Gene and method for producing multi-flower and multi-pod high-yield soybeans and application of gene and method

Abstract

The invention discloses a gene and a method for producing multi-flower and multi-pod high-yield soybean and application of the gene, and belongs to the field of plant biotechnology breeding. According to the soybean Glyma. 11G168800 gene and the soybean Glyma. 18G060200 gene provided by the invention, after a plant generates function loss mutation of endogenous double genes or the expression of the Glyma. 11G168800 gene and the Glyma. 18G060200 gene is inhibited, phenotypes with increased plant height, increased single plant pod number, increased single plant grain number, increased single plant yield, increased branch number, increased lateral branch node number and / or increased pod number of each node can be obtained; the gene and the application method thereof have wide application prospects in the field of soybean breeding.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology breeding, and specifically relates to a method for obtaining high-yield soybeans with more flowers and pods by generating loss-of-function mutations using the Glyma.11G168800 gene and the Glyma.18G060200 gene, and its application. Background Art

[0002] Soybean (Glycine max) is an important crop for both oil and grain, which is crucial for ensuring global food security and sustainable development. Data from the National Bureau of Statistics shows that in 2022, the soybean output in China was 20.28 million tons, while the total imported soybean volume reached as high as 91.08 million tons, that is, more than 80% of China's soybean demand relies on imports. Therefore, continuously increasing the domestic soybean output is an important measure to solve the dilemma of soybean supply and demand in China and ensure national food security. At the same time, the average yield per mu of soybeans in the United States in 2022 was as high as 500 jin, while the average yield per mu of soybeans in China was only 264 jin; therefore, there is a huge room for growth and potential in the average yield per unit of soybeans in China. The yield per plant of crops is closely related to the branching and inflorescence structure of the plant. Soybean flowers are clustered in the leaf axils or at the top, so the number of lateral branches and nodes directly determines the number of flowers that the plant can produce, further affecting the yield per plant. Exploring new gene resources that control traits such as the number of lateral branches or nodes is crucial for soybean improvement breeding. Summary of the Invention

[0003] All references mentioned herein are incorporated herein by reference. Unless otherwise specified, 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 invention belongs. Unless otherwise specified, the techniques used or mentioned herein are standard techniques well known to one of ordinary skill in the art. The materials, methods, and examples are for illustrative purposes only and not for limitation.

[0004] In the embodiments of the present application, through phylogenetic analysis, it is found that there are 12 MND1 homologous genes in soybeans, among which 7 genes are highly expressed in flowers or shoot apical meristems, including the orthologous gene of MND1 and the genes on two sister branches of HLS1 (HOOKLESS1) / MND1 (MND1S1 and MND1S2). The aforementioned 7 genes are determined as the target genes of this study, and their functions are studied by constructing CRISPR / Cas9 knockout mutants. Through a series of experiments, it is found that when the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene in soybean plants produce loss-of-function double mutations, unexpected technical effects are achieved. The loss-of-function double mutants have phenotypes such as increased plant height, increased number of pods per plant, increased number of seeds per plant, increased yield per plant, increased number of branches, increased number of nodes on lateral branches, and / or increased number of pods per node. The aforementioned phenotypes are of great significance for high-yield soybean breeding, etc. When the endogenous Glyma.07G240100 gene and Glyma.17G033300 gene in soybean plants produce loss-of-function double mutations, unexpected technical effects are also achieved. The double mutants of the Glyma.07G240100 gene and Glyma.17G033300 gene have phenotypes such as increased plant height, increased pod-setting height, increased number of effective branches, increased number of pods per plant, increased number of seeds per plant, increased number of pods per node, and / or increased total yield per plant, which is of great significance for soybean breeding.

[0005] The embodiments of the present application provide a method for producing a soybean plant, the soybean plant having phenotypes such as increased plant height, increased number of pods per plant, increased number of seeds per plant, increased yield per plant, increased number of branches, increased number of nodes on lateral branches, and / or increased number of pods per node, the method comprising the steps of:

[0006] (a) generating one or more soybean plants, each of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene in the soybean plant containing at least one loss-of-function mutation, or the expression of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene being inhibited;

[0007] (b) obtaining at least one seed of the soybean plant produced in step (a).

[0008] Optionally, the polynucleotide sequence of the Glyma.11G168800 gene is selected from one of the sequences of the following groups:

[0009] (a) the polynucleotide sequence shown in SEQ ID No: 1 or 2;

[0010] (b) the polynucleotide sequence whose encoded amino acid sequence is shown in SEQ ID No: 3;

[0011] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and a loss-of-function mutation of the endogenous polynucleotide sequence in a soybean plant has the function of increasing plant height, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the yield per plant, increasing the number of branches, increasing the number of nodes on lateral branches, and / or increasing the number of pods per node;

[0012] (d) A polynucleotide sequence having at least 90%, 95%, 98% or more similarity to the polynucleotide sequence shown in any one of (a)-(c), and a loss-of-function mutation of the endogenous polynucleotide sequence in a soybean plant has the function of increasing plant height, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the yield per plant, increasing the number of branches, increasing the number of nodes on lateral branches, and / or increasing the number of pods per node; or

[0013] (e) A polynucleotide sequence complementary to the sequence described in any one of (a)-(d).

[0014] Optionally, the polynucleotide sequence of the Glyma.18G060200 gene is selected from one of the sequences in the following group:

[0015] (a) The polynucleotide sequence shown in SEQ ID No: 5 or 6;

[0016] (b) The polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No: 7;

[0017] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and a loss-of-function mutation of the endogenous polynucleotide sequence in a soybean plant has the function of increasing plant height, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the yield per plant, increasing the number of branches, increasing the number of nodes on lateral branches, and / or increasing the number of pods per node;

[0018] (d) A polynucleotide sequence having at least 90%, 95%, 98% or more similarity to the polynucleotide sequence shown in any one of (a)-(c), and a loss-of-function mutation of the endogenous polynucleotide sequence in a soybean plant has the function of increasing plant height, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the yield per plant, increasing the number of branches, increasing the number of nodes on lateral branches, and / or increasing the number of pods per node; or

[0019] (e) A polynucleotide sequence complementary to the sequence described in any one of (a)-(d).

[0020] The embodiments of the present application also provide a method for producing a soybean plant, the soybean plant having a phenotype of increased plant height, increased pod-setting height, increased number of effective branches, increased number of pods per plant, increased number of seeds per plant, increased number of pods per node, and / or increased total yield per plant. The method includes the steps:

[0021] (a) Generating one or more soybean plants, wherein each of the endogenous Glyma.07G240100 gene and Glyma.17G033300 gene in the soybean plant contains at least one loss-of-function mutation, or the expression of the endogenous Glyma.07G240100 gene and Glyma.17G033300 gene is inhibited;

[0022] (b) Obtaining at least one seed of the soybean plant produced in step (a).

[0023] Optionally, the polynucleotide sequence of the Glyma.07G240100 gene is selected from one of the sequences in the following groups:

[0024] (a) The polynucleotide sequence shown in SEQ ID No: 9 or 10;

[0025] (b) The polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No: 11;

[0026] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and the loss-of-function mutation of the endogenous polynucleotide sequence in the soybean plant has the function of increasing plant height, increasing pod-setting height, increasing the number of effective branches, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the number of pods per node, and / or increasing the total yield per plant;

[0027] (d) A polynucleotide sequence having at least 90%, 95%, 98% or more similarity with the polynucleotide sequence shown in any of (a)-(c), and the loss-of-function mutation of the endogenous polynucleotide sequence in the soybean plant has the function of increasing plant height, increasing pod-setting height, increasing the number of effective branches, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the number of pods per node, and / or increasing the total yield per plant; or

[0028] (e) A polynucleotide sequence complementary to any of the sequences described in (a)-(d).

[0029] Optionally, the polynucleotide sequence of the Glyma.17G033300 gene is selected from one of the sequences in the following groups:

[0030] (a) The polynucleotide sequence shown in SEQ ID No: 13 or 14;

[0031] (b) A polynucleotide sequence whose encoded amino acid sequence is as shown in SEQ ID No: 15;

[0032] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and a loss-of-function mutation of the endogenous polynucleotide sequence in a soybean plant has the function of increasing plant height, pod-setting height, number of effective branches, number of pods per plant, number of seeds per plant, number of pods per node, and / or total yield per plant;

[0033] (d) A polynucleotide sequence having at least 90%, 95%, 98% or more similarity to the polynucleotide sequence shown in any one of (a)-(c), and a loss-of-function mutation of the endogenous polynucleotide sequence in a soybean plant has the function of increasing plant height, pod-setting height, number of effective branches, number of pods per plant, number of seeds per plant, number of pods per node, and / or total yield per plant; or

[0034] (e) A polynucleotide sequence complementary to any one of the sequences described in (a)-(d).

[0035] Optionally, the Glyma.11G168800, Glyma.18G060200, Glyma.07G240100 or Glyma.17G033300 genes provided in the embodiments of the present application further include homologous genes or the same genes of different varieties having at least 80%, 85%, 90%, 95%, 98% or 99% sequence similarity to their polynucleotide sequences, or homologous genes or the same genes of different varieties having at least 90%, 95% or 98% sequence similarity to the amino acid sequences of the Glyma.11G168800 or Glyma.18G060200 or Glyma.07G240100 or Glyma.17G033300 genes disclosed in the embodiments of the present invention. After a homozygous loss-of-function mutation of the endogenous homologous gene, it has the functions of increasing plant height, number of pods per plant, number of seeds per plant, yield per plant, number of branches, number of nodes on lateral branches, and / or number of pods per node. The homologous gene can be isolated from any plant.

[0036] Optionally, the method provided in the present application can be applied to any plant containing a homologous gene of Glyma.11G168800 or Glyma.18G060200 or Glyma.07G240100 or Glyma.17G033300. Preferably, the plants include monocotyledonous plants such as corn, millet, wheat, barley, rye, rice and sorghum, and dicotyledonous plants such as cotton, soybean, peanut, sunflower, sweet potato, potato, apple and tobacco.

[0037] The percentage of sequence similarity described in this application can be obtained through well-known bioinformatics algorithms, including the Myers and Miller algorithms, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the algorithms of Karlin and Altschul, which are well-known to those skilled in the art.

[0038] Those skilled in the art should be aware that there are single nucleotide polymorphisms (SNPs) in the same gene among different varieties of the same plant, that is, there are often differences in individual bases in the nucleotide sequence of the same gene. However, there are a large number of varieties of the same crop, and it is impossible for the inventor to list them one by one. The embodiments of this application only provide the sequences of representative varieties in soybean crops. Therefore, those skilled in the art should know that nucleotide sequences with SNPs in genes and their nucleotide sequences of Glyma.11G168800 or Glyma.18G060200 or Glyma.07G240100 or Glyma.17G033300 derived from different varieties, and methods and applications for obtaining traits such as increased plant height, increased number of pods per plant, increased number of seeds per plant, increased yield per plant, increased number of branches, increased number of nodes on lateral branches, and / or increased number of pods per node by using their endogenous loss-of-function mutations are also within the protection scope of the present invention.

[0039] Optionally, the loss-of-function mutation described in this application 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.

[0040] Optionally, the loss-of-function mutation includes, but is not limited to, being obtained by methods such as physical mutagenesis, chemical mutagenesis, and gene editing. Physical mutagenesis includes, but is not limited to, radiation mutagenesis, space breeding, etc.; chemical mutagenesis methods include mutagenesis caused by treating with mutagens such as EMS; gene editing methods include, but are not limited to, methods such as ZFN, TALEN, and / or CRISPR / Cas.

[0041] Those skilled in the art know that the main principle of the CRISPR / Cas gene editing system or gene editing method is to find the position to be gene-edited in the host genome through a nucleic acid fragment called guide RNA (gRNA), that is, the target DNA sequence, and then cut the DNA by the Cas protein. In this application, the Cas protein includes, but is not limited to, proteins such as Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13, and / or Cas14.

[0042] Optionally, when the gene editing system used is CRISPR / Cas9, the gene mutant sequence obtained by the CRISPR / Cas9 method, and the target sequence used in the CRISPR / Cas9 technology is selected from one of the sequences in the following group:

[0043] (a) A fragment conforming to the sequence arrangement rule of 5'-Nx-NGG-3' in the nucleotide sequences shown in SEQ ID No: 1, 3, 5, 9 or 10, where N represents any one of A, G, C, and T, 14 < X < 30, and X is an integer, and Nx represents X consecutive nucleotides; or

[0044] (b) A nucleotide sequence complementary to the polynucleotide sequence described in (a).

[0045] Optionally, in the CRISPR / Cas9 gene editing system disclosed in the embodiments of the present application, the target sequences of the Glyma.11G168800 or Glyma.18G060200 gene are shown in SEQ ID NO: 24 and SEQ ID NO: 23 respectively; the target sequences of the Glyma.07G240100 or Glyma.17G033300 gene are shown in SEQ ID NO: 22 and SEQ ID NO: 21 respectively.

[0046] Optionally, the soybean plant has double mutants of the Glyma.11G168800 and Glyma.18G060200 genes, and the nucleotide sequences of the double mutants are shown in SEQ ID NO: 4 or SEQ ID NO: 8.

[0047] Optionally, the soybean plant has double mutants of the Glyma.07G240100 and Glyma.17G033300 genes, and the nucleotide sequences of the double mutants are shown in SEQ ID NO: 12 or SEQ ID NO: 16.

[0048] Optionally, the loss-of-function mutation described in the embodiments of the present application can also be obtained by crossing with a soybean plant having this loss-of-function mutation.

[0049] Optionally, in the method described in the embodiments of the present application, the reduction or inhibition of the normal expression or protein function of the lodging-related gene is obtained by RNA interference (i.e., RNAi) and / or mutation. Those skilled in the art know that the RNAi technology is a conventional technology in the art. It specifically binds to the homologous region of the mRNA expressed by the target gene through short double-stranded RNA (siRNA: small interfering RNA) of 21-23 bp or long double-stranded RNA (dsRNA: double-strand RNA), degrades the mRNA, and achieves the effect of inhibiting gene expression.

[0050] Optionally, in the present application, the expression of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene in soybeans can be inhibited by the RNAi method, thereby affecting the activity of the aforementioned genes, inhibiting gene expression, and making the soybean plants have phenotypes such as increased plant height, increased number of pods per plant, increased number of seeds per plant, increased yield per plant, increased number of branches, increased number of nodes on lateral branches, and / or increased number of pods per node.

[0051] Optionally, the present application also provides an application of any of the aforementioned methods in soybean breeding. Preferably, the application includes, but is not limited to, the application in trait regulation such as increasing the plant height of soybean plants, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the yield per plant, increasing the number of branches, increasing the number of nodes on lateral branches, and / or increasing the number of pods per node.

[0052] Optionally, the embodiments of the present application also provide a feed, meal, protein, or oil product made from soybeans. The feed, meal, protein, or oil product contains loss-of-function mutants of the Glyma.11G168800 gene and Glyma.18G060200 gene. Each of the Glyma.11G168800 gene and Glyma.18G060200 gene contains at least one loss-of-function mutation, or the expression of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene is inhibited. The loss-of-function mutation includes substitution, deletion, and / or addition of one or more nucleotides in the nucleotide sequence of the aforementioned gene. Optionally, the polynucleotide sequence of the loss-of-function mutant is as shown in SEQ ID NO:4 or SEQ ID NO:8.

[0053] Optionally, the embodiments of the present application further provide a feed, meal, protein or oil product made from soybeans, wherein the feed, meal, protein or oil product contains a loss-of-function mutant of the Glyma.07G240100 or Glyma.17G033300 gene, each of the Glyma.07G240100 or Glyma.17G033300 contains at least one loss-of-function mutation, or the expression of the endogenous Glyma.07G240100 or Glyma.17G033300 gene is inhibited. The loss-of-function mutation includes substitution, deletion and / or addition of one or more nucleotides in the nucleotide sequence of the aforementioned gene. Optionally, the polynucleotide sequence of the loss-of-function mutant is as shown in SEQ ID NO:12 or SEQ ID NO:16.

[0054] The embodiments of the present application further provide a method for identifying mutants of the Glyma.11G168800 gene and the Glyma.18G060200 gene. After PCR amplification, the PCR product is sequenced to confirm the specific mutation site. The method is characterized in that the PCR amplification primers for the Glyma.11G168800 gene are as shown in SEQ ID NO:37 and SEQ ID NO:38; the PCR amplification primers for the Glyma.18G060200 gene are as shown in SEQ ID NO:35 and SEQ ID NO:36.

[0055] The embodiments of the present application further provide a method for identifying mutants of the Glyma.07G240100 or Glyma.17G033300 gene. After PCR amplification, the PCR product is sequenced to confirm the specific mutation site. The method is characterized in that the PCR amplification primers for the Glyma.07G240100 gene are as shown in SEQ ID NO:33 and SEQ ID NO:34; the PCR amplification primers for the Glyma.17G033300 gene are as shown in SEQ ID NO:31 and SEQ ID NO:32.

[0056] Optionally, in the embodiments of the present application, transferring a nucleotide sequence, a vector, a construct or an expression cassette into a plant or introducing it into a plant or transforming a plant all refer to transferring a target nucleotide sequence, a construct, a vector or an expression cassette into a recipient cell or a recipient plant by a conventional transgenic method or a method of hybridizing with a target transgenic plant. Any transgenic method known to those skilled in the art can be used to transform a recombinant expression vector into a plant cell to produce the transgenic plants or mutants of the embodiments of the present application. The transformation methods may include direct or indirect transformation methods. Specifically, the transformation methods include, but are not limited to, polyethylene glycol-induced DNA uptake, liposome-mediated transformation, gene gun introduction, electroporation, microinjection, and Agrobacterium-mediated plant transformation methods, etc.

[0057] Compared with the prior art, the present application has the following beneficial effects:

[0058] (1) The present application provides a method for producing soybean plants and its applications. By causing each of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene in soybeans to contain at least one loss-of-function mutation, or the expression of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene to be inhibited, plants with excellent agronomic traits such as increased plant height, increased number of pods per plant, increased number of seeds per plant, increased yield per plant, increased number of branches, increased number of nodes on lateral branches, and / or increased number of pods per node are obtained; or, by causing each of the endogenous Glyma.07G240100 or Glyma.17G033300 gene in soybeans to contain at least one loss-of-function mutation, or the expression of the endogenous Glyma.07G240100 or Glyma.17G033300 gene to be inhibited, plants with excellent agronomic traits such as increased plant height, increased pod-setting height, increased number of effective branches, increased number of pods per plant, increased number of seeds per plant, increased number of pods per node, and / or increased total yield per plant are obtained; the aforementioned genes, methods and their applications provide new germplasm resources and breeding ideas for soybean breeding, and are of great significance to global food security and agricultural sustainable development;

[0059] (2) The functions of double mutations of the Glyma.11G168800 gene and Glyma.18G060200 gene in soybean acyltransferase genes, and the functions of double mutations of the Glyma.07G240100 and Glyma.17G033300 genes are clarified, providing new gene resources for crop species;

[0060] (3) In the case where a single-gene mutation does not exhibit an obvious corresponding phenotype, by means of double mutations of two genes, the normal expression or protein function of the aforementioned genes is reduced or inhibited, thereby obtaining breeding materials with application value, which provides new ideas for crop breeding and the study of gene action mechanism networks.

[0061] Term Definitions Related to the Present Invention

[0062] 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 invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

[0063] In the context of the present application, the term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides in single-stranded or double-stranded form and their polymers. Unless specifically restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to those of the reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides.

[0064] The term "homologous gene" in the present application refers to two or more gene sequences with a sequence similarity of 80%, which includes orthologous genes (also known as vertical homologous genes, direct homologous genes, or orthologous genes by directed evolution), paralogous genes (also known as collateral homologous genes, co-paralogous genes, or paralogous genes by parallel evolution), and / or xenologous genes.

[0065] The term "sequence similarity" refers to the degree of similarity between two sequences, which is a quantitative concept used to compare the similarity between different sequences, thereby discovering and analyzing the association between two sequences. Sequence similarity can be used to compare gene sequences, protein sequences, DNA sequences, etc.

[0066] As used herein, "stringent hybridization conditions" refers to conditions of low ionic strength and high temperature known in the art. Typically, under stringent conditions, a probe hybridizes to its target sequence with a detectable level that is higher than that for hybridization to other sequences (e.g., at least 2-fold above background). Stringent hybridization conditions are sequence-dependent and will vary in different environmental conditions. Longer sequences hybridize specifically at higher temperatures. By controlling the stringency of hybridization or wash conditions, target sequences that are 100% complementary to the probe can be identified. Exhaustive guidance for nucleic acid hybridization can be found in 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 usually selected to be about 5 - 10 °C below the thermal melting point (T m ) of the specific sequence at a specified ionic strength and pH. T m is the temperature at which 50% of the probe that is complementary to the target hybridizes to the target sequence at equilibrium (at a specified ionic strength, pH, and nucleic acid concentration) (since the target sequence is present in excess, 50% of the probe is occupied at equilibrium at T m ). Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion concentration at pH 7.0 to 8.3, usually about 0.01 to 1.0 M sodium ion concentration (or other salts), 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 can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least twice the background hybridization, optionally 10-fold background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC, and 1% SDS, incubated at 42 °C; or 5×SSC, 1% SDS, incubated at 65 °C, washed in 0.2×SSC and washed in 0.1% SDS at 65 °C. The wash can be carried out for 5, 15, 30, 60, 120 minutes or longer.

[0067] The term "recombinant expression vector": one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, together with vectors having helper plasmids, are most commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be able to express in plant cells, heterologous DNA sequences to be transcribed, etc.

[0068] The term "number of pods per plant" refers to the total number of effective and ineffective pods on one soybean plant.

[0069] The term "number of pods per node" refers to the total number of pods borne at the axils of each alternate trifoliolate leaf of soybean (referred to as a node), which is the number of pods per node.

[0070] The term "hybridization", in a broad sense, refers to the process of gamete combination between individuals of different populations or genotypes to produce hybrids. According to the different genetic relationships of the parents, it includes related hybridization and distant hybridization.

[0071] The "mutation" described in this application refers to a "loss-of-function mutation" or "null mutation", which is a mutation in the coding sequence of a gene that causes a decrease or complete loss of the function of the gene product (usually a protein). A loss-of-function mutation can be caused, for example, by truncation of the gene product (resulting from frameshift or nonsense mutations), and the phenotype associated with an allele having a loss-of-function mutation can be recessive or dominant.

[0072] The term "RNA interference" (RNAi) is a gene-blocking technology, which is a process in which double-stranded RNA (dsRNA) molecules block the expression of specific genes or silence them at the mRNA level, that is, sequence-specific post-transcriptional gene silencing (PTGS). Brief Description of the Drawings

[0073] Figure 1 It is a maximum likelihood tree analysis of MND1 homologous sequences of different crops.

[0074] Figure 2 It is a gene sequencing gel diagram of mnd1s1 and mnd1s2 mutants.

[0075] Figure 3Comparative analysis of the expression levels of the Glyma.11G168800 and Glyma.18G060200 genes in Huachun 6 and the T2 generation mutants, where A and B are the comparative analysis of the expression of Glyma.11G168800 in the leaves and axillary buds of Huachun 6 and the mnd1s1 mutant; C and D are the comparative analysis of the expression of the Glyma.18G060200 gene in the leaves and axillary buds of Huachun 6 and the mnd1s1 mutant.

[0076] Figure 4 It is the comparison of the whole plant phenotypes of Huachun 6 and the T1 generation mutants.

[0077] Figure 5 It is the comparison of the yield per plant of Huachun 6 and the T1 generation mutants.

[0078] Figure 6 It is the comparison of the pod numbers at the corresponding nodes of Huachun 6 and the T1 generation mutants.

[0079] Figure 7 It is the analysis of the data from the variety inspection of Huachun 6 and the T1 generation mutants.

[0080] Explanation of the reference numerals: 11G refers to the Glyma.11G168800 gene; 18G refers to the Glyma.18G060200 gene; HC6 refers to Huachun 6. Detailed implementation manners

[0081] The following further describes the present invention in combination with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the present invention without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0082] The inbred lines used in the following embodiments can obtain relevant information from the "China Crop Germplasm Information Network" and apply for obtaining the corresponding seeds.

[0083] Example 1. Creation of soybean MND1 / HLS1 homologous gene mutants using gene editing technology

[0084] The inventors of the present application first carried out an evolutionary analysis of the MND1 gene family in land plants using the maximum likelihood method RAxML, and identified all 12 homologous genes annotated in the soybean genome. The results are as Figure 1 shown, from Figure 1It can be seen that the MND1 gene of barley is homologous to the HLS1 gene of Arabidopsis thaliana, and the gene family can be divided into four evolutionary branches, including the HLS1 branch, the MND1 branch, and the MND1s1 and MND1s2 branches that are sister branches to the HLS1+MND1 branch ( Figure 1 At the same time, the inventors used the public transcriptome database (database website link: Phytozome(doe.gov) , the soybean genome was selected "Glycine max Wm82.a4.v1") to determine the tissue and developmental stage specific expression patterns of 12 MND1 / HLS1 genes, and 7 MND1 / HLS1 genes specifically expressed in inflorescence and shoot apical meristem were identified. The 7 MND1 / HLS1 genes are Glyma.07G022800, Glyma.08G219000, Glyma.09G035900, Glyma.17G033300, Glyma.07G240100, Glyma.18G060200 and Glyma.11G168800. Then, the inventors used Huachun No. 6, a major national soybean variety in southern China, as the receptor and used CRISPR / Cas9 gene editing technology to target and edit these 7 MND1 / HLS1 homologous genes specifically expressed in inflorescence and shoot apical meristem.

[0085] The target design adopts the high-throughput CRISPR-Cas9 target design program developed by Weimi Biotechnology (Jiangsu) Technology Co., Ltd. The target design principles of this program are as follows: 1) The knockout site is in the coding (CDS) region and is as close to the front end of the protein or the important functional region as possible; 2) Try to cover a higher proportion of transcripts; 3) There is no off-target or the off-target is located in the intergenic region; 4) Targets with higher editing efficiency are preferred; 5) The sequence has a relatively balanced GC content and is not easy to form a secondary structure. The successful application of this program in the design of whole-genome targets in rice has proved its feasibility. The inventors have shown in experiments that the phenotypes of single-gene mutants of Glyma.18G060200 and Glyma.11G168800 are not obvious. Therefore, the embodiment of this application designs a gene editing method with two genes and two targets for the aforementioned evolutionary branch genes, in order to obtain double-gene mutants and analyze their phenotypes. The target information for the seven genes is shown in Table 1 below:

[0086] Table 1. Gene knockout target sequences

[0087]

[0088] Example 2. Detection of T0 generation gene-edited seedlings and other related molecular experiments

[0089] To identify the editing status of mutants, leaf DNA of mutants was extracted, and then the target gene was amplified by PCR (polymerase chain reaction) using gene-specific primers. Gene-specific amplification primers (Table 3) were designed on both sides of the double target, and the amplification products were sent to the company for first-generation Sanger sequencing to determine the editing status of the target gene near the double target. The specific operations are as follows:

[0090] (1) DNA extraction

[0091] We used SDS (Sodium dodecyl sulfate) to roughly extract the leaf DNA of Huachun 6 and mutants. The preparation of the extraction solution and the operation steps are as follows:

[0092] Table 2. Preparation of working solution

[0093]

[0094] Operation steps:

[0095] ① When two trifoliate leaves had grown on the plant, take about 2 mg of young leaves and put them into a grinding tube, add 2 grinding beads, and store them in liquid nitrogen.

[0096] ② Adjust the grinder to 60 HZ and 60 S to thoroughly grind the leaves into powder.

[0097] ③ Add 600 μL of working solution, invert it up and down to mix evenly, and then centrifuge at 12000 rpm for 10 min.

[0098] ④ Pipette 400 μL of the supernatant into a new 1.5 mL EP tube, and add 800 μL of 95% ethanol to wash away impurities.

[0099] ⑤ Place it in a -20 °C refrigerator and let it stand for 5 min, then centrifuge at 12000 rpm for 15 min.

[0100] ⑥ Pour out the supernatant, invert the EP tube on the paper to dry the water, and finally add 100 μL of double-distilled water to dissolve the DNA precipitate.

[0101] (2) Identification of gene editing status by PCR and first-generation sequencing (Sanger sequencing method)

[0102] The gene sequence at the target site was amplified using specific primers (Table 3), and then the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for first-generation sequencing to identify the editing status of the obtained T0-generation edited seedlings. A total of 109 T0-generation edited seedlings were obtained from 4 transformation events (Table 1), and no obvious phenotypes were found in the T0-generation seedlings.

[0103] The specific information and experimental operations are as follows:

[0104] Table 3. Gene detection primer information

[0105]

[0106]

[0107] Table 4. PCR reaction system

[0108]

[0109] The reaction procedure is as follows:

[0110]

[0111] Example 3. Genotype identification of T1 generation homozygous mutants

[0112] To further identify the genotypes and phenotypes of the mutants and ensure that at least 2 different independent mutant editing events can be screened for each mutant event and target gene, we randomly selected 4 transformation events, a total of 47 different T0 generation edited plant lines (ensuring at least 6 lines for each transformation event), and planted them in the school's scientific research and teaching base. Among the T1 generation mutants planted in the field on July 14, 2023, unexpectedly, it was found that one plant line had a phenotype of multiple flowers and pods ( Figure 4 D, E, F). The gene editing situation of this plant line was as follows: the Glyma.11G168800 gene had a homozygous deletion of 317 bp ( Figure 2 A), and the Glyma.18G060200 gene had a homozygous insertion of 214 bp ( Figure 2 B). Evolutionary analysis showed that the proteins encoded by the two genes, Glyma.11G168800 and Glyma.18G060200, were one of the two sister branches of MND1. Therefore, its double mutant was named mnd1s1; at the same time and location, another homozygous double knockout mutant (the Glyma.07G240100 gene had a homozygous deletion of 325 bp, and the Glyma.17G033300 gene had a homozygous deletion of 361 bp, Figure 2 C and Figure 2 D) did not have this phenotype of multiple flowers and pods ( Figure 4 G, H, I). Evolutionary analysis showed that the proteins encoded by the two genes, Glyma.07G240100 and Glyma.17G033300, were the other sister branch of MND1. Therefore, its mutant was named mnd1s2.

[0113] Table 5. The number of generations added for each transformation event in the T0 generation

[0114]

[0115] In the embodiments of the present application, the gDNA sequence of Glyma.11G168800 is as shown in SEQ ID NO:1, its CDS is as shown in SEQ ID NO:2, and the amino acid sequence encoded by it is as shown in SEQ ID NO:3. The nucleotide sequence of the loss-of-function mutant after the gene mutation of Glyma.11G168800 disclosed in the embodiments of the present application is as shown in SEQ ID NO:4. The gDNA sequence of Glyma.18G060200 is as shown in SEQ ID NO:5, its CDS is as shown in SEQ ID NO:6, and the amino acid sequence encoded by it is as shown in SEQ ID NO:7. The nucleotide sequence of the loss-of-function mutant after the gene mutation of Glyma.18G060200 discovered in the embodiments of the present application is as shown in SEQ ID NO:8. The gDNA sequence of Glyma.07G240100 is as shown in SEQ ID NO:9, its CDS is as shown in SEQ ID NO:10, and the amino acid sequence encoded by it is as shown in SEQ ID NO:11. The nucleotide sequence of the loss-of-function mutant after the gene mutation of Glyma.07G240100 disclosed in the embodiments of the present application is as shown in SEQ ID NO:12. The gDNA sequence of Glyma.17G033300 is as shown in SEQ ID NO:13, its CDS is as shown in SEQ ID NO:14, and the amino acid sequence encoded by it is as shown in SEQ ID NO:15. The nucleotide sequence of the loss-of-function mutant after the gene mutation of Glyma.17G033300 disclosed in the embodiments of the present application is as shown in SEQ ID NO:16.

[0116] To further confirm the specific biological functions of the homozygous deletion of Glyma.11G168800 and the inserted fragment in Glyma.18G060200 in the mnd1s1 mutant, we designed specific primers (Table 8) for the 5'-UTR regions of the aforementioned two genes and qPCR to detect whether the expression level of the gene increased or decreased, respectively indicating that the inserted fragment might result in a gain-of-function phenotype of the gene or a loss-of-function or loss-of-phenotype of the gene. The qPCR results showed that the expression levels of both Glyma.11G168800 and Glyma.18G060200 genes were significantly down-regulated, that is, the mnd1s1 homozygous double-knockout mutant was a loss-of-function mutant of the gene ( Figure 3 ).

[0117] In the embodiments of the present application, the actual expression levels of the mutant genes were detected by RNA extraction, reverse transcription and qPCR. The specific operations are as follows.

[0118] (1) RNA extraction:

[0119] ① Sampling: Place two grinding beads in a sterilized grinding tube, soak the grinding tube in liquid nitrogen, and use forceps to pick up about 2 mg of leaves or axillary buds and put them into the grinding tube.

[0120] ② Grinding: Adjust the grinder to 60 HZ for 30 s to thoroughly grind the material into powder. Immediately put it back into liquid nitrogen after grinding.

[0121] ③ Add 500 μL of Trizol extraction solution and place the tube on ice.

[0122] ④ Invert the tube up and down to mix and dissolve the powder, then let it stand horizontally for 10 min.

[0123] ⑤ Add 100 μL of chloroform, invert the tube up and down to mix, and let it stand on ice for 10 min.

[0124] ⑥ Centrifuge at 13000 rpm at 4 °C for 10 min.

[0125] ⑦ Pipette 250 μL of the supernatant into a new sterilized 1.5 mL centrifuge tube, add an equal volume of 250 μL of isopropanol, and let it stand on ice for 10 min.

[0126] ⑧ Centrifuge at 13000 rpm at 4 °C for 10 min.

[0127] ⑨ Discard the supernatant, add 500 μL of 75% ethanol to wash the precipitate.

[0128] ⑩ Centrifuge at 13000 rpm at 4 °C for 5 min. Discard the supernatant and wash the precipitate again.

[0129] Centrifuge at 13000 rpm at 4 °C for 2 min. Use a pipette tip to suck away the residual liquid and air-dry for 10 min.

[0130] Add 40 μL of sterilized water to dissolve the RNA. Then store it at -80 °C.

[0131] (2) Reverse transcription procedure (using Novoprotein kit):

[0132] ① Genomic DNA removal

[0133] After performing normalization according to the RNA concentration, remove genomic DNA according to the following table

[0134] Table 6. Preparation of genomic DNA removal system

[0135]

[0136]

[0137] Pipette to mix well and incubate at 42 °C for 2 min.

[0138] ② Prepare the first-strand cDNA synthesis reaction mixture

[0139] Table 7. Preparation of the first-strand cDNA synthesis reaction mixture

[0140]

[0141] Pipette up and down to mix

[0142] ③ First-strand cDNA synthesis

[0143] Table 8. First-strand cDNA synthesis program

[0144]

[0145] After the reaction, store the cDNA at -20 °C.

[0146] (3) qPCR operation steps

[0147] ① Primer design

[0148] Table 9. qPCR primer sequences

[0149]

[0150]

[0151] ② Reaction mixture preparation

[0152] Table 10. qPCR reaction mixture

[0153]

[0154] ③ The qPCR reaction program is as follows:

[0155]

[0156] After the reaction, calculate the expression level of the target gene by the fluorescence change value compared with the internal reference gene (2 -Δct )

[0157] (4) Result analysis

[0158] In the qPCR experiment, we took the apical young leaves and axillary buds of all nodes of Huachun 6 and T2 generation mutants at 24 days old under the same conditions on the same shelf in the same greenhouse. For result analysis, we used the 2^ -Δct value to calculate the relative expression level of the target gene to actin in Huachun 6 and T2 generation mutants, and then used Graphpad software to plot the graph. The results are as Figure 3As shown, the expression levels of Glyma.11G168800 and Glyma.18G060200 both decreased in the leaves and axillary buds of the mutants, indicating that the expression levels of both genes were significantly downregulated in the mutants, that is, the homozygous double-knockout mutant mnd1s1 was a mutant with loss of gene function( Figure 3 ).

[0159] In the qPCT experiment, 12 seeds of each of the T1 generation mutants mnd1s1-1, mnd1s1-2, and mnd1s1-3 were selected for planting. After detection, most of the editing types of the T2 generation were the same as those of the T1 generation, and the editing types of the 3 mutants sampled in the qPCR experiment were the same as those of the T1 generation.

[0160] Example 4. Specific field phenotype analysis of T1 generation mutants

[0161] Through field phenotype analysis, compared with Huachun 6 and the mnd1s2 mutant lines, the plant heights, the number of pods per plant, the number of grains per plant, and the grain weight per plant of the three double-knockout mutants mnd1s1-1, mnd1s1-2, and mnd1s1-3 with the same editing type were significantly increased, and the number of branches also increased slightly( Figure 7 ). However, the 100-seed weight of the double-knockout mutants decreased( Figure 7 H), but the total yield of the whole plant showed a significant increase( Figure 5 , Figure 7 G). At the same time, the phenotype analysis results also showed that the number of nodes on the lateral branches of the mnd1s1 mutant was more than that of Huachun 6 and the mnd1s2 mutant( Figure 4 D, E, F), and more pods were produced per node on the main stem( Figure 6 ), that is, the increase in the number of pods per node was the main reason for the significant increase in the yield of the mnd1s1 mutant plants. To sum up, the analysis of the yield traits of the mnd1s1 mutant provided in the embodiments of the present application shows that the growth period of this mutant is similar to that of the wild type (both flowering on August 14th), but the yield is significantly increased. Accordingly, it is shown that the homozygous double mutant obtained by simultaneously mutating the Glyma.11G168800 and Glyma.18G060200 genes has great practical production application value. The double gene mutation has the functions of increasing the plant height of soybeans, increasing the number of pods per plant, increasing the number of grains per plant, increasing the yield per plant, increasing the number of branches, increasing the number of nodes on the lateral branches, and / or increasing the number of pods per node, and can significantly improve the yield per plant and the yield per unit area of soybeans, and has great application potential in soybean breeding.

[0162] Compared with the wild type Huachun 6, the plant heights, pod-setting heights, numbers of effective branches, numbers of pods per plant, and numbers of seeds per plant of the single plants mnd1s2-1, mnd1s2-2, and mnd1s2-3 of the double-knockout mutants with the three same editing types of mnd1s2 increased ( Figure 7 ), and more pods were produced per node in the main stem nodes ( Figure 6 ), and the total yields of the entire single plants all showed significant increases ( Figure 5 ). This indicates that the homozygous double mutants obtained by simultaneously mutating the Glyma.07G240100 and Glyma.17G033300 genes have great production application value and great application potential in soybean breeding.

Claims

1. A method for producing a soybean plant having a phenotype of increased plant height, increased number of pods per plant, increased number of seeds per plant, increased yield per plant, increased number of branches, increased number of nodes on lateral branches, and / or increased number of pods per node, the method comprising the steps of: (a) generating one or more soybean plants in which each of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene contains at least one loss-of-function mutation, or the expression of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene is inhibited; (b) obtaining at least one seed of the soybean plant produced in step (a).

2. The method according to claim 1, wherein the polynucleotide sequence of the Glyma.11G168800 gene is selected from one of the sequences of the following groups: (a) the polynucleotide sequence shown in SEQ ID No: 1 or 2; (b) the polynucleotide sequence encoding an amino acid sequence shown in SEQ ID No: 3; (c) a polynucleotide sequence capable of hybridizing with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and the loss-of-function mutation of the endogenous polynucleotide sequence in the soybean plant has the function of increasing plant height, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the yield per plant, increasing the number of branches, increasing the number of nodes on lateral branches, and / or increasing the number of pods per node; (d) a polynucleotide sequence having at least 90%, 95%, 98% or more similarity to the polynucleotide sequence shown in any one of (a)-(c), and the loss-of-function mutation of the endogenous polynucleotide sequence in the soybean plant has the function of increasing plant height, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the yield per plant, increasing the number of branches, increasing the number of nodes on lateral branches, and / or increasing the number of pods per node; or (e) a polynucleotide sequence complementary to the sequence described in any one of (a)-(d); wherein the polynucleotide sequence of the Glyma.18G060200 gene is selected from one of the sequences of the following groups: (a) the polynucleotide sequence shown in SEQ ID No: 5 or 6; (b) the polynucleotide sequence encoding an amino acid sequence shown in SEQ ID No: 7; (c) a polynucleotide sequence capable of hybridizing with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, and the loss-of-function mutation of the endogenous polynucleotide sequence in the soybean plant has the function of increasing plant height, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the yield per plant, increasing the number of branches, increasing the number of nodes on lateral branches, and / or increasing the number of pods per node; (d) a polynucleotide sequence having at least 90%, 95%, 98% or more similarity to the polynucleotide sequence shown in any one of (a)-(c), and the loss-of-function mutation of the endogenous polynucleotide sequence in the soybean plant has the function of increasing plant height, increasing the number of pods per plant, increasing the number of seeds per plant, increasing the yield per plant, increasing the number of branches, increasing the number of nodes on lateral branches, and / or increasing the number of pods per node; or (e) A polynucleotide sequence complementary to any one of the sequences described in (a)-(d).

3. The method according to any one of claims 1-2, wherein the loss-of-function mutation includes substitution, deletion, and / or addition of one or more nucleotides in the polynucleotide sequence of the gene; wherein the gene expression is inhibited in a manner of RNA interference.

4. The method according to claim 3, wherein the loss-of-function mutation is obtained by techniques such as physical mutagenesis, chemical mutagenesis, ZFN, TALEN, and / or CRISPR / Cas gene editing, or obtained by hybridization with a soybean plant having the loss-of-function mutation.

5. The method according to claim 4, wherein the CRISPR / Cas is the CRISPR / Cas9 gene editing method, and the target sequence used is selected from one of the sequences in the following group: (a) Fragments conforming to the sequence arrangement rule of 5’-Nx-NGG-3’ in the nucleotide sequences shown in SEQ ID No: 1 or 2, and SEQ ID No: 5 or 6, wherein N represents any one of A, G, C, and T, 14 < X < 30, and X is an integer, and Nx represents X consecutive nucleotides; or (b) A nucleotide sequence complementary to the polynucleotide sequence described in (a).

6. The method according to claim 5, wherein the target sequence is as shown in SEQ ID NO: 24 or 23.

7. The method according to any one of claims 1-6, wherein the polynucleotide sequence containing at least one loss-of-function mutation is as shown in SEQ ID NO: 4 or SEQ ID NO:

8.

8. Use of the method according to any one of claims 1-7 for increasing the plant height, the number of pods per plant, the number of seeds per plant, the yield per plant, the number of branches, the number of nodes on lateral branches, and / or the number of pods per node of a soybean plant.

9. A feed, meal, protein or oil product made from soybeans, characterized in that, The feed, meal, protein, or oil product contains loss-of-function mutants of the Glyma.11G168800 gene and the Glyma.18G060200 gene, and each of the Glyma.11G168800 gene and the Glyma.18G060200 gene contains at least one loss-of-function mutation.

10. The feed, meal, protein, or oil product according to claim 8, wherein the polynucleotide sequence containing at least one loss-of-function mutation is as shown in SEQ ID NO: 4 or SEQ ID NO: 8.

Images