Corn gene dwf4 and functional sites and uses thereof

Abstract

The present application relates to the field of plant gene map cloning and molecular breeding, and particularly relates to a maize gene DWF4 and related functional sites and uses thereof. Specifically, the present application provides a method for regulating maize plant type based on the DWF4 gene, and a maize plant or part, seed, cell or offspring of the maize plant in which the endogenous DWF4 gene is disrupted, and thus the present application also provides a method for producing a maize plant or hybrid maize plant, and a maize yield-increasing planting method.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant gene mapping and molecular breeding, and in particular to a maize gene DWF4 and related functional sites and uses thereof. BACKGROUND

[0002] Maize is the largest grain crop in China, with a sowing area of about 603 million mu in 2022, a total output of 277 million tons, accounting for about 43.8% of the total grain output. Maize has become an important crop to ensure national food security and people's happy life. However, the space for increasing maize yield by simply increasing sowing area is very limited. The fundamental way out is to fully tap the great potential of maize genetic resources and significantly increase maize yield per mu, which is the main way to ensure the sustained increase of China's total maize production.

[0003] At present, the average yield per mu of maize in China is less than 450 kg, and the average planting density is 3500 plants per mu, with large regional differences. The highest planting density of summer maize in the Huanghuaihai region is mostly 4000 plants per mu, while the lowest planting density in the southwest region is mostly less than 2800 plants per mu. Therefore, planting density is an important reason for the insufficient yield per mu of maize in China.

[0004] Duvick and Cassman et al. found through research on maize hybrids released in the United States from 1930 to 1990 that the maize hybrids planted in the United States tended to be more and more compact, and continuously adapted to dense planting, which was significantly related to the continuous increase in yield. Maize plant type is mainly determined by the size and spatial distribution of maize leaf size, stem-leaf angle (leaf angle), plant height and ear height, female and male ears, and root organ morphology. Leaf angle is the main trait that determines the compactness of maize plants, directly affecting the light interception capacity of maize canopy and the light energy utilization rate of the population. Maize varieties with smaller leaf angles have compact plant types, high light transmittance in the upper part of the population, and the lower leaves of the population can be in a good light state, which facilitates the full and efficient use of light energy by leaves, thus allowing for larger planting populations and higher population yields. Therefore, cloning key genes that control leaf angle morphogenesis and studying the molecular regulation mechanism of maize leaf angle formation have important theoretical and practical guiding significance for breeding maize ideal plant types and cultivating high-yield varieties.

[0005] In summary, leaf angle is a main trait to measure the compactness of maize plant, and is a key factor to affect whether maize can adapt to high density planting, and further determine whether maize can increase yield by high density planting. A number of genes controlling maize leaf angle have been cloned through mutants, homologous alignment and QTL mapping, such as lg1, lg2, Wab1, kn1, Lgn, drl1, drl2, ZmTAC1, ZmCLA4, UPA1 (brd1), UPA2 (ZmRAVL1) and ZmILI1 (Moreno et al., 1997; Walsh et al., 1998; Moon et al., 2013; Strable et al., 2017; Ku et al., 2011; Zhang et al., 2014; Tian et al., 2019; Ren et al., 2019). Since the phenotypes of the genes cloned through mutants are relatively extreme, and at the same time carry other adverse phenotypes, these genes have not been widely used in production. The number of natural variants existing in maize population cloned through QTL mapping is still small, and the effect of a single QTL cannot effectively shape the ideal plant type of maize hybrids, which has certain limitations in breeding practice application. In addition, the cloned genes regulating maize compact plant type generally regulate the angle of all leaf positions of maize, while in maize production, the plant type with upper leaf angle compact and lower leaf angle relatively loose is more needed, which is more conducive to the distribution of light in different crown layers and more suitable for high density planting. SUMMARY

[0006] The present inventors have found, through extensive research, a key gene regulating the "upper compact and lower loose" density-tolerant ideal plant type of maize, and further obtained a method for regulating maize plant type by disrupting the gene, and a "upper compact and lower loose" density-tolerant ideal plant type obtained by the method, for improving the plant type of maize elite inbred lines and cultivating new maize varieties with high yield and density tolerance.

[0007] Method for regulating maize plant type

[0008] In a first aspect, the present application provides a method for regulating maize plant type, the method comprising:

[0009] (1) disrupting an endogenous DWF4 gene of a maize cell;

[0010] (2) developing or regenerating from the maize cell a maize plant or part, seed, cell or progeny thereof comprising the DWF4 gene disrupted.

[0011] I. Plant type

[0012] In certain embodiments, the plant type comprises a leaf angle, such as a leaf angle above the ear.

[0013] In some embodiments, the plant type is a compact upper leaf and loose lower leaf type. In this document, the term "compact upper leaf and loose lower leaf type" has the meaning commonly understood by those skilled in the art, referring to an ideal maize plant type with compact upper leaves and relatively flat lower leaves. In some embodiments, the upper leaf angle of the plant type described in this invention is less than 30 degrees, less than 25 degrees, or less than 20 degrees. In some embodiments, the upper leaf angle of the plant type is smaller than the lower leaf angle. In some embodiments, the lower leaf angle of the plant type is greater than 20 degrees, for example, 20–40 degrees, 20–30 degrees, or 30–40 degrees.

[0014] In some implementations, disrupting the DWF4 gene can reduce the angle between the upper leaves and the lower leaves by a greater degree, resulting in a plant type with a tight upper leaf and a loose lower leaf. The degree of reduction refers to the proportion of the reduction in the leaf angle compared to the corresponding reduction in the leaf angle of maize plants with an intact endogenous DWF4 gene, for example, [leaf angle]. (未破坏) -Leaf angle (破坏) Leaf angle (未破坏) *100%. In some embodiments, the reduction in the angle between the upper leaves and the lower leaves is at least 1.5 times, for example, at least 2 times, greater than the reduction in the angle between the upper leaves and the lower leaves.

[0015] In some implementations, disrupting the DWF4 gene can reduce the degree of decrease in the upper leaf angle (e.g., the angle between the second leaf below the flag leaf), the middle leaf angle (e.g., the angle between the first leaf above the ear), and the lower leaf angle (e.g., the angle between the first leaf below the ear) in that order, thereby forming a plant type with a tight upper section and a loose lower section. The degree of reduction refers to the proportion of reduction in the corresponding leaf angle compared to maize plants with an intact endogenous DWF4 gene. For example, [leaf angle...] (未破坏) -Leaf angle (破坏) Leaf angle (未破坏) *100%. In some embodiments, the reduction in the upper leaf angle is at least 1.2 times, for example, at least 1.5 times, greater than the reduction in the middle leaf angle. In some embodiments, the reduction in the middle leaf angle is at least 1.2 times, for example, at least 1.5 times, greater than the reduction in the lower leaf angle.

[0016] In some embodiments, the maize plant or parts thereof, seeds, cells or progeny obtained by the method have a reduced ear leaf angle compared to maize plants with intact endogenous DWF4 genes, for example, a reduction of at least 20%, at least 30%, at least 40% or at least 50%.

[0017] In some embodiments, the maize plant or parts thereof, seeds, cells or progeny obtained by the method also possess one or more traits selected from the following compared to maize plants with the endogenous DWF4 gene not destroyed: increased 100-kernel weight, increased number of kernels per ear, increased ear weight or increased yield per unit area.

[0018] II. Gene disruption

[0019] In certain embodiments, the corn cell is homozygous for the disruption, i.e., both copies of the DWF4 gene are disrupted.

[0020] In certain embodiments, the disruption comprises a disruption of the function, expression level, activity, or a combination thereof of the DWF4 gene.

[0021] A genetic disruption comprises any genetic alteration that renders the encoded gene product inactive. For example, the genetic alteration can be a deletion or knockout of the entire gene, a deletion of regulatory sequences required for transcription or translation, a deletion of a portion of the gene resulting in a truncated gene product, or any of a variety of mutagenic strategies that render the encoded gene product inactive. In certain embodiments, the disruption comprises introducing a modification (e.g., a deletion, a substitution, an insertion, an inversion, a duplication, or a combination thereof of one or more nucleotides) in the DWF4 gene. In other embodiments, the disruption comprises targeting a silencing molecule to the DWF4 gene to knock down or silence the DWF4 gene. In certain embodiments, the disruption is stably transferable to the next generation of cells.

[0022] Modification

[0023] In certain embodiments, the disruption comprises introducing a modification in the endogenous DWF4 gene.

[0024] In certain embodiments, the modification is in the coding region and / or non-coding region of the endogenous DWF4 gene, e.g., a promoter, a 5' UTR, an intron, an exon, a 3' UTR, a terminator, and any combination thereof.

[0025] In certain embodiments, the modification comprises a deletion, a substitution, an insertion, an inversion, a duplication, or any combination thereof.

[0026] In certain embodiments, the modification results in a nonsense mutation, a missense mutation, a frameshift mutation, a splice site mutation, or any combination thereof.

[0027] In certain embodiments, the corn cell is homozygous for the modification.

[0028] In certain embodiments, the modification comprises a first modification in a first allele and a second modification in a second allele, the first modification and the second modification being the same as or different from each other but both resulting in a disruption of the DWF4 gene.

[0029] Genome editing

[0030] In certain embodiments, the modification is introduced by genome editing.

[0031] The editing system that can be used in the present application can be any site-specific (sequence-specific) genome editing system now known that can introduce mutations in a target-specific manner. Genome editing can result in an insertion / deletion ("indel") mutation (i.e., "SDN1"), a base editing (i.e., "SDN2"), or an allele insertion or substitution (i.e., "SDN3"). SDN1 can be generated via targeted introduction of a DNA double-strand break without the use of a recombination template. Such a break can be repaired by a non-homologous end joining (NHEJ) process, which can result in a small (e.g., a few nucleotides) insertion or deletion (indel) at the site of repair. Such an indel can result in a frameshift mutation, causing a premature stop codon or other type of loss-of-function mutation in the targeted gene. SDN2 or SDN3 gene editing can involve providing one or more recombination templates (e.g., in a vector) that comprise a sequence of the gene of interest (i.e., to be introduced into the plant genome) that can be used for homology directed repair (HDR) within the plant. SDN2 or SDN3 can generate a double-stranded DNA break or a single-stranded DNA break (nick) (e.g., using a nickase Cas9 variant), both of which can induce HDR, whereby the target sequence is replaced by the sequence of the provided recombination template by HDR.

[0032] In certain embodiments, the genome editing comprises the use of at least one site-specific nuclease, such as an RNA-guided nuclease (e.g., a Cas nuclease), a zinc finger nuclease, a meganuclease, a TALE-nuclease, a recombinase, a transposase, and any combination thereof.

[0033] In certain embodiments, the genome editing is selected from the group consisting of CRISPR / Cas, TALEN, ZFN, transposon technology, PASTE technology, PE technology, base editor, and any combination thereof.

[0034] In certain embodiments, the site-specific nuclease is a site-specific endonuclease, such as an RNA-guided endonuclease, a zinc finger nuclease, a meganuclease, or a TALE-nuclease. The site-specific endonuclease targets the endogenous DWF4 gene, induces a DNA break at the target site, and the modification is completed by, for example, homologous recombination (HR) or non-homologous end joining (NHEJ).

[0035] In certain embodiments, the genome editing comprises an RNA-guided endonuclease and a guide RNA (gRNA). The RNA-guided endonuclease can be selected from: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 / Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1 (also known as Cas12a), CasX, CasY, and homologs or modified versions thereof, Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo)), and homologs or modified versions thereof.

[0036] In certain embodiments, the RNA-guided endonuclease is a Cas protein. Cas proteins referred to herein can be any known or later identified Cas effector protein, such as, but not limited to, Cas9, Cas12a (Cpf1), Cas12b (C2c1), Cas13a (C2c2), C2c3, Cas13b, which can be derived from any suitable source and thus can include different orthologs from a variety of (prokaryotic) organisms. In certain embodiments, the Cas protein is Cas9, such as Staphylococcus aureus Cas9 (SaCas9) or Streptococcus pyogenes Cas9 (SpCas9). In certain embodiments, the Cas protein is Cas12a, such as from Acidaminococcus sp., such as Acidaminococcus sp. BV3L6 Cpf1 (AsCas12a), or Lachnospiraceae bacterium Cas12a, such as Lachnospiraceae bacterium MA2020 or Lachnospiraceae bacterium MD2006 (LBCas12a).

[0037] In certain embodiments, the genome editing is a CRISPR / Cas system, which recruits a Cas enzyme protein to a target locus by a guide RNA (gRNA) to complete the modification, the gRNA comprises a guide sequence having complementarity to a target sequence in the target locus. In certain embodiments, the gRNA can be a chimeric guide RNA or a single guide RNA (sgRNA). In certain embodiments, the gRNA comprises a guide sequence and a tracr mate sequence (or direct repeat sequence). In certain embodiments, the gRNA comprises a guide sequence, a tracr mate sequence (or direct repeat sequence), and a tracr sequence. In certain embodiments, the CRISPR-Cas system described herein does not comprise and / or does not rely on the presence of a tracr sequence (e.g., if the Cas protein is Cas12a).

[0038] In certain embodiments, the RNA-guided endonuclease and its guide RNA can be introduced into the maize cell by a recombinant DNA construct or a vector. In certain embodiments, the RNA-guided endonuclease and its guide RNA are introduced into the maize cell by the same or different recombinant DNA constructs or vectors. In certain embodiments, the recombinant DNA construct or vector is integrated or not integrated into the genome of the maize cell.

[0039] Indel or point mutation

[0040] In certain embodiments, the modification comprises an indel mutation or a point mutation.

[0041] In certain embodiments, the modification is located in the coding region (e.g., exon and / or intron) of the DWF4 gene. In certain embodiments, the modification is located in the 1st exon of the DWF4 gene.

[0042] In certain embodiments, the modification is introduced by CRISPR / Cas.

[0043] In certain embodiments, step (1) comprises introducing into a maize cell a Cas proteinase (e.g., Cas9 proteinase) and a guide RNA (gRNA). In certain embodiments, the gRNA directs the Cas proteinase (e.g., Cas9 proteinase) to the endogenous DWF4 gene via base pairing, inducing a DNA break (e.g., a double-stranded DNA break or a single-stranded DNA break (nick)) at the target site, and the modification is completed by, e.g., homologous recombination (HR) or non-homologous end joining (NHEJ). In certain embodiments, the gRNA targets a coding region (e.g., an exon, such as exon 1) of the endogenous DWF4 gene. In certain embodiments, the gRNA targets the nucleotide sequence set forth in SEQ ID NO: 5. In certain embodiments, the gRNA and Cas proteinase (e.g., Cas9 proteinase) are introduced into the maize cell via one or more recombinant DNA constructs or vectors. In certain embodiments, the recombinant DNA construct or vector does not integrate into the genome of the maize cell.

[0044] In certain embodiments, the Cas proteinase can be linked to a base editing domain (e.g., a deaminase domain, such as an adenine deaminase and / or a cytosine deaminase) to form a base editor, and the base editor is guided / targeted to the target nucleic acid under the guidance of the gRNA, thereby editing the target nucleic acid.

[0045] In certain embodiments, the modification is introduced via TALEN technology.

[0046] In certain embodiments, step (1) comprises introducing into a maize cell a TALE-nuclease. In certain embodiments, the TALE-nuclease targets the endogenous DWF4 gene via base pairing by a nucleic acid binding domain contained therein, inducing a DNA break (e.g., a double-stranded break) at the target site, and the modification is completed by, e.g., homologous recombination (HR) or non-homologous end joining (NHEJ).

[0047] In certain embodiments, the modification is introduced via ZFN technology. In certain embodiments, step (1) comprises introducing into a maize cell a zinc finger nuclease. In certain embodiments, the zinc finger nuclease targets the endogenous DWF4 gene via base pairing by a zinc finger DNA binding domain contained therein, inducing a DNA break (e.g., a double-stranded break) at the target site, and the modification is completed by, e.g., homologous recombination (HR) or non-homologous end joining (NHEJ).

[0048] DNA fragment insertion

[0049] In certain embodiments, the modification comprises insertion of a DNA fragment, e.g., a large fragment insertion, such as an insertion of greater than 50 bp (e.g., greater than 60 bp, 70 bp, 80 bp, 90 bp, or 100 bp).

[0050] In certain embodiments, the modification is achieved by transposon technology (e.g., CRISPR-based transposase), Programmable Addition via Site-specific Targeting Elements (PASTE) technology, prime editing (PE) technology, or TnpB technology.

[0051] In certain embodiments, the insertion is at a position in the coding region (e.g., exon and / or intron) of the endogenous DWF4 gene. In certain embodiments, the insertion is at a position in the 2nd exon of the endogenous DWF4 gene. In certain embodiments, the insertion is at a position corresponding to between positions 1401 and 1402 of reference sequence SEQ ID NO: 4. In certain embodiments, the insertion is at a position corresponding to between Chr1:30516741 bp and 30516742 bp of the B73 reference genome sequence (NAM-5.0 version).

[0052] In certain embodiments, the inserted DNA fragment is greater than about 100 bp in length. In certain embodiments, the inserted DNA fragment is about 100-1000 bp, about 100-900 bp, about 100-800 bp, about 100-700 bp, about 100-600 bp, about 100-500 bp, e.g., about 100-400 bp, about 200-400 bp, about 200-300 bp, about 250-300 bp, about 250-290 bp, about 260-280 bp, about 270-280 bp, e.g., about 273 bp in length.

[0053] In certain embodiments, the inserted DNA fragment comprises the sequence set forth in SEQ ID NO: 1.

[0054] Knockdown or silencing

[0055] In certain embodiments, the disruption is achieved by knocking down or silencing the DWF4 gene.

[0056] In certain embodiments, the disruption is achieved by introducing a silencing molecule targeting the DWF4 gene, selected from an RNA interference molecule or an antisense nucleic acid molecule. In certain embodiments, the silencing molecule is complementary to at least a portion of the sequence of the DWF4 gene or an RNA transcript thereof.

[0057] In certain embodiments, the RNA interference molecule directly acts on the coding region of the DWF4 mRNA, inhibits translation of DWF4 or binds to the target mRNA and causes its degradation. In certain embodiments, the RNA interference molecule acts on the non-coding region of the DWF4 mRNA, inhibits translation of DWF4. In certain embodiments, the RNA interference molecule directly inhibits transcription of DWF4. In certain embodiments, the RNA interference molecule comprises dsRNA, siRNA, shRNA.

[0058] In certain embodiments, the antisense nucleic acid molecule base pairs with the DWF4 gene, inhibits expression of DWF4, in certain embodiments, the antisense nucleic acid molecule binds to DWF4 mRNA, inhibits translation of DWF4. In certain embodiments, the antisense nucleic acid molecule binds to the DNA sequence of DWF4, inhibits transcription of DWF4. In certain embodiments, the antisense nucleic acid molecule inhibits post-transcriptional processing and modification of DWF4 mRNA, thereby inhibiting translation of DWF4. In certain embodiments, the antisense nucleic acid molecule comprises antisense RNA, antisense DNA and ribozyme.

[0059] In certain embodiments, the nucleotide sequence encoding the silencing molecule is present on a vector. In certain embodiments, the vector is integrated or not integrated into the genome of the corn cell.

[0060] Exemplary protocol

[0061] In certain embodiments, step (1) comprises using gene editing to transform a vector comprising a nucleic acid sequence targeting the DWF4 gene in the corn cell, and the vector is capable of causing the DWF4 gene to be disrupted.

[0062] In certain embodiments, the vector is a CRISPR / Cas gene knockout vector comprising a nucleotide sequence encoding a Cas protease and a gRNA. In certain embodiments, the vector is pBUE411.

[0063] In certain embodiments, the vector comprises a nucleotide sequence encoding a gRNA targeting the sequence set forth in SEQ ID NO: 5. In certain embodiments, the vector further comprises a nucleotide sequence encoding a Cas nuclease (e.g., Cas9).

[0064] In another aspect, the present application also provides a guide RNA (gRNA) that binds to the nucleotide sequence set forth in SEQ ID NO: 5.

[0065] In another aspect, the present application also provides a vector comprising a gRNA as described above. In certain embodiments, the vector further comprises a nucleotide sequence encoding a Cas nuclease (e.g., Cas9).

[0066] III. DWF4 gene

[0067] In certain embodiments, the DWF4 gene: (i) comprises a nucleotide sequence as set forth in SEQ ID NO: 4, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) sequence identity thereto; or (ii) encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 11 or 24, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) sequence identity thereto.

[0068] Maize plants and products thereof

[0069] In a second aspect, the present application provides a maize plant or part, seed, cell, or progeny thereof in which the endogenous DWF4 gene is disrupted.

[0070] In certain embodiments, the maize plant or part, seed, cell, or progeny thereof has a reduced leaf angle, e.g., ear leaf angle, e.g., reduced by at least 20%, at least 30%, at least 40%, or at least 50%, as compared to a maize plant in which the endogenous DWF4 gene is not disrupted.

[0071] In certain embodiments, the maize plant or part, seed, cell, or progeny thereof is a tight- upper-loose-lower plant type.

[0072] In certain embodiments, the maize plant or part, seed, cell, or progeny thereof further has one or more traits selected from the group consisting of increased hundred kernel weight, increased kernel number per ear, increased ear weight, or increased yield per acre, as compared to a maize plant in which the endogenous DWF4 gene is not disrupted.

[0073] In certain embodiments, the maize cell is homozygous for the disruption.

[0074] In certain embodiments, the disruption comprises disrupting the function, expression level, activity, or a combination thereof of the DWF4 gene. In certain embodiments, the disruption comprises introducing a modification (e.g., a deletion, a substitution, an insertion, an inversion, a duplication, or a combination thereof of one or several amino acids) in the DWF4 gene or the DWF4 gene is knocked down or silenced.

[0075] In certain embodiments, the corn plant is artificially modified such that its endogenous DWF4 gene is disrupted.

[0076] In certain embodiments, the endogenous DWF4 gene of the corn plant or a part, seed, cell, or progeny thereof comprises a modification (e.g., an artificial modification).

[0077] In certain embodiments, the modification is located in the coding region and / or non-coding region of the endogenous DWF4 gene, e.g., a promoter, a 5’ UTR, an intron, an exon, a 3’ UTR, a terminator, and any combination thereof.

[0078] In certain embodiments, the modification comprises a deletion, a substitution, an insertion, an inversion, a duplication, or any combination thereof.

[0079] In certain embodiments, the modification results in a nonsense mutation, a missense mutation, a frameshift mutation, a splice site mutation, or any combination thereof.

[0080] In certain embodiments, the corn plant or a part, seed, cell, or progeny thereof is homozygous for the modification.

[0081] In certain embodiments, the modification comprises a first modification in a first allele and a second modification in a second allele, the first modification and the second modification being the same or different from each other but both resulting in disruption of the DWF4 gene.

[0082] In certain embodiments, the corn plant or a part, seed, cell, or progeny thereof further comprises a genome editing system for introducing the modification. In other embodiments, the corn plant or a part, seed, cell, or progeny thereof does not comprise a genome editing system for introducing the modification.

[0083] In certain embodiments, the genome editing system comprises at least one site-specific nuclease, e.g., an RNA-guided nuclease (e.g., a Cas nuclease), a zinc finger nuclease, a meganuclease, a TALE nuclease, a recombinase, a transposase, and any combination thereof.

[0084] In certain embodiments, the genome editing system is selected from the group consisting of CRISPR / Cas, TALEN, ZFN, transposon technology, PASTE technology, PE technology, base editor, and any combination thereof.

[0085] In certain embodiments, the genome editing system is as defined in the first aspect.

[0086] In certain embodiments, the genome editing system is present on one or more vectors. In certain embodiments, the vectors are integrated or not integrated into the genome of the com cell.

[0087] In certain embodiments, the modification comprises an indel mutation or a point mutation. In certain embodiments, the modification is located in the coding region (e.g., exons and / or introns) of the DWF4 gene. In certain embodiments, the modification is located in exon 1 of the DWF4 gene.

[0088] In certain embodiments, the com plant or part, seed, cell, or progeny thereof comprises a CRISPR / Cas system (e.g., a Cas nuclease (e.g., Cas9 nuclease) and a guide RNA (gRNA)) or one or more vectors encoding the CRISPR / Cas system used to introduce the modification. In certain embodiments, the gRNA targets the nucleotide sequence set forth in SEQ ID NO: 5.

[0089] In certain embodiments, the com plant or part, seed, cell, or progeny thereof does not comprise the CRISPR / Cas system or one or more vectors encoding the CRISPR / Cas system.

[0090] In certain embodiments, the modification comprises an insertion of a DNA fragment, for example a large fragment insertion, such as an insertion of greater than 50 bp (e.g., greater than 60 bp, 70 bp, 80 bp, 90 bp, or 100 bp). In certain embodiments, the insertion is located in the coding region (e.g., exons and / or introns) of the endogenous DWF4 gene. In certain embodiments, the insertion is located in the 2nd exon of the endogenous DWF4 gene. In certain embodiments, the insertion is located corresponding to between positions 1401 and 1402 of reference sequence SEQ ID NO: 4. In certain embodiments, the insertion is located corresponding to between Chr1 :30516741 bp and 30516742 bp of the B73 reference genome sequence (NAM-5.0 version). In certain embodiments, the inserted DNA fragment is greater than about 100 bp in length. In certain embodiments, the inserted DNA fragment is about 100-1000 bp, about 100-900 bp, about 100-800 bp, about 100-700 bp, about 100-600 bp, about 100-500 bp, e.g., about 100-400 bp, about 200-400 bp, about 200-300 bp, about 250-300 bp, about 250-290 bp, about 260-280 bp, about 270-280 bp, e.g., about 273 bp in length. In certain embodiments, the inserted DNA fragment comprises the sequence set forth in SEQ ID NO: 1.

[0091] In certain embodiments, the corn plant or part, seed, cell, or progeny thereof comprises an editing system (e.g., a transposon system, a PASTE system, or other DNA fragment site-directed insertion system) for introducing the insertion of the DNA fragment or a vector encoding the editing system.

[0092] In certain embodiments, the corn plant or part, seed, cell, or progeny thereof does not comprise the editing system or a vector encoding the editing system.

[0093] In certain embodiments, the endogenous DWF4 gene of the corn plant or part, seed, cell, or progeny thereof is knocked down or silenced. In certain embodiments, the corn plant or part, seed, cell, or progeny thereof comprises or does not comprise a silencing molecule targeting the DWF4 gene or a vector encoding the silencing molecule. In certain embodiments, the silencing molecule is as defined in the first aspect.

[0094] Exemplary protocol

[0095] In certain embodiments, the endogenous DWF4 gene in the com plant or part, seed, cell, or progeny thereof comprises an insertion sequence set forth in SEQ ID NO: 1 and a 5' flanking region consisting of positions 1-1401 of SEQ ID NO: 4 and a 3' flanking region consisting of positions 1402-7564 of SEQ ID NO: 4.

[0096] In certain embodiments, the disrupted endogenous DWF4 gene in the com plant or part, seed, cell, or progeny thereof comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 13-23.

[0097] In certain embodiments, the com plant or part, seed, cell, or progeny thereof is obtained by the method of the first aspect of the application.

[0098] In a third aspect, the application provides an article of manufacture comprising the com plant or part, seed, cell, or progeny thereof of the second aspect of the application.

[0099] In certain embodiments, the article of manufacture comprises genomic DNA of the com plant or part, seed, cell, or progeny thereof.

[0100] In certain embodiments, the article of manufacture is a food product, an agricultural product (e.g., a feed), a cosmetic product, a pharmaceutical product, or an industrial product.

[0101] In certain embodiments, the article of manufacture is selected from one or more of com ear, com on the cob, com silk, com pollen, com grits, com flour, crushed com, com meal, com oil, com starch, com steep liquor, com malt, com sugar, com syrup, margarine produced from com oil, unsaturated com oil, saturated com oil, com flakes, popcorn, ethanol and / or liquor produced from com, dried distillers grains with solubles (DDGS) produced from com fermentation, animal feed from com, cosmetics, and fillers.

[0102] In another aspect, the application also provides a method of producing the article of manufacture of the third aspect of the application, comprising obtaining the com plant or part, seed, cell, or progeny thereof of the second aspect of the application and producing the article of manufacture of the third aspect of the application therefrom. In certain embodiments, the com plant or part, seed, cell, or progeny thereof is obtained by the method of the first aspect of the application.

[0103] In certain embodiments, the part of the com plant of any of the preceding aspects is selected from the group consisting of kernels, pollen, ovules, flowers, shoots, roots, stalks, silks, tassels, ears, and leaves.

[0104] The present application also relates to a corn plant or part, seed, cell or progeny thereof of any of the preceding aspects for use in the production of a food product, an agricultural product (e.g., a feed), a cosmetic product, a pharmaceutical product or an industrial product.

[0105] In certain embodiments, the corn plant or part, seed, cell or progeny thereof is used in the production of a food product, a feed, a starch or for brewing.

[0106] In certain embodiments, the part of the corn plant is selected from the group consisting of a kernel, pollen, ovule, flower, shoot, root, stem, silk, ear, and leaf.

[0107] In certain embodiments, the corn plant or part, seed, cell or progeny thereof is used in the production of an article selected from the group consisting of a corn ear, corn on the cob, corn silk, corn pollen, corn grits, corn flour, crushed corn, corn meal, corn oil, corn starch, corn syrup, corn malt, corn sugar, corn syrup, margarine produced from corn oil, unsaturated corn oil, saturated corn oil, corn flakes, popcorn, ethanol and / or liquor produced from corn, dried distillers grains with solubles (DDGS) produced from corn fermentation, animal feed from corn, cosmetics, and fillers.

[0108] Methods of producing a corn plant

[0109] In a fourth aspect, the present application provides a method of producing a corn plant, the method comprising:

[0110] (1) providing a corn plant having a disrupted endogenous DWF4 gene as a first plant and a second plant;

[0111] (2) crossing the first plant and the second plant to obtain a progeny plant.

[0112] In certain embodiments, the first plant and the second plant are each independently selected from a corn plant as described in the second aspect of the present application or obtained from the method described in the first aspect of the present application.

[0113] In certain embodiments, the disruption of the endogenous DWF4 gene confers an altered plant architecture to the progeny plant, for example a reduced leaf angle, for example a leaf angle on the ear, for example a reduction of at least 20%, at least 30%, at least 40% or at least 50% compared to a corn plant in which the endogenous DWF4 gene is not disrupted.

[0114] In certain embodiments, the altered plant architecture is a tight upper loose lower plant architecture.

[0115] In certain embodiments, the disruption of the endogenous DWF4 gene also confers one or more altered traits to the progeny plants, such as increased hundred kernel weight, increased kernel number per ear, increased ear weight, or increased yield per acre compared to a maize plant in which the endogenous DWF4 gene is not disrupted.

[0116] In certain embodiments, the endogenous DWF4 gene of the first plant and the second plant comprises different modifications, respectively.

[0117] In certain embodiments, the endogenous DWF4 gene of the first plant comprises a modification comprising an indel mutation or a point mutation. In certain embodiments, the modification is in the coding region of the endogenous DWF4 gene. In certain embodiments, the modification is in exon 1 of the endogenous DWF4 gene. In certain embodiments, the first plant comprises or does not comprise one or more vectors encoding a CRISPR / Cas system (e.g., a Cas protease (e.g., Cas9 protease) and a guide RNA (gRNA)) used to introduce the modification. In certain embodiments, the gRNA targets the nucleotide sequence set forth in SEQ ID NO: 5. In certain embodiments, the endogenous DWF4 gene of the first plant that is disrupted comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 13-23.

[0118] In certain embodiments, the endogenous DWF4 gene of the second plant comprises a modification comprising an insertion of a DNA fragment. In certain embodiments, the location of the insertion is in the coding region of the endogenous DWF4 gene. In certain embodiments, the location of the insertion is in exon 2 of the endogenous DWF4 gene. In certain embodiments, the location of the insertion corresponds to between positions 1401 and 1402 of reference sequence SEQ ID NO: 4. In certain embodiments, the location of the insertion corresponds to between Chr1:30516741 bp and 30516742 bp of the B73 reference genome sequence (NAM-5.0 version). In certain embodiments, the inserted DNA fragment is about 100-500 bp in length, such as about 100-400 bp, about 200-400 bp, about 200-300 bp, about 250-300 bp, about 250-290 bp, about 260-280 bp, about 270-280 bp, such as about 273 bp. In certain embodiments, the inserted DNA fragment comprises the sequence set forth in SEQ ID NO: 1. In certain embodiments, the endogenous DWF4 gene of the second plant comprises the inserted sequence set forth in SEQ ID NO: 1 and a 5' flanking region consisting of positions 1-1401 of SEQ ID NO: 4 and a 3' flanking region consisting of positions 1402-7564 of SEQ ID NO: 4.

[0119] In a fifth aspect, the application provides a method of producing a hybrid corn plant, the method comprising crossing a first corn plant having a disruption of an endogenous DWF4 gene with a second corn plant lacking the disruption to produce a hybrid corn plant, wherein the disruption of the endogenous DWF4 gene confers an altered plant architecture to the hybrid corn plant.

[0120] In certain embodiments, the altered plant architecture comprises a reduced leaf angle, e.g., ear leaf angle, e.g., reduced by at least 20%, at least 30%, at least 40%, or at least 50%, as compared to the second corn plant.

[0121] In certain embodiments, the altered plant architecture is a tight upper loose lower plant architecture.

[0122] In certain embodiments, the disruption of the endogenous DWF4 gene also confers one or more altered traits to the hybrid corn plant, e.g., increased hundred kernel weight, increased kernel number per ear, increased ear weight, or increased yield per acre, as compared to the second corn plant.

[0123] In certain embodiments, the first corn plant is selected from the corn plants described in the second aspect of the application or obtained from the method described in the first aspect of the application.

[0124] In certain embodiments, the hybrid corn plant is homozygous for the disruption of the endogenous DWF4 gene.

[0125] In certain embodiments, the second corn plant is a corn inbred, e.g., W22, M017, B104, PH207, OSL476, TXB2-3, X1C14A, Z58, HuangC, C7-2, 9058, DMY1, DMY2, or DMY3.

[0126] In a sixth aspect, the application provides a method of producing a corn plant having an altered plant architecture, the method comprising the steps of:

[0127] (1) providing a first corn plant having a disruption of an endogenous DWF4 gene;

[0128] (2) crossing the first corn plant with a second corn plant lacking the disruption, optionally further comprising backcrossing and / or selfing; and

[0129] (3) selecting a progeny plant from step (2) by detecting in the progeny plant the presence of the disruption of the endogenous DWF4 gene, or the presence of a molecular marker linked to the disruption, thereby producing a corn plant having an altered plant architecture.

[0130] In certain embodiments, the altered plant architecture comprises a reduced leaf angle, e.g., a reduced leaf angle above the ear, e.g., at least 20%, at least 30%, at least 40%, or at least 50% reduction compared to a maize plant in which the endogenous DWF4 gene is not disrupted. In certain embodiments, the altered plant architecture is a tight upper loose lower plant architecture.

[0131] In certain embodiments, the first maize plant is selected from a maize plant defined in the second aspect or obtained from the method of the first aspect.

[0132] In the present context, a disrupted DWF4 gene can be referred to as a "lac1 allele", thus the expression "a plant in which the endogenous DWF4 gene is disrupted" also refers to a plant comprising a lac1 allele.

[0133] Detection of disruption

[0134] In certain embodiments, step (3) comprises detecting whether the endogenous DWF4 gene is disrupted. Methods for detecting gene disruption are well known to the person skilled in the art, including but not limited to nucleic acid sequencing, hybridization methods, amplification methods, etc.

[0135] In certain embodiments, the disruption comprises introducing a modification in the endogenous DWF4 gene, the modification comprising an indel mutation or a point mutation. In certain embodiments, step (3) comprises: (a) providing a sample comprising genomic DNA of the progeny plant; (b) performing a nucleic acid amplification reaction on the sample; and (c) sequencing the amplicon. In certain embodiments, step (b) comprises using a primer pair flanking the region where the modification is targeted.

[0136] In certain embodiments, the disruption comprises introducing a modification in the endogenous DWF4 gene, the modification comprising an insertion of a DNA fragment. In certain embodiments, step (3) comprises: (a) providing a sample comprising genomic DNA of the progeny plant; (b) performing a nucleic acid amplification reaction on the sample; and (c) detecting the length of the amplification product by gel electrophoresis. In certain embodiments, step (b) comprises using a primer pair flanking the region where the modification is targeted. In certain embodiments, step (b) comprises using the primer pair set forth in SEQ ID NOs: 2 and 3.

[0137] Detection of molecular marker

[0138] A molecular marker can be any nucleic acid sequence that is linked to the disrupted endogenous DWF4 gene, and can be derived from genomic nucleotide sequences or expressed nucleotide sequences (e.g., from spliced RNA, cDNA, etc.), and can also refer to nucleic acids used to detect the marker sequence (e.g., complementary to the marker sequence or to nucleic acids flanking the same, such as probes or primers (e.g., primer pairs) used to amplify and / or hybridize to the marker sequence).

[0139] A molecular marker can be detected by methods well recognized in the art, including but not limited to nucleic acid sequencing, hybridization methods, amplification methods (e.g., PCR-based sequence-specific amplification methods), restriction fragment length polymorphism detection (RFLP), isozyme marker detection, polynucleotide polymorphism detection by allele-specific hybridization (ASH), amplified variable sequence detection of plant genomes, self-sustained sequence replication detection, simple sequence repeat detection (SSR), single nucleotide polymorphism detection (SNP), and / or amplified fragment length polymorphism detection (AFLP).

[0140] In certain embodiments, the molecular marker is selected from alleles of presently known polymorphisms, and alleles of other candidate polymorphisms can be readily identified by one skilled in the art using any of the techniques well known in the art for discovering polymorphisms.

[0141] In certain embodiments, the molecular marker is selected from single nucleotide polymorphisms (SNPs), insertion-deletions (InDels), single feature polymorphisms (SFPs), simple sequence repeats (SSRs), amplified fragment length polymorphisms (AFLPs), random amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs).

[0142] In certain embodiments, the molecular marker is genetically less than 20 cM from the endogenous DWF4 gene. In certain embodiments, the molecular marker is genetically 1 cM, 2 cM, 3 cM, 4 cM, 5 cM, 6 cM, 7 cM, 8 cM, 9 cM, 10 cM, 11 cM, 12 cM, 13 cM, 14 cM, 15 cM, 16 cM, 17 cM, 18 cM, 19 cM, 19.5 cM, or 19.9 cM from the endogenous DWF4 gene.

[0143] In certain embodiments, step (3) comprises: (a) providing a sample comprising genomic DNA of the progeny plant; (b) detecting a molecular marker linked to the disrupted endogenous DWF4 gene.

[0144] In certain embodiments, step (b) comprises providing a specific primer pair for the molecular marker, and amplifying the molecular marker, and detecting the amplification product.

[0145] In certain embodiments, step (b) comprises using a specific probe and detecting hybridization signal.

[0146] In certain embodiments, step (b) comprises sequencing.

[0147] In certain embodiments, step (b) comprises gene chip (e.g., SNP chip) detection of the molecular marker.

[0148] In certain embodiments, the molecular marker is a SNP. Non-limiting examples of detecting a SNP include hybridization, amplification, sequencing, etc. In certain embodiments, the detecting comprises a SNP chip.

[0149] In certain embodiments, the progeny plant is homozygous for the disrupted endogenous DWF4 gene or a molecular marker linked thereto.

[0150] In certain embodiments, the method is used to introgress a lac1 allele into a maize inbred to impart altered plant architecture (e.g., reduced leaf angle above the ear, e.g., tight lower loose upper plant architecture) thereto. Accordingly, in certain embodiments, the second maize plant is a maize inbred. A maize inbred refers to an agronomically elite maize line that typically has commercially significant yield and / or commercially susceptible vigor, seed set, standability, shattering, abiotic / biotic resistance, or herbicide tolerance. Numerous maize inbreds are available and known to one of ordinary skill in the art of maize breeding. In certain embodiments, non-limiting examples of the maize inbred include W22, Mo 17, B104, PH207, OSL476, TXB2-3, X1C14A, Z58, Huang C, C7-2, 9058, DMY1, DMY2, or DMY3.

[0151] Methods of identifying a maize plant having altered plant architecture

[0152] In a seventh aspect, the present application provides a method of identifying a maize plant or a part, seed, cell, or progeny thereof having altered plant architecture, the method comprising detecting whether an endogenous DWF4 gene of the maize plant or a part, seed, cell, or progeny thereof is disrupted or whether a molecular marker linked to the disruption is present.

[0153] In certain embodiments, the altered plant architecture comprises a reduced leaf angle, e.g., a reduced leaf angle above the ear, e.g., a reduction of at least 20%, at least 30%, at least 40%, or at least 50%, as compared to a maize plant in which the endogenous DWF4 gene is not disrupted.

[0154] In certain embodiments, the altered plant architecture is a tight lower loose upper plant architecture.

[0155] In certain embodiments, the maize plant or part, seed, cell, or progeny thereof further comprises one or more traits selected from the group consisting of increased hundred seed weight, increased kernel number per ear, increased ear weight, or increased yield per acre compared to a maize plant in which the endogenous DWF4 gene has not been disrupted.

[0156] In certain embodiments, the disruption is as defined in the first aspect of the application.

[0157] In certain embodiments, the method comprises detecting whether the endogenous DWF4 gene has been disrupted. The detection of the disruption is as defined in the sixth aspect.

[0158] In certain embodiments, the method comprises detecting a molecular marker that is linked to the disruption. The molecular marker and the detection thereof are as defined in the sixth aspect. In certain embodiments, the gene disruption linked to the molecular marker comprises a modification in the endogenous DWF4 gene. The modification can be selected from the group consisting of an indel mutation or a point mutation, or an insertion of a DNA fragment as defined hereinabove.

[0159] In another aspect, the present application also provides a primer pair comprising a first primer and a second primer, wherein:

[0160] the first primer comprises: (i) a nucleotide sequence consisting of at least 15 contiguous nucleotides (e.g., 15-30 contiguous nucleotides, such as 15-25 contiguous nucleotides, 18-25 contiguous nucleotides) of a first target region on chromosome 1 of maize or the complement thereof, or (ii) a sequence having at least 80% identity compared to the nucleotide sequence of (i); wherein the first target region is located before the position corresponding to 30516741 bp of Chr1 of the B73 reference genome sequence (NAM-5.0 version);

[0161] the second primer comprises: (iii) a nucleotide sequence consisting of at least 15 contiguous nucleotides (e.g., 15-30 contiguous nucleotides, such as 15-25 contiguous nucleotides, 18-25 contiguous nucleotides) of a second target region on chromosome 1 of maize or the complement thereof, or (iv) a sequence having at least 80% identity compared to the nucleotide sequence of (iii); wherein the second target region is located after the position corresponding to 30516742 bp of Chr1 of the B73 reference genome sequence (NAM-5.0 version).

[0162] In certain embodiments, the length of the sequence amplified by the first primer and the second primer is no more than 8000 bp, for example, no more than 7000 bp, no more than 6000 bp, no more than 5000 bp, no more than 4000 bp, no more than 3000 bp, no more than 2000 bp.

[0163] In certain embodiments, the first primer comprises a sequence set forth in SEQ ID NO: 2 or a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) identical thereto, and the second primer comprises a sequence set forth in SEQ ID NO: 3 or a sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) identical thereto.

[0164] A method of planting for corn yield increase

[0165] In an eighth aspect, the present application provides a method of planting for corn yield increase, the method comprising: planting (i) the corn plant, or a part, seed, cell, or progeny thereof, of the second aspect of the present application, or (ii) the corn plant, or a part, seed, cell, or progeny thereof, obtained by the method of the first aspect of the present application, or (iii) the corn plant, or a part, seed, cell, or progeny thereof, obtained by the method of the fourth aspect, the fifth aspect, or the sixth aspect of the present application, or (iv) the corn plant, or a part, seed, cell, or progeny thereof, identified by the method of the seventh aspect, at a planting density no less than about 5000 plants per acre.

[0166] In certain embodiments, the planting density is no less than about 5500 plants per acre, no less than about 6000 plants per acre, no less than about 6500 plants per acre, no less than about 7000 plants per acre, or no less than about 7500 plants per acre. In certain embodiments, the planting density is about 5000 plants per acre to about 15000 plants per acre (e.g., about 5000 plants per acre to about 12000 plants per acre, about 6000 plants per acre to about 12000 plants per acre, about 7000 plants per acre to about 12000 plants per acre; e.g., about 7500 plants per acre, about 8000 plants per acre, about 9000 plants per acre, about 10000 plants per acre, or about 12000 plants per acre).

[0167] Definitions of terms

[0168] In the present application, unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be generally understood by one of ordinary skill in the art. Also, the methods of cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, and the like, described herein are in accordance with conventional techniques. In addition, for purposes of the present application, the following terms are defined with the following meanings.

[0169] As used herein, the term "DWF4 gene" has the meaning commonly known to those skilled in the art and encodes a cytochrome P450 protein, which is one of the catalyzing enzymes in the BR synthesis pathway and can be found in various public databases (e.g., GenBank: EF519871, Maize GDB: GRMZM2G065635) or can be referenced to the amino acid sequence set forth in SEQ ID NO: 11 or 24 and has at least 80% sequence identity thereto. The location of the DWF4 gene in the corn genome corresponds to 30515341:30522904 of chromosome 1 of the corn reference genome Zm-B73-REFERENCE-NAM-5.0. An exemplary sequence of the DWF4 gene can be referenced to SEQ ID NO: 4 or a sequence having at least 80% sequence identity thereto and can also be found in various public databases (e.g., Maize GDB: Zm00001eb009730). The DWF4 gene can also be referred to as ZmDWF4 gene in corn. Furthermore, the corn DWF4 gene is also referred to as lac1 (leaf angle architecture of smart canopy 1) gene according to the definition herein. Thus, in the context of the present application, "DWF4 gene" and "lac1 gene" have the same meaning and can be used interchangeably.

[0170] As used herein, the term "corresponding to" in the context of nucleic acid sequences or protein sequences means that, when a sequence to be compared (e.g., a nucleic acid sequence or an amino acid sequence) is aligned with a reference sequence, the nucleic acids or amino acids "corresponding to" the positions recited in the present application are those that align with these positions in the reference sequence, but are not necessarily located in these exact numerical positions with respect to the sequence to be compared. For example, the expression "corresponding to position 1401 of reference sequence SEQ ID NO: 4" means that, when the sequence to be compared is optimally aligned (i.e., to obtain the highest percentage of identity) with SEQ ID NO: 4, the position in the sequence to be compared that is equivalent to this position of SEQ ID NO: 4. The optimal alignment of sequences for comparison can be done by computerized implementations of known algorithms or by visual inspection. Existing sequence alignment and multiple sequence alignment algorithms include BLAST, ClustalW / ClustalW2 / Clustal Omega, etc.

[0171] As used herein, the term "plant" includes an explant, plant part, seedling, seedling plant, or whole plant at any stage of regeneration or development. The term "plant part" refers to any organ or intact tissue of a plant, such as a root, a shoot structure (e.g., stem, leaf), a flower (e.g., pollen, ovule, ear, inflorescence, calyx, petal, stamen, carpel, anther), a fruit, a seed (e.g., embryo, endosperm, seed coat), a protective tissue, a conducting tissue, a nutritive tissue, a mechanical tissue, a meristem, a propagule. The term "propagule" includes any plant part capable of growing into a whole plant.

[0172] As used herein, the term "maize" refers to Zea mays or corn and includes all plant varieties that can be bred with corn, including wild Zea species.

[0173] As used herein, the term "haploid plant" refers to a plant individual that contains only a haploid nuclear genome in its somatic cells. The haploid plants in the present invention can be maternal haploid plants, i.e., that have lost the paternal nuclear genome while retaining the maternal nuclear genome. Alternatively, the haploid plants in the present invention can be paternal haploid plants, i.e., that have lost the maternal nuclear genome while retaining the paternal nuclear genome. Typically, the maternal mitochondrial and plastid (e.g., chloroplast) genomes are retained in both maternal and paternal haploid plants.

[0174] As used herein, the term "gene" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term includes double- and single- stranded DNA and RNA. It also includes known types of modifications, for example, methylation, "capping," substitution of one or more of the naturally occurring nucleotides with an analog. Preferably, a gene contains coding sequences comprising DNA segments coding for a polypeptide. A "coding sequence" is a nucleotide sequence, which when transcribed into mRNA and translated into a polypeptide, a protein, or a precursor thereof. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, mRNA, cDNA, recombinant nucleic acid sequences, or genomic DNA, and in some instances can include introns.

[0175] As used herein, the term "locus" refers to one or more specific locations or sites on a chromosome at which a gene (e.g., a DWF4 gene) or genetic marker is found.

[0176] As used herein, the term "endogenous / endogenous" refers to a gene or allele that is present at its natural genomic location. The term "endogenous / endogenous" can be used interchangeably with "native." In certain embodiments, the endogenous gene or allele is a wild-type gene or allele. In certain embodiments, the endogenous gene is mutagenized. In certain embodiments, the expression, activity, and / or stability of the endogenous gene is modified.

[0177] As used herein, the term "plant genome" refers to the nuclear genome, mitochondrial genome, or plastid (e.g., chloroplast) genome of a plant cell.

[0178] As used herein, the term "gene editing" refers to a modification at a specific site in the genome of an organism by gene editing technology, including substitution, deletion, or addition of one or more bases, resulting in an increase or loss of function, thereby changing the genetic information and phenotypic characteristics of the organism. The term "gene editing technology" refers to a technology that modifies a specific site in the genome of an organism by a synthetic nucleotide fragment, common gene editing technologies include gene knockout, gene insertion, gene mutation, and gene recombination, etc.

[0179] As used herein, the term "nucleic acid" can be any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, which is not subject to any particular limitation in length. For nucleic acids used in the construction of recombinant constructs, it is preferred that the nucleic acid is DNA, as DNA is more stable and easier to manipulate than RNA.

[0180] As used herein, the term "primer" is an isolated nucleic acid that is annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then is extended along the target DNA strand by a polymerase (e.g., a DNA polymerase). Multiple pairs or sets of primers can be used for amplification of nucleic acid molecules, for example, by polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

[0181] As used herein, the term "transformation" is the process of introducing a heterologous nucleic acid (e.g., a nucleic acid construct, vector, expression cassette, etc.) into a host cell or organism. In particular, "transformation" refers to the process by which a DNA molecule is transiently transformed into an organism, or stably transformed or integrated into the genome of an organism or is capable of autonomous replication. Transformation techniques for plants and plant cells are well known in the art and can include, for example, electroporation, microinjection, Agrobacterium-mediated transformation, and ballistic transformation.

[0182] As used herein, the term "transformed / transgenic / recombinant" refers to a host organism, such as a bacterium or a plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such an extrachromosomal molecule is capable of autonomous replication. A transformed cell, tissue, or plant is understood to include not only the end product of the transformation process, but also transgenic progeny thereof. A "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism or naturally occurring organism, such as a bacterium or a plant.

[0183] As used herein, the term "event" refers to a recombinant plant produced by transforming and regenerating plant cells or tissues with heterologous DNA (e.g., an expression cassette including a gene of interest). The term "event" includes the original transformant of the heterologous DNA and / or progeny of the transformant. The term "event" also includes progeny produced by sexual outcrossing between the transformant and another maize line. Even after repeated backcrossing to a recurrent parent, the heterologous DNA (e.g., insert DNA and flanking DNA) from the transformed parent is present at the same chromosomal location in the hybrid progeny or is capable of maintaining autonomous replication in the hybrid progeny. Typically, transformation of plant tissue results in multiple events, and a particular event is selected based on expression of the heterologous DNA or other desirable characteristics.

[0184] As used herein, the term "transposition" refers to the action of a transposase in excising a transposon from one polynucleotide and then integrating it into a different site in the same polynucleotide or a second polynucleotide. A "transposase" is a polypeptide that catalyzes excision of a corresponding transposon from a donor polynucleotide (e.g., a vector) and, assuming the transposase is not integration-defective, subsequent integration of the transposon into a target nucleic acid.

[0185] As used herein, the term "transposon" refers to a polynucleotide that can be excised from a first polynucleotide (e.g., a vector) and integrated into a second location in the same polynucleotide or a second polynucleotide (e.g., the genome of a cell or extrachromosomal DNA) by the action of a corresponding transposase in trans. A transposon comprises a first transposon end and a second transposon end, which are polynucleotide sequences recognized and transposed by the transposase. A transposon typically also comprises a first polynucleotide sequence located between the two transposon ends, such that the first polynucleotide sequence is transposed along with the two transposon ends by the action of the transposase. A natural transposon typically comprises DNA encoding a transposase that acts on the transposon.

[0186] As used herein, the term "CRISPR" refers to a locus containing multiple short direct repeat sequences found in approximately 40% of sequenced bacteria and 90% of sequenced archaea. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) proteins and non-coding RNA elements that can program the specificity of CRISPR-mediated nucleic acid cleavage. The CRISPR system is divided into three classes, with class I and class III requiring multiple CRISPR-associated (Cas) proteins to function together, whereas class II systems require only one Cas protein (e.g., Cas9) to exhibit endonuclease activity. Thus, the type II CRISPR / Cas system (e.g., Cas9) is most widely used in many cases. The term "guide RNA (gRNA)" refers to an RNA molecule used to guide Cas protein in the CRISPR / Cas system for targeted editing in the genome to direct an endonuclease to a target site in the genome of a plant by base pairing or hybridization to cause a double-stranded DNA break or nick at or near the target site. The gRNA can be transformed or introduced into a plant cell or tissue in the form of a gRNA molecule, or in the form of a recombinant DNA molecule, construct or vector comprising a transcribable DNA sequence encoding the gRNA operably linked to a plant-expressible promoter. The gRNA can include, for example, a CRISPR RNA (crRNA), a single guide RNA (sgRNA), or any other RNA molecule that can guide or direct an endonuclease to a specific target site in the genome. The term "single guide RNA (sgRNA)" refers to an RNA molecule comprising a crRNA covalently linked to a tracrRNA by a linker sequence, which can be expressed as a single RNA transcript or molecule. The term "Cas9" protein is an endonuclease that cleaves nucleic acids, in the CRISPR / Cas9 system, the sgRNA recognizes and binds to the target DNA, and Cas9 mediates cleavage of the target DNA. The requirement for cleavage is the presence of a conserved protospacer adjacent motif (PAM) downstream of the DNA target site, which typically has the sequence 5'-NGG-3', and less commonly the sequence 5'-NAG-3'.

[0187] As used herein, the term "TALEN" (synonymous with "TAL-enzyme") refers to a transcription activator-like (TAL) effector nuclease, an artificial restriction enzyme created by fusing a transcription activator-like effector (TAL, i.e., TALE) DNA binding domain to a nuclease domain. The carboxy-terminal end of the TALEN contains a nuclease domain (e.g., PvuII, MutH, TevI, FokI, AlwI, MlyI, SbfI, SdaI, StsI, CleDORF, Clo051, or Pept071) that recognizes specific DNA base pairs with the aid of a TALE (derived from plant pathogenic Xanthomonas bacteria) and cleaves the DNA strand at the specific site.

[0188] As used herein, the term "ZFN" refers to a Zinc finger nuclease, an artificially synthesized restriction enzyme that was the first to be used for genome editing, which is a heterodimer comprising a DNA-binding zinc finger protein (ZFP) domain and a non-specific Fokl nuclease domain. The Fokl nuclease of the DNA cleavage domain must dimerize to cleave DNA. If two zinc finger binding sites are palindromic, then a zinc finger nuclease monomer can cleave the target site. The term "zinc finger nuclease" is broad and includes monomeric zinc finger nucleases that can cleave double-stranded DNA without assistance from another zinc finger nuclease. The term "zinc finger nuclease" is also used to refer to one or both members of a pair of zinc finger nucleases engineered to work together to cleave DNA at the same site. ZFN technology has been used to modify endogenous genes in a variety of organisms.

[0189] As used herein, the term "megabase meganuclease" refers to a unique enzyme with high activity and long recognition sequence (>14 bp) that results in site-specific cleavage of a target DNA. Engineered versions of naturally occurring megabase meganucleases typically have extended DNA recognition sequences (e.g., 14 to 40 bp). Megabase meganucleases can comprise a backbone or base enzyme selected from the group consisting of I-Crel, I-Ceul, I-Msol, I-Scel, I-Anil, and I-Dmol.

[0190] As used herein, the term "RNA interference" or "RNAi" refers to the silencing or reduction of gene expression mediated by small double-stranded RNA. It is a process of sequence-specific post-transcriptional gene silencing in plants and animals initiated by inhibitory RNA (iRNA) that is homologous in its duplex region to the sequence of the silenced gene. The silenced gene can be an endogenous or exogenous gene of an organism, can be integrated into a chromosome, or can be present in a transfection vector that is not integrated into the genome. The expression of the silenced gene is inhibited completely or partially. RNAi can also be viewed as the ability to inhibit the function of a target RNA; the function of the target RNA can be complete or partial. The term "RNAi molecule" refers to a single- or double-stranded RNA molecule that comprises both a sense and an antisense sequence. For example, an RNAi molecule can be a double-stranded polynucleotide molecule comprising a sense and an antisense region that are self-complementary, wherein the antisense region has complementarity to a target nucleic acid molecule. An RNAi molecule can also be a single-stranded hairpin polynucleotide having a sense and an antisense region that are self-complementary, wherein the antisense region has complementarity to a target nucleic acid molecule, or can be a circular single-stranded polynucleotide having two or more loop structures, and a stem comprising a sense and an antisense region that are self-complementary, wherein the antisense region has complementarity to a target nucleic acid molecule, and wherein the circular polynucleotide can be processed in vivo or in vitro to generate an active molecule capable of mediating RNAi.

[0191] As used herein, the term "operably linked" refers to functional linkage between a promoter or other regulatory element and a relevant transcribable DNA sequence or coding sequence of a gene (or transgene) such that the promoter, etc. exerts the necessary effects or functions to initiate, assist, influence, cause, and / or promote the transcription and expression of the relevant transcribable DNA sequence or coding sequence, at least in some cell, tissue, developmental stage, and / or conditions. Two transcribable DNA sequences can also be "operably linked" to each other if their transcription is controlled by a common promoter or other regulatory element.

[0192] As used herein, "linkage" of genes refers to different genes on the same chromosome being linked in groups with each other and segregating together into the same gamete in inheritance. The "physical distance" between genes that are linked to each other is the actual physical distance, expressed in base pairs (bp), kilobase pairs (kb), or megabase pairs (Mb). The "genetic distance" between genes on the same chromosome is measured by the frequency of crossing over or recombination frequency (RF) and is expressed in centimorgans (cM). One cM corresponds to a recombination frequency of about 1%. If no recombinants are found, the RF is zero and the genes are physically very close together or they are identical. The farther apart two genes are, the higher the RF.

[0193] As used herein, the term "molecular marker" is a DNA sequence (e.g., a gene or a partial fragment thereof) that displays polymorphism between two or more plants of the same species and is located on a chromosome near the target gene. The statement "molecular marker linked to the target allele" means that the molecular marker and the target allele segregate together into the same gamete during inheritance, thus the presence of the molecular marker indicates the presence of the target allele in the plant containing the molecular marker. Useful polymorphisms include single nucleotide polymorphisms (SNPs), insertions / deletions (InDels), single-feature polymorphisms (SFPs), simple sequence repeats (SSRs), amplified fragment length polymorphisms (AFLPs), random amplified polymorphic DNA (RAPDs), restriction fragment length polymorphisms (RFLPs), etc. Molecular markers can be used to identify specific DNA sequences, or locations on the genome or chromosome, or to identify introgression fragments of genes. "Gene introgression" refers to a chromosome segment (or part or region of chromosome) that has been introduced into another plant of the same or closely related species through hybridization or traditional breeding techniques (such as backcrossing). In other words, gene introgression is the result of breeding methods (such as backcrossing) expressed by the verb "gene introgression".

[0194] As used herein, the term "backcross" refers to a method by which progeny plants are repeatedly backcrossed with one of their parents. In a backcross scheme, the "donor" parent is a parental plant that possesses the desired gene or locus to be introgressed. The "recipient" parent (used once or multiple times) or "recurrent" parent (used twice or more) is a parental plant in which the gene or locus is introgressed.

[0195] As used in this article, the term "hybridization" refers to the fusion of gametes through pollination to produce offspring (e.g., cells, seeds, or plants).

[0196] As used herein, the terms “desired allele” and “target allele” are used interchangeably to refer to an allele associated with a desired trait (e.g., reduced leaf angle). In this context, the desired allele may refer to a disrupted DWF4 gene that confers a reduced leaf angle (e.g., a compact top and loose bottom growth habit) on maize plants; the disrupted DWF4 gene may also be referred to as the “lac1 allele.”

[0197] As used in this article, the term "SNP (single nucleotide polymorphism)" refers to a single nucleotide (base) difference site that exists between two parents at the genomic level.

[0198] As used in this article, the term "InDel (insertion-deletion)" refers to the difference in the genomes of two parents, that is, a certain number of nucleotide insertions or deletions in the genome of one parent relative to the other parent.

[0199] As used herein, the term “about” is intended to cover a specified amount of variation of ±10%, ±5%, ±1%, ±0.5%, or ±0.1% when referring to a measurable value.

[0200] Beneficial effects of the invention

[0201] This invention provides for the first time the key gene for cloning and regulating the ideal plant type of maize with "tight top and loose bottom" to tolerate high density. This allows for the rapid and precise targeted improvement of modern maize backbone inbred lines using technologies such as molecular marker-assisted selection, gene editing, and haploid breeding, providing a theoretical basis and technical support for the breeding of new high-yielding maize varieties that tolerate high density.

[0202] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0203] Figure 1 Phenotypic distribution in the F2 population: the angle between the second leaf on the ear of maize.

[0204] Figure 2 The phenotypic distribution of the leaf angle of the upper leaves of maize in the F2 population, among which, Figure 2 A shows the phenotypic distribution of the leaf angle in the upper leaves of maize in the F2 population constructed from wild-type (WT) and the maize inbred line Mo17. Figure 2 B is a phenotypic distribution of the leaf angle of the upper leaves of maize in the F2 population constructed from the mutant lac1 and the maize inbred line Mo17.

[0205] Figure 3 Linkage analysis of extreme phenotypic amplification.

[0206] Figure 4 : fine positioning of lac1, where, Figure 4 A represents the initial location of lac1 on chromosome 1.

[0207] Figure 4 B is the fine location of lac1 on chromosome 1.

[0208] Figure 5 Genotyping results in the F2 segregating population.

[0209] Figure 6 : NEWMOL vector map.

[0210] Figure 7 : pBUE411 vector map.

[0211] Figure 8 : Leaf angle phenotype analysis between wild type (WT) and leaf angle mutant lac1, in which, Figure 8 A is the whole plant phenotype picture of wild type (WT) and leaf angle mutant lac1, Figure 8 B is the phenotype data statistics of wild type (WT) and leaf angle mutant lac1.

[0212] Figure 9 : Comparison of leaf ear area phenotype between wild type (WT) and leaf angle mutant lac1, in which, Figure 9 A is the mature leaf ear picture of flag leaf second leaf, upper ear leaf and lower ear leaf of wild type (WT) and leaf angle mutant lac1, Figure 9 B is the phenotype data statistics of mature leaf ear area of flag leaf second leaf, upper ear leaf and lower ear leaf of wild type (WT) and leaf angle mutant lac1.

[0213] Figure 10 : Scanning electron microscope analysis of immature leaf collar and mature leaf ear of wild type (WT) and leaf angle mutant lac1, in which, Figure 10 A is the scanning electron microscope picture of early immature leaf collar (right), early immature leaf collar (middle) and mature leaf ear (left) of wild type (WT) and leaf angle mutant lac1, Figure 10 B is the width of early immature leaf collar, Figure 10 C is the width of middle immature leaf collar, Figure 10 D is the width of inner edge of mature leaf ear, Figure 10 E is the width of middle part of mature leaf ear, Figure 10 F is the width of outer edge of mature leaf ear, Figure 10 G is the length of mature leaf ear cell, Figure 11 H is the width of mature leaf ear cell.

[0214] Figure 11 : Analysis of the number of sclerenchyma cells at the proximal and distal ends of the leaf collar region of wild type and lac1, in which, Figure 11 A is the number of sclerenchyma cells at the proximal and distal ends of the leaf collar region, Figure 12 B is the number of sclerenchyma cells at the proximal and distal ends of the leaf collar region.

[0215] Figure 12 : Increase of leaf angle of lac1 overexpression plants, in which, Figure 12A is the whole plant phenotype photos of wild type (B104) and 2 lac1 overexpression transgenic events (DWF4-OE#1 and DWF4-OE#2), Figure 12 B is the relative expression of lac1 in wild type (B104) and 2 lac1 overexpression transgenic events (DWF4-OE#1 and DWF4-OE#2), Figure 13 C is the comparison of phenotype statistics of wild type (B104) and 2 lac1 overexpression transgenic events (DWF4-OE#1 and DWF4-OE#2).

[0216] Figure 13 : The leaf angle of lac1 knockout induction lines is reduced, wherein, Figure 13 A is the CRISPR / Cas9 editing site, Figure 13 B is the knockout target sequence of 3 homozygous editing lines, Figure 13 C is the whole plant phenotype photos of wild type (B104) and 3 homozygous editing lines, Figure 14 D is the comparison of leaf angle statistics of wild type (B104) and 3 homozygous editing lines.

[0217] Figure 14 : The leaf angle of the progeny plants of the cross between lac1 knockout induction lines and their respective cross lines is reduced, wherein, Figure 14 A is the CRISPR / Cas9 editing site and the knockout target sequence of the progeny plants of the cross between lac1 knockout induction lines and their respective cross lines, Figure 15 B-14K is the whole plant phenotype photos and the comparison of leaf angle statistics of the progeny plants of the cross between lac1 knockout induction lines and their respective cross lines: (14B-14C) B104, (14D-14E) PH207, (14F-14G) OSL476, (14H-14I) TXB2-3, (14J-14K) X1C14A.

[0218] Figure 16 : Aerial photos of high-density planting test of mutant lac1 and wild type (WT) (2021, Tieling). Figure 17 : Plants in high-density planting plots of wild type (WT) and leaf angle mutant lac1 (2021, Tieling, Liaoning).

[0219] Figure 17 : Yield test results of wild type (WT) and leaf angle mutant lac1 (2021, Tieling, Liaoning), wherein, Figure 18 A-17D is the comparison of ear traits between wild type (WT) and leaf angle mutant lac1 under 5 different planting densities: (17A) hundred-grain weight, (17B) single ear grain number, (17C) single ear weight, (17D) unit area yield.

[0220] Figure 18: Yield test results of wild type (WT) and leaf angle mutant lac1 (2021, Sanya, Hainan), wherein, Figure 19 A-18D is the comparison of ear traits between wild type (WT) and leaf angle mutant lac1 at 5 different planting densities: (18A) 100-grain weight, (18B) kernel number per ear, (18C) ear weight, (18D) yield per unit area.

[0221] Figure 19 : Hybrid constructed by lac1 x lac1-KO#2 increases maize yield under high planting density (2022, Shangzhuang, Beijing), wherein, Figure 20 A-19D is the comparison of ear traits between wild type hybrid (W22 x LH244) and improved hybrid (lac1 x lac1-KO#2) at 3 different planting densities: (19A) 100-grain weight, (19B) kernel number per ear, (19C) ear weight, (19D) yield per unit area.

[0222] Figure 20 : Hybrid constructed by lac1 x lac1-KO#2 increases maize yield under high planting density (2022, Tongzhou, Beijing), wherein, Figure 21 A-20D is the comparison of ear traits between wild type hybrid (W22 x LH244) and improved hybrid (lac1 x lac1-KO#2) at 3 different planting densities: (20A) 100-grain weight, (20B) kernel number per ear, (20C) ear weight, (20D) yield per unit area.

[0223] Figure 22 : Amino acid sequence alignment of wild type (WT) and leaf angle mutant lac1.

[0224] Figure 22 : Effect of mutant dpa1 backcross-improved inbred line, wherein, 1-1. Discovery of mutant lac1 A-22G is the statistical analysis of leaf angle phenotype of 7 maize improved inbred lines with wild type (WT) and mutant dpa1 alleles: (22A) Z58, (22B) HC, (22C) C7-2, (22D) 9058, (22E) DMY1, (22F) DMY2, (22G) DMY3.

[0225] Sequence information

[0226] Table 1: Information of sequences involved in the present application is described in the following table:

[0227]

[0228] DETAILED DESCRIPTION

[0229] The present application will now be described in the following non-limiting examples.

[0230] Those skilled in the art will appreciate that the examples describe the application by way of example only, and are not intended to limit the scope of the application as claimed. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of reagents or instruments is not specified, conventional products available commercially are used.

[0231] Example 1: Acquisition of lac1 gene

[0232] 1-2. Genetic analysis of mutant lac1

[0233] In summer 2018, in a test field in Tieling, Liaoning, more than ten mutant plants with compact leaf angle were found in a maize inbred line W22 background material. The mutant plants showed significantly reduced leaf angle in the upper, middle and lower parts, with the greatest reduction in the upper leaf angle, the middle leaf angle, and the least reduction in the lower leaf angle. The whole plant showed an ideal compact canopy with the upper leaves being compact and the middle and lower leaves being relatively flat. The mutant was named lac1 (leaf angle architecture of smart canopy 1). The mutant did not show the adverse phenotypes that mutants usually carry in important agronomic traits, and therefore, it was considered to have certain breeding value.

[0234] Figure 1

[0235] The mutant lac1 was crossed with its corresponding wild type inbred line W22 to produce an F2 segregation population. The obtained F2 segregation population was planted in the field, and about 10-15 days after pollination, the upper leaf angle of 112 maize plants was measured when the leaf angle was basically fixed. Data analysis found that in the F2 segregation population, there were 26 plants of lac1 type with reduced upper leaf angle and compact plant type, and 86 plants of wild type W22 leaf angle type, showing a bimodal distribution of leaf angle size, which is a typical characteristic of single gene mutation. Figure 1 The chi-square test results showed that the segregation ratio of wild type WT and mutant lac1 in the F2 segregation population was in accordance with 3:1 (χ 2 = 0.19 < χ 2 (0.05, 1) = 3.84; Table 2). 1-3. Preliminary mapping and fine mapping of lac1 gene The results of Table 1 and Table 2 showed that the lac1 mutant phenotype was controlled by a single gene, and the mutant was a recessive mutant.

[0236] Table 2: Genetic analysis of mutant lac1

[0237]

[0238] χ 2 (0.05, 1) = 3.84

[0239] Figure 2

[0240] Genetic mapping of the lac1 mutant. First, the compact leaf angle mutant lac1 and the wild-type (WT) were used as male parents and crossed with Mo17 to produce the F1 generation. In the F1 plants, the leaf angle of the maize plants was normal, indicating that this mutant is recessive. In 2020, F2 populations (7400 plants each) of wild-type (WT) and Mo17, as well as mutants lac1 and Mo17, were planted at the Tieling experimental base. Fifteen days after pollination, after the plant architecture was fixed, the leaf angle phenotype above the ear was investigated and statistically analyzed. The phenotypic distribution showed that the leaf angle phenotype distribution in the F2 populations constructed from wild-type (WT) and Mo17 conformed to a normal distribution. Figure 2 A); while in the F2 populations generated by the mutants lac1 and Mo17, the leaf angle phenotypic distribution showed a bimodal distribution. 1-3-1. Chromosome landing B) This indicates that the lac1 mutant phenotype is regulated by a single gene in this F2 population. Based on the above results, it can be inferred that the F2 population constructed from the mutants lac1 and Mo17 is suitable for lac1 gene localization, and based on the leaf angle phenotype distribution of the two populations, it can be preliminarily determined that individual plants with an ear leaf angle of less than 20 degrees are the extreme leaf angle phenotype, that is, the individual plant phenotype controlled by the homozygous lac1 genotype.

[0241] 1-3-2. Fine mapping

[0242] Chromosome landing analysis was performed on 200 individual plants from the F2 segregating population generated by the leaf angle compact mutants lac1 and Mo17. First, two pairs of Indel molecular markers were developed on each maize chromosome, located at the head and tail ends of the chromosome, respectively. For the 10 maize chromosomes, homozygous Mo17 and homozygous W22 genotypes were screened using the two pairs of molecular markers, and linkage analysis was performed with the leaf angle phenotype of the F2 segregating population. The results showed a significant association between the markers located on chromosome 1 and the phenotype (Table 3).

[0243] Table 3: Validation of the Chromosome 1 effect

[0244]

[0245] Figure 3

[0246] The functional site of mutant lac1 was located on the first chromosome by chromosome landing method. Then, 7400 F2 single plants of lac1 and Mo17 were planted in Tieling in 2020, and 500 extreme small angle single plants (<20°) and 427 extreme large angle single plants (>20°) were screened according to the extreme phenotype of leaf angle.

[0247] In order to further confirm that the gene controlling the phenotype of lac1 is located on the first chromosome, a pair of Indel markers located on the first chromosome were selected, and the extreme phenotype of the F2 segregation population of lac1 and Mo17 was amplified and linked (agarose gel electrophoresis), and the results are shown in Figure 4

[0248] On the first chromosome, a plurality of Indel markers were developed. Using 114 extreme single plants in the F2 segregation population of lac1 and Mo17, the target gene was located between molecular markers M23 and M50 (A) by screening the cross single plants with extreme phenotype and genotype linkage analysis. Further molecular markers were added between M23 and M50, and a total of 43 extreme small angle cross single plants were screened therebetween. Combined with extreme small angle and genotype analysis, the positioning interval was finally narrowed to a positioning interval of 144 kb, and the interval contains a candidate gene (B). Figure 4 Figure 5 The candidate gene encodes a cytochrome P450 protein, which is a catalytic enzyme in the BR synthesis pathway.

[0249] By aligning the lac1 mutant and wild type sequences, it was found that the lac1 mutant gene sequence contains a 273 bp transposon insertion, and the insertion sequence is as follows:

[0250] GGAATAAAATTCTCTGCAGCGCTGACGCACGCCATTATCCGTACAGCACTATATCGATGAATGACACGCGTCCAAAATCAATAGCTAATGATGCTGGCTAATCGAGCCATTGATGACACGGCTAATCGAGCCGTTCATTCCTTAGGCAAAAAGCCAGGACGTCTGGACGAGCGGCTTTGAGATGTGAAGACAGACATATGTCATTCATCGAAGTGGTGCCGCACGGGTGACTGCATATATCACCGCTGCAGAGAATTTTATTCCCCCGCGCAG (SEQ ID NO: 1)​​

[0251] Mutant lac1 is crossed with wild type W22, and F2 segregation population is obtained by selfing. In F2 segregation population, plants are grouped by the phenotype of leaf angle, and two groups are obtained, one group with similar phenotype of mutant lac1, and one group with phenotype of wild type. DNA of plants in two groups is extracted, and molecular markers lac1-TE-2F (GCTGAGGAAGTTGAGCGAGA, SEQ ID NO: 2) and lac1-TE-1R (AACCGGTACATCCTGCAGAA, SEQ ID NO: 3) are used for amplification and agarose gel electrophoresis. As shown in the results, 1-4. Nucleic acid molecular information of lac1 gene insertion and deletion of 273 bp are carried in plants with phenotype of mutant lac1, while plants with phenotype of wild type carry 273 bp heterozygous genotype or 273 bp homozygous wild type. The above data show that the insertion site of 273 bp is the functional site leading to the phenotype of lac1 mutant, and the insertion of 273 bp leads to the premature termination of amino acid sequence on the protein of the gene.

[0252] 2-1. Construction of lac1 overexpression vector

[0253] The nucleic acid sequence of lac1 is obtained from maize GDB (https: / / www.maizegdb.org / ), for example, see chromosome: Zm-B73-REFERENCE-NAM-5.0: 1:30515341:30522904:-1, and the details are as follows:

[0254] (1) Promoter region: upstream of 5' UTR

[0255] (2) Gene region: as shown in the following genomic sequence, the sequence marked with double underlines is the coding region of the genome

[0256] (3) UTR region: the 5' UTR region is the nucleic acid sequence upstream of ATG, which is marked with single underlines; the 3' UTR is the nucleic acid sequence downstream of TGA, which is marked with single underlines.

[0257] The sequence of lac1 gene (SEQ ID NO: 4) is as follows:

[0258]

[0259]

[0260] Among them, the insertion site of 273 bp in lac1 mutant is located between the 1401th base and the 1402th base of the above lac1 gene sequence.

[0261] The sequence of the protein encoded by the wild-type lac1 gene can be found in the maize GDB database under accession number GRMZM2G065635, or the sequence shown below:

[0262] MGAMMASITSELLFFLPFILLALLALYTTTVAKCHGTHPWRRQKKKRPNLPPGARGWPLV GETFGYLRAHPATSVGRFMERHVARYGKIYRSSLFGERTVVSADAGLNRYILQNEGRLFECS YPRSIGGILGKWSMLVLVGDAHREMRAISLNFLSSVRLRAVLLPEVERHTLLVLRSWPP SDGTFSAQHEAKKFTFNLMAKNIMSMDPGEEETERLRLEYITFMKGVVSAPLNFPGTAYWK ALKSRASILGVIERKMEDRLEKMSREKSSVEEDDLLGWALKQSNLSKEQILDLLLSLLFAG HETSSMALALAIFFLEGCPKAVQELREEHLLIARRQRLRGASKLSWEDYKEMVFTQCVIN ETLRLGNVVRFLHRKVIRDVHYNGYDIPRGWKILPVLAAVHLDSSLYEDPSRFNPWRWKS NAPSSFMPYGGGPRLCAGSELAKLEMAIFLHHLVLNFRWELAEPDQAFVYPFVDFPKGL PI RVQRVADDQGHRSVLTESTRG (SEQ ID NO: 11).

[0263] Example 2: Construction of lac1 transgenic vector

[0264] Component

[0265] (1) Using the genome of B73 as a reference sequence, design amplification primers lac1-CDS-F (ATGGGCGCCATGATGGCCTC, SEQ ID NO: 6) / lac1-CDS-R (TCAGCCTCTTGTGCTCTCGG, SEQ ID NO: 7) containing the full-length coding region (CDS) of the lac1 gene, and using the cDNA of inbred line B73 as the amplification template, amplify the full-length CDS of lac1. Purify the amplification product using a purification kit (OMEGA). The above amplification uses high-fidelity enzyme;

[0266] (2) Use endonucleases BamHI and SmalI to double-enzyme cut the vector NEWMOL. The enzyme cutting system is shown in Table 4 below:

[0267] Table 4: NEWMOL enzyme digestion system

[0268] Amount added Plasmid 2 μg BamHI 2 μl SmalI 2 μl 2.5 μl 10×T BSA 5 μl Distilled water To 50 μl Figure 6

[0269] Enzymatic digestion at 37°C for 6h, and storage at -20°C for standby;

[0270] (3) Recombination and ligation of the purified fragment and the enzyme-digested vector NEWMOL( Component ) as shown in Table 5 below:

[0271] Table 5: NEWMOL ligation system

[0272] Amount added Fragment 100 ng Vector after enzyme digestion 1 μl 2 × MultiF Seanless 5 μl Total 10 μl 2-2. Construction of Cas9 vector

[0273] Recombination and ligation at 50°C for 15min;

[0274] (4) Transformation of the ligated vector into E. coli DH5a for expansion, selection of single clones for sequencing and identification of positive single clones, extraction of positive single clone plasmids, and standby transformation of Agrobacterium.

[0275] Component

[0276] (1) Selection of target points. Log in to the website http: / / crispr.hzau.edu.cn / CRISPR2 / to select the lac1 gene target site, and the selection principles mainly include that the target site is located on the CDS and is as close to the start codon ATG as possible, the target site has high specificity, low off-target rate, high evaluation efficiency of the target site combined with the target sequence, and the GC content of the target site is 55%-60%, and finally the nucleic acid sequence GGAGGG CCAGCAGGATGAA (SEQ ID NO: 5) is selected as the target sequence of gRNA;

[0277] (2) Primer annealing and double-stranded synthesis. When synthesizing the target point, the sequence GGCG is added at the 5' end of the forward primer and the sequence AAAC is added at the 5' end of the reverse complementary sequence, 5ul of the forward primer and the reverse complementary primer, a total of 10ul system, and the annealing conditions are as follows:

[0278] (i) Denaturation at 95°C for 10min;

[0279] (ii) Annealing (Tm value minus 3-5°C) °C for 10min, and the slope (R) is 3-5%;

[0280] (iii) Incubation at 16°C;

[0281] (3) Enzyme digestion system of the vector pBUE411 as shown in Table 6:

[0282] Table 6: pBUE411 enzyme digestion system

[0283] Amount added pBUE411 1 μg 1 μl BsaI HF ]] cutsmart 5 μl To 50 μl ddH2O Figure 7

[0284] Incubate at 37°C for 4 hours or more;

[0285] (4) Target sequence and vector connection. Use Takara T4 ligase to introduce the target into the pBUE411 vector after enzyme digestion Component ), and the connection system is shown in Table 7:

[0286] Table 7: pBUE411 connection system

[0287] Amount added Target fragment 4.5 μl pBUE411 after enzyme digestion 0.5 μl T4 Ligase 0.5 μl 10 × T4 Ligase Buffer 1 μl To 10 μl ddH2O 2-3. Agrobacterium-mediated maize immature embryo transformation

[0288] Incubate at 16°C overnight;

[0289] (5) Transform the ligation product into E. coli DH5a. Select single colonies and use primers OsU3-FD3 (GACAGGCGTCTTCTACTGGTGCTAC, SEQ ID NO: 8) and the reverse complement of the target to identify colony PCR, and use OsU3-FD3 to sequence to verify the target sequence. Expand the positive single colony, extract the plasmid, and prepare for transformation of Agrobacterium.

[0290] 2-3-1. Preparation of Agrobacterium competence

[0291] Component

[0292] (1) Prepare YEB medium (pH = 7.0). The formula of liquid YEB medium is shown in Table 8:

[0293] Table 8: Formula of liquid YEB medium

[0294] Amount of reagent Yeast extract Beef extract 1.0g Peptone 5.0g Sucrose 5.0g Magnesium sulfate 5.0g Total 0.5g 2-3-2. Agrobacterium transformation (liquid nitrogen freezing method) 1L

[0295] For solid YEB medium, after the above reagents are fully dissolved, add 15 g of agar powder;

[0296] (2) Activation of Agrobacterium. Take Agrobacterium EHA105 strain stored at -80°C, streak on YEB solid plate (containing 0.1 mg / ml of rifampicin), and incubate at 28°C in the dark for 48 h;

[0297] (3) Agrobacterium expansion. Select Agrobacterium single colonies with a diameter of 2-3 mm (regular shape), inoculate into 5 ml of YEB medium containing rifampicin, set the culture temperature to 28°C and the rotation speed to 220 rpm, and shake culture overnight;

[0298] (4) Agrobacterium transfer. Take 2 ml of Agrobacterium overnight culture and transfer it to 50 ml of YEB liquid medium containing rifampicin. Set the culture temperature to 28°C and the shaking speed to 220 rpm for 3-4 h until the OD value of the bacterial solution is between 0.5 and 1.0.

[0299] (5) Agrobacterium collection and washing. Transfer the Agrobacterium solution to a 50 ml centrifuge tube and centrifuge at 5000 rpm for 5 min at 4°C to collect the bacterial cells. Remove the supernatant and add pre-cooled 100 mM NaCl solution to resuspend the bacterial cells. Centrifuge at 5000 rpm for 5 min at 4°C to collect the bacterial cells.

[0300] (6) Preparation and aliquotting of Agrobacterium competence. After removing the supernatant, add 1 ml of pre-cooled 20 mM CaCl2 solution to resuspend the bacterial cells. Aliquot 50 μl of the bacterial solution per tube, freeze in liquid nitrogen, and store at -80°C for later use.

[0301] 2-3-3. Agrobacterium-mediated maize immature embryo genetic transformation

[0302] (1) Thaw the Agrobacterium competence on ice (about 10 min) and add 1 μg of plasmid to the Agrobacterium competence. Mix well, and ice-bath for 30 min.

[0303] (2) After ice-bathing, quickly transfer to liquid nitrogen for 5 min, then quickly transfer to a 37°C water bath for 5 min, and finally ice-bath for 2 min.

[0304] (3) Then add 1 ml of YEB culture solution without antibiotics, and shake at 200 rpm in the dark at 8°C for 3-4 h.

[0305] (4) Centrifuge at 5000 rpm for 2 min at room temperature to collect the bacterial cells. After removing most of the supernatant, resuspend the bacterial cells and evenly spread them on the YEB solid medium containing antibiotics.

[0306] (5) Place the YEB plate with the bacterial cells on it in a 28°C constant temperature incubator and cultivate in the dark for 48 h.

[0307] 3-1. Phenotype analysis of wild type and mutant lac1

[0308] (1) Shake the bacteria. The bacteria stored at -80℃ were inoculated into 25ml YEB liquid medium containing antibiotics (0.05mg / ml rifampicin and 0.1mg / ml kanamycin) and cultured at 28℃ with 180rpm shaking overnight in the dark. On the second day, 10ml of the overnight culture was added to 40ml YEB liquid medium containing antibiotics (1:5 dilution) and cultured at 28℃ with 180rpm shaking until about 5pm. The bacteria were collected by centrifugation at 4000rpm for 10min, the supernatant was removed, and 25ml of induction medium was added to resuspend the bacterial pellet to an OD 660 = 0.1, and cultured at 28℃ with 180rpm shaking overnight in the dark. On the third day, the overnight culture was collected by centrifugation at 4000rpm for 10min, and an appropriate amount of infection medium (Inf + 200μM AS) was added to make the OD 660 = 0.8, and 2ml aliquots were prepared and stored in a 22℃ dark incubator for use.

[0309] (2) Embryo dissection. The 10-13 day old tassels of the B73-329 inbred line were removed from the cross, and the top 1cm was removed. A forceps was inserted into the upper axis and soaked in a sterilization solution (50% commercial bleach and one drop of Tween20) for 20min, making sure that the tassel was submerged. The tassel was then washed three times with sterile ddH2O or 70% ethanol for 45-60s and sterile water twice. The surface of the kernel was removed by one-third using a sterile scalpel, and the embryo was removed using the back of a sterile scalpel. The 1.2-2mm embryo was selected to ensure its integrity and placed in the infection medium. When the volume of the embryo reached 0.5ml, it was stored in a 22℃ dark incubator for use.

[0310] (3) Infection. The embryo in the infection solution was inverted and washed with a syringe, and the infection solution was removed. The embryo was washed with fresh infection solution 1-2 times, and the infection solution was finally added to submerge the embryo. The embryo was heat shocked at 45℃ for 5min, and the infection solution was removed with a syringe. The infection medium containing Agrobacterium was added to the embryo, and the embryo was inverted several times. The embryo was placed in a centrifuge tube at room temperature for 30min to complete the infection process. The infection solution was removed, and the integrity of the embryo was ensured.

[0311] (4) Co-culture. After the infection was completed, the embryo was gently transferred to the Co-culture medium with a long-handled spoon. The embryo was evenly separated with the back of the long-handled spoon (care was taken not to damage the embryo), and the embryo was turned into an arch shape with the tip of a sterile scalpel. The embryo was sealed and labeled, and cultured at 22℃ in the dark for 20-24h, not exceeding 24h. This step required attention, as the Co-culture medium contained AgNO3, and the operation should be performed as quickly as possible in the light.

[0312] (5) Screening culture. The young embryos on Co medium were transferred to S medium with sterilized small scalpel tips, 25 young embryos were evenly placed on each plate, the wounded or incomplete young embryos were discarded to avoid contaminating the medium, the plates were sealed and numbered, and cultured at 30°C in the dark for 2 weeks. AgNO3 was also contained in S medium, and the operation under light should be as fast as possible;

[0313] (6) Pre-differentiation. The callus in S medium was transferred to PR medium with sterilized tweezers, 15 calli were evenly distributed on each plate, and the non-growing young embryos, too small and brown calli could not be transferred to PR medium. The culture plates were sealed and numbered, and cultured at 26°C, 55% humidity, and light (light: dark 16:8) for one week, and a layer of white gauze was covered on the culture dish during culture.

[0314] (7) Differentiation. The well pre-differentiated and green calli in PR medium were gently transferred to R medium, 10 calli were evenly distributed on each culture plate, the culture plates were sealed and numbered, and cultured at 26°C for 1-2 months, and the medium was replaced every 2 weeks. During the replacement of the medium, larger seedlings or small seedlings could be transferred to rooting medium. The calli with dead leaves or brown calli were carefully removed, and the calli too large could be divided into small pieces with a diameter of about 5 mm;

[0315] (8) Rooting. The well differentiated corn seedlings in R medium were transferred to Rooting medium, the roots of the seedlings with roots were gently placed in the medium, covered with a lid, and cultured at 26°C for 2 weeks. If the seedlings grew well, the next step of transplanting was carried out, and if the seedlings did not grow roots for 20 days, they were discarded;

[0316] (9) Transplanting. The well-grown and rooted seedlings were transplanted into soil and placed in a greenhouse for careful management.

[0317] Example 3: Functional verification of lac1 gene

[0318] 3-1-1. Leaf angle variation analysis of wild type and mutant lac1

[0319] Figure 8

[0320] This study utilized the ideal high-density maize plant type lac1, whose genetic background is W22. The mutant lac1 and wild-type (WT) were intercropped in the field, and the phenotypic characteristics of leaf angles at maturity were measured. The angles of the upper leaf (two leaves below the flag leaf), the middle leaf (first leaf above the ear), and the lower leaf (first leaf below the ear) were measured. These three angles were used as indicators representing the leaf angles of the upper, middle, and lower parts of the maize plant to investigate maize plant type and leaf angles. Figure 8 A). Statistical analysis showed that, compared to the wild type, the mutant lac1 exhibited a highly significant decrease in the angles of the flag leaf and the second leaf from the top, the first leaf above the spike, and the first leaf below the spike, with the decrease being more pronounced in the upper leaves. 3-1-2. Histological and cytological analysis of leaf ear B). These results indicate that there were highly significant differences in the upper leaf angle, middle leaf angle, and lower leaf angle among near-isogenic lines, suggesting that the mutant lac1 is involved in the morphogenesis of maize plant architecture.

[0321] 3-1-2-1. Analysis of leaf ear area and proximal and distal axial leaf ear width

[0322] Figure 9

[0323] To investigate the reasons for the phenotypic differences in leaf angle between the mutant lac1 and the wild type, the auricles of the wild type and the mutant lac1 under natural conditions were examined. It was found that the auricles of the mutant lac1 had a smaller spatial extension and a significantly smaller auricle area compared to the wild type. Figure 9 A). Further statistical analysis of the auricle area in the leaf ring region showed that, compared to the wild-type LH244, the auricle area of ​​the flag leaf, second leaf from the top, upper leaf, and lower leaf of the lac1 knockout line was significantly reduced. 3-1-2-2. Scanning electron microscope analysis of leaf collar region B). The above results indicate that, compared to the wild type, the auricle area of ​​the lac1 mutant is significantly reduced in both its natural extension state and its actual auricle area, thereby causing differences in the leaf angle at the same leaf position.

[0324] Figure 10

[0325] To investigate the histological differences that cause the phenotypic differences in leaf angle between wild-type (WT) and mutant lac1, the auricles and ligules of early immature leaves (L4), mid-stage immature leaves (L3), and mature leaves (L2) were observed using scanning electron microscopy (SEM). The histological changes that lead to the phenotypic differences between the two types were studied, and the specific experiments are as follows:

[0326] Wild-type (WT) and mutant lac1 were grown in a glass greenhouse and matured to the V2 development stage. Leaf annulus samples from the early 4th leaf, mid-development 3rd leaf, and mature 2nd leaf of both wild-type (WT) and mutant lac1 were collected and fixed for scanning electron microscopy (SEM) observation. The histological development of the leaf annulus of the early immature leaf (L4), mid-development immature leaf (L3), and mature leaf auricle (L2) was mainly observed. First, the leaf annulus of the early immature leaf (L4) and mid-development immature leaf (L3) of wild-type (WT) and mutant lac1 were scanned. Figure 10 A, 10C). Scanning electron microscopy revealed that the leaf annulus of the early immature leaves (L4) and mid-immature leaves (L3) of the mutant lac1 was significantly narrower compared to the wild type (WT). Secondly, the auricles of mature leaves (L2) of both the wild type (WT) and the mutant lac1 were scanned. Figure 10 A). Scanning results showed that, compared to the wild type (WT), the mature auricle area of ​​the mutant was significantly reduced; the width of the mature auricles of the mutant lac1 was significantly smaller than that of the wild type (WT) near the midrib, in the middle, and at the margins. Figure 10 D-10F). The edge region of mature leaf auricles was magnified and scanned to observe changes in cell morphology. ImageJ software was used to statistically analyze the length and width of mature leaf auricle cells. A significance test showed no significant difference in cell length and width between the mutant lac1 and the wild-type (WT) mature leaf auricles. 3-1-2-3. Analysis of the number of layers and thickness of the sclerenchyma at the proximal and distal axial ends G-10H).

[0327] The above analysis revealed that the difference in mature auricle area between the wild-type (WT) and the mutant lac1 is mainly due to the difference in the width of the annulus, thus showing the difference in auricle area between the two. Since there was no significant difference in auricle cell size between the two, the difference in auricle area is mainly due to the number of cells in the annulus region. The difference in early annulus width further indicates that the difference in the number of cells in the mature annulus region stems from the number of cells that differentiate into the auricle region early in development. In conclusion, the difference in auricle area between the wild-type (WT) and the mutant lac1 is caused by the difference in the number of cells in the auricle, and this occurs in the early stages of auricle development.

[0328] Figure 11

[0329] Studies have shown that the unbalanced development of the proximal or distal end of the leaf ring cells, the development and mechanical strength of the mechanical tissue, the formation and size of the vascular bundle, and the composition of the cell wall can all affect the size of the leaf angle (Zhao et al., 2010; Ning et al., 2011; Zhao et al., 2013; Zhang et al., 2015). Therefore, it is particularly important to analyze the histocytology of the transverse section at the leaf ring. At the V3, V9, and V13 stages, the leaf rings (fully expanded leaves) of mature leaves V3, V9, and V13 were taken and fixed in FAA fixing solution to prepare paraffin samples for the differential study of the wild type and lac1 transverse section histocytology.

[0330] The paraffin sections of the wild type and lac1 leaf ring transverse sections were observed under a light microscope, and the number of layers of the proximal and distal end of the leaf ring area was counted at the V3, V9, and V13 stages. The results of the leaf ring transverse section analysis showed that, relative to the wild type, the number of layers of the proximal and distal end of the leaf ring area was significantly increased in the lac1 mutant ( 3-2. Expression or editing type and phenotype analysis of transgenic lines ). Therefore, the difference in the leaf angle between the wild type and the lac1 mutant is related to the thickness and number of layers of the proximal and distal end of the leaf ring area. In the transverse sections of both the wild type and the lac1 mutant, the more layers and the greater thickness of the proximal and distal end of the leaf ring area, the stronger the support of the leaf vein to the leaf, thereby making the lac1 mutant maintain a smaller leaf angle. The above results show that the difference in the leaf angle between the wild type and the lac1 mutant is partially caused by the number of layers or the thickness of the proximal end of the leaf ring area.

[0331] 3-2-1. Expression and phenotype analysis of overexpression transgenic lines

[0332] The genetic transformation of maize was completed by Beijing Auri Seeds Co., Ltd. and the Maize Functional Genomics Platform of China Agricultural University. The main transformation was the overexpression of the lac1 gene and the CRISPR / Cas9 gene knockout vector. In the spring of 2021, the DNA samples of the lac1 gene CRISPR / Cas9 knockout line T0 generation transformation strain were obtained for PCR amplification, identification of editing types, and self-crossing for breeding. In the autumn of 2022, the T1 generation seeds of the lac1 gene overexpression transgenic positive event were obtained, and the homozygous T2 generation family was obtained for phenotype detection in the winter of the same year by planting in the Hainan South Base of China Agricultural University.

[0333] Figure 12

[0334] This study first validated the biological function of the candidate gene lac1 using overexpression technology. First, the full-length coding region of lac1 was amplified, and an overexpression vector was constructed using the NEWMOL vector as the backbone. The NEWMOL vector carries an eGFP tag, and the kernels fluoresce red; positive kernels can be selected by color in the genetically transformed offspring. Next, the constructed overexpression vector containing the lac1 coding region was used for maize genetic transformation. In this study, maize genetic transformation was completed by Beijing ORG Seed Industry Co., Ltd., resulting in two Bar-positive lac1 gene overexpression T0 generation transgenic events (DWF4-OE#1 and DWF4-OE#2). Target gene amplification detection confirmed that both lac1 overexpression transgenic events contained the target gene, and no segregation occurred between the lines. Simultaneously, observation of the kernel color of the offspring from the two lac1 overexpression transgenic events revealed that the kernels carried red fluorescence, indicating positive kernels carrying the target vector; the observation results were consistent with the target gene PCR detection. Two pure lines overexpressing lac1 were subjected to adult plant phenotypic identification and molecular biological experiments, and self-pollination was carried out for seed production.

[0335] In the winter of 2022, lac1 overexpression materials were planted at the China Agricultural University Experimental Station in Hainan Province. Expression analysis and phenotypic investigation were conducted on two lac1 overexpression transgenic events. Leaf rings of immature leaves (L3 and L4) from V2 seedlings at the developmental stage were collected to detect the expression level of the lac1 gene. The results showed that, compared with wild-type B104, the expression level of the lac1 gene was significantly increased in both overexpression events. Figure 12 B); Approximately 15 days after pollination, the leaf angle phenotype of mature plants was measured. The results showed that, compared to wild-type B104, the leaf angles below the spike, above the spike, and between the flag and second-to-last leaves were significantly increased in both lac1 overexpression events, resulting in a more spreading plant architecture. 3-2-2. Mutation site and phenotype analysis of lac1 edited by CRISPR / Cas9 technology C).

[0336] The above results indicate that increased expression of the lac1 gene significantly increases the leaf angle in maize, resulting in a looser plant structure. Therefore, expression and phenotypic analysis of lac1-overexpressing lines confirms that lac1 participates in the regulatory pathway of maize leaf angle formation, thus clarifying the function of the lac1 gene.

[0337] Figure 13

[0338] The biological function of the candidate gene lac1 was verified using CRISPR / Cas9 gene editing technology. A specific sequence near the ATG of the lac1 coding region was selected, and a target site was designed using the online CRISPR / Cas9 target site design tool CRISPR P2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ). A specific CRISPR / Cas9 target site (same as SEQ ID NO: 5) was selected to construct a knockout vector for genetic transformation of the maize inducer line. Positive and editing site screening was performed on the positive transformation events of the maize inducer line, and three homozygous editing lines HI3-KO#1, HI3-KO#2 and HI3-KO#3 were obtained. Among them, the sequence of the unedited natural lac1 gene fragment contained in the HI3 selfing line is shown in SEQ ID NO: 12, and the sequences of the editing sites contained in the homozygous editing lines HI3-KO#1, HI3-KO#2 and HI3-KO#3 are shown in SEQ ID NO: 13-15, respectively. The upper, middle and lower leaf angles of the three editing lines were measured, and it was found that the leaf angles of the homozygous editing inducer lines at the three leaf positions were significantly reduced relative to the wild type inducer line, and the angle of the upper leaf angle reduction was greater Figure 14 A-13D). Thus, a "tight upper and loose lower" density-tolerant plant type similar to the lac1 mutant phenotype was produced, confirming that DWF4 is a functional gene that regulates leaf angle.

[0339] To further verify the function of lac1 gene and whether the plant type of elite inbred lines can be improved by induced editing, the induced line carrying lac1 editing vector (target sequence same as SEQ ID NO: 5) was crossed with B104, PH207, OSL476, TXB2-3 and X1C14A, and single haploids were screened in the offspring. DH lines were obtained by chromosome doubling with colchicine. In the DH lines, primers cas9-F (TCCAGAGATAAAGGCGCTTC, SEQ ID NO: 9) and cas9-R (GGCCGCTTCTTCTTCTGAC, SEQ ID NO: 10) were designed to amplify the genomic DNA of the DH lines and perform single-strand sequencing. Homozygous or heterozygous DH lines with edited target sites were selected for self-crossing or crossing with wild type. The selected DH lines were screened for genetic background using primers (primer sequences are shown in Table S1 in Zhong et al. (2019)) that exist polymorphisms between the induced line and the induced inbred line. DH lines that do not carry the genomic sequence of the induced line were selected, and the offspring of the DH lines were selected as seed, and homozygous knockout inbred lines with only the target site edited were screened. Using the above method, improved inbred lines of B104, PH207, OSL476, TXB2-3 and X1C14A inbred lines with only lac1 site edited were obtained, and were named as DH-B104-KO#1, DH-B104-KO#2, DH-PH207-KO#1, DH-PH207-KO#2, DH-OSL476-KO#1, DH-OSL476-KO#2, DH-TXB2-3-KO#1 and DH-X1C14A-KO#1, respectively. Figure 14 A). Wherein the B104, PH207, OSL476, TXB2-3 and X1C14A inbred lines contain the sequence of the unedited natural lac1 gene fragment as shown in SEQ ID NO: 12, and the DH-B104-KO#1, DH-B104-KO#2, DH-PH207-KO#1, DH-PH207-KO#2, DH-OSL476-KO#1, DH-OSL476-KO#2, DH-TXB2-3-KO#1 and DH-X1C14A-KO#1 contain the sequence of the edited site as shown in SEQ ID NO: 16-23, respectively.

[0340] The lac1 site of the improved inbred line with homozygous editing is planted in the field with the wild type inbred line, about 15-20 days after the plants are pollinated, the improved inbred line and the wild type are phenotyped, mainly measuring the leaf angle of the lower ear leaf, the upper ear leaf and the second leaf from the bottom. Data analysis shows that the leaf angle of the lower ear leaf, the upper ear leaf and the second leaf from the bottom of DH-B104-KO#1, DH-B104-KO#2, DH-PH207-KO#1, DH-PH207-KO#2, DH-OSL476-KO#1, DH-OSL476-KO#2, DH-TXB2-3-KO#1 and DH-X1C14A-KO#1 are significantly smaller than the wild type, especially the upper leaf angle, which is more different, showing a "tight upper and loose lower" plant type characteristic 3-3. Small plot yield test of wild type and mutant lac1 B-14K). The above results further show that lac1 is an important gene for regulating leaf angle, and the loss of function of the gene mainly affects the size of the upper leaf angle; the function of lac1 gene can be quickly modified by induced line-mediated gene editing technology, and large-scale improved inbred lines with "tight upper and loose lower" dense compact plant type can be created.

[0341] 3-3-1. TL small plot test in 2021

[0342] Figure 15

[0343] (1) Test design of plots

[0344] In 2021 summer and 2022 winter, the mutation lac1 and wild type (WT) were planted in Liaoning Province Tieling City Tieling County and Hainan Province Sanya City Yizhou District Nanbin Farm, the test scheme was split plot test, the main area was different planting density, and the sub-area was two different genotypes. The biological repetition was 3, 5 planting densities, 5 rows of mutation lac1 and wild type (WT) were continuously sown under each density, the row length was 5 meters, and the row width was 0.5 meters Figure 16 ). Consistent field management measures were taken to ensure normal growth of corn. The plots were pollinated in a natural state to reduce the impact of human activities on the growth of corn in the plots. Artificial fine harvesting was adopted, and the middle 3 rows of each genotype were harvested and all the ears were completely harvested. The corn ears were fully dried, attention was paid to reduce grain shedding, and the ear traits and yield data were analyzed. The main investigation was 100-grain weight, ear grain number, single plant yield and plot yield per mu. The 100-grain weight was the weight of the middle 100 grains of the ear; the ear grain number was the number of developed grains on a single ear; the single plant yield was the weight of all the grains on a single corn plant; and the plot yield per mu was the average single plant yield calculated by the investigation of the same genotype under the same density. The formula for yield per mu is: (M=(m1+m2+m3…mx) / x*X, M: corn grain yield per mu, mx: single ear grain weight, X: number of corn plants per mu).

[0345] (2) Factor analysis of panicle size and yield in the plot

[0346] In the summer of 2021 and the winter of 2022, field dense planting plot experiments were conducted on wild-type (WT) and leaf angle mutant lac1 in Tieling County, Liaoning Province and Nanbin Farm, Sanya, Hainan Province, respectively. Figure 17 A split-plot field trial design was adopted, with the main plot representing different planting densities and the subplot representing two different genotypes. Analysis showed that as planting density increased, the yield per plant of both the mutant lac1 and the wild-type (WT) gradually decreased, but the decrease was significantly less than that of the wild-type. Therefore, at lower planting densities (e.g., 3000 plants / mu, 4500 plants / mu, and 6000 plants / mu), the yield per plant and yield per mu of the mutant lac1 were lower than those of the wild-type (WT); while at higher planting densities (e.g., above 7500 plants / mu), the yield per plant and yield per mu of the mutant lac1 were significantly higher than those of the wild-type (WT). Figure 18 A-17D). Analysis of yield-related traits revealed that, under high-density planting conditions, the mutant lac1, exhibiting a reduced leaf angle, had a significantly greater 100-grain weight than the wild type (WT); simultaneously, data from the winter 2022 crop evaluation showed that the mutant lac1 also had significantly more grains per ear than the wild type (A-17D). 3-3-2. Yield test of Tongzhou F1 hybrid in 2022 The results indicate that differences in 100-grain weight or number of grains per ear are the main reasons for yield differences between the wild-type (WT) and the leaf angle mutant lac1 under high-density planting conditions. Therefore, the mutant lac1 exhibits a reduced leaf angle and has a yield-increasing effect under higher-density planting conditions, making this gene of significant value in breeding applications.

[0347] Figure 19

[0348] (1) Experimental design of the community

[0349] The lac1 material and the knockout line lac1-KO#2 were used to construct the F1 generation improved hybrid, and the wild type materials W22 and LH244 were used to construct the F1 generation wild type hybrid. For the constructed F1 generation hybrid materials, a dense planting yield test was carried out in Beijing, and a split plot experimental design was adopted. The main area was three biological repeats of planting density, and the sub-area was two genotypes under different planting densities. Three biological repeats were set, and each biological repeat was set under three densities of 3000, 5000 and 7000 plants per mu; 5 rows of wild type and 5 rows of improved hybrid were planted under each density; the row length was set to 5 meters, and the row width was set to 0.5 meters. During the growth period of corn, the same and consistent field management measures were implemented between different repeats, and unified fertilization, weeding, disease and pest control, etc. were carried out. Pollination was completed under natural open conditions, and no artificial assisted pollination was carried out. When the physiological state of corn kernels reached the mature period, harvesting was carried out, and the middle 3 rows of each genotype were used for variety examination and yield test.

[0350] (2) Factor analysis of ear and yield of plot

[0351] In the summer of 2022, the wild type and the improved hybrid were planted in the dense planting plot yield test in Haidian and Tongzhou of Beijing. After harvesting, the corn ears were fully dried, and then the variety examination was carried out. The main traits investigated include 100-grain weight, ear grain number, ear grain weight and unit area yield. The analysis of plot variety data found that under higher planting conditions (such as 5000 plants per mu), the yield of the improved hybrid was significantly higher than that of the wild type; the analysis of yield factor traits found that under higher planting density, the improved hybrid had more fertile grains, higher single ear grain weight, and finally higher total yield Figure 20 A-19D, ​ A-20D).

[0352] Example 4: Verification of functional site of lac1 gene

[0353] 4-1. Nucleotide sequence analysis of functional sites

[0354] As described in Example 1, the F2 genetic mapping population constructed by lac1 x Mo17 was used to locate the functional site regulating the generation of lac1 mutant phenotype to a 273 bp insertion through primary positioning and fine positioning. Sequencing of the 273 bp (as shown in Figure 21 A) found that the insertion of the nucleotide sequence caused the premature termination of the coding region, thus causing the mutation of the gene DWF4 and producing the ideal plant type of “tight upper and loose lower”. Figure 21 B shows the amino acid sequence alignment results of the wild type DWF4 coding product of W22 (SEQ ID NO: 24) and the coding product of the leaf clip angle mutant lac1 (SEQ ID NO: 25).

[0355] To further verify that 273bp is the functional variation causing the "up tight and down loose" plant type, the F2 population of lac1 x W22 was constructed for molecular marker linkage analysis, and it was found that the genotype of wild type with large angle was homozygous or heterozygous genotype, while the genotype of plants with small angle was homozygous genotype with 273bp insertion. Further indicated that the insertion of 273bp is the functional site leading to the mutation of gene DWF4 and causing the functional variation of "up tight and down loose" plant type.

[0356] 4-2. Genetic analysis of functional sites

[0357] The mutant lac1 was crossed with its corresponding wild type inbred line W22 to generate F2 segregation population. The obtained F2 segregation population was planted in the field, and about half a month after pollination, the leaf angle phenotype of 112 corns was investigated. The analysis of the phenotype data distribution of the leaf angle of 112 corns found that it was bimodal distribution, which was the typical phenotype distribution of single gene controlled traits. Figure 1 Among them, 26 strains of lac1 type showed compact plant type with reduced leaf angle, and 86 strains of W22 type showed wild type leaf angle. Chi-square test was performed. The test results showed that the segregation ratio of wild type WT and mutant lac1 in F2 segregation population was consistent with 3:1 (χ 2 =0.19<χ 2 (0.05,1)=3.84; Table 2 of Example 1). The above results showed that the lac1 mutant phenotype was controlled by single gene inheritance, and the mutant was a recessive mutant.

[0358] 4-3. Phenotypic verification of genetically backcrossed improved material

[0359] The natural mutant lac1 has the ideal plant type of "up tight and down loose", and its natural variation site is a 273bp insertion deletion in gene DWF4, which causes premature termination of gene expression. Specific primers lac1-TE-2F (GCTGAGGAAGTTGAGCGAGA, SEQ ID NO: 2) and lac1-TE-1R (AACCGGTACATCCTGCAGAA, SEQ ID NO: 3) were designed near the insertion site. Through hybridization or backcrossing with different inbred lines, combined with specific primer assisted selection technology of molecular marker, the function and phenotype effect of lac1 gene regulation in different backgrounds were verified.

[0360] To verify the potential value of mutation lac1 in breeding practice, seven elite inbred lines of maize (Z58, HuangC, C7-2, 9058, DMY1, DMY2 and DMY3) were selected for backcross improvement. The mutant lac1 was crossed with the seven elite inbred lines of maize, and after two generations of backcrossing, the mutant lac1 allele was introgressed into the seven elite inbred lines of maize by combining molecular marker-assisted selection. The obtained BC2F2 segregation population was planted in a good environment, and about 15 days after pollination, the leaf angle phenotype of the upper and middle leaves of single plants carrying the lac1 allele and not carrying the lac1 allele in the segregation population was investigated, and a significance test was performed. The results showed that the upper and middle leaf angles of the maize improved inbred lines carrying the mutant lac1 allele were significantly reduced compared to the segregation lines not carrying the lac1 allele Figure 22 ). This indicates that the mutant lac1 can significantly reduce the leaf angle of inbred lines, and can be used to breed dense-tolerant maize lines, and has the potential to improve backbone inbred lines.

[0361] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details in accordance with all the teachings of the disclosure, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

[0362] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.

[0363] REFERENCES

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[0365] 2. Walsh J, Waters CA, Freeling M (1998) The maize gene liguleless2 encodes a basic leucine zipper protein involved in the establishment of the leaf blade-sheath boundary. Genes Dev. 12:208-218.

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Claims

1. A method of modulating plant architecture of a maize plant, the method comprising: (1) disrupting an endogenous DWF4 gene in a maize cell; (2) developing or regenerating a maize plant from the maize cell in which the endogenous DWF4 gene is disrupted; wherein the plant architecture is a tight upper and loose lower architecture.

2. The method of claim 1, wherein the disrupting comprises disrupting a function, expression level, activity, or a combination thereof of the DWF4 gene.

3. The method of claim 1 or 2, wherein the disrupting comprises introducing a modification in the endogenous DWF4 gene.

4. The method of claim 3, wherein, the modification is in a coding region and / or a non-coding region of the endogenous DWF4 gene.

5. The method of claim 3, wherein, the modification comprises a deletion, a substitution, an insertion, an inversion, a duplication, or any combination thereof.

6. The method of claim 3, wherein, the modification results in a nonsense mutation, a missense mutation, a frameshift mutation, a splice site mutation, or any combination thereof.

7. The method of claim 3, wherein, the maize cell is homozygous for the modification.

8. The method of claim 3, wherein, the modification comprises a first modification in a first allele and a second modification in a second allele, the first modification and the second modification being the same as or different from each other.

9. The method of claim 3, wherein, the modification is introduced by genome editing.

10. The method of claim 9, wherein, the genome editing comprises using at least one site-specific nuclease.

11. The method of claim 10, wherein, the site-specific nuclease is an RNA-guided nuclease, a zinc finger nuclease, a meganuclease, a TALE-nuclease, a recombinase, a transposase, and any combination thereof.

12. The method of claim 11, wherein, the RNA-guided nuclease is a Cas nuclease.

13. The method of claim 9, wherein, the genome editing is selected from the group consisting of CRISPR / Cas, TALEN, ZFN, transposon technology, PASTE technology, PE technology, base editor, and any combination thereof.

14. The method of claim 3, wherein the modification comprises an indel mutation or a point mutation.

15. The method of claim 14, wherein, the modification is introduced by CRISPR / Cas.

16. The method of claim 14, wherein, step (1) comprises introducing a Cas proteinase and a guide RNA (gRNA) into the maize cell.

17. The method of claim 16, wherein, the Cas proteinase is a Cas9 proteinase.

18. The method of claim 16, wherein, the gRNA targets a nucleotide sequence set forth in SEQ ID NO:

5.

19. The method of claim 3, wherein the modification comprises an insertion of a DNA fragment.

20. The method of claim 19, wherein, the modification is achieved by transposon technology, PASTE technology, PE technology, or TnpB technology.

21. The method of claim 20, wherein, the transposon technology is a CRISPR-based transposase.

22. The method of claim 19, wherein, the inserted DNA fragment is greater than 100 bp in length.

23. The method of claim 22, wherein, the inserted DNA fragment is 100-500 bp, 100-400 bp, 200-400 bp, 200-300 bp, 250-300 bp, 250-290 bp, 260-280 bp, or 270-280 bp in length.

24. The method of claim 23, wherein, the inserted DNA fragment is 273 bp in length.

25. The method of claim 19, wherein, the inserted DNA fragment has a sequence set forth in SEQ ID NO:

1.

26. The method of claim 2, wherein the disrupting is achieved by knocking down or silencing the DWF4 gene.

27. The method of claim 26, wherein, the disrupting is achieved by introducing a silencing molecule targeting the DWF4 gene, the silencing molecule being selected from the group consisting of an RNA interference molecule or an antisense nucleic acid molecule.

28. The method of claim 1, wherein, The DWF4 gene: (i) a nucleotide sequence as set forth in SEQ ID NO: 4; or (ii) encodes a polypeptide, the amino acid sequence of which is as set forth in SEQ ID NO: 11 or 24.

29. A method of producing a maize plant, the method comprising: (1) providing a maize plant having a disrupted endogenous DWF4 gene as a first plant and a second plant; (2) crossing the first plant and the second plant to obtain a progeny plant; wherein the disruption of the endogenous DWF4 gene confers an altered plant architecture to the progeny plant, the altered plant architecture being a tight upper and loose lower plant architecture.

30. The method of claim 29, wherein, each of the first plant and the second plant is independently a maize plant obtained from the method of any one of claims 1-28.

31. A method of producing a hybrid maize plant, the method comprising crossing a first maize plant having a disrupted endogenous DWF4 gene with a second maize plant lacking the disruption to produce a hybrid maize plant, wherein the disruption of the endogenous DWF4 gene confers an altered plant architecture to the hybrid maize plant; the altered plant architecture being a tight upper and loose lower plant architecture.

32. The method of claim 31, wherein, the first maize plant is obtained from the method of any one of claims 1-28.

33. The method of claim 31, wherein, the hybrid maize plant is homozygous for the disruption of the endogenous DWF4 gene.

34. The method of claim 31, wherein, the second maize plant is a maize inbred.

35. The method of claim 34, wherein, the maize inbred is W22, M017, B104, PH207, OSL476, TXB2-3, Z58, HuangC, C7-2, 9058, DMY1, DMY2, or DMY3.

36. A method of identifying a maize plant or a part, seed, cell, or progeny thereof having an altered plant architecture, the method comprising detecting whether the endogenous DWF4 gene of the maize plant or a part, seed, cell, or progeny thereof is disrupted or a molecular marker linked to the disruption is present; the altered plant architecture being a tight upper and loose lower plant architecture.

37. The method of claim 36, wherein, the disruption is as defined in any one of claims 2-28.

38. The method of claim 36, wherein, the disruption is as defined in any one of claims 14-18, the method comprising: (1) providing a sample comprising genomic DNA of the maize plant or a part, seed, cell, or progeny thereof; (2) performing a nucleic acid amplification reaction on the sample; and (3) sequencing the amplicon.

39. The method of claim 38, wherein, Step (2) comprises using a primer pair flanking the region targeted for modification.

40. The method of claim 36, wherein, the disruption is as defined in any one of claims 19-25, the method comprising: (1) providing a sample comprising genomic DNA of the maize plant or a part, seed, cell, or progeny thereof; (2) performing a nucleic acid amplification reaction on the sample; and (3) detecting the length of the amplification product by gel electrophoresis.

41. The method of claim 40, wherein, Step (2) comprises using the primer pair set forth in SEQ ID NO: 2 and 3.

42. The method of claim 36, wherein, the method comprising: (1) providing a sample comprising genomic DNA of the maize plant or a part, seed, cell, or progeny thereof; (2) detecting a molecular marker linked to the disrupted endogenous DWF4 gene.

43. The method of claim 42, wherein, the disruption is as defined in any one of claims 14-25.

44. The method of claim 42, wherein, The molecular marker is selected from the group consisting of single nucleotide polymorphism (SNP), insertion-deletion (InDel), single feature polymorphism (SFP), simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and restriction fragment length polymorphism (RFLP).

45. A method of producing a maize plant having an altered plant architecture, the method comprising the steps of: (1) providing a first maize plant having a disruption of an endogenous DWF4 gene; (2) crossing the first maize plant with a second maize plant that is free of the disruption; and (3) selecting a progeny plant from step (2) by detecting the presence of the disruption of the endogenous DWF4 gene, or the presence of a molecular marker that is linked to the disruption, in the progeny plant, thereby producing a maize plant having an altered plant architecture; wherein the altered plant architecture is a tight upper loose lower plant architecture.

46. The method of claim 45, wherein, The progeny plant is homozygous for the disruption of the endogenous DWF4 gene or the molecular marker.

47. The method of claim 45, wherein, The molecular marker is selected from the group consisting of single nucleotide polymorphism (SNP), insertion-deletion (InDel), single feature polymorphism (SFP), simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and restriction fragment length polymorphism (RFLP).

48. The method of claim 45, wherein, The first maize plant is obtained by the method of any one of claims 1-28.

49. The method of claim 45, wherein, The second maize plant is a maize inbred.

50. The method of claim 49, wherein, The maize inbred is W22, M017, B104, PH207, OSL476, TXB2-3, Z58, HuangC, C7-2, 9058, DMY1, DMY2, or DMY3.

51. A method of increasing yield of corn, the method comprising: Planting (i) a maize plant or part, seed, cell, or progeny thereof obtained by the method of any one of claims 1-28, or (ii) a maize plant or part, seed, cell, or progeny thereof obtained by the method of any one of claims 29-35, 45-50, or (iii) a maize plant or part, seed, cell, or progeny thereof identified by the method of any one of claims 36-44 at a planting density of no less than 5000 plants per acre.

52. The method of claim 51, wherein, The planting density is no less than 5500 plants per acre, no less than 6000 plants per acre, no less than 6500 plants per acre, no less than 7000 plants per acre, or no less than 7500 plants per acre.

53. The method of claim 52, wherein, The planting density is 5000 plants per acre to 15000 plants per acre.

54. The method of claim 53, wherein, The planting density is 5000 plants per acre to 12000 plants per acre, 6000 plants per acre to 12000 plants per acre, or 7000 plants per acre to 12000 plants per acre.

55. The method of claim 54, wherein, The planting density is 7500 plants per acre, 8000 plants per acre, 9000 plants per acre, 10000 plants per acre, or 12000 plants per acre.

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