Method for improving maize yield in density-tolerant manner and use thereof

Abstract

Disclosed are a method for improving maize yield in a density-tolerant manner and the use thereof, which belong to the field of plant biotechnical breeding. The homozygous lg1 mutant has adverse agronomic traits such as small ears, low yield and high empty stem rate. Provided is a new density-tolerant breeding method. An LG1 / lg1 heterozygous genotype material is obtained by hybridizing an lg1 mutant strain with an LG1 wild-type strain. Compared with the LG1 wild-type strain, the LG1 / lg1 heterozygous genotype material reduces the maize leaf angle and the tassel branch number, and increases the yield under high density conditions; compared with the lg1 mutant, the LG1 / lg1 heterozygous genotype material has excellent agronomic traits such as wider leaves, larger ears, reduced empty stem rate, dense-planting increased yield; and the breeding method has wide application prospects in a density-tolerant and high-yield maize breeding field.

Description

A method for improving corn yield tolerance and its application Technical Field

[0001] The present application belongs to the field of plant biotechnology breeding, and specifically relates to a method and application of using the LG1 / lg1 heterozygous genotype to improve corn yield tolerance. Background Art

[0002] Corn is my country's top crop, crucial to the country's food security and agricultural development. However, limited arable land (China's arable land accounts for only 7% of the world's total, yet it must feed approximately 20% of the world's population) forces us to focus on increasing yield per unit area as the primary means of boosting corn production. Furthermore, my country's average corn yield is only 60% of that in the United States, leaving enormous potential for improvement.

[0003] Research has shown that improving cultivar tolerance to dense planting and planting density are key to increasing corn yields. Over the past 80 years, corn planting density in the United States has increased from ~30,000 plants / hectare in the 1930s to ~70,000 plants / hectare today (62,000-104,000 plants / hectare). Concurrently, corn yields have risen from 1,287 kg / hectare in the 1930s to 9,595 kg / hectare in 2010 (USDA-NASS, 2012). However, increases in yield per plant and heterosis have not been significant during this period; the yield increase is primarily due to increased dense planting tolerance and planting density. Studies of Chinese corn varieties from different eras have also shown that modern varieties are becoming more densely tolerant than earlier varieties in terms of photosynthetic efficiency, lodging resistance, empty stalk rate, and yield. Therefore, improving dense planting tolerance and planting density are key goals and trends in modern corn breeding and production.

[0004] Reducing the plant's stem-leaf angle and reducing the number of tassel branches are key to improving corn's tolerance to dense planting. A smaller stem-leaf angle and fewer tassel branches can minimize mutual shading between corn plants, improve the overall canopy structure in the field, enhance ventilation and light transmission between plants, and better facilitate sunlight capture and photosynthesis by corn's functional leaves (the ear leaf, the first leaf above the ear, and the first leaf below the ear), contributing to high corn yields. Furthermore, good ventilation and light transmission significantly increase the red light / far-red light (R / FR) ratio in the lower layers of the plant, reducing the adverse effects of dense planting and shade avoidance, such as excessive stem growth, weakened roots, and reduced stem strength, thereby promoting stable corn yields. Smaller tassels reduce excessive pollen shedding, thereby reducing dust adhesion to lower leaves, which in turn facilitates photosynthesis and improves the supply of "sources." Furthermore, the tassel is a crucial "sink" organ, developing earlier than the female ear. This significantly outperforms the female ear in nutrient competition, and smaller tassels conserve energy, which in turn increases the yield of the female ear. Research from various institutions has shown that modern corn breeding is progressing toward smaller leaf angles and smaller tassel branch numbers, a trend that is highly correlated with improved corn density tolerance. This further demonstrates the importance of leaf angles and tassel branch numbers in corn breeding for density tolerance.

[0005] Reducing the empty stalk rate of corn is also crucial to improving corn density tolerance. Empty stalks of corn refer to the phenomenon that there are no fruiting ears on the stalk, or there are fruit ears but they are poorly developed and have no grains. Dense planting of empty stalks will directly affect the three elements of yield (number of ears per unit area, number of grains per ear, and 100-grain weight), thereby seriously affecting corn yield. Dense planting of corn will cause a significant increase in the empty stalk rate. An analysis of the relationship between agronomic traits and yield of corn at different densities found that the impact of the empty stalk rate on yield will increase rapidly with the increase in planting density. The higher the planting density, the greater the negative impact of the empty stalk rate on yield. Dense planting of empty stalks can generally reduce corn yield by 15-20%, and in severe cases it can be reduced by more than 60%. It is one of the important factors limiting the increase in corn dense planting yield. Therefore, reducing the empty stalk rate under dense planting conditions is also crucial to improving corn density tolerance.

[0006] Studies have found that the LG1 gene is a key gene that regulates the changes in leaf angles of gramineous crops such as corn, rice, and wheat. Existing studies have shown that homozygous lg1 can simultaneously reduce the leaf angle of the plant and the number of tassel branches, which is conducive to the plant type to tolerate dense planting. However, the inventors found that its lack of leaf ear and ligule easily causes pests and pathogens to invade from the leaf sheath, increasing the risk of pest damage, and previous experiments have shown that homozygous lg1 mutants do not have the effect of increasing yield under high density conditions (5000 plants / mu and 6000 plants / mu, reference Lambert RJ, Johnson RR: Leaf Angle, Tassel Morphology, and the Performance of Maize Hybrids. Crop Science. 1978, 18 (3): 499-502). Therefore, a breeding strategy and method that can reduce the leaf angle and the number of tassel branches in production, but can produce leaf ear and ligule normally, while not significantly reducing the ear and not increasing the empty stalk rate, will have broad application prospects in the field of corn dense planting.

[0007] Summary of the Invention

[0008] All references cited herein are incorporated by reference. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, the techniques used or described herein are standard techniques known to one of ordinary skill in the art. The materials, methods, and examples are illustrative only and not limiting.

[0009] The embodiments of the present application provide an improved method for breeding for dense-density tolerance, wherein the method hybridizes an lg1 mutant strain with an LG1 wild-type strain to obtain an LG1 / lg1 heterozygous genotype strain, wherein the heterozygous genotype strain has excellent dense-density tolerance traits.

[0010] Optionally, the excellent dense-tolerance traits include: reduced corn leaf angle, reduced number of tassel branches, smaller tassel branch angle, wider leaves, larger ears, reduced empty stalk rate and / or dense planting to increase yield. Specifically, compared with the LG1 wild-type strain, the LG1 / lg1 heterozygous genotype material reduces the corn leaf angle and the number of tassel branches, and dense planting to increase yield; compared with the lg1 mutant, the leaves become wider, the ears become larger, the empty stalk rate is reduced, the tassel branch angle becomes larger, dense planting to increase yield and / or the ability to resist diseases and pests is enhanced. Those skilled in the art know that the widening of plant leaves can enable the leaves to intercept sunlight more effectively, which is beneficial to photosynthesis and thus conducive to increased yield.

[0011] Optionally, the polynucleotide sequence of the wild-type LG1 gene is selected from one of the following groups of sequences:

[0012] (a) a polynucleotide sequence as shown in SEQ ID No: 1, 2 or 3;

[0013] (b) a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID No: 4;

[0014] (c) a polynucleotide sequence capable of hybridizing with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, wherein a homozygous mutation of the polynucleotide sequence has the function of causing complete loss of the plant's ligule and auricles, a smaller leaf angle, upright leaves, narrower leaves, a reduced number of tassel branches, a smaller tassel branch angle, an increased empty stalk rate, and / or smaller fruit ears;

[0015] (d) a polynucleotide sequence that is at least 90%, 95%, 98% or more similar to the polynucleotide sequence shown in any one of (a) to (c), wherein a homozygous mutation of the polynucleotide sequence has the effect of causing complete loss of the ligule and auricle of the plant, a smaller leaf angle, upright leaves, narrower leaves, a reduced number of tassel branches, a smaller tassel branch angle, an increased empty stalk rate, and / or a smaller ear; or

[0016] (e) A polynucleotide sequence complementary to any one of the sequences described in (a) to (d).

[0017] Optionally, the LG1 gene provided in the embodiments of the present application also includes a homologous gene having at least 80%, 85%, 90%, 95%, 98% or 99% sequence similarity with the polynucleotide sequence disclosed in the embodiments of the present application, or a homologous gene having at least 90%, 95% or 98% sequence similarity with the amino acid sequence of LG1 disclosed in the embodiments of the present invention, and after endogenous homozygous mutation, the homologous gene has the function of causing the complete loss of the plant's ligule and auricle, a smaller leaf angle, upright leaves, narrower leaves, a reduced number of tassel branches, a smaller tassel branch angle, an increased empty stem rate and / or smaller ears. The homologous gene can be isolated and obtained from any plant.

[0018] Alternatively, the methods provided in the examples of this application can be applied to any plant containing an LG1 homologous gene. Preferably, the plant includes monocotyledonous plants such as corn, millet, wheat, barley, rye, rice, and sorghum.

[0019] The percentages of sequence similarity described in this application can be obtained by well-known bioinformatics algorithms, including the Myers and Miller algorithm, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0020] Those skilled in the art should be aware that there is single nucleotide polymorphism (SNP) in the same gene between different varieties of the same plant, that is, the nucleotide sequence of the same gene often has differences in individual bases, but there are many varieties of the same crop, and it is impossible for the inventors to list them one by one. The embodiments of this application only provide sequences of representative varieties in corn crops. Therefore, those skilled in the art should be aware that the nucleotide sequences of different varieties with SNPs in the LG1 gene and its nucleotide sequence disclosed in this application, the homozygous mutant strains obtained by mutation thereof are hybridized with the wild type to obtain methods and applications of traits such as wider leaves, larger ears, reduced empty stalk rate and / or dense planting to increase yield, are also within the scope of protection of this application.

[0021] Optionally, the lg1 mutant strain is obtained by mutation, and the mutation includes substitution, deletion and / or addition of one or more nucleotides in the nucleotide sequence of the gene.

[0022] Optionally, the mutation is obtained by physical mutagenesis, chemical mutagenesis, ZFN, TALEN and / or CRISPR / Cas gene editing.

[0023] Optionally, when using CRISPR / Cas for corn plant gene editing, the target site sequence is shown as SEQ ID NO:5 or SEQ ID NO:6.

[0024] Optionally, the lg1 mutant has a genomic mutant nucleotide sequence selected from one of the following groups:

[0025] (a) a genomic mutant nucleotide sequence in which the entire nucleotide sequence of SEQ ID No. 1 is deleted;

[0026] (b) a genomic mutant nucleotide sequence in which the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2, bp 743-764, or the nucleotide sequence of SEQ ID No. 3, bp 229-250 is replaced with 5'-TTCCGCCGCCGTCC-3';

[0027] (c) the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2, 766-835 bp, or the nucleotide sequence of a genomic mutant with a sequence deletion of SEQ ID No. 3, 252-321 bp;

[0028] (d) The nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2 at 836 bp, or the nucleotide sequence of a genomic mutant having a single nucleotide deletion at SEQ ID No. 3 at 322 bp.

[0029] Optionally, the dense planting refers to a corn planting density greater than or equal to 4,000 plants / mu, preferably greater than or equal to 5,000 plants / mu.

[0030] Optionally, the embodiments of the present application also provide the application of any of the aforementioned methods in corn breeding, preferably including but not limited to applications in reducing the leaf angle of the plant, widening the leaves, reducing the number of male spike branches, reducing the angle of the male spike branches, reducing the empty stalk rate, and increasing yields by dense planting.

[0031] Specifically, the dense planting and yield increase mentioned herein refers to the increase in corn yield under high-density planting conditions, and the high density refers to greater than or equal to 4,000 plants / mu, preferably greater than or equal to 5,000 plants / mu.

[0032] The present invention also provides a plant cell, tissue, organ, or commercial product that is not used as a propagation material, wherein the plant cell, tissue, organ, or commercial product contains a heterozygous LG1 / lg1 genotype. The heterozygous LG1 / lg1 genotype refers to a plant cell, tissue, organ, or commercial product that contains both a wild-type LG1 gene and a mutant lg1.

[0033] The present application also provides a method for regulating agronomic traits of corn plants, characterized in that a homozygous mutant is obtained by mutating an endogenous gene of the plant, wherein the homozygous mutant has a phenotype of complete absence of ligule and auricle, reduced leaf angle, upright leaves, narrowed leaves, reduced number of tassel branches, reduced tassel branch angle, increased empty stalk rate and / or smaller ears, and the polynucleotide sequence of the endogenous gene is selected from one of the following groups of sequences:

[0034] (a) a polynucleotide sequence as shown in SEQ ID No: 1, 2 or 3;

[0035] (b) a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID No: 4;

[0036] (c) a polynucleotide sequence capable of hybridizing with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, wherein a homozygous mutation of the polynucleotide sequence has the function of causing complete loss of the ligule and auricle of the plant, a smaller leaf angle, upright leaves, narrower leaves, a reduced number of tassel branches, a smaller tassel branch angle, an increased empty stalk rate, and / or smaller ears;

[0037] (d) a polynucleotide sequence that is at least 90%, 95%, 98% or more similar to the polynucleotide sequence shown in any one of (a) to (c), wherein a homozygous mutation of the polynucleotide sequence has the function of causing the plant to completely lose its ligule and auricle, reduce the leaf angle, make the leaves upright, narrow the leaves, reduce the number of tassel branches, reduce the angle of tassel branches, increase the empty stem rate and / or reduce the size of the fruit ear; or

[0038] (e) A polynucleotide sequence complementary to any one of the sequences described in (a) to (d).

[0039] Alternatively, the nucleotide sequence, vector, construct or expression cassette described in the embodiments of the present application is transferred into a plant or introduced into a plant or the plant is transformed, all referring to a method of transferring the target nucleotide sequence, construct, vector or expression cassette into a recipient cell or recipient plant by conventional transgenic methods or hybridization with a target transgenic plant. Any transgenic method known to those skilled in the art can be used to transform the recombinant expression vector into a plant cell to produce the transgenic plant or mutant of the embodiments of the present application. The transformation method may include direct or indirect transformation methods. Specifically, the transformation method includes but is not limited to polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, microinjection, and Agrobacterium-mediated plant transformation methods, etc.

[0040] Compared with the prior art, this application has the following beneficial effects:

[0041] (1) The embodiment of the present application provides a method for improving corn yield and its application, by hybridizing an lg1 mutant line with an LG1 wild-type line to obtain an LG1 / lg1 heterozygous genotype line. Compared with the lg1 mutant line, the LG1 / lg1 heterozygous genotype line has excellent agronomic traits such as wider leaves, larger ears, reduced empty stalk rate and / or increased yield due to dense planting; compared with the LG1 wild-type line, the LG1 / lg1 heterozygous genotype material reduces the corn leaf angle and the number of tassel branches, and has the effect of increased yield due to dense planting; in the breeding of dense-tolerant and high-yield varieties, the technical problem that the lg1 mutant has the dense-tolerant traits of smaller leaf angle and upright leaves, but reduced yield, reduced disease and pest resistance, and increased empty stalk rate is solved.

[0042] (2) The method provided in the embodiments of the present application makes it possible to cultivate new varieties of crops that are tolerant to high density and high yield, especially new varieties of corn, which is of great significance to global food security and sustainable agricultural development.

[0043] Definitions of terms used in this application

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of this application, the preferred methods, devices, and materials are now described.

[0045] In the context of this application, the term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof, in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0046] The term "homologous genes" in this application refers to two or more gene sequences with a sequence similarity of 80%, including orthologous genes (also known as vertical homologous genes, orthologous genes or directed evolutionary homologous genes), horizontal homologous genes (also known as paralogous genes, parallel homologous genes or parallel evolutionary homologous genes) and / or heterologous genes.

[0047] The term sequence "similarity" refers to the degree of similarity between two sequences. It is a quantitative concept used to compare the degree of similarity between different sequences, thereby discovering and analyzing the relationship between the two sequences. Sequence similarity can be used to compare gene sequences, protein sequences, DNA sequences, etc. "Stringent hybridization conditions" as described in this application refer to conditions of low ionic strength and high temperature known in the art. Generally, under stringent conditions, the detectable degree of hybridization of a probe to its target sequence is higher than the detectable degree of hybridization to other sequences (for example, at least 2 times greater than the background). Stringent hybridization conditions are sequence-dependent and will vary under different environmental conditions. Longer sequences will hybridize specifically at higher temperatures. By controlling the stringency of hybridization or washing conditions, target sequences that are 100% complementary to the probe can be identified. For detailed guidance on nucleic acid hybridization, reference can be made to the relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes," Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are generally selected to be below the thermal melting point (TP) of the specific sequence at a specified ionic strength pH. m ) about 5-10℃. mThe temperature at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (under specified ionic strength, pH, and nucleic acid concentration) (because the target sequence is present in excess, the target sequence is present at T m Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salts), at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes (including but not limited to, 10 to 50 nucleotides) and at least about 60°C for long probes (including but not limited to, greater than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least twice the background hybridization, optionally 10 times the background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5× SSC, and 1% SDS, incubation at 42°C; or 5× SSC, 1% SDS, incubation at 65°C, wash in 0.2× SSC, and wash in 0.1% SDS at 65°C. The washing may be performed for 5, 15, 30, 60, 120 minutes or longer.

[0048] The term "recombinant expression vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to in the art as binary vectors. Binary vectors, along with vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation. Binary vectors typically include the cis-acting sequences required for T-DNA transfer, a selectable marker engineered for expression in plant cells, and the heterologous DNA sequence to be transcribed.

[0049] The term "leaf angle" refers to the angle formed by the midrib of a leaf blade and the upper stem where the pulvinus of the leaf blade contacts.

[0050] The term "hybridization" is a broad term that refers to the process of hybridization between gametes of different populations or genotypes to produce hybrids. Depending on the relationship between the parents, it includes close hybridization and distant hybridization.

[0051] In this application, lg1 mutant, lg1 homozygous mutant or lg1 / lg1 mutant all refer to lg1 homozygous mutant. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 shows a comparative analysis of wild-type and lg1 mutants. Figure 1A shows the sequences of the B73 inbred line and the lg1 mutant derived from the B73 background. LG1-WT is the B73 wild-type, and LG1-MT is the lg1 mutant derived from the B73 background. Figure 1B shows a comparison of the overall plant shape of the B73 inbred line and the lg1 mutant derived from the B73 background. Figure 1C shows a comparison of the leaf angles of the B73 inbred line and the lg1 mutant derived from the B73 background. Figure 1D shows a comparison of the auricles and ligules of the B73 inbred line and the lg1 mutant derived from the B73 background.

[0053] Figure 2 shows a comparative analysis of the traits of LG1 wild-type, heterozygous, and homozygous mutant materials. Figure 2A shows a comparison of whole-plant morphology for the LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under the B73 background; Figures 2B and 2E show a comparison of leaf angles for the LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under the B73 background, respectively; Figures 2C and 2F show a comparison of tassels and statistics of tassel branch numbers for the LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under the B73 background; and Figures 2D and 2G show a comparison of ear weights for the LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under the B73 background.

[0054] FIG3 shows the mutation of the LG1 gene in the PH4CV, PH6WC and Jing724 maize inbred line backgrounds.

[0055] Figure 4 shows the comparative photos and statistical data of leaf angles of LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under different planting density conditions (LD: 3000 plants / mu, MD: 6000 plants / mu, HD: 9000 plants / mu) in the PH6WC, PH4CV, and B73 inbred lines.

[0056] Figure 5 shows comparative photographs of tassels and statistical data on the number of tassel branches for the LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under different planting densities (LD: 3000 plants / mu, MD: 6000 plants / mu, HD: 9000 plants / mu).

[0057] Figure 6 shows photographs and statistical data of leaf angles of LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under different planting density conditions in the hybrid B73 / PH6WC, B73 / PH4CV, and PH6WC / PH4CV backgrounds. LD: 3000 plants / mu, MD: 5000 plants / mu, and HD: 7000 plants / mu.

[0058] Figure 7 shows comparative photographs of tassels and statistical data on the number of tassel branches for the LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under different planting densities (LD: 3000 plants / mu, MD: 5000 plants / mu, HD: 7000 plants / mu).

[0059] Figure 8 shows the statistical analysis of empty stem rates for different strains under different planting density conditions. Figures 8A, 8B, and 8C show the empty stem rates for the LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under different planting density conditions in the B73, PH4CV, and PH6WC inbred lines, respectively. Figures 8D, 8E, and 8F show the empty stem rates for the LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under different planting density conditions in the B73 / PH4CV, B73 / PH6WC, and PH6WC / PH4CV hybrid lines, respectively. LD: 3000 plants / mu, MD: 6000 plants / mu, and HD: 9000 plants / mu.

[0060] Figure 9 Plot yield statistics of LG1 / LG1 wild type, LG1 / lg1 heterozygous genotype and lg1 / lg1 homozygous mutant materials under different density conditions in the hybrid B73 / PH6WC, B73 / PH4CV and PH6WC / PH4CV backgrounds.

[0061] Figure 10 shows the statistical analysis of leaf width across different inbred and hybrid backgrounds. Figures 10A and 10B show the leaf width statistics for the LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under different planting density conditions in the PH4CV and PH6WC inbred backgrounds, respectively. Figures 10C and 10D show the leaf width statistics for the LG1 / LG1 wild-type, LG1 / lg1 heterozygous, and lg1 / lg1 homozygous mutant materials under different planting density conditions in the B73 / PH4CV and PH6WC / PH4CV hybrid backgrounds, respectively. LD: 3000 plants / mu, MD: 6000 plants / mu, and HD: 9000 plants / mu.

[0062] Figure 11 shows the different phenotypic statistics of LG1 / LG1 wild type and LG1 / lg1 heterozygous genotype materials under the hybrid Jing724 / Jing92 and Jing724-lg1 / Jing92 backgrounds under 3000 plant conditions in the field. Among them, Figure 11A shows the plant types of LG1 / LG1 wild type and LG1 / lg1 heterozygous genotype materials under the hybrid Jing724 / Jing92 and Jing724-lg1 / Jing92 backgrounds under the condition of 3000 plants per mu in the field; Figure 11B shows the photos of tassels of LG1 / LG1 wild type and LG1 / lg1 heterozygous genotype materials under the hybrid Jing724 / Jing92 and Jing724-lg1 / Jing92 backgrounds under the condition of 3000 plants per mu in the field (the LG1 / lg1 heterozygous genotype materials have fewer tassel branches and a smaller tassel angle than the LG1 / LG1 wild type); Figure 11C shows the plot yield statistics of LG1 / LG1 wild type and LG1 / lg1 heterozygous genotype materials under different field density conditions under the hybrid Jing724 / Jing92 and Jing724-lg1 / Jing92 backgrounds. DETAILED DESCRIPTION

[0063] The present application will be further described below with reference to specific embodiments, and the advantages and features of the present application will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not limit the scope of the present application in any way. It should be understood by those skilled in the art that the details and forms of the present application may be modified or replaced without departing from the spirit and scope of the present application, and such modifications and replacements are intended to fall within the scope of protection of the present application.

[0064] The inbred lines used in the following examples can be obtained from the "China Crop Germplasm Information Network" by obtaining relevant information and applying for the corresponding seeds.

[0065] Example 1. Rapid creation of lg1 gene mutants in the B73 inbred background using haploid-mediated gene editing (IMGE)

[0066] The inventors previously cleverly combined haploid induction with gene editing techniques to develop "haploid-mediated gene editing (IMGE)" (Reference: Baobao Wang et al., Development of a Haploid-Inducer Mediated Genome Editing System for Accelerating Maize Breeding, Molecular Plant 12, 597–602, April 2019). This method involves introducing the CRISPR / Cas9 vector into a haploid-induced line through backcrossing or genetic transformation. This haploid-induced line, carrying the CRISPR / Cas9 vector, is then used as the male parent to cross with a commercial inbred line to obtain haploids. Haploids with edited target sites are then screened based on changes in target traits and sequencing analysis. Finally, homozygous, gene-edited doubled haploid material is obtained through artificial or natural chromosome doubling. This allows the creation of commercial doubled haploid lines with improved traits and no transgenic components within two generations (one year), greatly accelerating the progress of crop breeding. IMGE technology breaks the problem of traditional gene editing being limited by the genetic background and genetic transformation capacity of the material to be improved. It can achieve targeted improvement of agronomic traits within two generations, quickly and efficiently. At the same time, the improved materials do not carry exogenous genetically modified components and are easy to develop commercially. It is especially friendly to breeders and easy to operate: gene editing can be achieved through pollination.

[0067] To rapidly create lg1 mutants in the B73 background using IMGE technology, we constructed a gene-editing vector targeting the LG1 gene (target sites are SEQ ID No. 5: 5'-GCGGAGACTAAGTGGCTGTAGGG-3' and SEQ ID No. 6: 5'-GGGGGAGCATCACCATCAACTGC-3'). This vector was then genetically transformed into the ZC01 inbred line. The resulting transgenic event with a high mutation rate was hybridized with the maize haploid inducer line CAU5, which was then backcrossed with CAU5 for three generations to obtain the BC3F1 generation of maize haploid inducer line CAU5 containing the LG1 gene-editing vector. LG1-IMGE There are two key technical points in this process: the first is to use the Bar gene on the gene vector as a screening marker to ensure that the gene editing vector targeting the LG1 gene is retained in the backcross progeny; the second is to ensure that the haploid induction alleles Zmmtl and Zmdmp derived from CAU5 are retained in the backcross progeny in a homozygous state through sequencing identification.

[0068] Next, use CAU5 LG1-IMGEThe B73 inbred line was subjected to IMGE haploid induction, successfully obtaining a haploid containing the lg1 mutation. The lg1 mutant was also obtained in the B73 inbred line background through natural doubling (Figure 1). This mutant does not contain exogenous transgenic components (does not contain a CRISPR vector). Sequencing analysis revealed that the 8.8kb sequence containing the entire LG1 gene coding region was deleted in this mutant (Figure 1A), and the rest of the genetic background was almost the same as that of B73. Phenotypic analysis revealed that the B73-lg1 homozygous mutant completely lacked the ligule and auricle, resulting in a smaller leaf angle and upright leaves (Figures 1B-1D).

[0069] Example 2. Phenotypic analysis of LG1 / LG1 wild-type, LG1 / lg1 heterozygous genotype, and lg1 / lg1 homozygous mutant materials under the B73 background

[0070] Comparison of lg1 homozygous mutants in the B73 and B73 backgrounds revealed that, in addition to the absence of auricles and a reduced leaf angle, the homozygous lg1 / lg1 mutants had narrower leaves (Figures 1C and 1D), fewer tassel branches, and significantly smaller ears (Figure 2). Generally, simultaneously reducing the leaf angle and tassel branch number is beneficial for maize plant architecture to tolerate high density. However, the lack of auricles and ligules in the lg1 homozygous mutants makes it easier for pests and pathogens to invade through the leaf sheaths, increasing the risk of pest damage. Furthermore, the ears are smaller. These factors are extremely detrimental to high and stable yields in maize under dense planting conditions, indicating that the lg1 / lg1 homozygous mutant cannot be truly applied in breeding for high density tolerance.

[0071] Next, we created LG1 / lg1 heterozygous genotypes in the B73 background through hybridization. Analysis of these hybrids, combined with LG1 / LG1 wild-type and lg1 / lg1 homozygous mutants, revealed (Figure 2) that at a normal maize planting density of 4500 plants / mu, the leaf angle and tassel branch number of the LG1 / lg1 heterozygous genotypes were intermediate between those of the lg1 / lg1 homozygous mutant and the LG1 / LG1 wild-type. Furthermore, the LG1 / lg1 heterozygous genotypes possessed normal auricles and ligules, effectively preventing pests and pathogens from invading the leaf sheaths. More interestingly, we found that at 4500 plants / mu, the cob size of the LG1 / lg1 heterozygous genotypes was virtually identical to that of the homozygous LG1 / LG1 wild-type, without exhibiting cob size reduction or yield loss. While the leaf angle was more compact and the tassel branch number was reduced, the cob size remained unchanged. These results suggest that the LG1 / lg1 heterozygous genotypes have unexpected potential for breeding for high-density tolerance.

[0072] Example 3. Testing the density tolerance effect of LG1 / LG1 wild type, LG1 / lg1 heterozygous genotype and lg1 / lg1 homozygous mutant materials under multiple inbred line backgrounds

[0073] In order to test the application potential and universality of LG1 / lg1 heterozygous genotype materials in high-density breeding, we used the maize haploid induction line CAU5 containing the LG1 gene editing vector created earlier. LG1-IMGE The backbone maize inbred lines PH6WC, PH4CV and Jing724 were subjected to IMGE haploid induction, and haploids containing the lg1 mutation were successfully obtained. The lg1 mutants in the PH6WC, PH4CV and Jing724 inbred line backgrounds were obtained by natural doubling. Among them, one lg1 gene mutant in the PH4CV background was obtained ( FIG3 ), which was named PH4CV-lg1. The sequence of its mutation site is shown in FIG3 , and the nucleotide sequence characteristics of its genome mutant are as follows: the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2, 743-764 bp, or the nucleotide sequence of SEQ ID No. 3, 229-250 bp was replaced with 5'-TTCCGCCGCCGTCC-3' (SEQ ID NO: 7); two lg1 gene mutants in the PH6WC background were obtained ( FIG3 ), which were named PH6WC-lg1#1 and PH6WC-lg1#2, respectively. The sequence of their mutation sites is shown in FIG3 , wherein the nucleotide sequence characteristics of the PH6WC-lg1#1 genome mutant are as follows: a single nucleotide deletion occurred in the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2, 836 bp, or SEQ ID No. 3, 322 bp; the nucleotide sequence characteristics of the PH6WC-lg1#2 genome mutant are as follows: a single nucleotide deletion occurred in the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. A sequence deletion occurred in the nucleotide sequence of SEQ ID No. 2, bp 766-835, or SEQ ID No. 3, bp 252-321. A mutant of the lg1 gene in the Jing724 background (Figure 3) was identified, designated Jing724-lg1. The sequence of the mutation site is shown in Figure 3. The genomic mutant nucleotide sequence is characterized by a genomic mutant nucleotide sequence in which the sequence of SEQ ID No. 1 or SEQ ID No. 2, bp 822-837, is replaced with 5'-GGA-3'. LG1 / lg1 heterozygous genotypes were then generated from the PH6WC and PH4CV inbred line backgrounds through hybridization, with PH6WC-lg1#1 used for the PH6WC line.

[0074] Then, strict field density experiments were carried out on LG1 / LG1 wild type, LG1 / lg1 heterozygous genotype and lg1 / lg1 homozygous mutant materials under the background of PH6WC, PH4CV and B73 inbred lines. The experiment was carried out at the Langfang Experimental Station in Hebei Province in 2022. Three planting densities were set up for each material, namely 3,000 plants / mu, 6,000 plants / mu and 9,000 plants / mu. Three replicates were set up for each material and density, and 4-row areas were planted. The middle two rows were selected for the investigation of various agronomic traits. Phenotypic analysis found that under different inbred line backgrounds and different planting density conditions, the leaf angle and number of male spike branches of the LG1 / lg1 heterozygous genotype materials were between the lg1 / lg1 homozygous mutant and the LG1 / LG1 wild type (Figures 4 and 5, the materials with the B73 background were not repeated), and the LG1 / lg1 heterozygous genotype materials had normal leaf ears and ligules, which could effectively prevent pests and pathogens from invading the leaf sheaths. Furthermore, we found that, under different inbred line backgrounds and planting densities, the empty stalk rate of the lg1 / lg1 homozygous mutant was significantly higher than that of the LG1 / lg1 heterozygous genotype and the LG1 / LG1 wild-type. Furthermore, the leaf width of the lg1 / lg1 homozygous mutant was significantly narrower than that of the LG1 / lg1 heterozygous genotype and the LG1 / LG1 wild-type, indicating that the LG1 gene also plays a significant role in regulating the empty stalk rate and leaf width in maize. More interestingly, under the same planting density across different inbred lines, the empty stalk rate and leaf width of the LG1 / lg1 heterozygous genotype did not differ significantly from those of the LG1 / LG1 wild-type (Figures 8A, 8B, 8C, Figures 10A and 10B). The LG1 / lg1 heterozygous genotype exhibited a more compact leaf angle and a reduced number of tassel branches, without increasing the empty stalk rate or narrowing the leaf width. This suggests that the LG1 / lg1 heterozygous genotype has unexpected potential for breeding for high-density tolerance.

[0075] Example 4 Testing the density tolerance effect of LG1 / LG1 wild type, LG1 / lg1 heterozygous genotype and lg1 / lg1 homozygous mutant materials in multiple hybrid backgrounds

[0076] To test the application potential and universality of LG1 / lg1 heterozygous genotype materials in high-density breeding of maize hybrids, different LG1 genetic materials under the backgrounds of PH6WC, PH4CV, and B73 inbred lines were crossed to create different hybrid combinations. Field rigorous density experiments were then conducted on LG1 / LG1 wild-type, LG1 / lg1 heterozygous genotype, and lg1 / lg1 homozygous mutant materials under the hybrid backgrounds of B73 / PH6WC, B73 / PH4CV, and PH6WC / PH4CV. The experiment was carried out at the Langfang Experimental Station in Hebei Province in 2022. Three planting densities were set up for each material: 3,000 plants / mu, 5,000 plants / mu, and 7,000 plants / mu. Each material and density was set up with three replicates, planted in four rows, and the two middle rows were selected for the examination of various agronomic traits. Phenotypic analysis revealed that, similar to inbred lines, under different hybrid backgrounds and planting densities, the leaf angle, number of tassel branches, and tassel branch angle of the LG1 / lg1 heterozygous genotype materials were between those of the lg1 / lg1 homozygous mutant and the LG1 / LG1 wild type (Figures 6 and 7, the number of tassel branches was not counted for materials with the PH6WC / PH4CV hybrid background), and the LG1 / lg1 heterozygous genotype materials had normal auricles and ligules, which could effectively prevent pests and The pathogen invaded the leaf sheath; the empty stem rate of the lg1 / lg1 homozygous mutant was significantly increased compared with that of the LG1 / lg1 heterozygous genotype and the LG1 / LG1 wild-type materials, and the leaf width of the lg1 / lg1 homozygous mutant was significantly narrower than that of the LG1 / lg1 heterozygous genotype and the LG1 / LG1 wild-type materials, while there was no significant difference in the empty stem rate and leaf width between the LG1 / lg1 heterozygous genotype and the LG1 / LG1 wild-type materials (Figures 8D, 8E, 8F, Figures 10C and 10D).

[0077] An examination of plot yield (yield per unit area) in various hybrids revealed that, across all hybrids and planting density conditions, the lg1 / lg1 homozygous mutant exhibited significantly lower plot yield than the LG1 / LG1 wild-type material, with the reduction being even greater at high density (Figure 9). This suggests that, despite its plant architecture advantages, the lg1 / lg1 homozygous mutant cannot be truly used in conventional maize yield breeding and density tolerance breeding. Under low density, the plot yield of the LG1 / lg1 heterozygous genotype was slightly lower than that of the LG1 / LG1 wild-type, but gradually surpassed it with increasing planting density. Under high density (≥5000 plants / mu or above), the plot yield of the LG1 / lg1 heterozygous genotype was significantly higher than that of the LG1 / LG1 wild-type (Figure 9), demonstrating that it has the yield-increasing effect of dense planting and is applicable across different hybrid backgrounds.

[0078] In addition, we also created the hybrids Jing724 / Jing92 and Jing724-lg1 / Jing92 by hybridizing Jing724, Jing724-lg1 and Jing92. Similar density tests were conducted at the Langfang Experimental Station in Hebei Province in 2023. Two planting densities were set up for each material, namely 3,000 plants / mu and 7,000 plants / mu respectively. Three replicates were set up for each material and density, and the plants were planted in four rows. The two middle rows were selected to examine various agronomic traits. Phenotypic and yield analysis revealed that the leaf angle, number of tassel branches, and tassel branch angle of the LG1 / lg1 heterozygous genotype were significantly smaller than those of the LG1 / LG1 wild type (Figure 11). The LG1 / lg1 heterozygous genotype also had normal auricles and ligules, which could effectively prevent pests and pathogens from invading the leaf sheaths. Under low-density conditions, the plot yield of the LG1 / lg1 heterozygous genotype was comparable to that of the LG1 / LG1 wild type, but under high-density conditions (≥7,000 plants / mu or above), the plot yield of the LG1 / lg1 heterozygous genotype was significantly higher than that of the LG1 / LG1 wild type.

[0079] In summary, we have demonstrated that the LG1 / lg1 heterozygous genotype material (compared with the LG1 / LG1 wild type) has the effect of making the leaf angle of corn more compact, reducing the number of tassel branches, and reducing the tassel branch angle under different density conditions, while the leaf width does not become narrower and the empty stalk rate does not increase. In addition, the LG1 / lg1 heterozygous genotype material can significantly increase the unit area yield of corn hybrids under high density conditions (≥5000 plants / mu).

Claims

1. A method for improving corn breeding for high density tolerance, comprising hybridizing an lg1 mutant strain with an LG1 wild-type strain to obtain an LG1 / lg1 heterozygous genotype strain, wherein the heterozygous genotype strain has excellent high density tolerance traits of reduced leaf angle, reduced number of tassel branches, reduced tassel angle, and / or increased dense planting compared with the LG1 wild-type strain; or, compared with the lg1 mutant strain, has excellent high density tolerance traits of wider leaves, larger ears, larger tassel angle, reduced empty stalk rate, and / or increased dense planting; The polynucleotide sequence of the LG1 wild-type gene is selected from one of the following groups of sequences: (a) a polynucleotide sequence as shown in SEQ ID No: 1, 2 or 3; (b) a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID No: 4; (c) a polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, wherein a homozygous mutation of the polynucleotide sequence has the function of causing the plant's leaf tongue and leaf ear to be completely missing, the leaf angle to be reduced, the leaves to be upright, the leaves to be narrowed, the number of tassel branches to be reduced, the angle of tassel branches to be reduced, the empty stem rate to be increased, and / or the ear to be smaller; (d) a polynucleotide sequence that is at least 90%, 95%, 98% or more similar to any of the polynucleotide sequences shown in (a) to (c), wherein a homozygous mutation of the polynucleotide sequence has the function of causing the plant's leaf tongue and leaf ear to be completely missing, the leaf angle to be smaller, the leaves to be upright, the leaves to be narrower, the number of tassel branches to be reduced, the angle of tassel branches to be smaller, the empty stem rate to be increased and / or the ear to be smaller; or (e) A polynucleotide sequence complementary to any one of the sequences described in (a) to (d).

2. The method according to claim 2, wherein the lg1 mutant strain is obtained by mutation, and the mutation comprises substitution, deletion and / or addition of one or more nucleotides in the nucleotide sequence of the gene.

3. The method according to claim 2, wherein the mutation is obtained by natural mutation, physical mutagenesis, chemical mutagenesis, ZFN, TALEN and / or CRISPR / Cas gene editing technology.

4. The method according to claim 1 or 2, wherein the lg1 mutant has a genomic mutant nucleotide sequence selected from one of the following groups of sequences: (a) a genomic mutant nucleotide sequence in which the entire nucleotide sequence of SEQ ID No. 1 is deleted; (b) a genomic mutant nucleotide sequence in which the nucleotide sequence of 743-764 bp of SEQ ID No. 1 or SEQ ID No. 2 is replaced with 5'-TTCCGCCGCCGTCC-3'; (c) a genomic mutant nucleotide sequence in which a single nucleotide is deleted at bp 836 of SEQ ID No. 1 or SEQ ID No. 2; or (d) a genomic mutant nucleotide sequence in which nucleotide sequence 766-835 of SEQ ID No. 1 or SEQ ID No. 2 is deleted; or (e) The genomic mutant nucleotide sequence in which the sequence 822-837 bp of SEQ ID No. 1 or SEQ ID No. 2 is replaced by 5'-GGA-3'.

5. The method according to any one of claims 1-3, wherein the dense planting refers to greater than or equal to 4000 plants / mu.

6. Use of the method according to any one of claims 1 to 5 for reducing the leaf angle of a plant, widening the leaf blade, reducing the number of tassel branches, reducing the angle of tassel branches, reducing the empty stem rate and / or increasing yield by dense planting.

7. The use according to claim 6, wherein the dense planting and yield increase refers to an increase in corn yield under high density conditions, and the high density refers to greater than or equal to 4,000 plants / acre.

8. A method for regulating agronomic traits of corn plants, wherein the homozygous mutant is obtained by mutating an endogenous gene of the plant, wherein the homozygous mutant has a phenotype of complete absence of ligule and auricle, reduced leaf angle, upright leaves, narrowed leaves, reduced number of tassel branches, reduced tassel branch angle, increased empty stalk rate and / or smaller ears, wherein the polynucleotide sequence of the endogenous gene is selected from one of the following groups of sequences: (a) a polynucleotide sequence as shown in SEQ ID No: 1, 2 or 3; (b) a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID No: 4; (c) a polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, wherein a homozygous mutation of the polynucleotide sequence has the function of causing the plant's leaf tongue and leaf ear to be completely missing, the leaf angle to be reduced, the leaves to be upright, the leaves to be narrowed, the number of tassel branches to be reduced, the angle of tassel branches to be reduced, the empty stem rate to be increased, and / or the ear to be smaller; (d) a polynucleotide sequence that is at least 90%, 95%, 98% or more similar to any of the polynucleotide sequences shown in (a) to (c), wherein a homozygous mutation of the polynucleotide sequence has the function of causing the plant's leaf tongue and leaf ear to be completely missing, the leaf angle to be smaller, the leaves to be upright, the leaves to be narrower, the number of tassel branches to be reduced, the angle of tassel branches to be smaller, the empty stem rate to be increased and / or the ear to be smaller; or (e) A polynucleotide sequence complementary to any one of the sequences described in (a) to (d).

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