynucleotide comprises a restoration gene, and the restoration gene can restore the male fertility of a plant that is male sterile due to the recessive male sterility gene; the second polynucleotide comprises a screening gene that can regulate the grain shape and / or endosperm development morphology, wherein the grain shape comprises the size, length, width, or / and thickness of the seed, and the endosperm development morphology comprises whether the endosperm is sunken, wrinkled, or / and powdery; the sterile line A is a sterile line comprising a homozygous recessive male sterility gene; The screening gene silences the Su1 gene; the Su1 gene encodes the Su1 protein, and the Su1 protein is a protein of C1, C2 or C3 as follows: C1. A protein having an amino acid sequence as shown in SEQ ID No.10; C2. A protein having more than 80% identity with the protein shown in C1) and having the same function as the protein obtained by replacing and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No.10; C3. A fusion protein obtained by connecting a protein tag at the N-terminus or / and C-terminus of C1) or C2); b) Fertilizing the female gametes of the sterile line A with the male gametes of the maintenance line B, and distinguishing the obtained seeds into seeds of the sterile line A and seeds of the maintenance line B according to the external characteristics of the screening gene, thereby completing the propagation of the male sterile line and the corresponding maintenance line.

2. The method according to claim 1, characterized in that: The recessive male sterility gene is selected from at least one of ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, ms10, ms11, ms12, ms13, ms14, ms15, ms16, ms17, ms18, ms19, ms20, ms21, ms22, ms23, ms24, ms25, ms26, ms27, ms28, ms29, ms30, ms31, ms32, ms33, ms34, ms35, ms36, ms37, ms38, ms43, ms45, ms47, ms48, ms49, ms50 and ms52; The restoration gene is selected from at least one of Ms1, Ms2, Ms3, Ms4, Ms5, Ms6, Ms7, Ms8, Ms9, Ms10, Ms11, Ms12, Ms13, Ms14, Ms15, Ms16, Ms17, Ms18, Ms19, Ms20, Ms21, Ms22, Ms23, Ms24, Ms25, Ms26, Ms27, Ms28, Ms29, Ms30, Ms31, Ms32, Ms33, Ms34, Ms35, Ms36, Ms37, Ms38, Ms43, Ms45, Ms47, Ms48, Ms49, Ms50 and Ms52.

3. The method according to claim 2, characterized in that: The recessive male sterility gene is ms45, and the restoration gene is Ms45; the restoration gene Ms45 encodes Ms45 protein, and the Ms45 protein is the following A1, A2 or A3 protein: A1, a protein having an amino acid sequence as shown in SEQ ID No. 2; A2, a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by replacing and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No.2; A3. A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

4. The method according to claim 3, characterized in that: The sequence of the first polynucleotide is SEQ ID No.

3.

5. The method according to claim 4, characterized in that: The screening gene comprises a forward interference segment, a reverse interference segment, and an intron forming a hairpin structure between the forward interference segment and the reverse interference segment; the sequence of the forward interference segment is SEQ ID No.4; the sequence of the reverse interference segment is SEQ ID No.5; and the sequence of the intron forming the hairpin structure is SEQ ID No.

6.

6. A method for constructing a maintenance system, characterized in that: The method comprises introducing the nucleic acid molecule of any one of claims 1 to 5 into a sterile line comprising a homozygous recessive male sterility gene, to obtain a maintainer line in which the nucleic acid molecule exists in a heterozygous state and the recessive male sterility gene is homozygous.

7. An isolated nucleic acid molecule characterized in that: The nucleic acid molecule comprises a first polynucleotide and a second polynucleotide; the first polynucleotide comprises a restoration gene, which can restore the male fertility of a plant that is male sterile due to a recessive male sterility gene; the second polynucleotide comprises a screening gene that can regulate the grain shape and / or endosperm development morphology, the grain shape comprising the size, length, width and / or thickness of the seed, and the endosperm development morphology comprising whether the endosperm is sunken, wrinkled and / or powdery.

8. The biological material related to the nucleic acid molecule according to claim 7, which is any one of the following B1) to B8): B1) an expression cassette containing the nucleic acid molecule; B2) a recombinant vector containing the nucleic acid molecule, or a recombinant vector containing the expression cassette described in B1); B3) a recombinant microorganism containing the nucleic acid molecule, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2); B4) a transgenic plant cell line containing the nucleic acid molecule, or a transgenic plant cell line containing the expression cassette described in B1); B5) transgenic plant tissue containing the nucleic acid molecule, or transgenic plant tissue containing the expression cassette described in B1); B6) A transgenic plant organ containing the nucleic acid molecule, or a transgenic plant organ containing the expression cassette described in B1).

9. Use of the method according to any one of claims 1 to 5, and / or the construction method according to claim 6, and / or the nucleic acid molecule according to claim 7, and / or the biological material according to claim 8 in plant hybrid seed production.

Images