Genes, methods and applications for producing high-yield soybean with more flowers and more pods

Abstract

The application discloses a kind of production of multi-flower multi-lodging high-yield soybean gene, method and application thereof, belong to the field of plant biotechnology breeding.The soybean Glyma.11G168800 gene and Glyma.18G060200 gene provided in the application can obtain the phenotype with increased plant height, increased number of pods per plant, increased number of grains per plant, increased yield per plant, increased number of branches, increased number of lateral branch nodes and / or increased number of pods per node after the functional loss mutation of endogenous double genes is generated in the plant, or after the expression of Glyma.11G168800 gene and Glyma.18G060200 gene is inhibited.The aforementioned gene and application method have broad application prospects in the field of soybean breeding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant biotechnology breeding, and particularly relates to a method for obtaining a soybean with more flowers, more pods and high yield by using Glyma.11G168800 gene and Glyma.18G060200 gene to produce functional loss mutation and application thereof. BACKGROUND

[0002] Soybean (Glycine max) is an important food and oil crop for global food security and sustainable development. The average yield per hectare of soybean in China has great growth space and potential. Crop yield per plant is closely related to plant branching and inflorescence structure. Soybean flower clusters are born in leaf axils or at the top, so the number of lateral branches and nodes directly determines the number of flowers that a plant can produce, which further affects the yield per plant. It is crucial to explore new gene resources that control the number of lateral branches or nodes for soybean improvement and breeding. SUMMARY

[0003] All references mentioned in this text are incorporated herein by reference. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs. Unless otherwise indicated, all techniques or methods used or mentioned in this text are standard techniques or methods known to those of ordinary skill in the art. Materials, methods, and examples are illustrative only and not limiting.

[0004] The present application provides a method for producing a soybean plant having the phenotype of increased plant height, increased pod number per plant, increased grain number per plant, increased yield per plant, increased branch number, increased lateral branch number, and / or increased pod number per node, the method comprising the steps of:

[0005] The present application provides a method for producing a soybean plant having the phenotype of increased plant height, increased pod number per plant, increased grain number per plant, increased yield per plant, increased branch number, increased lateral branch number, and / or increased pod number per node, the method comprising the steps of:

[0006] (a) producing one or more soybean plants each having at least one loss-of-function mutation in the endogenous Glyma.11G168800 gene and

[0007] Glyma.18G060200 gene, or the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene being suppressed;

[0008] (b) obtaining at least one seed of the soybean plant produced in step (a).

[0009] Optionally, the polynucleotide sequence of the Glyma.11G168800 gene is selected from one of the following group of sequences:

[0010] (a) the polynucleotide sequence as set forth in SEQ ID No: 1 or 2;

[0011] (b) a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID No: 3;

[0012] (c) a polynucleotide sequence capable of hybridizing under stringent hybridization conditions to the polynucleotide sequence of (a) or (b), and a loss-of-function mutation in the polynucleotide sequence endogenous to a soybean plant has a function of increasing plant height, increasing pod number per plant, increasing seed number per plant, increasing yield per plant, increasing branch number, increasing lateral node number, and / or increasing pod number per node;

[0013] (d) a polynucleotide sequence having at least 90%, 95%, 98%, or more similarity to the polynucleotide sequence of any one of (a) to (c), and a loss-of-function mutation in the polynucleotide sequence endogenous to a soybean plant has a function of increasing plant height, increasing pod number per plant, increasing seed number per plant, increasing yield per plant, increasing branch number, increasing lateral node number, and / or increasing pod number per node; or

[0014] (e) a polynucleotide sequence complementary to the sequence of any one of (a) to (d).

[0015] Optionally, wherein the polynucleotide sequence of Glyma.18G060200 gene is selected from one of the following group of sequences:

[0016] (a) a polynucleotide sequence as shown in SEQ ID No: 5 or 6;

[0017] (b) a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID No: 7;

[0018] (c) a polynucleotide sequence capable of hybridizing under stringent hybridization conditions to the polynucleotide sequence of (a) or (b), and a loss-of-function mutation in the polynucleotide sequence endogenous to a soybean plant has a function of increasing plant height, increasing pod number per plant, increasing seed number per plant, increasing yield per plant, increasing branch number, increasing lateral node number, and / or increasing pod number per node;

[0019] (d) a polynucleotide sequence having at least 90%, 95%, 98%, or more similarity to the polynucleotide sequence of any one of (a) to (c), and a loss-of-function mutation in the polynucleotide sequence endogenous to a soybean plant has a function of increasing plant height, increasing pod number per plant, increasing seed number per plant, increasing yield per plant, increasing branch number, increasing lateral node number, and / or increasing pod number per node; or

[0020] (e) a polynucleotide sequence complementary to the sequence of any one of (a) to (d).

[0021] The application also provides a method for producing a soybean plant having a phenotype of increased plant height, increased pod height, increased effective branch number, increased pod number per plant, increased seed number per plant, increased pod number per node, and / or increased total yield per plant, the method comprising the steps of:

[0022] (a) producing one or more soybean plants each having at least one loss-of-function mutation in an endogenous Glyma.07G240100 gene and

[0023] Glyma.17G033300 gene, or each having suppressed expression of an endogenous Glyma.07G240100 gene and Glyma.17G033300 gene;

[0024] (b) obtaining at least one seed of the soybean plant produced in step (a).

[0025] Optionally, the polynucleotide sequence of the Glyma.07G240100 gene is selected from one of the following sequences:

[0026] (a) a polynucleotide sequence as set forth in SEQ ID No: 9 or 10;

[0027] (b) a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID No: 11;

[0028] (c) a polynucleotide sequence capable of hybridizing to the polynucleotide sequence of (a) or (b) under stringent hybridization conditions, and a loss-of-function mutation in the polynucleotide sequence endogenous to the soybean plant has a function of increasing plant height, increasing pod height, increasing effective branch number, increasing pod number per plant, increasing seed number per plant, increasing pod number per node, and / or increasing total yield per plant;

[0029] (d) a polynucleotide sequence having at least 90%, 95%, 98% or more similarity to the polynucleotide sequence of any one of (a)-(c), and a loss-of-function mutation in the polynucleotide sequence endogenous to the soybean plant has a function of increasing plant height, increasing pod height, increasing effective branch number, increasing pod number per plant, increasing seed number per plant, increasing pod number per node, and / or increasing total yield per plant; or

[0030] (e) a polynucleotide sequence complementary to the sequence of any one of (a)-(d).

[0031] Optionally, the polynucleotide sequence of the Glyma.17G033300 gene is selected from one of the following sequences:

[0032] (a) a polynucleotide sequence as set forth in SEQ ID No: 13 or 14;

[0033] (b) its encoding amino acid sequence is as shown in SEQ ID No: 15;

[0034] (c) a polynucleotide sequence capable of hybridizing to the polynucleotide sequence of (a) or (b) under stringent hybridization conditions, and a loss-of-function mutation of the polynucleotide sequence endogenous to the soybean plant has the function of increasing plant height, pod height, effective branch number, pod number per plant, seed number per plant, pod number per node, and / or total yield per plant;

[0035] (d) a polynucleotide sequence having at least 90%, 95%, 98% or more similarity to the polynucleotide sequence of any one of (a)-(c), and a loss-of-function mutation of the polynucleotide sequence endogenous to the soybean plant has the function of increasing plant height, pod height, effective branch number, pod number per plant, seed number per plant, pod number per node, and / or total yield per plant; or

[0036] (e) a polynucleotide sequence complementary to the sequence of any one of (a)-(d).

[0037] Alternatively, the Glyma.11G168800, Glyma.18G060200, Glyma.07G240100 or Glyma.17G033300 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 to the polynucleotide sequence thereof, or the same gene in different varieties, or a homologous gene having at least 90%, 95% or 98% sequence similarity to the amino acid sequence of Glyma.11G168800 or Glyma.18G060200 or Glyma.07G240100 or Glyma.17G033300 gene disclosed in the embodiments of the present application, or the same gene in different varieties, and the homologous gene has the function of increasing plant height, pod number per plant, seed number per plant, yield per plant, branch number, lateral branch number and / or pod number per node after a loss-of-function mutation of the endogenous homozygous gene, and the homologous gene can be isolated from any plant.

[0038] Alternatively, the Glyma.11G168800, Glyma.18G060200, Glyma.07G240100 or Glyma.17G033300 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 to the polynucleotide sequence thereof, or the same gene in different varieties, or a homologous gene having at least 90%, 95% or 98% sequence similarity to the amino acid sequence of Glyma.11G168800 or Glyma.18G060200 or Glyma.07G240100 or Glyma.17G033300 gene disclosed in the embodiments of the present application, or the same gene in different varieties, and the homologous gene has the function of increasing plant height, pod number per plant, seed number per plant, yield per plant, branch number, lateral branch number and / or pod number per node after a loss-of-function mutation of the endogenous homozygous gene, and the homologous gene can be isolated from any plant.

[0039] Alternatively, the Glyma.11G168800, Glyma.18G060200, Glyma.07G240100 or Glyma.17G033300 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 to the polynucleotide sequence thereof, or the same gene in different varieties, or a homologous gene having at least 90%, 95% or 98% sequence similarity to the amino acid sequence of Glyma.11G168800 or Glyma.18G060200 or Glyma.07G240100 or Glyma.17G033300 gene disclosed in the embodiments of the present application, or the same gene in different varieties, and the homologous gene has the function of increasing plant height, pod number per plant, seed number per plant, yield per plant, branch number, lateral branch number and / or pod number per node after a loss-of-function mutation of the endogenous homozygous gene, and the homologous gene can be isolated from any plant.

[0040] Glyma.18G060200 or Glyma.07G240100 or Glyma.17G033300 homologous genes. 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, tobacco, etc.

[0041] The percentage of sequence similarity described in the present application can be obtained by known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul's algorithm, which are known to those skilled in the art.

[0042] Those skilled in the art should know that there are single nucleotide polymorphisms (SNPs) between different varieties of the same plant for the same gene, i.e. the nucleotide sequences of the same gene often have individual base differences, but there are many varieties of the same crop, and the inventors cannot list them one by one, and the sequences of representative varieties of soybean crops are only provided in the embodiments of the present application. Therefore, those skilled in the art should know that the nucleotide sequences of different varieties of Glyma.11G168800 or Glyma.18G060200 or Glyma.07G240100 or

[0043] The nucleotide sequences of Glyma.17G033300 gene and its nucleotide sequences with SNPs, and the method and application of obtaining the traits of increased plant height, increased number of pods per plant, increased number of grains per plant, increased yield per plant, increased number of branches, increased number of lateral branch nodes, and / or increased number of pods per node, etc. by using the endogenous loss-of-function mutation thereof, are also within the protection scope of the present application.

[0044] Alternatively, the loss-of-function mutation described in the present 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.

[0045] Alternatively, the loss-of-function mutation includes but is not limited to being obtained by physical mutagenesis, chemical mutagenesis, gene editing, etc. The physical mutagenesis includes but is not limited to radiation mutagenesis, space breeding, etc.; the method of chemical mutagenesis includes mutagenesis caused by treatment with mutagenic agents such as EMS; the method of gene editing includes but is not limited to ZFN, TALEN and / or

[0046] CRISPR / Cas, etc.

[0047] As known by those skilled in the art, the main principle of the CRISPR / Cas gene editing system or gene editing method is to find the location of the gene editing to be performed, i.e., the target DNA sequence, in the host genome by a nucleic acid fragment called guide RNA (gRNA), and then to cut the DNA by a Cas protein. In the present application, the Cas protein includes but is not limited to Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13 and / or Cas14, etc.

[0048] Optionally, when the gene editing system used is CRISPR / Cas9, the gene mutant sequence obtained by the CRISPR / Cas9 method has a target sequence selected from one of the following sequences:

[0049] (a) a fragment in the nucleotide sequence shown in SEQ ID No: 1, 3, 5, 9 or 10, which conforms to the sequence arrangement rule of 5'-Nx-NGG-3', wherein N represents any one of A, G, C and T, 14 < X < 30, and X is an integer, Nx represents X consecutive nucleotides; or

[0050] (b) a nucleotide sequence complementary to the polynucleotide sequence of (a).

[0051] Optionally, in the CRISPR / Cas9 gene editing system disclosed in the embodiments of the present application, the target sequence of the Glyma.11G168800 or Glyma.18G060200 gene is shown in SEQ ID NO: 24 and SEQ ID NO: 23, respectively; and the target sequence of the Glyma.07G240100 or Glyma.17G033300 gene is shown in SEQ ID NO: 22 and SEQ ID NO: 21, respectively.

[0052] Optionally, the soybean plant has a double mutant of Glyma.11G168800 and Glyma.18G060200 genes, and the nucleotide sequence of the double mutant is shown in SEQ ID NO: 4 or SEQ ID NO: 8.

[0053] Optionally, the soybean plant has a double mutant of Glyma.07G240100 and Glyma.17G033300 genes, and the nucleotide sequence of the double mutant is shown in SEQ ID NO: 12 or SEQ ID NO: 16.

[0054] Optionally, the loss-of-function mutation can also be obtained by crossing a soybean plant having the loss-of-function mutation.

[0055] Optionally, the method described in the embodiments of the present application, wherein the reduction or inhibition of the normal expression or protein function of the lodging-related gene comprises obtaining by means of RNA interference (i.e. RNAi) and / or mutation. Those skilled in the art know that the RNAi technology is a routine technology in the art, which specifically binds to the homologous region of the mRNA expressed by the target gene through 21-23 bp short-chain double-stranded RNA (siRNA: small interfering RNA) or long-chain double-stranded RNA (dsRNA: double-stranded RNA), degrades the mRNA, and achieves the effect of inhibiting gene expression.

[0056] Optionally, the expression of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene in soybean can be inhibited by the method of RNAi in the present application, thereby affecting the activity of the aforementioned genes, inhibiting the expression of the genes, and making the soybean plants have the phenotypes of increased plant height, increased pod number per plant, increased grain number per plant, increased yield per plant, increased branch number, increased lateral branch number, and / or increased pod number per node.

[0057] Optionally, the present application also provides an application of the aforementioned method in soybean breeding, preferably, the application includes but is not limited to the application in the regulation of traits such as increased plant height, increased pod number per plant, increased grain number per plant, increased yield per plant, increased branch number, increased lateral branch number, and / or increased pod number per node.

[0058] Optionally, the embodiments of the present application also provide a feed, meal, protein or oil product made of soybean, which contains a loss-of-function mutant of Glyma.11G168800 gene and Glyma.18G060200 gene, each of which 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 genes. Optionally, the polynucleotide sequence of the loss-of-function mutant is as shown in SEQ ID NO: 4 or SEQ ID NO: 8.

[0059] Optionally, the application further provides a soybean-made feed, meal, protein or oil product, which contains a functional loss mutant of Glyma.07G240100 or Glyma.17G033300, each of which contains at least one functional loss mutation, or the endogenous Glyma.07G240100 or Glyma.17G033300 gene expression is inhibited. The functional loss mutation includes substitution, deletion and / or addition of one or more nucleotides on the nucleotide sequence of the aforementioned gene. Optionally, the polynucleotide sequence of the functional loss mutant is shown as SEQ ID NO: 12 or SEQ ID NO: 16.

[0060] The application further provides a method for identifying a Glyma.11G168800 gene and a Glyma.18G060200 gene mutant, which confirms the specific mutation site by sequencing the PCR product after PCR amplification, and is characterized in that the PCR amplification primer of the Glyma.11G168800 gene is shown as SEQ ID NO: 37 and SEQ ID NO: 38; and the PCR amplification primer of the Glyma.18G060200 gene is shown as SEQ ID NO: 35 and SEQ ID NO: 36.

[0061] The application further provides a method for identifying a Glyma.07G240100 or Glyma.17G033300 gene mutant, which confirms the specific mutation site by sequencing the PCR product after PCR amplification, and is characterized in that the PCR amplification primer of the Glyma.07G240100 gene is shown as SEQ ID NO: 33 and SEQ ID NO: 34; and the PCR amplification primer of the Glyma.17G033300 gene is shown as SEQ ID NO: 31 and SEQ ID NO: 32.

[0062] Optionally, the methods described in the embodiments of this application for transferring nucleotide sequences, vectors, constructs, or expression cassettes into plants, introducing them into plants, or transforming plants all refer to transferring the target nucleotide sequence, construct, vector, or expression cassette into recipient cells or recipient plants through conventional transgenic methods or methods of hybridization with target transgenic plants. Any transgenic method known to those skilled in the art can be used to transform recombinant expression vectors into plant cells to produce transgenic plants or mutants of the embodiments of this application. 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.

[0063] Compared with the prior art, this application has the following beneficial effects:

[0064] (1) This application provides a method for producing soybean plants and its application, by enabling soybean endogenous

[0065] The expression of the Glyma.11G168800 and Glyma.18G060200 genes each contains at least one loss-of-function mutation, or the expression of endogenous Glyma.11G168800 and Glyma.18G060200 genes is suppressed, thereby obtaining plants with superior agronomic traits such as increased plant height, increased number of pods per plant, increased number of grains per plant, increased yield per plant, increased number of branches, increased number of lateral branch nodes, and / or increased number of pods per node; or, by inducing the expression of endogenous soybean Glyma.07G240100 or Glyma... Each of the .17G033300 genes contains at least one loss-of-function mutation, or the expression of the endogenous Glyma.07G240100 or Glyma.17G033300 gene is suppressed, thereby obtaining plants with superior agronomic traits such as increased plant height, increased pod height, increased number of effective branches, increased number of pods per plant, increased number of grains per plant, increased number of pods per node, and / or increased total yield per plant. The aforementioned genes, methods, and their applications provide new germplasm resources and breeding ideas for soybean breeding, which are of great significance to global food security and sustainable agricultural development.

[0066] (2) The functions of the double mutations of Glyma.11G168800 and Glyma.18G060200 genes and Glyma.07G240100 and Glyma.17G033300 genes in soybean acyltransferase genes were clarified, providing new gene resources for crop species.

[0067] (3) In the case where a single gene mutation does not have a clear corresponding phenotype, by double mutation of two genes, the normal expression or protein function of the aforementioned genes is reduced or inhibited, thereby obtaining breeding materials with application value, and providing new ideas for crop breeding and research on gene action mechanism network.

[0068] Definitions of terms involved in the invention

[0069] 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 the present application, the preferred methods, devices and materials are now described.

[0070] In the context of the present application, the term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides in single or double-stranded form and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0071] 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, positive homologous genes or directional evolution homologous genes), paralogous genes (also known as paralogous genes, parallel evolution homologous genes) and / or heterologous homologous genes.

[0072] The term "sequence similarity" refers to the degree of similarity between two sequences, which is a quantitative concept for comparing the degree of similarity between different sequences, so as to find and analyze the relationship between two sequences. Sequence similarity can be used to compare gene sequences, protein sequences, DNA sequences, etc.

[0073] The term "strict hybridization conditions" as used in this application refers to conditions of low ionic strength and high temperature known in the art. Typically, under strict conditions, the detectability of a probe hybridizing with its target sequence is significantly higher than that hybridizing with other sequences (e.g., at least twice the background level). Strict hybridization conditions are sequence-dependent and will vary under different environmental conditions; longer sequences hybridize specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the strictness of hybridization or washing conditions. Detailed guidance on 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 strict conditions are typically chosen to be below the melting point (T0) of the specific sequence at a specified ionic strength pH. m Approximately 5-10℃. m The temperature at which 50% of the probe complementary to the target sequence hybridizes to the target sequence under equilibrium conditions (at specified ionic strength, pH, and nucleic acid concentration) (because the target sequence is in excess, therefore at T...). m (Under equilibrium conditions, 50% of the probe is occupied). Strict conditions may include: a salt concentration of less than about 1.0 M sodium ions at pH 7.0 to 8.3, typically about 0.01 to 1.0 M sodium ions (or other salts), and a temperature of 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) more than 50 nucleotides). Strict conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, the positive signal may be at least twice the background hybridization, and, where appropriate, 10 times the background hybridization. Exemplary strict hybridization conditions may be: 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 washing can be performed for 5, 15, 30, 60, 120 minutes or longer.

[0074] The term "recombinant expression vector" is one or more DNA vectors used to effect plant transformation; these vectors are often referred to in the art as binary vectors. Binary vectors, along 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, a selectable marker engineered to be expressible in plant cells, a heterologous DNA sequence to be transcribed, etc.

[0075] The term "pod number per plant" refers to the total number of pods on a soybean plant.

[0076] The term "pod number per node" refers to the total number of pods that are borne on each of the trifoliolate leaf axils (referred to as nodes) of a soybean plant.

[0077] The term "crossing" refers to the broadest sense of sexual mating between different populations or genotypes of individuals, resulting in the production of offspring, and includes both crosses between related and unrelated individuals.

[0078] As used herein, "mutation" refers to a "loss-of-function mutation" or "loss-of-function mutation" is a mutation in the coding sequence of a gene that results in a reduction or complete loss of function of the gene product, typically a protein. A loss-of-function mutation can result, for example, from a truncation of the gene product (as a result of a frameshift or nonsense mutation), and the phenotype associated with an allele having a loss-of-function mutation can be recessive or dominant.

[0079] The term "RNA interference" (RNAi) is a gene-silencing technology that uses double-stranded RNA (dsRNA) molecules to block the expression of specific genes at the mRNA level, i.e., sequence-specific post-transcriptional gene silencing (PTGS). BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 is a maximum likelihood tree analysis of MND1 homologs from different crops.

[0081] Figure 2 is a gel map of the gene sequencing of mndlsi and mndls2 mutants.

[0082] Figure 3Figure 1 is a comparison of the expression levels of Glyma.11G168800 and Glyma.18G060200 in Huachun 6 and T2 mutant, wherein A and B are a comparison of the expression of Glyma.11G168800 in the leaves and axillary buds of Huachun 6 and mnd1s1 mutant, respectively; C and D are a comparison of the expression of Glyma.18G060200 in the leaves and axillary buds of Huachun 6 and mnd1s1 mutant, respectively.

[0083] Figure 4 Figure 2 is a comparison of the whole-plant phenotypes of Huachun 6 and T1 mutant.

[0084] Figure 5 Figure 3 is a comparison of the yield of single plants of Huachun 6 and T1 mutant.

[0085] Figure 6 Figure 4 is a comparison of the number of pods at corresponding nodes of Huachun 6 and T1 mutant.

[0086] Figure 7 Figure 5 is an analysis of the test data of Huachun 6 and T1 mutant.

[0087] Figure 6 is a legend of the symbols used in the figures, wherein 11G refers to Glyma.11G168800 gene; 18G refers to Glyma.18G060200 gene; HC6 refers to Huachun 6. DETAILED DESCRIPTION

[0088] The present application will be further described below in conjunction with specific examples, and the advantages and features of the present application will become more apparent as the description proceeds. However, these examples are merely exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the present application without departing from the spirit and scope of the present application, and such modifications and substitutions fall within the scope of the present application.

[0089] The inbred lines used in the following examples can be obtained from the "China Crop Germplasm Information Network" for relevant information and corresponding seeds can be obtained by application.

[0090] Example 1. Creation of soybean MND1 / HLS1 homologous gene mutant using gene editing technology

[0091] The present inventors first performed evolutionary analysis on the MND1 gene family of land plants using the maximum likelihood method RAxML, and identified all 12 homologous genes annotated in the soybean genome, and the results are shown in Figure 1 Figure 1 ​It can be seen that the MND1 gene of barley is homologous to the HLS1 gene of Arabidopsis, and the gene family can be divided into four evolutionary branches, including the HLS1 branch, the MND1 branch, and the MND1s1 and MND1s2 branches which form a sister branch with the HLS1+MND1 branch. Figure 1 Meanwhile, the inventors determined the tissue and developmental stage-specific expression patterns of 12 MND1 / HLS1 genes using the public transcriptome database (database website link: Phytozome (doe.gov), soybean genome selection "Glycine max Wm82.a4.v1") and identified 7 MND1 / HLS1 genes specifically expressed in inflorescences and shoot apical meristems, which are Glyma.07G022800, Glyma.08G219000, Glyma.09G035900, Glyma.17G033300, Glyma.07G240100, Glyma.18G060200 and Glyma.11G168800, respectively. Then, the inventors used the CRISPR / Cas9 gene editing technology to target edit the 7 MND1 / HLS1 homologous genes specifically expressed in inflorescences and shoot apical meristems using the southern soybean main cultivar national variety Huachun No. 6 as the receptor.

[0092] The target design uses the high-throughput CRISPR-Cas9 target site design program developed by Unimibio (Jiangsu) Co., Ltd. The target site design principles of the program are as follows: 1) the knockout site is in the coding (CDS) region and is as far as possible at the front end of the protein or in the important functional region; 2) as far as possible to cover a higher proportion of transcripts; 3) no off-target or off-target in the intergenic region; 4) preferentially editing target sites with higher efficiency; 5) the sequence has a relatively balanced GC content and is not easy to form a secondary structure. The successful application of the program in rice whole genome target site design has proved its feasibility. The inventors have experiments showing that the single gene mutant phenotypes of Glyma.18G060200 and Glyma.11G168800 are not obvious, so the application examples design a double gene double target gene editing method for the aforementioned partial evolutionary branch genes in order to obtain double gene mutants and analyze their phenotypes. The target information for the 7 genes is shown in Table 1:

[0093] Table 1. Gene knockout target site sequence

[0094]

[0095]

[0096] Example 2. Detection of T0 generation gene editing seedlings and related molecular experiments

[0097] To identify the editing status of the mutant, leaf DNA was extracted from the mutant, and then PCR (polymerase chain reaction) amplification of the target gene was performed using gene-specific primers. The gene-specific amplification primers (Table 3) were designed on both sides of the two target sites. The amplification products were sent to the company for first-generation Sanger sequencing to determine the editing status of the target gene near the two target sites. The specific procedures are as follows:

[0098] (1) DNA extraction

[0099] We used SDS (Sodium dodecyl sulfate) to crudely extract leaf DNA from Huachun 6 and its mutant. The preparation and operation steps of the extraction solution are as follows:

[0100] Table 2. Working fluid preparation

[0101]

[0102] Operating steps:

[0103] ① When the plant has 2 leaves with 3 leaflets, take about 2mg of tender leaves into a grinding tube, put in 2 grinding beads, and freeze in liquid nitrogen.

[0104] ② Set the grinder to 60Hz and 60 seconds to grind the blades into powder.

[0105] ③ Add 600 μL of working solution, invert to mix well, and then centrifuge at 12000 rpm for 10 min.

[0106] ④ Pipette 400 μL of supernatant into a new 1.5 mL EP tube and add 800 μL of 95% ethanol to wash away impurities.

[0107] ⑤ Place in a -20℃ refrigerator and let stand for 5 minutes, then centrifuge at 12000 rpm for 15 minutes.

[0108] ⑥ Discard the supernatant, invert the EP tube onto paper to dry, and finally add 100μL of double-distilled water to dissolve the DNA precipitate.

[0109] (2) Identification of gene editing by PCR and first-generation sequencing (Sanger sequencing)

[0110] The gene sequence at the target site was amplified using specific primers (Table 3), and the PCR product was then 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 the four transformation events (Table 1), and no obvious phenotype was found in the T0 generation seedlings.

[0111] The specific information and experimental procedures are as follows:

[0112] Table 3. Information of primers for gene detection

[0113]

[0114] Table 4. PCR reaction system

[0115]

[0116] The reaction procedure was as follows:

[0117]

[0118] Example 3. Genotype identification of T1 generation homozygous mutants

[0119] Further genotype and phenotype identification of the mutants and ensuring that each mutation event and target gene can be screened for at least 2 different independent mutation editing events, we randomly selected 4 transformation events totaling 47 different T0 generation edited seedling strains (ensuring at least 6 strains for each transformation event) to be planted in the school scientific research and teaching base. In the T1 generation mutants planted in the field on July 14, 2023, it was unexpectedly found that one strain appeared the phenotype of more flowers and more pods ( Figure 4 D, E, F), and the gene editing of this strain was as follows: Glyma.11G168800 gene homozygous deletion of 317 bp ( Figure 2 A), Glyma.18G060200 homozygous insertion of 214 bp ( Figure 2 B). Evolutionary analysis showed that the proteins encoded by Glyma.11G168800 and Glyma.18G060200 are one of the two sister branches of MND1, so the double mutant of this strain was named mnd1s1; another homozygous double knockout mutant (Glyma.07G240100 homozygous deletion of 325 bp, Glyma.17G033300 homozygous deletion of 361 bp, Figure 2 C and Figure 2 D) planted at the same time and place did not have this phenotype of more flowers and more pods ( Figure 4 G, H, I), and evolutionary analysis showed that the proteins encoded by Glyma.07G240100 and Glyma.17G033300 are another sister branch of MND1, so the mutant of this strain was named mnd1s2.

[0120] Table 5. Number of generations of each transformation event in T0 generation

[0121]

[0122] In the embodiments of the present application, the gDNA sequence of Glyma.11G168800 is shown as SEQ ID NO:1, the CDS is shown as SEQ ID NO:2, the encoded amino acid sequence is shown as SEQ ID NO:3, the nucleotide sequence of the functional deletion mutant of Glyma.11G168800 gene after mutation is shown as SEQ ID NO:4. The gDNA sequence of Glyma.18G060200 is shown as SEQ ID NO:5, the CDS is shown as SEQ ID NO:6, the encoded amino acid sequence is shown as SEQ ID NO:7, the nucleotide sequence of the functional deletion mutant of Glyma.18G060200 gene after mutation is shown as SEQ ID NO:8. The gDNA sequence of Glyma.07G240100 is shown as SEQ ID NO:9, the CDS is shown as SEQ ID NO:10, the encoded amino acid sequence is shown as SEQ ID NO:11, the nucleotide sequence of the functional deletion mutant of Glyma.07G240100 gene after mutation is shown as SEQ ID NO:12. The gDNA sequence of Glyma.17G033300 is shown as SEQ ID NO:13, the CDS is shown as SEQ ID NO:14, the encoded amino acid sequence is shown as SEQ ID NO:15, the nucleotide sequence of the functional deletion mutant of Glyma.17G033300 gene after mutation is shown as SEQ ID NO:16.

[0123] To further confirm the specific biological function of Glyma.11G168800 homozygous deletion and Glyma.18G060200 insertion fragment in mnd1s1 mutant, we designed specific primers (Table 8) in the 5'-UTR region of the two genes and performed qPCR to detect whether the expression level of the gene is up-regulated or down-regulated, which indicates that the insertion fragment may cause the gain-of-function phenotype of the gene or cause the loss-of-function or loss phenotype of the gene. The qPCR results show that

[0124] The expression levels of Glyma.11G168800 and Glyma.18G060200 are significantly down-regulated, i.e., the mnd1s1 homozygous double knock-in mutant is a loss-of-function mutant Figure 3

[0125] In the embodiments of the present application, the actual expression amount of the mutant gene is detected by RNA extraction, reverse transcription and qPCR, and the specific operation is as follows.

[0126] (1) RNA extraction:​

[0127] ① Sampling: Put two grinding beads into a sterile grinding tube, immerse the tube in liquid nitrogen, and use tweezers to put about 2 mg of leaf or axillary bud into the tube.

[0128] ② Grinding: Set the grinder to 60 HZ, 30 S, and grind the material into powder. Immediately put the tube back into liquid nitrogen after grinding.

[0129] ③ Add 500 μL Trizol extraction solution, and place the tube on ice.

[0130] ④ After dissolving the powder by inverting the tube, place it on ice for 10 min.

[0131] ⑤ Add 100 μL chloroform, invert the tube, and place it on ice for 10 min.

[0132] ⑥ Centrifuge at 13,000 rpm, 4°C for 10 min.

[0133] ⑦ Take 250 μL supernatant into a new sterile 1.5 mL centrifuge tube, add 250 μL isopropanol, and place it on ice for 10 min.

[0134] ⑧ Centrifuge at 13,000 rpm, 4°C for 10 min.

[0135] ⑨ Discard the supernatant, and add 500 μL 75% ethanol to wash the precipitate.

[0136] ① 13,000 rpm, 4°C, centrifuge for 5 min. Discard the supernatant, and wash the precipitate again.

[0137] 13,000 rpm, 4°C, centrifuge for 2 min. Use a syringe to remove residual liquid, and air dry for 10 min.

[0138] Add 40 μL sterile water to dissolve the RNA. Then store it at -80°C.

[0139] (2) Reverse transcription step (using Novagen kit):

[0140] ① Removal of genomic DNA

[0141] After uniform treatment according to the RNA concentration, remove the genomic DNA according to the following table

[0142] Table 6. Preparation of genomic DNA removal system

[0143]

[0144] Mix by blowing, and incubate at 42°C for 2 min.

[0145] Preparation of the first strand cDNA synthesis reaction solution

[0146] Table 7. Preparation of the first strand cDNA synthesis reaction solution

[0147]

[0148] Mix by pipetting

[0149] Synthesis of the first strand cDNA

[0150] Table 8. Synthesis procedure of the first strand cDNA

[0151]

[0152] After the reaction, the cDNA was stored at -20°C.

[0153] (3) qPCR operation steps

[0154] ① Primer design

[0155] Table 9. qPCR primer sequences

[0156]

[0157] ② System preparation

[0158] Table 10. qPCR reaction system

[0159]

[0160] ③ The qPCR reaction procedure is as follows:

[0161]

[0162] After the reaction, the expression amount of the target gene was calculated by comparing the fluorescence change value with that of the internal reference gene (4) Result analysis

[0163] In the qPCR experiment, the top young leaves and all axillary buds of 24-day-old Huachun 6 and T2 mutant plants were taken under the same conditions in the same greenhouse. The expression amount of the target gene relative to actin in Huachun 6 and T2 mutant was calculated using 2^- Δct , and the results were plotted using Graphpad software, as shown in Figure 3 . The expression levels of Glyma.11G168800 and Glyma.18G060200 in the leaves and axillary buds of the mutant were decreased, indicating that the expression levels of Glyma.11G168800 and Glyma.18G060200 in the mutant were significantly down-regulated, i.e., the mnd1s1 homozygous double-knockout mutant was a loss-of-function mutant (Figure 3 ).

[0164] In the qPCT experiment, 12 seeds of each of the T1 generation mutant mnd1s1-1, mnd1s1-2 and mnd1s1-3 were planted, and it was detected that most of the T2 generation editing types were the same as those of the T1 generation. In the qPCR experiment, the editing types of the three mutants were the same as those of the T1 generation.

[0165] Example 4. Specific field phenotype analysis of T1 generation mutants

[0166] Through field phenotype analysis, compared with Huachun 6 and mnd1s2 mutant lines, the plant height, pod number per plant, grain number per plant and grain weight per plant of the three double knock-out mutants mnd1s1-1, mnd1s1-2 and mnd1s1-3 with the same editing type were significantly increased, and the branch number was also slightly increased. Figure 7 However, the hundred-grain weight of the double knock-out mutants was reduced Figure 7 H), but the total yield per plant was significantly increased Figure 5 , Figure 7 G). At the same time, the phenotype analysis results also showed that the node number of the lateral branches of the mnd1s1 mutant was more than that of Huachun 6 and mnd1s2 mutants Figure 4 D, E, F), and more pods were produced per node of 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 plant. In summary, the analysis of the yield traits of the mnd1s1 mutant provided in the application examples showed that the growth period of the mutant was similar to that of the wild type (both flowering on August 14), but the yield was significantly increased. Accordingly, it was indicated that the homozygous double mutant obtained by simultaneously mutating the Glyma.11G168800 and Glyma.18G060200 genes had great practical production application value, and the double gene mutation had the functions of increasing the plant height, increasing the pod number per plant, increasing the grain number per plant, increasing the yield per plant, increasing the branch number, increasing the node number of the lateral branches and / or increasing the number of pods per node of soybean, and could significantly increase the yield per plant and the yield per unit area of soybean, and had great application potential in soybean breeding.

[0167] Compared with the wild type Huachun 6, the plant height, pod height, effective branch number, pod number per plant, grain number per plant Figure 7 of the three double knock-out mutants mnd1s2-1, mnd1s2-2 and mnd1s2-3 with the same editing type were increased, and more pods were produced per node of the main stem Figure 6 , and the total yield per plant was significantly increased Figure 5). Therefore, the homozygous double mutant obtained by simultaneously mutating Glyma.07G240100 and Glyma.17G033300 genes has 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 pod number per plant, increased seed number per plant, increased yield per plant, increased branch number, increased lateral branch number, and / or increased pod number per node, the method comprising the steps of: producing one or more soybean plants, wherein each of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene in the soybean plant contains at least one loss-of-function mutation, or the expression of the endogenous Glyma.11G168800 gene and Glyma.18G060200 gene is inhibited; obtaining at least one seed of the soybean plant produced in the above step; wherein the polynucleotide sequence of the Glyma.11G168800 gene is selected from one of the following sequences: (a) a polynucleotide sequence as set forth in SEQ ID No: 1 or 2; (b) a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID No: 3; or (c) a polynucleotide sequence complementary to any of the sequences of (a) to (b) ; and wherein the polynucleotide sequence of the Glyma.18G060200 gene is selected from one of the following sequences: (I) a polynucleotide sequence as set forth in SEQ ID No: 5 or 6; (II) a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID No: 7; or (III) a polynucleotide sequence complementary to any of the sequences of (I) to (II). 2.The method of claim 1, wherein the loss-of-function mutation comprises one or more substitutions, deletions and / or additions of nucleotides in the polynucleotide sequence of the gene; and wherein the expression of the gene is inhibited by RNA interference. 3.The method of claim 2, wherein the loss-of-function mutation is obtained by physical mutagenesis, chemical mutagenesis, ZFN, TALEN and / or CRISPR / Cas gene editing technology, or obtained by crossing with a soybean plant having the loss-of-function mutation. 4.The method of claim 3, wherein the CRISPR / Cas is a CRISPR / Cas9 gene editing method, and the target sequence used is selected from one of the following sequences: (a) a fragment of the nucleotide sequence as set forth in SEQ ID No: 1 or 2, and SEQ ID No: 5 or 6, which conforms to the sequence arrangement rule of 5’-Nx-NGG-3’, 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 of (a). 5.The method of claim 4, wherein the target sequence is as set forth in SEQ ID NO: 24 and 23. 6.The method of any one of claims 1 to 5, wherein the polynucleotide sequence containing at least one loss-of-function mutation is as set forth in SEQ ID NO: 4 and SEQ ID NO:

8. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 7. Use of the method according to any one of claims 1 to 6 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 per branch and / or the number of pods per node of a soybean plant.

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