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WO2019038417A1 - Méthodes pour augmenter le rendement en grain - Google Patents

Méthodes pour augmenter le rendement en grain Download PDF

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Publication number
WO2019038417A1
WO2019038417A1 PCT/EP2018/072854 EP2018072854W WO2019038417A1 WO 2019038417 A1 WO2019038417 A1 WO 2019038417A1 EP 2018072854 W EP2018072854 W EP 2018072854W WO 2019038417 A1 WO2019038417 A1 WO 2019038417A1
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plant
nucleic acid
mads1
grain
mutation
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Xiangdong Fu
Qian Liu
Ruixi HAN
Kun Wu
Yafeng YE
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Institute of Genetics and Developmental Biology of CAS
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Institute of Genetics and Developmental Biology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to methods for increasing plant yield, and in particular grain yield, and even more particularly, to a method that increases grain size without reducing grain number, the method comprising modulating the expression or activity of a MADS- domain transcription factor 1 (MADS1 ). Also described are genetically altered plants characterised by the above phenotype and methods of producing such plants.
  • MADS1 MADS- domain transcription factor 1
  • a substantial increase in grain yield of the major crops is required to feed a growing world population.
  • the prime breeding target is to increase both grain size and number as two direct components of ultimate grain yield.
  • this simultaneous improvement is a major challenge because of the well-established negative correlation between these two traits 6 , all of which is controlled by quantitative trait loci (QTL) and influenced by environmental changes.
  • Grain yield is also often associated with reduced grain quality.
  • natural variants of the rice G protein ⁇ subunits DENSE AND ERECT PANICLE1 (DEP1 ) and GRAIN SIZE3 (GS3) have been shown to boost grain yield 2-4 , but typically show only a mediocre quality.
  • the nature of variations associated with higher yield potential is also quite different for each of the two subunits 7 .
  • the gs3 allele causes an increase in the size of grain 2
  • the dep1 -1 allele increases grain number 3,4 .
  • Grain size and number are inherently connected with floral organ identity and growth, which are tightly regulated by various combinations of MADS-domain transcription factors 8 .
  • MADS-domain transcription factors 8 Although a number of genes have been shown to control grain number and size in rice 9-17 , the molecular mechanisms underlying the interplay between floral organ identity specification and growth capacity still remain unclear.
  • the G protein ⁇ subunits GS3 2 and DEP1 3,4 interact with the evolutionarily conserved keratin-like domain of MADS-domain proteins, and act as cofactors to enhance OsMADSI transcriptional activity and promote the co-operative transactivation of common target genes, thereby regulating grain size and shape.
  • OsMADS1 lgy3 allele with high yield-associated dep1-1 and gs3 alleles represents an effective strategy for simultaneously improving both the productivity and end-use quality of rice.
  • a method of increasing grain yield in a plant comprising modulating the expression of at least one nucleic acid encoding a MADS-domain transcription factor 1 (MADS1 ) and/or modulating the activity of a MADS-domain transcription factor 1 (MADS1 ).
  • a method of increasing grain quality comprising modulating the expression of at least one nucleic acid encoding a MADS-domain transcription factor 1 (MADS1 ) and/or modulating the activity of a MADS-domain transcription factor 1 (MADS1 ).
  • modulating the expression and/or activity of MADS1 comprises reducing or abolishing the expression and/or activity of a MADS1 polypeptide. More preferably, modulating the expression and/or activity of MADS1 comprises expressing or increasing the expression of a dominant negative gain of function allele of MADS1 .
  • the increase in grain yield comprises an increase in at least one of grain size, grain number, grain length, grain weight and thousand kernel weight.
  • the method increases grain size without reducing grain number. Accordingly, in a preferred embodiment, the method increases grain size and grain number.
  • the method comprises introducing at least one mutation into at least one nucleic acid sequence encoding a MADS1 transcription factor or the promoter of a MADS1 transcription factor.
  • the mutation is a loss of function mutation or a dominant negative gain of function mutation. More preferably, the mutation is an insertion, deletion and/or substitution.
  • the mutation is in at least one conserved domain, preferably the MADS-domain, the I domain, the K domain and/or the C-domain.
  • the mutation results in at least one truncated and/or non-functional domain. More preferably, the mutation results in a C-terminal truncated protein.
  • the method comprises using RNA interference to reduce or abolish the expression of a MADS1 nucleic acid.
  • the increase in yield is relative to a wild-type or control plant.
  • the method further comprises reducing or abolishing the expression of at least one nucleic acid encoding a DENSE AND ERECT PANICLE1 (DEP1 ) and/or GRAIN SIZE3 (GS3) polypeptide and/or modulating the activity of a DENSE AND ERECT PANICLE1 (DEP1 ) and/or GRAIN SIZE3 (GS3) polypeptide.
  • a genetically altered plant, part thereof or plant cell wherein said plant comprises at least one mutation in at least one nucleic acid encoding a MADS1 polypeptide and/or the MADS1 promoter.
  • a genetically altered plant, part thereof or plant cell wherein said plant is characterised by altered expression or activity of the MADS1 polypeptide.
  • the plant is characterised by an increase in grain yield, preferably when said plant is compared to a control or wild-type plant.
  • the increase in grain yield comprises an increase in at least one of grain size, grain number, grain length, grain weight and thousand kernel weight. More preferably, the increase in grain size is not accompanied by a reduction in grain number.
  • the genetically altered plant, part thereof or plant cell comprises an RNA interference construct that reduces the expression of a MADS1 polypeptide.
  • the plant part is a grain or a seed.
  • a method of producing a plant with increased grain yield comprising introducing at least one mutation into at least one nucleic acid sequence encoding a MADS1 polypeptide and/or the promoter of the MADS1 polypeptide.
  • a method of increasing grain yield and/or grain quality comprising introducing at least one mutation into at least one nucleic acid sequence encoding a MADS1 polypeptide and/or the promoter of the MADS1 polypeptide.
  • a method of producing a plant with increased grain yield comprising introducing and expressing in said plant an RNA interference construct that reduces or abolishes the expression of a MADS1 nucleic acid.
  • the method further comprises measuring an increase in grain yield, wherein an increase in grain yield comprises an increase in at least one of grain number, grain length, grain weight and thousand kernel weight.
  • the method further comprises measuring a reduction or absence of the expression of a MADS1 nucleic acid and/or measuring a reduction or absence of activity of a MADS1 polypeptide. More preferably, the method may further comprise regenerating a plant and screening for an increase in grain yield.
  • the nucleic acid encodes a MADS1 polypeptide wherein the MADS1 polypeptide comprises or consists of SEQ ID NO: 1 or a functional variant or homologue thereof.
  • the nucleic acid comprises or consists of SEQ ID NOs 2 to 3 or a functional variant or homologue thereof.
  • the nucleic acid encoding a MADS1 promoter comprises or consists of SEQ ID NO: 176 or a functional variant or homologue thereof.
  • the mutation is a loss of function mutation or a dominant negative gain of function mutation.
  • the mutation is an insertion, deletion and/or substitution.
  • the mutation is introduced using targeted genome modification, preferably ZFNs, TALENs or CRISPR/Cas9.
  • the mutation is introduced using mutagenesis, preferably TILLING or T-DNA insertion.
  • a method for identifying and/or selecting a plant that will have increased grain yield, preferably compared to a wild-type or control plant comprising detecting in the plant or plant germplasm at least one polymorphism in the MADS1 gene and selecting said plant or progeny thereof.
  • the polymorphism is an insertion, deletion and/or substitution.
  • the method further comprises introgressing the chromosomal region comprising the at least one polymorphism in the MADS1 gene into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.
  • a nucleic acid construct comprising a nucleic acid sequence encoding at least one DNA-binding domain that can bind to at least one MADS1 gene.
  • the nucleic acid sequence encodes at least one protospacer element, and wherein the sequence of the protospacer element is selected from SEQ ID Nos 24, 29, 34, 39, 44, 49, 54, 59, 64, 69, 74, 79, 84, 89, 94, 99, 104, 109, 1 14, 1 19, 124, 129, 134, 139, 144, 149, 154 and 159 or a sequence that is at least 90% identical to SEQ I D Nos 24, 29, 34, 39, 44, 49, 54, 59, 64, 69, 74, 79, 84, 89, 94, 99, 104, 109, 1 14, 1 19, 124, 129, 134, 139, 144, 149, 154 and 159.
  • the construct further comprises a nucleic acid sequence encoding a CRISPR RNA (crRNA) sequence, wherein said crRNA sequence comprises the protospacer element sequence and additional nucleotides. More preferably the construct further comprises a nucleic acid sequence encoding a transactivating RNA (tracrRNA), wherein preferably the tracrRNA is defined in SEQ ID NO.163 or a functional variant thereof.
  • crRNA CRISPR RNA
  • tracrRNA transactivating RNA
  • the construct encodes at least one single-guide RNA (sgRNA), wherein said sgRNA comprises the tracrRNA sequence and the crRNA sequence, wherein the sgRNA comprises or consists of a sequence selected from SEQ ID NOs 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155 and 160.
  • sgRNA single-guide RNA
  • the construct is operably linked to a promoter.
  • the promoter is a constitutive promoter.
  • the nucleic acid construct further comprises a nucleic acid sequence encoding a CRISPR enzyme.
  • the CRISPR enzyme is a Cas protein. More preferably, the Cas protein is Cas9 or a functional variant thereof.
  • the nucleic acid construct encodes a TAL effector.
  • the nucleic acid construct further comprises a sequence encoding an endonuclease or DNA-cleavage domain thereof. More preferably, the endonuclease is Fokl.
  • a single guide (sg) RNA molecule wherein said sgRNA comprises a crRNA sequence and a tracrRNA sequence, wherein the crRNA sequence can bind to at least one sequence selected from SEQ ID Nos 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, 73, 78, 83, 88, 93, 98, 103, 108, 1 13, 1 18, 123, 128, 133, 138, 143, 148, 153 and 158 or a variant thereof.
  • an isolated plant cell transfected with at least one nucleic acid construct as described herein.
  • the isolated plant cell is transfected with at least one nucleic acid construct as described herein and a second nucleic acid construct, wherein said second nucleic acid construct comprises a nucleic acid sequence encoding a Cas protein, preferably a Cas9 protein or a functional variant thereof.
  • the second nucleic acid construct is transfected before, after or concurrently with the first nucleic acid construct described herein.
  • a genetically modified plant wherein said plant comprises the transfected cell as described herein.
  • the nucleic acid encoding the sgRNA and/or the nucleic acid encoding a Cas protein is integrated in a stable form.
  • a method of increasing grain yield in a plant comprising introducing and expressing in a plant a nucleic acid construct as described herein, wherein preferably said increase is relative to a control or wild-type plant.
  • nucleic acid construct as described herein to increase grain yield in a plant.
  • the nucleic acid construct reduces or abolishes the expression and/or activity of MADS1 in a plant.
  • a method for obtaining the genetically modified plant as described herein comprising:
  • a method of modifying, preferably decreasing the levels of at least one nucleic acid selected from the GARP family of genes, the MADS-box family genes, an auxin efflux carrier gene and an auxin response factor gene comprising modulating the expression or activity of MADS1.
  • a short interfering nucleic acid (siNA) molecule wherein said molecule targets SEQ ID NO: 167 or variant thereof.
  • the plant is preferably a monocot or dicot.
  • the monocot plant is selected from rice, wheat, maize, sorghum and millet.
  • the dicot plant is selected from soybean and brassica.
  • the plant is rice, more preferably, the indica or japonica variety.
  • the plant carries a mutation in the DENSE AND ERECT PANICLE1 (DEP1 ) and/or GRAIN SIZE3 (GS3) gene. DESCRIPTION OF THE FIGURES
  • Figure 1 shows that the interaction between DEP1 and OsMADSI regulates grain size and yield potential of rice,
  • (b) QTL mapping for grain length and grain yield
  • M represents the MADS domain
  • I represents the intervening domain
  • K represents the keratin-like domain
  • C represents the C-terminal domain
  • Figure 2 shows the physical interaction between DEP1 and OsMADSI .
  • BiFC assays nLUC-tagged DEP1 (or dep1 -1 ) was co-transformed into tobacco leaves along with either cLUC-targeted OsMADSI or cLUC-targeted OsMADSI lgy3 . The interaction of the rice GA receptor GID1 and the DELLA protein SLR1 was used as a positive control,
  • the ratio of LUC to REN activity was monitored in rice protoplasts co-transfected with various effector and reporter constructs.
  • the DEP1 -OsMADSI interaction promotes the OsMADS1-induced target gene transactivation activity.
  • FIG. 4 shows that the G ⁇ dimmer is a functional OsMADSI cofactor in regulating grain size
  • (c) Effect of the GS3- OsMADSI interaction on OsMADSI -induced transactivation activity. The LUC/REN activity obtained from a co-transfection with an empty effector construct and indicated reporter constructs was set to be one. Data shown as mean ⁇ s.e.m. (n 6).
  • (d-l) A field-based comparison of RD23-LGY3-gs3 and RD23-lgy3- gs3 plants: (d) Plant morphology. Scale bar: 20 cm; (e) Grain size and shape. Scale bar: 5 mm; (f) Plant height; (g) Heading date; (h) The number of tillers per plant; (i) The number of grains per panicle; (j) Grain length; (k) Grain width; (I) The overall grain yield per plant, (m-p) A field trial of indica hybrid rice: (m) Grain size and shape.
  • Figure 5 shows the semi-dominant qlgy3 allele from the japonica variety L-204 was associated with the formation of long-grain and high-yield, a, Grain length, b, Grain width, c, 1 ,000-grain weight, d, Grain yield per plant. All phenotypic data were measured from the paddy-grown BC4F2 progenies derived from the cross between RIL186 and HJX74 (the recurrent parent) under normal cultivation conditions.
  • Figure 6 shows the effect of the truncating splice site mutation on gene expression and function of OsMADSI
  • b Immunoblot of OsMADSL The total proteins were extracted from young panicles (3 to 6 cm length). The abundance of HSP90 protein detected by anti-HSP90 antibodies was used as loading control, c, Localization of OsMADS1 -GFP and OsMADS1 lgy3 -GFP fusion proteins. Scale bar, 10 ⁇ m.
  • Figure 8 shows grain chalkiness and endosperm transparency of the NILs plants, a, Grains formed by the WYJ7-LGY3-dep1-1 and WYJ7-igy3-dep1-1 plants, b, Grains formed by the RD23-LGY3-gs3 and WYJ7-lgy3-gs3 plants, c, Grain chalkiness and endosperm transparency of the two hybrid combinations (PA64S/931 1-LGY3-gs3 and PA64S/931 1-lgy3-gs3).
  • PA64S a photo-thermosensitive genie male sterile line Peiai64S.
  • Figure 9 shows scanning electron microscopy images of the transverse sections of starch granules from the NILs plants, a, W YY7-LGY3-dep 1-1 and WYJ7-lgy3-dep1-1 grains, b, RD23-LGY3-gs3 and RD23-lgy3-gs3 grains, c, PA64S/931 1 -LGY3-gs3 and PA64S/931 1-lgy3-gs3 grains.
  • the endosperm of the NILs plants carrying the Igy3 allele comprised largely sharp edged, compactly arranged polygonal starch granules.
  • PA64S a photo-thermosensitive genie male sterile line Peiai64S.
  • Figure 10 shows that the DEP1 protein interacts directly with a number of MADS- domain transcription factors, a, Yeast two-hybrid assays.
  • the transformant co- transformed with empty vectors pGADT7 and pGBDT7 was used as a negative control.
  • b BiFC assays.
  • the nLUC-tagged OsMADSs were co-transformed into tobacco leaves along with the cLUC-targeted DEP1.
  • Figure 11 shows the effect of the constitutive knockdown of OsMADSI on grain size and shape in WYJ7-LGY3-DEP1 and WYJ-LGY3-dep1-1 plants.
  • the transgenic plants that had been RNAi-silenced for OsMADSI under control of the rice Actin promoter exhibited an open flower with the elongated leafy palea and lemma.
  • Figure 12 is a table showing the effects of the Igy3 allele on the physiochemical characteristics of milled rice.
  • Figure 13 is a table of the primer sequences used for transgene constructs.
  • Figure 14 shows a sequence alignment between OsMADSI and homologues.
  • Figure 15 shows grains formed by transgenic NPB-LGY3 plants in which OsMADSI had been knocked down by RNAi.
  • nucleic acid As used herein, the words “nucleic acid”, “nucleic acid sequence”, “nucleotide”, “nucleic acid molecule” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products.
  • genes also encompass a gene.
  • gene or “gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.
  • polypeptide and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
  • the aspects of the invention involve recombination DNA technology and exclude embodiments that are solely based on generating plants by traditional breeding methods.
  • a method of increasing yield in a plant comprising modulating the expression and/or activity of a MADS-domain transcription factor 1 (MADS1 ).
  • MADS1 may also be known as LEAFY HULL STERILE 1 , AGAMOUS-LIKE 4, AGL4, SEP2, or SEPALLATA-like protein, and such terms may be used interchangeably.
  • modulating the expression and/or activity of MADS1 comprises reducing or abolishing the expression and/or activity of a MADS1 polypeptide.
  • modulating the expression and/or activity of MADS1 comprises expressing or increasing the expression of a dominant negative gain of function allele of MADS1.
  • an "increase” as used herein may refer to an increase of at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 or 95% or more compared to a control plant.
  • yield in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight. The actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square metres.
  • increased yield can be taken to comprise any or at least one of the following and can be measured by assessing one or more of (a) increased biomass (weight) of one or more parts of a plant, aboveground (harvestable parts), or increased root biomass, increased root volume, increased root length, increased root diameter or increased root length or increased biomass of any other harvestable part.
  • Increased biomass may be expressed as g/plant or kg/hectare, (b) increased seed yield per plant, which may comprise one or more of an increase in seed biomass (weight) per plant or on an individual basis, (c) increased seed filling rate, (d) increased number of filled seeds, (e) increased harvest index, which may be expressed as a ratio of the yield of harvestable parts such as seeds over the total biomass, (f) increased viability/germination efficiency, (g) increased number or size or weight of seeds or pods or beans or grain (h) increased seed volume (which may be a result of a change in the composition (i.e.
  • lipid also referred to herein as oil
  • carbohydrate total content and composition (i) increased (individual or average) seed area, (j) increased (individual or average) seed length, (k) increased (individual or average) seed perimeter, (I) increased growth or increased branching, for example inflorescences with more branches, (m) increased fresh weight or grain fill (n) increased ear weight (o) increased thousand kernel weight (TKW), which may be taken from the number of filled seeds counted and their total weight and may be as a result of an increase in seed size and/or seed weight (p) decreased number of barren tillers per plant and (q) sturdier or stronger culms or stems. All parameters are relative to a wild-type or control plant.
  • said increased yield comprises an increase in at least one of tiller number, grain size, grain number per panicle, grain length, grain width, grain weight and thousand kernel weight.
  • Yield is increased relative to a control or wild-type plant.
  • the yield is increased by 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to a control or wild-type plant.
  • Yield may alternatively be increased by between 20-50%, and more preferably between 5 and 15% or more compared to a control plant.
  • an increase in grain yield can be measured by assessing one or more of grain number, grain length, grain weight andthousand kernel weight.
  • the skilled person would be able to measure any of the above yield parameters using known techniques in the art.
  • a method of increasing at least one of grain size, grain number, grain length, grain weight and thousand kernel weight comprising modulating the expression and/or activity of a MADS-domain transcription factor 1 (MADS1 ), as described herein.
  • MADS1 MADS-domain transcription factor 1
  • a reduction as used herein may be at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the grain number in a control plant.
  • a method of increasing grain quality comprising modulating the expression and/or activity of a MADS- domain transcription factor 1 (MADS1 ), as described herein. Accordingly, in one embodiment there is provided a method of increasing grain yield and grain quality.
  • MADS1 MADS- domain transcription factor 1
  • grain quality may refer to the quality of the grain.
  • grain quality may refer to any or at least one of (i) milling quality (ii) cooking, eating and processing quality, (iii) nutritional quality, (iv) cleanliness, soundness and purity, (v) colour grain size, shape, weight, uniformity and general appearance and (vi) grain chalkiness, translucency and colour.
  • said increase in grain quality comprises a decrease or reduction in grain chalkiness and/or an increase in translucency.
  • a “reduction” or “increase” as used herein may be up to or more than 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a wild-type or control plant.
  • seed and “grain” as used herein can be used interchangeably.
  • increase means a decrease in the levels of MADS1 expression and/or activity by up to or more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a wild-type or control plant.
  • bolish means that no expression of MADS1 is detectable or that no functional MADS1 polypeptide is produced. Methods for determining the level of MADS1 expression and/or activity would be well known to the skilled person. In particular reductions can be measured by any standard technique known to the skilled person.
  • a reduction in the expression and/or content levels of MADS1 may compromise a measure of protein and/or nucleic acid levels and can be measured by any technique known to the skilled person, such as, but not limited to, any form of gel electrophoresis or chromatography (e.g. HPLC).
  • the mutation reduces the ability of MADS1 to bind and express downstream target genes.
  • the method may comprise measuring the transcriptional profile of the mutated protein (versus wild-type or a control) using techniques standard in the art, such as, but not limited to, RNA-seq and CHIP-seq.
  • At least one mutation is meant that where the MADS1 gene is present as more than one copy or homoeologue (with the same or slightly different sequence) there is at least one mutation in at least one gene.
  • all genes are mutated.
  • the "dominant negative gain of function allele of MADS1" comprises or consists of SEQ ID NO: 4 or a functional variant or homologue thereof.
  • any mutation that results in a dominant negative gain of function as described herein is encompassed within the scope of the invention.
  • dominant negative gain of function is meant that the mutated protein acts antagonistically to the wild-type allele. Alternatively, the gain of function allele may wholly or partially disrupt the activity of the wild-type allele when expressed or overexpressed.
  • a dominant negative allele may be assessed by measuring and comparing the transcriptional profile of a heterozygous plant (MADS1 -wild-type/MADS1 -mutant allele) with a wild-type (homozygous) or a control plant.
  • the transcriptional profile of a plant can be measured using any technique standard in the art, such as, but not limited to, RNA-seq and CHIP-seq.
  • the dominant negative gain of function allele of MADS1 may be referred to herein as MADS1 lgy3 .
  • “expression” means that the allele is expressed in a plant that did not previously express the allele.
  • “increasing expression” means increasing the expression of the allele by at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a wild-type or control plant.
  • the method comprises introducing at least one mutation into the, preferably endogenous, gene encoding MADS1 .
  • the method may comprise introducing at least one mutation into the MADS1 promoter. Preferably said mutation is in the coding region of the MADS1 gene.
  • said mutation is at least one splice site.
  • said mutation is in an intronic sequence, the 5'UTR, the 3'UTR, the termination signal, the splice acceptor site or the ribosome binding site.
  • at least one mutation or structural alteration may be introduced into the MADS1 promoter or MADS1 enhancer elements such that the MADS1 gene is either not expressed (i.e. expression is abolished) or expression is reduced, as defined herein.
  • at least one mutation may be introduced into the MADS1 gene such that the altered gene does not express a full- length (i.e. expresses a truncated) MADS1 protein or does not express a fully functional MADS1 protein.
  • the activity of the MADS1 polypeptide can be considered to be reduced or abolished as described herein.
  • Such an altered MADS1 polypeptide may act antagonistically or wholly or partially disrupt the activity of wild-type MADS1 .
  • Such a protein can be considered to be a dominant negative gain of function allele as defined herein.
  • the mutation may result in the expression of a MADS1 polypeptide with no, significantly reduced or altered (i.e. a dominant negative) biological activity in vivo.
  • MADS1 may not be expressed at all.
  • the sequence of the MADS1 gene comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 2 or 3, or a functional variant or homologue thereof and encodes a polypeptide as defined in SEQ ID NO: 1 or a functional variant or homologue thereof.
  • MADS1 promoter is meant a region extending for at least 5kbp, preferably at least 2.5kbp upstream of the ATG codon of the MADS1 ORF (open reading frame).
  • sequence of the MADS1 promoter comprises or consists of a nucleic acid sequence as defined in SEQ ID No: 176 or a functional variant or homologue thereof.
  • an 'endogenous' nucleic acid may refer to the native or natural sequence in the plant genome.
  • the endogenous sequence of the MADS1 gene comprises or consists of SEQ ID NO: 2 or 3 and encodes an amino acid sequence as defined in SEQ ID NO: 1 or homologs thereof.
  • functional variants as defined herein
  • homologs are shown in SEQ ID NOs 5 to 22.
  • the homolog encodes a polypeptide selected from SEQ ID NOs 5, 7, 9, 1 1 , 13, 15, 17, 19 and 21 or the homolog comprises or consists of a nucleic acid sequence selected from SEQ ID NOs 6, 8, 10, 12, 14, 16, 18, 20 and 22.
  • a functional variant of a nucleic acid sequence refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full non-variant sequence.
  • a functional variant also comprises a variant of the gene of interest, which has sequence alterations that do not affect function, for example in non-conserved residues.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product.
  • a "variant” or a “functional variant” has at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at
  • homolog also designates a MADS1 promoter or MADS1 gene orthologue from other plant species.
  • a homolog may have, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 9
  • overall sequence identity is at least 37%. In one embodiment, overall sequence identity is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
  • the "MADS1 " polypeptide (for MADS-domain transcription factor 1 ) encodes a transcription factor, characterised by a conserved sequence motif, the "MADS-box", which encodes the 56-60 amino acid DNA-binding domain, the "MADS-domain”.
  • the MADS domain generally binds to DNA sequences comprising a "CArG-box", which are sequences comprising the motif CC[A T]6GG.
  • the MADS-domain may be known as a MEF-2 (Myocyte-enhancer factor 2) or a Type II MADS-domain protein.
  • MEF2-like MADS-domain proteins may also be known as MIKC proteins, which further refer to their conserved domain structure.
  • the MADS (M) domain is followed by an Intervening (I), a Keratin-like (K) and a C-terminal (C) domain.
  • I and K domains are required for protein interaction, while the C domain activates transcription.
  • sequence of the conserved domains of MADS1 comprise or consist of the following: MADS domain:
  • the MADS1 nucleic acid (coding) sequence encodes a MADS1 protein comprising at least, at least two, at least three or all four conserved domains or variants thereof, selected from a MADS-domain, I domain, K domain and C domain, as defined herein.
  • the MADS protein comprises all four conserved domains or a variant thereof.
  • the variant has at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to any of the conserved domains identified in SEQ.
  • the mutation is in the MADS-domain and leads to a loss of function mutation.
  • the mutation is in the C terminal domain and leads to a decrease in transcriptional activity.
  • the mutation may be considered a gain of function mutation.
  • the mutation may be in the I and/or K domains. Two nucleic acid sequences or polypeptides are said to be "identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • percentage of sequence identity is used in reference to proteins or peptides, it is recognised that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • sequences differ in conservative substitutions
  • percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.
  • Suitable homologues can be identified by sequence comparisons and identifications of conserved domains. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function, for example when overexpressed in a plant.
  • nucleotide sequences of the invention and described herein can also be used to isolate corresponding sequences from other organisms, particularly other plants, for example crop plants.
  • methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein.
  • Topology of the sequences and the characteristic domains structure can also be considered when identifying and isolating homologs.
  • Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof.
  • hybridization techniques all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen plant.
  • the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker.
  • Hybridization of such sequences may be carried out under stringent conditions.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background).
  • Stringent conditions are sequence dependent and will be different in different circumstances.
  • target sequences that are 100% complementary to the probe can be identified (homologous probing).
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
  • stringent conditions will be those in which the salt concentration is less than about 1 .5 M Na ion, typically about 0.01 to 1 .0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a variant as used herein can comprise a nucleic acid sequence encoding a MADS1 polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to a nucleic acid sequence as defined in SEQ ID NO: 2 or 3.
  • a method of increasing yield and/or increasing grain quality in a plant comprising modulating the expression and/or activity of a MADS1 , as described herein, wherein the method comprises introducing at least one mutation into a MADS1 gene, wherein the MADS1 gene comprises or consists of a. a nucleic acid sequence encoding a polypeptide as defined in SEQ ID NO:1 ; or b. a nucleic acid sequence as defined in SEQ ID NO: 2 or 3; or
  • a nucleic acid sequence encoding a polypeptide wherein the polypeptide comprises at least one, at least two, at least three or all four conserved domains selected from a MADS-domain, a I domain, a K domain and a C domain as defined in SEQ ID NO:1 17 to 120 respectively, or a variant thereof, wherein the variant has at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to any of SEQ ID NO: 182 to 185; or
  • nucleic acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to either (a) or (b); or
  • nucleic acid sequence encoding a MADS1 polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to the nucleic acid sequence of any of (a) to (d).
  • the mutation that is introduced into the endogenous MADS1 gene or promoter thereof to silence, reduce, inhibit or modulate the biological activity and/or expression levels of the MADS1 gene or protein may be selected from the following mutation types
  • a "missense mutation” which is a change in the nucleic acid sequence that results in the substitution of an amino acid for another amino acid
  • a "nonsense mutation” or "STOP codon mutation” which is a change in the nucleic acid sequence that results in the introduction of a premature STOP codon and, thus, the termination of translation (resulting in a truncated protein); plant genes contain the translation stop codons "TGA” (UGA in RNA), "TAA” (UAA in RNA) and “TAG” (UAG in RNA); thus any nucleotide substitution, insertion, deletion which results in one of these codons to be in the mature mRNA being translated (in the reading frame) will terminate translation.
  • a frameshift mutation resulting in the nucleic acid sequence being translated in a different frame downstream of the mutation.
  • a frameshift mutation can have various causes, such as the insertion, deletion or duplication of one or more nucleotides.
  • splice site which is a mutation that results in the insertion, deletion or substitution of a nucleotide at the site of splicing.
  • an "insertion”, “deletion” or “substitution” may refer to the insertion, deletion or substitution of at least one, two, three, four, five, six, seven, eight, nine or ten nucleotides.
  • said mutation results in a loss of function.
  • the mutation results inreduced or no expression of the MADS1 nucleic acid.
  • said mutation may result in a non-functional protein. That is, the MADS polypeptide will have reduced or abolished activity. In other words, the normal function of the MADS1 gene is lost or reduced.
  • Such a mutation may also be referred to as a "null" mutation.
  • said mutation may result in a dominant negative gain of function. This term is defined above.
  • At least one mutation is introduced into at least one conserved domain of the MADS polypeptide.
  • said mutation is a loss of function mutation such as a premature stop codon, or an amino acid change in a highly conserved region that is predicted to be important for protein structure.
  • at least one mutation is introduced into at least one domain selected from the MADS- domain, the I domain, the K domain and the C domain.
  • the mutation results in a non-functional domain.
  • the mutation may result in a non-functional truncated domain.
  • the mutation is a splice site mutation.
  • the mutation may be an insertion-deletion mutation at the splice site of the intron 7/exon 8 junction.
  • the plant is rice.
  • the mutation will result in the truncation of the terminal 37 residues and the addition of a further 5 residues to the C domain. Accordingly, the mutation may result in a truncated C domain.
  • the mutation is at least one of the following mutations: a T to A subsiution at position 8056 of SEQ ID NO:2 or a homologous position thereof;
  • the mutation is all of the above mutations in SEQ ID NO: 2 or a functional variant or homologue thereof.
  • At least one mutation as defined above and which leads to the insertion, deletion or substitution of at least one nucleic acid or amino acid compared to the wild-type MADS1 promoter or MADS1 nucleic acid or protein sequence can affect the biological activity of the MADS1 protein.
  • the mutation may be the insertion of a dominant negative gain of function allele.
  • the mutation is the insertion of a nucleic acid sequence comprising or consisting of SEQ ID NO: 4 or a variant thereof. A variant is defined elsewhere.
  • the mutation is introduced into the MADS1 promoter and/or a MADS1 enhancer and is at least the deletion and/or insertion of at least one nucleic acid.
  • Other major changes such as deletions that remove functional regions of the promoter or enhancers are also included as these will reduce the expression of MADSL
  • a mutation may be introduced into the MADS1 promoter and/or enhancer and at least one mutation is introduced into the MADS1 gene.
  • the mutation is introduced using mutagenesis or targeted genome editing. That is, in one embodiment, the invention relates to a method and plant that has been generated by genetic engineering methods as described above, and does not encompass naturally occurring varieties.
  • mutagenesis preferably targeted genome modification
  • mutagenesis is used to create a loss-of function or null MADS1 allele.
  • mutagenesis, preferably targeted genome modification is used to create a dominant negative gain-of function MADS1 allele.
  • Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events.
  • DSBs DNA double-strand breaks
  • HR homologous recombination
  • customisable DNA binding proteins can be used: meganucleases derived from microbial mobile genetic elements, ZF nucleases based on eukaryotic transcription factors, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, and the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats).
  • ZF and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate nuclease and DNA-binding domains, ZF and TALE proteins consist of individual modules targeting 3 or 1 nucleotides (nt) of DNA, respectively. ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of Fokl to direct nucleolytic activity toward specific genomic loci.
  • TAL effectors Upon delivery into host cells via the bacterial type III secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in host gene promoters and activate transcription. Their targeting specificity is determined by a central domain of tandem, 33-35 amino acid repeats. This is followed by a single truncated repeat of 20 amino acids. The majority of naturally occurring TAL effectors examined have between 12 and 27 full repeats.
  • RVD repeat- variable di-residue
  • Naturally occurring recognition sites are uniformly preceded by a T that is required for TAL effector activity.
  • TAL effectors can be fused to the catalytic domain of the Fokl nuclease to create a TAL effector nuclease (TALEN) which makes targeted DNA double-strand breaks (DSBs) in vivo for genome editing.
  • TALEN TAL effector nuclease
  • Assembly of a custom TALEN or TAL effector construct involves two steps: (i) assembly of repeat modules into intermediary arrays of 1-10 repeats and (ii) joining of the intermediary arrays into a backbone to make the final construct. Accordingly, using techniques known in the art it is possible to design a TAL effector that targets a MADS1 gene or promoter sequence as described herein.
  • CRISPR Another genome editing method that can be used according to the various aspects of the invention is CRISPR.
  • CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids.
  • CRISPR loci in microbial hosts contain a combination of CRISPR- associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid
  • each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers).
  • the non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer).
  • the Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus.
  • tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences.
  • the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition.
  • Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.
  • CRISPR-Cas9 compared to conventional gene targeting and other programmable endonucleases is the ease of multiplexing, where multiple genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different gene.
  • the intervening section can be deleted or inverted (Wiles et al., 2015).
  • Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
  • the Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases.
  • the HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA.
  • sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms.
  • DSBs site-specific double strand breaks
  • codon optimized versions of Cas9 which is originally from the bacterium Streptococcus pyogenes, have been used.
  • the single guide RNA is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease.
  • sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA.
  • the sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities.
  • the canonical length of the guide sequence is 20 bp.
  • sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3. Accordingly, using techniques known in the art it is possible to design sgRNA molecules that targets a MADS1 gene or promoter sequence as described herein.
  • Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.
  • mutagenesis methods can be used to introduce at least one mutation into a MADS1 gene or MADS1 promoter sequence. These methods include both physical and chemical mutagenesis. A skilled person will know further approaches can be used to generate such mutants, and methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds.
  • insertional mutagenesis is used, for example using T-DNA mutagenesis (which inserts pieces of the T-DNA from the Agrobacterium tumefaciens T-Plasmid into DNA causing either loss of gene function or gain of gene function mutations), site-directed nucleases (SDNs) or transposons as a mutagen. Insertional mutagenesis is an alternative means of disrupting gene function and is based on the insertion of foreign DNA into the gene of interest (see Krysan et al, The Plant Cell, Vol. 1 1 , 2283-2290, December 1999).
  • T-DNA is used as an insertional mutagen to disrupt the MADS1 gene or MADS1 promoter expression.
  • T- DNA not only disrupts the expression of the gene into which it is inserted, but also acts as a marker for subsequent identification of the mutation. Since the sequence of the inserted element is known, the gene in which the insertion has occurred can be recovered, using various cloning or PCR-based strategies.
  • the insertion of a piece of T-DNA in the order of 5 to 25 kb in length generally produces a disruption of gene function. If a large enough population of T-DNA transformed lines is generated, there are reasonably good chances of finding a transgenic plant carrying a T-DNA insert within any gene of interest. Transformation of spores with T-DNA is achieved by an Agrobacterium-mediated method which involves exposing plant cells and tissues to a suspension of Agrobacterium cells.
  • mutagenesis is physical mutagenesis, such as application of ultraviolet radiation, X-rays, gamma rays, fast or thermal neutrons or protons.
  • the method comprises mutagenizing a plant population with a mutagen.
  • the mutagen may be a fast neutron irradiation or a chemical mutagen, for example selected from the following non-limiting list: ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N- nitrosurea (ENU), triethylmelamine (1 ⁇ ), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N'-nitro- Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 di
  • the targeted population can then be screened to identify a MADS1 gene or promoter mutant.
  • the method used to create and analyse mutations is targeting induced local lesions in genomes (TILLING), reviewed in Henikoff et al, 2004.
  • seeds are mutagenised with a chemical mutagen, for example EMS.
  • the resulting M1 plants are self-fertilised and the M2 generation of individuals is used to prepare DNA samples for mutational screening.
  • DNA samples are pooled and arrayed on microtiter plates and subjected to gene specific PCR.
  • the PCR amplification products may be screened for mutations in the MADS1 target gene using any method that identifies heteroduplexes between wild type and mutant genes.
  • dHPLC denaturing high pressure liquid chromatography
  • DCE constant denaturant capillary electrophoresis
  • TGCE temperature gradient capillary electrophoresis
  • the PCR amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild type and mutant sequences.
  • Cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image-processing program.
  • Any primer specific to the MADS1 nucleic acid sequence may be utilized to amplify the MADS1 nucleic acid sequence within the pooled DNA sample.
  • the primer is designed to amplify the regions of the MADS1 gene where useful mutations are most likely to arise, specifically in the areas of the MADS1 gene that are highly conserved and/or confer activity as explained elsewhere.
  • the PCR primer may be labelled using any conventional labelling method.
  • the method used to create and analyse mutations is EcoTILLING.
  • EcoTILLING is molecular technique that is similar to TILLING, except that its objective is to uncover natural variation in a given population as opposed to induced mutations. The first publication of the EcoTILLING method was described in Comai et al.2004.
  • Rapid high-throughput screening procedures thus allow the analysis of amplification products for identifying a mutation conferring the reduction or inactivation of the expression of the MADS1 gene or alternatively, a dominant negative gain of function as compared to a corresponding non-mutagenised wild type plant.
  • the expression of the MADS1 gene may be reduced at either the level of transcription or translation.
  • expression of a MADS1 nucleic acid or MADS1 promoter sequence, as defined herein can be reduced or silenced using a number of gene silencing methods known to the skilled person, such as, but not limited to, the use of small interfering nucleic acids (siNA) against MADS1.
  • silencing is a term generally used to refer to suppression of expression of a gene via sequence-specific interactions that are mediated by RNA molecules. The degree of reduction may be so as to totally abolish production of the encoded gene product, but more usually the abolition of expression is partial, with some degree of expression remaining. The term should not therefore be taken to require complete "silencing" of expression.
  • the siNA molecule may include, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), antagomirs and short hairpin RNA (shRNA) capable of mediating RNA interference.
  • the RNAi molecule targets a fragment of the MADS1 RNA sequence, preferably a sequence within a 317bp fragment as defined in SEQ ID NO: 296.
  • the below forward and reverse primers were used to amplify the fragment of OsMADSI , which was subsequently inserted into a pLGY3::RNAi-LGY3 construct leading to the formation of a stem-loop structure and the generation of siRNA.
  • the grains formed by the transgenic NPB NPB-LGY3 plants in which OsMADSI had been knocked down by RNAi were longer than those formed by non-transgenic control plants, as well as those produced by transgenic NPB-LGY3 plants expressing the L-204 Igy3 cDNA driven by its native promoter.
  • the inhibition of expression and/or activity can be measured by determining the presence and/or amount of MADS1 transcript using techniques well known to the skilled person (such as Northern Blotting, RT-PCR and so on).
  • Transgenes may be used to suppress endogenous plant genes. This was discovered originally when chalcone synthase transgenes in petunia caused suppression of the endogenous chalcone synthase genes and indicated by easily visible pigmentation changes. Subsequently it has been described how many, if not all plant genes can be "silenced" by transgenes. Gene silencing requires sequence similarity between the transgene and the gene that becomes silenced. This sequence homology may involve promoter regions or coding regions of the silenced target gene.
  • the transgene able to cause gene silencing may have been constructed with a promoter that would transcribe either the sense or the antisense orientation of the coding sequence RNA. It is likely that the various examples of gene silencing involve different mechanisms that are not well understood. In different examples there may be transcriptional or post-transcriptional gene silencing and both may be used according to the methods of the invention.
  • RNA-mediated gene suppression or RNA silencing includes co-suppression wherein over-expression of the target sense RNA or mRNA, that is the MADS1 sense RNA or mRNA, leads to a reduction in the level of expression of the genes concerned.
  • RNAs of the transgene and homologous endogenous gene are co-ordinately suppressed.
  • Other techniques used in the methods of the invention include antisense RNA to reduce transcript levels of the endogenous target gene in a plant. In this method, RNA silencing does not affect the transcription of a gene locus, but only causes sequence-specific degradation of target mRNAs.
  • an “antisense” nucleic acid sequence comprises a nucleotide sequence that is complementary to a “sense” nucleic acid sequence encoding a MADS1 protein, or a part of the protein, i.e. complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA transcript sequence.
  • the antisense nucleic acid sequence is preferably complementary to the endogenous MADS1 gene to be silenced.
  • the complementarity may be located in the "coding region” and/or in the "non-coding region” of a gene.
  • the term “coding region” refers to a region of the nucleotide sequence comprising codons that are translated into amino acid residues.
  • non-coding region refers to 5' and 3' sequences that flank the coding region that are transcribed but not translated into amino acids (also referred to as 5' and 3' untranslated regions).
  • Antisense nucleic acid sequences can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid sequence may be complementary to the entire MADS1 nucleic acid sequence as defined herein, but may also be an oligonucleotide that is antisense to only a part of the nucleic acid sequence (including the mRNA 5' and 3' UTR).
  • the antisense nucleic acid may be designed to the C-domain, or the terminal 37 residues thereof.
  • the antisense oligonucleotide sequence may be complementary to the region surrounding the translation start site of an mRNA transcript encoding a polypeptide.
  • a suitable antisense oligonucleotide sequence is known in the art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less.
  • An antisense nucleic acid sequence according to the invention may be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art.
  • an antisense nucleic acid sequence may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acid sequences, e.g., phosphorothioate derivatives and acridine-substituted nucleotides may be used.
  • modified nucleotides that may be used to generate the antisense nucleic acid sequences are well known in the art.
  • the antisense nucleic acid sequence can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest.
  • production of antisense nucleic acid sequences in plants occurs by means of a stably integrated nucleic acid construct comprising a promoter, an operably linked antisense oligonucleotide, and a terminator.
  • the nucleic acid molecules used for silencing in the methods of the invention hybridize with or bind to mRNA transcripts and/or insert into genomic DNA encoding a polypeptide to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid sequence which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • Antisense nucleic acid sequences may be introduced into a plant by transformation or direct injection at a specific tissue site. Alternatively, antisense nucleic acid sequences can be modified to target selected cells and then administered systemically.
  • antisense nucleic acid sequences can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid sequence to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid sequences can also be delivered to cells using vectors.
  • RNA interference is another post-transcriptional gene-silencing phenomenon which may be used according to the methods of the invention. This is induced by double-stranded RNA in which mRNA that is homologous to the dsRNA is specifically degraded. It refers to the process of sequence-specific post-transcriptional gene silencing mediated by short interfering RNAs (siRNA).
  • siRNA short interfering RNAs
  • the process of RNAi begins when the enzyme, DICER, encounters dsRNA and chops it into pieces called small- interfering RNAs (siRNA). This enzyme belongs to the RNase III nuclease family.
  • RNAs are typically single stranded small RNAs typically 19-24 nucleotides long. Most plant miRNAs have perfect or near-perfect complementarity with their target sequences. However, there are natural targets with up to five mismatches. They are processed from longer non- coding RNAs with characteristic fold-back structures by double-strand specific RNases of the Dicer family.
  • RISC RNA-induced silencing complex
  • miRNAs serve as the specificity components of RISC, since they base-pair to target nucleic acids, mostly mRNAs, in the cytoplasm. Subsequent regulatory events include target mRNA cleavage and destruction and/or translational inhibition. Effects of miRNA overexpression are thus often reflected in decreased mRNA levels of target genes.
  • Artificial microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (http://wmd.weigelworld.org).
  • a plant may be transformed to introduce a RNAi, shRNA, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule that has been designed to target the expression of an MADS1 nucleic acid sequence and selectively decreases or inhibits the expression of the gene or stability of its transcript.
  • the RNAi, snRNA, dsRNA, shRNA siRNA, miRNA, amiRNA, ta-siRNA or cosuppression molecule used according to the various aspects of the invention comprises a fragment of at least 17 nt, preferably 22 to 26 nt and can be designed on the basis of the information shown in any of SEQ ID Nos.1 to 22. Guidelines for designing effective siRNAs are known to the skilled person. Briefly, a short fragment of the target gene sequence (e.g., 19-40 nucleotides in length) is chosen as the target sequence of the siRNA of the invention. The short fragment of target gene sequence is a fragment of the target gene mRNA.
  • the criteria for choosing a sequence fragment from the target gene mRNA to be a candidate siRNA molecule include 1 ) a sequence from the target gene mRNA that is at least 50-100 nucleotides from the 5' or 3' end of the native mRNA molecule, 2) a sequence from the target gene mRNA that has a G/C content of between 30% and 70%, most preferably around 50%, 3) a sequence from the target gene mRNA that does not contain repetitive sequences (e.g., AAA, CCC, GGG, TTT, AAAA, CCCC, GGGG, TTTT), 4) a sequence from the target gene mRNA that is accessible in the mRNA, 5) a sequence from the target gene mRNA that is unique to the target gene, 6) avoids regions within 75 bases of a start codon.
  • a sequence from the target gene mRNA that is at least 50-100 nucleotides from the 5' or 3' end of the native mRNA molecule 2) a sequence from the target gene
  • the sequence fragment from the target gene mRNA may meet one or more of the criteria identified above.
  • the selected gene is introduced as a nucleotide sequence in a prediction program that takes into account all the variables described above for the design of optimal oligonucleotides.
  • This program scans any mRNA nucleotide sequence for regions susceptible to be targeted by siRNAs.
  • the output of this analysis is a score of possible siRNA oligonucleotides. The highest scores are used to design double stranded RNA oligonucleotides that are typically made by chemical synthesis.
  • degenerate siRNA sequences may be used to target homologous regions.
  • siRNAs according to the invention can be synthesized by any method known in the art. RNAs are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA RNA synthesizer. Additionally, siRNAs can be obtained from commercial RNA oligonucleotide synthesis suppliers. siRNA molecules according to the aspects of the invention may be double stranded. In one embodiment, double stranded siRNA molecules comprise blunt ends. In another embodiment, double stranded siRNA molecules comprise overhanging nucleotides (e.g., 1 -5 nucleotide overhangs, preferably 2 nucleotide overhangs).
  • the siRNA is a short hairpin RNA (shRNA); and the two strands of the siRNA molecule may be connected by a linker region (e.g., a nucleotide linker or a non- nucleotide linker).
  • the siRNAs of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the siRNA. The skilled person will be aware of other types of chemical modification which may be incorporated into RNA molecules.
  • the silencing RNA molecule is introduced into the plant using conventional methods, for example a vector and Agrobacterium-mediated transformation. Stably transformed plants are generated and expression of the MADS1 gene compared to a wild type control plant is analysed.
  • Silencing of the MADS1 nucleic acid sequence may also be achieved using virus- induced gene silencing.
  • the plant expresses a nucleic acid construct comprising a RNAi, shRNA snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or co- suppression molecule that targets the MADS1 nucleic acid sequence as described herein and reduces expression of the endogenous MADS1 nucleic acid sequence.
  • a gene is targeted when, for example, the RNAi, snRNA, dsRNA, siRNA, shRNA miRNA, ta-siRNA, amiRNA or cosuppression molecule selectively decreases or inhibits the expression of the gene compared to a control plant.
  • RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule targets a MADS1 nucleic acid sequence when the RNAi, shRNA snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule hybridises under stringent conditions to the gene transcript.
  • a further approach to gene silencing is by targeting nucleic acid sequences complementary to the regulatory region of the gene (e.g., the promoter and/or enhancers) of MADS1 to form triple helical structures that prevent transcription of the gene in target cells.
  • Other methods such as the use of antibodies directed to an endogenous polypeptide for inhibiting its function in planta, or interference in the signalling pathway in which a polypeptide is involved, will be well known to the skilled man.
  • manmade molecules may be useful for inhibiting the biological function of a target polypeptide, or for interfering with the signalling pathway in which the target polypeptide is involved.
  • the suppressor nucleic acids may be anti-sense suppressors of expression of the MADS1 polypeptides.
  • a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene.
  • An anti-sense suppressor nucleic acid may comprise an anti-sense sequence of at least 10 nucleotides from the target nucleotide sequence. It may be preferable that there is complete sequence identity in the sequence used for down-regulation of expression of a target sequence, and the target sequence, although total complementarity or similarity of sequence is not essential.
  • One or more nucleotides may differ in the sequence used from the target gene.
  • a sequence employed in down-regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a variant of such a sequence.
  • the sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective anti-sense and sense RNA molecules to hybridise. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene. Effectively, the homology should be sufficient for the down-regulation of gene expression to take place.
  • Suppressor nucleic acids may be operably linked to tissue-specific or inducible promoters.
  • tissue-specific or inducible promoters For example, integument and seed specific promoters can be used to specifically down-regulate an MADS1 nucleic acid in developing ovules and seeds to increase grain number, weight, length or size.
  • Nucleic acid which suppresses expression of an MADS1 polypeptide as described herein may be operably linked to a heterologous regulatory- sequence, such as a promoter, for example a constitutive, inducible, tissue-specific or developmental specific promoter.
  • a heterologous regulatory- sequence such as a promoter, for example a constitutive, inducible, tissue-specific or developmental specific promoter.
  • the construct or vector may be transformed into plant cells and expressed as described herein. Plant cells comprising such vectors are also within the scope of the invention.
  • the invention in another aspect, relates to a silencing construct obtainable or obtained by a method as described herein and to a plant cell comprising such construct.
  • aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and in a preferred embodiment exclude embodiments that are solely based on generating plants by traditional breeding methods.
  • the method may comprise reducing and/or abolishing the activity of MADS1 .
  • this may comprise reducing MADSVs ability to bind and modulate (increase or decrease) transcription of its target genes.
  • MADSVs target genes include AP2/ERF family members, MAS-box family members, GARP family members, the auxin efflux carrier and auxin response factor genes. This is not an exhaustive list.
  • the method may further comprise reducing or abolishing the expression of at least one MADS1 co-factor.
  • the method further comprises introducing at least one mutation into a MADS1 co-factor such that preferably, the mutation reduces or abolishes the expression of the MADS1 co-factor or reduces or abolishes the activity of the MADS-1 co-factor.
  • the mutation may result in the expression of dominant negative gain of function MADS co-factor allele.
  • the MADS-co-factor is a Ggamma (Gy) subunit.
  • the Gy subunit may be a canonical or non-canonical subunit. Examples of canonical Gy subunits in rice include RGG1 and RGG2.
  • non-canonical Gy subunits in rice examples include GS3, DEP1 and OsGGC2.
  • the MADS-cofactor is DENSE AND ERECT PANICLE1 (DEP1 ) and/or GRAIN SIZE3 (GS3).
  • the method comprises introducing at least one mutation, as defined herein, into a DEP1 and/or GS3 gene or a functional variant or homologue thereof.
  • a functional variant or homologue is defined elsewhere.
  • the at least one mutation in a dep-1 gene involves the replacement of a 637-bp stretch of the middle of exon 5 by a 12-bp sequence, which has the effect of creating a premature stop codon and consequently a loss of 230 residues from the C terminus.
  • the at least one mutation in a GS3 gene is a single nucleotide mutation at the second exon, which results in a change of a cysteine codon (TGC) to a termination codon (TGA). This premature termination results in a 178-aa truncation in the C-terminus of the predicted protein.
  • the wild-type DEP1 gene encodes a polypeptide as defined in SEQ ID NO: 169 or a functional variant or homologue thereof.
  • said DEP1 gene comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 168 or a functional variant or homologue thereof.
  • the wild-type GS3 gene encodes a polypeptide as defined in SEQ ID NO: 172 or a functional variant or homologue thereof.
  • said GS3 gene comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 173 or a functional variant or homologue thereof.
  • a method of increasing cell proliferation resulting in an increase in grain length comprising modulating the expression and/or activity of at least one nucleic acid encoding a MADS1 polypeptide in said plant.
  • the terms "increase”, “improve” or “enhance” as used herein are interchangeable.
  • cell proliferation is increased by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40% or 50% compared to a control plant.
  • a genetically altered plant, part thereof or plant cell characterised in that the plant has an altered expression or activity of the MADS1 nucleic acid or polypeptide, for example, the plant does not express MADS1 , has reduced levels of MADS1 expression, does not express a functional MADS1 protein, expresses a MADS1 protein with reduced function and/or activity or expresses a MADS1 protein with a dominant negative gain of function.
  • the plant is a reduction (knock down) or loss of function (knock out) mutant wherein the function or expression of the MADS1 nucleic acid sequence is reduced or lost compared to a wild type control plant.
  • a mutation is introduced into either the MADS1 gene sequence or the corresponding promoter/enhancer sequence which disrupts the transcription of the gene. Therefore, preferably said plant comprises at least one mutation in the promoter and/or gene for MADS1 . In one embodiment the plant may comprise a mutation in both the promoter and gene for MADS1 .
  • the plant carries or expresses a dominant negative gain of function allele of MADS1.
  • a mutation is introduced into the MADS1 gene sequence, preferably into at least one conserved domain, as described above, or at a splice site, to produce a MADS1 protein that can act partially or wholly antagonistically to the wild-type allele (when present as a heteozygote).
  • said mutation may result in a protein with reduced, altered or no wild-type function (wherein the wild-type function may be considered to be the transcriptional regulation (decrease or increase) of down-stream target genes.
  • a plant, part thereof or plant cell characterised by an increased grain yield compared to a wild-type or control pant, wherein preferably, the plant comprises at least one mutation in the MADS1 gene and/or its promoter.
  • said increase in grain yield comprises at least one of grain size, grain number, grain length, grain weight and thousand kernel weight.
  • the plant may be produced by introducing a mutation, preferably a deletion, insertion or substitution into the MADS1 gene and/or promoter sequence by any of the above described methods.
  • a mutation preferably a deletion, insertion or substitution into the MADS1 gene and/or promoter sequence by any of the above described methods.
  • said mutation is introduced into a least one plant cell and a plant regenerated from the at least one mutated plant cell.
  • the plant or plant cell may comprise a nucleic acid construct expressing an RNAi molecule targeting the MADS1 gene as described herein.
  • said construct is stably incorporated into the plant genome.
  • These techniques also include gene target using vectors that target the gene of interest and which allows for integration of a transgene at a specific site.
  • the targeting construct is engineered to recombine with the target gene, which is accomplished by incorporating sequences from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene will be translated into a nonfunctional protein, if it is translated at all.
  • a short interfering nucleic acid molecule as described elsewhere, wherein the molecule targets SEQ ID NO: 167 or a variant thereof.
  • the siNA comprises a sense and an anti-sense strand.
  • a nucleic acid construct comprising a nucleic acid sequence encoding a siNA as described above. Also included in the scope of the invention is the use of the siNAs and nucleic acid constructs described above to modulate MADS1 expression and/or activity as described herein. Alternatively, also included is the use of the siNAs and nucleic acid constructs described above to increase grain yield and/or quality in a plant.
  • a method of reducing or abolishing the expression of MADS1 wherein the method uses RNA interference, and more preferably, the method comprises introducing and expressing in a plant the siNA or nucleic acid construct described above. The method may further comprise measuring the levels of MADS1 expression. A reduction is as described herein.
  • the method comprises introducing at least one mutation into the MADS1 gene and/or MADS1 promoter of preferably at least one plant cell using any mutagenesis technique described herein. Preferably said method further comprising regenerating a plant from the mutated plant cell.
  • the method may further comprise selecting one or more mutated plants, preferably for further propagation.
  • said selected plants comprise at least one mutation in the MADS1 gene and/or promoter sequence.
  • said plants are characterised by abolished or a reduced level of MADS1 expression and/or a reduced level of MADS1 polypeptide activity.
  • said plants are characterised by a dominant negative gain of function.
  • Expression and/or activity levels of MADS1 can be measured by any standard technique known to the skilled person. A reduction is as described herein.
  • the selected plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1 ) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
  • a "genetically altered plant” or “mutant plant” is a plant that has been genetically altered compared to the naturally occurring wild type (WT) plant.
  • a mutant plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant using a mutagenesis method, such as any of the mutagenesis methods described herein.
  • the mutagenesis method is targeted genome modification or genome editing.
  • the plant genome has been altered compared to wild type sequences using a mutagenesis method. Such plants have an altered phenotype as described herein, such as an increased grain yield.
  • increased grain yield is conferred by the presence of an altered plant genome, for example, a mutated endogenous MADS1 gene or promoter.
  • the endogenous promoter or gene sequence is specifically targeted using targeted genome modification and the presence of a mutated gene or promoter sequence is not conferred by the presence of transgenes expressed in the plant.
  • the genetically altered plant can be described as transgene-free.
  • a plant according to the various aspects of the invention, including the transgenic plants, methods and uses described herein may be a monocot or a dicot plant.
  • the plant is a crop plant.
  • crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use.
  • the plant is a cereal. In another embodiment the plant is Arabidopsis. In a most preferred embodiment, the plant is selected from rice, maize, wheat, sorghum, brassica, soybean and millet. In a most preferred embodiment the plant is rice, preferably the japonica or indica varieties. More preferably, the japonica variety is Wuyunjing 7. Preferably, the indica variety is RD23. In a further preferred embodiment, the plant carries a mutation in a canonical or non-canonical Gy subunit. Examples of canonical Gy subunits in rice include RGG1 and RGG2. Examples of non-canonical Gy subunits in rice include GS3, DEP1 and OsGGC2.
  • the plant carries or expresses a mutant dep-1 and/or gs-3 allele or a functional variant or homologue thereof.
  • the plant (endogenously) carries or expresses a nucleic acid sequence comprising or consisting of SEQ ID NO: 170 and/or 175 or a nucleic acid that encodes a polypeptide as defined in SEQ ID NO: 171 or 175 respectively.
  • plant as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned comprise the nucleic acid construct as described herein.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the nucleic acid construct as described herein.
  • the invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs.
  • the aspects of the invention also extend to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
  • Another product that may derived from the harvestable parts of the plant of the invention is biodiesel.
  • the invention also relates to food products and food supplements comprising the plant of the invention or parts thereof. In one embodiment, the food products may be animal feed.
  • a product derived from a plant as described herein or from a part thereof there is provided.
  • the plant part or harvestable product is a seed or grain. Therefore, in a further aspect of the invention, there is provided a seed produced from a genetically altered plant as described herein.
  • the plant part is pollen, a propagule or progeny of the genetically altered plant described herein. Accordingly, in a further aspect of the invention there is provided pollen, a propagule or progeny of the genetically altered plant as described herein.
  • a control plant as used herein according to all of the aspects of the invention is a plant which has not been modified according to the methods of the invention. Accordingly, in one embodiment, the control plant does not have altered expression of a MADS1 nucleic acid and/or altered activity of a MADS1 polypeptide, as described above. In an alternative embodiment, the plant has not been genetically modified, as described above. In one embodiment, the control plant is a wild type plant. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant. Genome editing constructs for use with the methods for targeted genome modification described herein
  • crRNA or CRISPR RNA is meant the sequence of RNA that contains the protospacer element and additional nucleotides that are complementary to the tracrRNA.
  • tracrRNA transactivating RNA
  • protospacer element is meant the portion of crRNA (or sgRNA) that is complementary to the genomic DNA target sequence, usually around 20 nucleotides in length. This may also be known as a spacer or targeting sequence.
  • sgRNA single-guide RNA
  • sgRNA single-guide RNA
  • gRNA single-guide RNA
  • the sgRNA or gRNA provide both targeting specificity and scaffolding/binding ability for a Cas nuclease.
  • a gRNA may refer to a dual RNA molecule comprising a crRNA molecule and a tracrRNA molecule.
  • TAL effector transcription activator-like (TAL) effector
  • TALE transcription activator-like (TAL) effector
  • a TALE protein is composed of a central domain that is responsible for DNA binding, a nuclear-localisation signal and a domain that activates target gene transcription.
  • the DNA-binding domain consists of monomers and each monomer can bind one nucleotide in the target nucleotide sequence.
  • Monomers are tandem repeats of 33-35 amino acids, of which the two amino acids located at positions 12 and 13 are highly variable (repeat variable diresidue, RVD). It is the RVDs that are responsible for the recognition of a single specific nucleotide.
  • HD targets cytosine; Nl targets adenine, NG targets thymine and NN targets guanine (although NN can also bind to adenine with lower specificity).
  • nucleic acid construct wherein the nucleic acid construct encodes at least one DNA-binding domain, wherein the DNA- binding domain can bind to a sequence in the MADS1 gene, wherein said sequence is selected from SEQ ID Nos 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, 73, 78, 83, 88, 93, 98, 103, 108, 1 13, 1 18, 123, 128, 133, 138, 143, 148, 153 and 158.
  • said construct further comprises a nucleic acid encoding a (SSN) sequence-specific nuclease, such as Fokl or a Cas protein.
  • SSN sequence-specific nuclease
  • the nucleic acid construct encodes at least one protospacer element wherein the sequence of the protospacer element is selected from SEQ ID No 24, 29, 34, 39, 44, 49, 54, 59, 64, 69, 74, 79, 84, 89, 94, 99, 104, 109, 1 14, 1 19, 124, 129, 134, 139, 144, 149, 154 and 159 or a variant thereof.
  • the nucleic acid construct comprises a crRNA-encoding sequence.
  • a crRNA sequence may comprise the protospacer elements as defined above and preferably additional nucleotides that are complementary to the tracrRNA.
  • An appropriate sequence for the additional nucleotides will be known to the skilled person as these are defined by the choice of Cas protein.
  • the nucleic acid construct further comprises a tracrRNA sequence.
  • a tracrRNA sequence an appropriate tracrRNA sequence would be known to the skilled person as this sequence is defined by the choice of Cas protein. Nonetheless, in one embodiment said sequence comprises or consists of a sequence as defined in SEQ ID NO: 103 or a variant thereof.
  • the nucleic acid construct comprises at least one nucleic acid sequence that encodes a sgRNA (or gRNA).
  • sgRNA typically comprises a crRNA sequence, a tracrRNA sequence and preferably a sequence for a linker loop.
  • the nucleic acid construct comprises at least one nucleic acid sequence that encodes a sgRNA sequence as defined in any of SEQ ID Nos 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155 and 160 or variant thereof.
  • the nucleic acid construct comprises or consists of a sequence selected from SEQ ID NO: 27, 32, 37, 42, 7, 52, 57, 62, 67, 72, 77, 82, 87, 92, 97, 102, 107, 1 12, 1 17, 122, 127, 132, 137, 142, 147, 152, 157 and 162.
  • the nucleic acid construct may further comprise at least one nucleic acid sequence encoding an endoribonuclease cleavage site.
  • the endoribonuclease is Csy4 (also known as Cas6f).
  • the nucleic acid construct comprises multiple sgRNA nucleic acid sequences the construct may comprise the same number of endoribonuclease cleavage sites.
  • the cleavage site is 5' of the sgRNA nucleic acid sequence. Accordingly, each sgRNA nucleic acid sequence is flanked by a endoribonuclease cleavage site.
  • the term 'variant' refers to a nucleotide sequence where the nucleotides are substantially identical to one of the above sequences.
  • the variant may be achieved by modifications such as insertion, substitution or deletion of one or more nucleotides.
  • the variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of the above described sequences.
  • sequence identity is at least 90%.
  • sequence identity is 100%. Sequence identity can be determined by any one known sequence alignment program in the art.
  • the invention also relates to a nucleic acid construct comprising a nucleic acid sequence operably linked to a suitable plant promoter.
  • a suitable plant promoter may be a constitutive or strong promoter or may be a tissues-specific promoter.
  • suitable plant promoters are selected from, but not limited to, cestrum yellow leaf curling virus (CmYLCV) promoter or switchgrass ubiquitin 1 promoter (PvUbil ) wheat U6 RNA polymerase III (TaU6) CaMV35S, wheat U6 or maize ubiquitin (e.g. Ubi 1 ) promoters.
  • CmYLCV cestrum yellow leaf curling virus
  • PvUbil switchgrass ubiquitin 1 promoter
  • TaU6 wheat U6 RNA polymerase III
  • CaMV35S wheat U6 or maize ubiquitin (e.g. Ubi 1 ) promoters.
  • expression can be specifically directed to particular tissues of wheat seeds through gene expression-regulating sequences.
  • the promoter is selected from the U3 promoter (SEQ ID NO: 177), the U6a promoter (SEQ ID NO: 178), the U6b promoter (SEQ ID NO: 179), the U3b promoter in dicot plants (SEQ ID NO: 180) and the U6-1 promoter in dicot plants (SEQ ID NO: 181 ).
  • the nucleic acid construct of the present invention may also further comprise a nucleic acid sequence that encodes a CRISPR enzyme.
  • CRISPR enzyme is meant an RNA-guided DNA endonuclease that can associate with the CRISPR system. Specifically, such an enzyme binds to the tracrRNA sequence.
  • the CRIPSR enzyme is a Cas protein ("CRISPR associated protein), preferably Cas 9 or Cpf1 , more preferably Cas9.
  • Cas9 is codon-optimised Cas9, and more preferably, has the sequence described in SEQ ID NO: 104 or a functional variant or homolog thereof.
  • the CRISPR enzyme is a protein from the family of Class 2 candidate x proteins, such as C2c1 , C2C2 and/or C2c3.
  • the Cas protein is from Streptococcus pyogenes.
  • the Cas protein may be from any one of Staphylococcus aureus, Neisseria meningitides, Streptococcus thermophiles or Treponema den ti cola.
  • the term "functional variant” as used herein with reference to Cas9 refers to a variant Cas9 gene sequence or part of the gene sequence which retains the biological function of the full non-variant sequence, for example, acts as a DNA endonuclease, or recognition or/and binding to DNA.
  • a functional variant also comprises a variant of the gene of interest which has sequence alterations that do not affect function, for example non-conserved residues.
  • Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active.
  • a functional variant of SEQ ID NO.104 has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the amino acid represented by SEQ ID NO: 104.
  • the Cas9 protein has been modified to improve activity. Suitable homologs or orthologs can be identified by sequence comparisons and identifications of conserved domains. The function of the homolog or ortholog can be identified as described herein and a skilled person would thus be able to confirm the function when expressed in a plant.
  • the Cas9 protein has been modified to improve activity.
  • the Cas9 protein may comprise the D10A amino acid substitution, this nickase cleaves only the DNA strand that is complementary to and recognized by the gRNA.
  • the Cas9 protein may alternatively or additionally comprise the H840A amino acid substitution, this nickase cleaves only the DNA strand that does not interact with the sRNA.
  • Cas9 may be used with a pair (i.e. two) sgRNA molecules (or a construct expressing such a pair) and as a result can cleave the target region on the opposite DNA strand, with the possibility of improving specificity by 100-1500 fold.
  • the Cas9 protein may comprise a D1 135E substitution.
  • the Cas 9 protein may also be the VQR variant.
  • the Cas protein may comprise a mutation in both nuclease domains, HNH and RuvC-like and therefore is catalytically inactive. Rather than cleaving the target strand, this catalytically inactive Cas protein can be used to prevent the transcription elongation process, leading to a loss of function of incompletely translated proteins when co-expressed with a sgRNA molecule.
  • An example of a catalytically inactive protein is dead Cas9 (dCas9) caused by a point mutation in RuvC and/or the HNH nuclease domains (Komor et al., 2016 and Nishida et al., 2016).
  • a Cas protein such as Cas9 may be further fused with a repression effector, such as a histone-modifying/DNA methylation enzyme or a Cytidine deaminase ( Komor et al.2016) to effect site-directed mutagenesis.
  • a repression effector such as a histone-modifying/DNA methylation enzyme or a Cytidine deaminase ( Komor et al.2016) to effect site-directed mutagenesis.
  • the cytidine deaminase enzyme does not induce dsDNA breaks, but mediates the conversion of cytidine to uridine, thereby effecting a C to T (or G to A) substitution.
  • the nucleic acid construct comprises an endoribonuclease.
  • the endoribonuclease is Csy4 (also known as Cas6f) and more preferably a codon optimised csy4, for example as defined in SEQ ID NO: 166.
  • the nucleic acid construct may comprise sequences for the expression of an endoribonuclease, such as Csy4 expressed as a 5' terminal P2A fusion (used as a self-cleaving peptide) to a cas protein, such as Cas9, for example, as defined in SEQ ID NO: 164
  • an endoribonuclease such as Csy4 expressed as a 5' terminal P2A fusion (used as a self-cleaving peptide)
  • a cas protein such as Cas9
  • the cas protein, the endoribonuclease and/or the endoribonuclease-cas fusion sequence may be operably linked to a suitable plant promoter.
  • suitable plant promoters are already described above, but in one embodiment, may be the Zea Mays Ubiquitin 1 promoter.
  • Suitable methods for producing the CRISPR nucleic acids and vectors system are known, and for example are published in Molecular Plant (Ma et al., 2015, Molecular Plant, DOI:10.1016/j.molp.2015.04.007), which is incorporated herein by reference.
  • the nucleic acid construct comprises at least one nucleic acid sequence that encodes a TAL effector, wherein said effector targets a MADS1 sequence selected from SEQ ID NO 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, 73, 78, 83, 88, 93, 98, 103, 108, 1 13, 1 18, 123, 128, 133, 138, 143, 148, 153 and 158.
  • Methods for designing a TAL effector would be well known to the skilled person, given the target sequence. Examples of suitable methods are given in Sanjana et al., and Cermak T et al, both incorporated herein by reference.
  • said nucleic acid construct comprises two nucleic acid sequences encoding a TAL effector, to produce a TALEN pair.
  • the nucleic acid construct further comprises a sequence-specific nuclease (SSN).
  • SSN is an endonuclease such as Fokl.
  • the TALENs are assembled by the Golden Gate cloning method in a single plasmid or nucleic acid construct.
  • a sgRNA molecule wherein the sgRNA molecule comprises a crRNA sequence and a tracrRNA sequence and wherein the crRNA sequence can bind to at least one sequence selected from SEQ ID Nos 26, 31,36,41,46, 51,56,61,66,71,76,81,86,91,96, 101 , 106, 111 , 116, 121 , 126, 131, 136, 141, 146, 151, 156 and 161 or a variant thereof.
  • the sequence of the sgRNA molecule is defined in any of SEQ ID NO: 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156 and 161 or variant thereof.
  • a "variant" is as defined herein.
  • the sgRNA molecule may comprise at least one chemical modification, for example that enhances its stability and/or binding affinity to the target sequence or the crRNA sequence to the tracrRNA sequence.
  • the crRNA may comprise a phosphorothioate backbone modification, such as 2'-fluoro (2'-F), 2'-0-methyl (2'-0-Me) and S-constrained ethyl (cET) substitutions.
  • the CRISPR constructs described herein can be used to create a loss-of function or null MADS1 allele or alternatively, a dominant negative gain-of function MADS1 allele. Table 1 details the target sequences, protospacer sequences, sgRNA sequence, sgRNA molecule and full nucleic acid construct sequences for the loss and gain-of function alleles respectively.
  • Table 1 SEQ ID NOs of loss- of function and gain-of function MADS1 CRISPR constructs
  • an isolated nucleic acid sequence that encodes for a protospacer element (as defined in any of SEQ ID Nos 24, 29, 34, 39, 44, 49, 54, 59, 64, 69, 74, 79, 84, 89, 94, 99, 104, 109, 1 14, 1 19, 124, 129, 134, 139, 144, 149, 154 and 159), or a sgRNA (as described in any of SEQ I D NO: 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155 and 160).
  • a protospacer element as defined in any of SEQ ID Nos 24, 29, 34, 39, 44, 49, 54, 59, 64, 69, 74, 79, 84, 89, 94, 99, 104, 109, 1 14, 1 19, 124, 129, 134, 139
  • Cas9 and sgRNA may be combined or in separate expression vectors (or nucleic acid constructs, such terms are used interchangeably).
  • an isolated plant cell is transfected with a single nucleic acid construct comprising both sgRNA and Cas9 as described in detail above.
  • an isolated plant cell is transfected with two nucleic acid constructs, a first nucleic acid construct comprising at least one sgRNA as defined above and a second nucleic acid construct comprising Cas9 or a functional variant or homolog thereof.
  • the second nucleic acid construct may be transfected below, after or concurrently with the first nucleic acid construct.
  • the advantage of a separate, second construct comprising a cas protein is that the nucleic acid construct encoding at least one sgRNA can be paired with any type of cas protein, as described herein, and therefore is not limited to a single cas function (as would be the case when both cas and sgRNA are encoded on the same nucleic acid construct).
  • the nucleic acid construct comprising a cas protein is transfected first and is stably incorporated into the genome, before the second transfection with a nucleic acid construct comprising at least one sgRNA nucleic acid.
  • a plant or part thereof or at least one isolated plant cell is transfected with mRNA encoding a cas protein and co-transfected with at least one nucleic acid construct as defined herein.
  • Cas9 expression vectors for use in the present invention can be constructed as described in the art.
  • the expression vector comprises a nucleic acid sequence as defined in SEQ ID NO: 104 or a functional variant or homolog thereof, wherein said nucleic acid sequence is operably linked to a suitable promoter.
  • suitable promoters include the Actin, CaMV35S, wheat U6 or maize ubiquitin (e.g. Ubi1 ) promoter.
  • CRISPR constructs nucleic acid constructs
  • sgRNA molecules any of the above described methods.
  • the CRISPR constructs may be used to create loss of function (i.e. "null") alleles or dominant negative gain of function alleles. Therefore, in a further aspect of the invention, there is provided a method of modulating MADS1 expression and/or activity, the method comprising introducing and expressing a CRISPR construct as described above or introducing a sgRNA molecule as also described above into a plant.
  • a method of modulating MADS1 expression and/or activity comprises introducing at least one mutation into the endogenous MADS1 gene and/or promoter using CRISPR/Cas9, and specifically, the CRISPR constructs described herein.
  • a method of producing a plant with a dominant negative gain of function MADS1 allele comprising introducing and expressing a gain-of function nucleic acid construct as defined above or introducing a gain-of function sgRNA molceule, as also defined above, in a plant.
  • a genetically modified or edited plant comprising the transfected cell described herein.
  • the nucleic acid construct or constructs may be integrated in a stable form.
  • the nucleic acid construct or constructs are not integrated (i.e. are transiently expressed).
  • the genetically modified plant is free of any sgRNA and/or Cas protein nucleic acid. In other words, the plant is transgene free.
  • introduction encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a genetic construct of the present invention and a whole plant regenerated there from.
  • the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • the resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
  • transformation Transformation of plants is now
  • Any of several transformation methods known to the skilled person may be used to introduce the nucleic acid construct or sgRNA molecule of interest into a suitable ancestor cell.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation.
  • Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant (microinjection), gene guns (or biolistic particle delivery systems (bioloistics)) as described in the examples, lipofection, transformation using viruses or pollen and microprojection.
  • Methods may be selected from the calcium/polyethylene glycol method for protoplasts, ultrasound-mediated gene transfection, optical or laser transfection, transfection using silicon carbide fibers, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like.
  • Transgenic plants can also be produced via Agrobacterium tumefaciens mediated transformation, including but not limited to using the floral dip/ Agrobacterium vacuum infiltration method as described in Clough & Bent (1998) and incorporated herein by reference.
  • At least one nucleic acid construct or sgRNA molecule as described herein can be introduced to at least one plant cell using any of the above described methods.
  • any of the nucleic acid constructs described herein may be first transcribed to form a preassembled Cas9- sgRNA ribonucleoprotein and then delivered to at least one plant cell using any of the above described methods, such as lipofection, electroporation or microinjection.
  • the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
  • the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying.
  • a further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants.
  • a suitable marker can be bar-phosphinothricin or PPT.
  • the transformed plants are screened for the presence of a selectable marker, such as, but not limited to, GFP, GUS ( ⁇ -glucuronidase). Other examples would be readily known to the skilled person.
  • putatively transformed plants may also be evaluated, for instance using PCR to detect the presence of the gene of interest, copy number and/or genomic organisation.
  • integration and expression levels of the newly introduced DNA may be monitored using Southern, Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1 ) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • T1 first generation
  • T2 homozygous second-generation
  • Specific protocols for using the above described CRISPR constructs would be well known to the skilled person.
  • a suitable protocol is described in Ma & Liu ("CRISPR/Casp-based multiplex genome editing in monocot and dicot plants") incorporated herein by reference.
  • the method also comprises the step of screening the genetically modified plant for SSN (preferably CRISPR)-induced mutations in the MADS1 gene or promoter sequence.
  • the method comprises obtaining a DNA sample from a transformed plant and carrying out DNA amplification to detect a mutation in at least one MADS1 gene or promoter sequence.
  • the methods comprise generating stable T2 plants preferably homozygous for the mutation (that is a mutation in in at least one MADS1 gene or promoter sequence). Plants that have a mutation in at least one MADS1 gene or promoter sequence can also be crossed with another plant also containing at least one different mutation in at least one MADS1 gene or promoter sequence to obtain plants with additional mutations in the MADS1 gene or promoter sequence.
  • this method can be used to generate a T2 plants with mutations on all or an increased number of homoelogs, when compared to the number of homoeolog mutations in a single T1 plant transformed as described above.
  • a genetically altered plant of the present invention may also be obtained by transference of any of the sequences of the invention by crossing, e.g., using pollen of the genetically altered plant described herein to pollinate a wild-type or control plant, or pollinating the gynoecia of plants described herein with other pollen that does not contain a mutation in at least one of the MADS1 gene or promoter sequence.
  • the methods for obtaining the plant of the invention are not exclusively limited to those described in this paragraph; for example, genetic transformation of germ cells from the ear of wheat could be carried out as mentioned, but without having to regenerate a plant afterward.
  • Method of screening plants for naturally occurring increased grain yield phenotypes there is provided a method for screening a population of plants and identifying and/or selecting a plant that carries or expresses a dominant negative gain of function allele of MADS.
  • a method for screening a population of plants and identifying and/or selecting a plant that has an increased grain yield and/or quality phenotype preferably an increase in at least one of grain size, grain number, grain length, grain weight and thousand kernel weight.
  • the method comprises detecting in the plant or plant germplasm at least one polymorphism in the MADS1 gene or promoter.
  • said screening comprises determining the presence of at least one polymorphism, wherein said polymorphism is at least one insertion and/or at least one deletion and/or substitution.
  • said polymorphism may comprise an insertion-deletion polymorphism in the splice site of intron 7/exon 8 of rice MADS1.
  • said polymorphism is at least one of the following: a T to A subsiution at position 8056 of SEQ ID NO:2 or a homologous position thereof;
  • Suitable tests for assessing the presence of a polymorphism would be well known to the skilled person, and include but are not limited to, Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats (SSRs-which are also referred to as Microsatellites), and Single Nucleotide Polymorphisms (SNPs).
  • RFLPs Restriction Fragment Length Polymorphisms
  • RAPDs Randomly Amplified Polymorphic DNAs
  • AP-PCR Arbitrarily Primed Polymerase Chain Reaction
  • DAF Sequence Characterized Amplified Regions
  • AFLPs Am
  • the method may further comprise introgressing the chromosomal region comprising a MADS1 polymorphism into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.
  • the second plant will express a dominant negative gain of function allele of MADS1 , and more preferably said second plant will display an increase in grain yield, preferably an increase in at least one of grain size, grain number, grain length, grain weight and thousand kernel weight.
  • the plant may endogenously express SEQ ID NO: 4 such that the resulting plant is homozygous for the dominant negative gain of function allele of MADS1.
  • the inventors have surprisingly identified a new MADS1 allele that acts as a dominant or semi-dominant gain of function allele. Accordingly, overexpression of this allele in a wild-type or control plant will also increase grain yield and/or quality.
  • nucleic acid construct comprising a nucleic acid sequence encoding a polypeptide as defined in SEQ ID NO: 4 or a functional variant or homolog thereof, wherein said sequence is operably linked to a regulatory sequence.
  • said regulatory sequence is a tissue-specific promoter or a constitutive promoter.
  • a functional variant or homolog is as defined above.
  • the promoter may be the endogenous MADS1 promoter, for example, as defined in SEQ ID NO: 176 or a variant thereof.
  • operably linked refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
  • a “plant promoter” comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The "plant promoter” can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed in the inventive process and described herein. This also applies to other “plant” regulatory signals, such as "plant” terminators.
  • the promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3'-regulatory region such as terminators or other 3' regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms.
  • the nucleic acid molecule For expression in plants, the nucleic acid molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern.
  • the term "operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
  • the promoter is a constitutive promoter.
  • a "constitutive promoter” refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ.
  • constitutive promoters include but are not limited to actin, HMGP, CaMV19S, GOS2, rice cyclophilin, maize H3 histone, alfalfa H3 histone, 34S FMV, rubisco small subunit, OCS, SAD1 , SAD2, nos, V-ATPase, super promoter, G-box proteins and synthetic promoters.
  • a vector comprising the nucleic acid sequence described above.
  • a host cell comprising the nucleic acid construct.
  • the host cell may be a bacterial cell, such as Agrobacterium tumefaciens, or an isolated plant cell.
  • the invention also relates to a culture medium or kit comprising a culture medium and an isolated host cell as described below.
  • transgenic plant expressing the nucleic acid construct as described above.
  • said nucleic acid construct is stably incorporated into the plant genome.
  • the nucleic acid sequence is introduced into said plant through a process called transformation as described above.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1 ) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
  • a suitable plant is defined above.
  • the invention relates to the use of a nucleic acid construct as described herein to increase at least one of grain size, grain number, grain length, grain weight and thousand kernel weight.
  • a method of increasing at least one of grain size, grain number, grain length, grain weight and thousand kernel weight comprising introducing and expressing in said plant the nucleic acid construct described herein.
  • a method of producing a plant with an increased in at least one of grain size, grain number, grain length, grain weight and thousand kernel weight comprising introducing and expressing in said plant the nucleic acid construct described herein.
  • Said increase is relative to a control or wild-type plant.
  • the japonica rice variety Wuyunjing7 carrying the dep1-1 allele (hereafter WYJ7-dep1- 1) produces more, but smaller grains than its near-isogenic line (NIL) WYJ7-DEP1 4 (Fig. 1 a).
  • NIL near-isogenic line
  • WYJ7-DEP1 4 Fig. 1 a
  • a set of 250 recombinant inbred lines (RILs) were developed from the cross between Wuyunjing7 and the American japonica rice variety L-204 which produces long and slender grains, one line (RIL186) carrying the dep1-1 allele formed grains which were bigger than that formed by the WYJ7-dep1-1 parent (Fig. 1a).
  • Positional cloning of qlgy3 was performed by using BC 2 F 2 and BC 3 F 2 populations developed from the cross between R186 and Thai cultivar RD23 (the recurrent parent), and a candidate region was narrowed to a -8.8 Kbp stretch flanked by markers XP21 and XP22 (Fig. 1 c).
  • This segment only contains the coding and 3' untranslated regions of OsMADSI, a gene encoding MADS-domain transcription factor 1 .
  • Sequence analysis indicated that an insertion-deletion polymorphism in the splice site of the intron 7/exon 8 junction was differentiated between L-204 and RD23 (Fig.
  • NPB-lgy3 was created in a Nipponbare background by introgressing a -274 Kbp segment from L-204 (Fig. 7a).
  • the NPB-lgy3 plants formed grains which were longer than those formed by Nipponbare (hereafter NPB-LGY3), but there was little difference with respect to grain width (Fig. 7b-d).
  • the mean length of NPB-lgy3 outer epidermal cells was indistinguishable from that of the equivalent cells in NPB-LGY3 (Fig. 7e, f), indicating that the Igy3 allele enhanced longitudinal grain growth by promoting cell proliferation.
  • the grains formed by the transgenic plants expressing the L-204 Igy3 cDNA driven either by its native promoter or by the Actin promoter were longer than that formed by non-transgenic plants, as were those grains produced by transgenic plants in which OsMADSI had been knocked down by RNAi (Fig. 7g, h).
  • the expression of the Nipponbare LGY3 cDNA driven by its native promoter had no effect on grain length, but constitutive expression of the Nipponbare LGY3 cDNA driven by the Actin promoter induced abnormalities in grain development 1 (Fig. 7h).
  • the product of OsMADS1 lgy3 appears to act as a dominant negative regulator of OsMADSI function.
  • the L-204 haplotype involving truncating splice site mutation is common within O. nivara accessions and tropical japonica germplasm, but it does not appear to occur within the elite indica and temperate japonica rice varieties, indicating that the Igy3 allele has not been exploited in high-yielding rice breeding programs.
  • the dep1-1 allele on the other hand has been widely used to develop high-yielding rice varieties by Chinese breeders 3,4 , therefore, it was of interest to characterize the effect on grain yield of combining elite alleles at the LGY3 and DEP1 loci.
  • OsMADSI is one of E-class MADS-box genes involved in the ABC the DE model of flower development 19, 20 .
  • Fig. 10a To uncover the molecular mechanism underlying genetic interaction between DEP1 and OsMADSI, we performed a yeast two-hybrid screen to identify DEP1 -interacting proteins, and found that both DEP1 and dep1 -1 proteins interacted with OsMADSI (Fig. 10a).
  • the DEP1 -OsMADSI interactions in planta were further confirmed by bimolecular fluorescence complementation (BiFC) and co- immunoprecipitation assays (Fig. 2a, b).
  • BiFC bimolecular fluorescence complementation
  • Fig. 2a, b co- immunoprecipitation assays
  • OsMADS1 lgy3 also interacted with DEP1 and dep1 -1 proteins (Fig.
  • RNA-seq A comparison of RNA-seq and ChlP-seq data 22 revealed a total of 451 genes which were co-operatively regulated by the DEP1 -OsMADSI interaction (Fig. 3a).
  • ChIP and EMSA assays revealed that both OsMADSI and OsMADSI lgy3 were able to bind the promoter regions of OsMADS55, OsKANADI4, OsPINIa and OsARF9, and that their DNA binding affinity was unaffected by variation in the C domain sequence (Fig. 3c, d). Further ChIP assays suggested that the in vivo association of OsMADSI with a same promoter region of target genes examined was unaffected by the presence or absence of dep1 -1 (Fig. 3e), indicating that changes in DNA-binding affinity of OsMADSI were not responsible for the direct effect of the DEP1 -OsMADSI interaction on target gene expression.
  • OsARF9 is homologous to A. thaliana ARF2 and S. lycopersicum SIARF9, which negatively control, respectively, seed size in A thaliana 23 and fruit weight in tomato 24 .
  • Transient expression assays in rice protoplasts showed that LUC activity driven by the OsARF9 promoter was moderately induced by OsMADSI on its own, but was substantially induced when OsMADSI and DEP1 were combined (Fig. 3f), consistent with OsMADSI and DEP1 acting as repressors of grain size (Fig. 1 a, f).
  • LUC activity was moderately induced by OsMADSI ⁇ K on its own, but not induced when DEP1 and OsMADS1 ⁇ K were combined (Fig.
  • G ⁇ dimer acts as a functional monomer 25 .
  • the rice genome encodes two canonical G ⁇ subunits (RGG1 and RGG2) and three non-canonical Gy subunits (GS3, DEP1 and OsGGC2) 26 .
  • BiFC assays showed that all of the Gy subunits interacted with OsMADSI (Fig. 4a).
  • the G ⁇ subunit RGB1 but not the Ga subunit RGA1 , was associated with OsMADSI in vivo (Fig. 4a), consistent with the previous observation that RGB1 -GFP fusion proteins could be detected in the nuclei of transgenic rice root cells 4 .
  • the suggestion is that the detached G ⁇ dimer associates with OsMADSI .
  • the transgenic plants overexpressing either RGB1, RGG1, RGG2 or GS3 formed grains which were smaller than those formed by non-transgenic plants, a phenotype reminiscent of that of dep1-1 overexpressors 4 (Fig. 4b).
  • the introduction of the Igy3 allele into these transgenic plants boosted grain length (Fig. 4b), indicating that the G ⁇ dimer controls grain size via its regulation of OsMADSI.
  • transient expression assays showed that OsMADSI -induced LUC activities of the common target genes examined were substantially enhanced when GS3 and OsMADSI were combined (Fig. 4c), representing a response similar to that regulated by the DEP1 -OsMADSI interaction (Fig. 3g).
  • GS3 also appears to be a cofactor for OsMADSI in the control of grain size.
  • the gs3 allele has been used widely in indica rice breeding programs 2, 5 .
  • the effect of allelic combinations on grain yield and quality were explored by generating NILs carrying allelic combinations of qGS3 and qLGY3 loci in the indica variety RD23 (hereafter RD23-LGY3-gs3) (Fig. 4d).
  • the two NILs RD23-LGY3-gs3 and RD23-lgy3- gs3 did not differ from one another with respect to either heading date, plant height or tiller number (Fig. 4f-h), although the number of grains per panicle was slightly reduced (Fig. 4i).
  • Both RD23-LGY3-gs3 and RD23-lgy3-GS3 plants formed longer grains than those formed by RD23-LGY3-GS3 plants (Fig. 4e), while RD23-lgy3-gs3 plants formed substantially longer and heavier grains than those formed by either RD23-LGY3-gs3 or RD23-lgy3-GS3 plants (Fig. 4e, j, k).
  • RD23- Igy3-gs3 proved to be on average -10.9% more productive than RD23-LGY3-gs3 (Fig. 4I).
  • the introduction of the Igy3 allele also improved grain appearance quality (Fig. 8b, 9b and Fig. 12).
  • the two-line hybrid combinations were developed by crossing a photo-thermosensitive genie male sterile PA64S either with a restorer line 931 1 (which carries the LGY3 and gs3 alleles, hereafter 931 1 -LGY3-gs3) or with a 931 1 NIL plants (which carries the Igy3 and gs3 alleles, hereafter 931 1 -lgy3-gs3).
  • the effect of the Igy3 allele was to enhance both grain length and weight (Fig. 4m-o), to increase grain yield by a mean of 7.1 % (Fig. 4p), and to substantially improve the grain quality (Fig.
  • the heterotrimeric G proteins are membrane-associated signal transduction molecules known to mediate a broad range of extracellular stimuli in plants (e.g., CLAVATA3- mediated stem cell niche maintenance) 27, 28 .
  • MADS-domain proteins have been shown to play important roles in the control of floral organ identity and flower development in response to changes in the external environment (e.g., temperature) 29, 30 .
  • the interaction between ⁇ subunits and MADS-domain proteins establishes a new molecular framework for the control of stem cell function and floral organ development under fluctuating environmental conditions. Combining Igy3 allele with high yield potential-associated dep1-1 and gs3 alleles also provides a new strategy in breeding simultaneously for higher grain yield and better grain quality in rice.
  • RESULTS Plant materials and growing conditions A set of 250 RILs population was bred from the cross between L-204 and WYJ7. Details of the germplasm used for the positional cloning and haplotype analysis have been described elsewhere 7, 18 .
  • the NILs plants carrying contrasting combinations of the qLGY3 and qDEP1 loci, qLGY3 and qGS3 loci were bred by crossing RIL186 six times with the recurrent parents WYJ7, RD23 and 931 1 , respectively.
  • Field-grown NIL plants were raised under standard paddy conditions at two experimental stations, one located in Lingshui (Hainan province) and the other in Hefei (Anhui province).
  • the primer sequences used for map-based cloning and genotyping assays were given in Extended Data Table 4. Transgene constructs.
  • the OsMADS1 lgy3 coding sequence was amplified from cv. L- 204, the OsMADSI coding sequence and its promoter regions lying 1 .9 Kbp upstream of the transcription start site and 3'-UTR lying 1.5 Kbp downstream of the termination site were amplified from cv.
  • RNA Quantitative real time PCR
  • the total RNA was extracted using TRIzol reagent (Invitrogen, New York, USA), and then treated with RNase-free DNase I (Invitrogen, New York, USA) according to the manufacturer's protocol.
  • the RNA was reverse-transcribed using a cDNA synthesis kit (TRANSGEN, Beijing, China).
  • qRT- PCR was performed as previously described 18 , each assay was replicated by at least three time with three biological replicates (independent RNA preparations).
  • the rice Actin3 (LOC_Os03g61970) was used as a reference.
  • Yeast two-hybrid assays Yeast two-hybrid assays were performed as described elsewhere 4 ' 18 .
  • the bait and prey vectors were co-transformed into yeast strain AH 109, and ⁇ -galactosidase assays were performed according to the manufacturer's protocol (Takara Bio Inc. Otsu, Japan).
  • the dep1 -1 protein was used as a bait to screen a cDNA library from poly(A)-containing RNA isolated from young (0.2 cm to 6 cm length) panicles.
  • Experimental procedures for screening and plasmid isolation were performed following the manufacturer's instructions. Relevant primer sequences were given in Fig 13.
  • BiFC assays Full length cDNAs of RGA1, RGB1, RGG1, RGG2, DEP1, dep1-1, GS3, OsGGC2, OsMADSI, OsMADSI, OsMADS4, OsMADS13, OsMADS13, OsMADS34, OsMADS55, OsMADS58, OsMADS1 lgy3 , and deleted and non-deleted versions of OsMADS1 were amplified and the amplicons inserted into pCAMBIA1300-35S-Cluc- RBS or pCAMBIA1300-35S-HA-Nluc-RBS vector to generate the required fusion transgenes.
  • Two plasmid vectors for testing the protein-protein interactions (such as DEP1 -nL UC and OsMADS1 -cLUC), together with the p19 silencing plasmid, were co- transfected into tobacco (N. benthamiana) leaves via Agrobacterium tuiefaciens infiltration, and LUC activity was measured as described elsewhere 31, 32 .
  • the injected leaves were detached after 36-48 hours later and sprayed with 1 mM luciferin (Promega, Wisconsin, USA). LUC signal was captured using a cooled CCD imaging apparatus. Each assay was repeated at least three times. Relevant primer sequences were given in Fig.13.
  • the lysates were incubated with agarose- conjugated anti-Flag antibodies (Sigma -Aldrich, St. Louis, U.S.A. ) at 4°C for at least 4 hours, then rinsed 5-6 times in the extraction buffer and eluted with 3xFLAG peptide (Sigma-Aldrich, St. Louis, U.S.A.).
  • the immunoprecipitates were electrophoretically separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Pittsburgh, USA). Proteins were detected by treating the membranes with anti-HA or anti-DDDDK-tag mAb-HRP-DirectT antibodies (MBL, Nagoya,Japan). ChlP- PCR analysis was performed as described elsewhere 18 . The extent of enrichment of specific DNA fragments was determined by qRT-PCR.
  • ChIP assays ChlP-PCR analysis was performed as described elsewhere. A 1 ⁇ 2 g aliquot of young panicles (3 cm length) was grindded with liquid nitrogen and fixed with 1 % formaldehyde under vacuum. After nuclei Nuclei were isolated and lysed as described, chromatin was ultrasonic fragmented on ice to an average size of 500 bp. The supernatant was firstly blocked with protein A agarose beads pre-absorbed with sheared salmon sperm DNA (Millipore pstate, USA), and then a small part of the buffer was setted aside to serve as input. Immunoprecipitation was performed with anti- OsMADSI antibodies (ABclonal, Woburn, USA) at 4°C.
  • Transactivation activity assays The ⁇ 2 Kbp DNA fragment of the promoter region of either OsMADS55, OsKANADI4, OsPINIa, OsARF9 or OsARF14 was amplified from cv. Nipponbare, and used to generate reporter plasmids containing a specific promoter fused to LUC. Full length cDNAs of OsMADSI, OsMADS1 lgy3 and deleted versions of OsMADSI were amplified and fused to GAL4BD, and inserted into PUC19 vector 7 to generate effector plasmid vectors (e.g., GAL4BD-OsMADS1). Transactivation analysis was performed using rice protoplasts as described elsewhere 18 .

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Abstract

L'invention concerne des méthodes pour augmenter le rendement des plantes, et en particulier le rendement en grains, et encore plus particulièrement, une méthode qui augmente à la fois le nombre de grains et la taille du grain, la méthode comprenant la modulation de l'expression ou de l'activité d'un facteur de transcription à domaine MADS 1 (MADS1). L'invention concerne également des plantes génétiquement modifiées caractérisées par les phénotypes ci-dessus ainsi que des méthodes de production de telles plantes.
PCT/EP2018/072854 2017-08-25 2018-08-24 Méthodes pour augmenter le rendement en grain Ceased WO2019038417A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110564761A (zh) * 2019-09-23 2019-12-13 中国农业科学院作物科学研究所 小麦wlsh1基因在调控植物的穗和籽粒发育中的应用
CN111172179A (zh) * 2020-01-19 2020-05-19 武汉艾迪晶生物科技有限公司 泛素连接酶基因OsNLA2、蛋白及其在水稻选育中的应用
CN111253480A (zh) * 2020-03-04 2020-06-09 宁波大学 水稻转录因子OsARF17基因及其在抗黑条矮缩病毒植物育种中的应用
CN111876414A (zh) * 2020-06-24 2020-11-03 湖南文理学院 一种改良酵母上游激活元件及其在鱼类中的应用
WO2020223642A1 (fr) * 2019-05-02 2020-11-05 Monsanto Technology Llc Compositions et procédés permettant de générer une diversité au niveau de séquences d'acide nucléique ciblées
CN112779280A (zh) * 2019-11-08 2021-05-11 中国科学院成都生物研究所 一种包含pOsOle18启动子的种子特异性干扰载体及其应用
CN113881652A (zh) * 2020-11-11 2022-01-04 山东舜丰生物科技有限公司 新型Cas酶和系统以及应用
CN114480443A (zh) * 2022-03-14 2022-05-13 华南农业大学 一种水稻株高株型调节基因OsUBR7的应用
WO2022251904A1 (fr) * 2021-06-02 2022-12-08 The University Of Adelaide Procédés pour améliorer le rendement des plantes
WO2023216046A1 (fr) * 2022-05-09 2023-11-16 中国科学院遗传与发育生物学研究所 Gène pour réguler et contrôler le nombre de branches de soja, et son utilisation
CN118421648A (zh) * 2024-05-14 2024-08-02 三明市农业科学研究院 水稻裂颖基因OsSG2、相关InDel连锁标记及其应用
WO2025178902A1 (fr) * 2024-02-22 2025-08-28 Pairwise Plants Services, Inc. Procédés et compositions pour améliorer les caractéristiques de rendement de plantes

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WO2020223642A1 (fr) * 2019-05-02 2020-11-05 Monsanto Technology Llc Compositions et procédés permettant de générer une diversité au niveau de séquences d'acide nucléique ciblées
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CN110564761A (zh) * 2019-09-23 2019-12-13 中国农业科学院作物科学研究所 小麦wlsh1基因在调控植物的穗和籽粒发育中的应用
CN112779280A (zh) * 2019-11-08 2021-05-11 中国科学院成都生物研究所 一种包含pOsOle18启动子的种子特异性干扰载体及其应用
CN111172179A (zh) * 2020-01-19 2020-05-19 武汉艾迪晶生物科技有限公司 泛素连接酶基因OsNLA2、蛋白及其在水稻选育中的应用
CN111172179B (zh) * 2020-01-19 2020-09-08 武汉艾迪晶生物科技有限公司 泛素连接酶基因OsNLA2、蛋白及其在水稻选育中的应用
CN111253480A (zh) * 2020-03-04 2020-06-09 宁波大学 水稻转录因子OsARF17基因及其在抗黑条矮缩病毒植物育种中的应用
CN111876414A (zh) * 2020-06-24 2020-11-03 湖南文理学院 一种改良酵母上游激活元件及其在鱼类中的应用
CN113881652A (zh) * 2020-11-11 2022-01-04 山东舜丰生物科技有限公司 新型Cas酶和系统以及应用
CN113881652B (zh) * 2020-11-11 2022-11-22 山东舜丰生物科技有限公司 新型Cas酶和系统以及应用
WO2022251904A1 (fr) * 2021-06-02 2022-12-08 The University Of Adelaide Procédés pour améliorer le rendement des plantes
CN114480443A (zh) * 2022-03-14 2022-05-13 华南农业大学 一种水稻株高株型调节基因OsUBR7的应用
CN114480443B (zh) * 2022-03-14 2023-06-20 华南农业大学 一种水稻株高株型调节基因OsUBR7的应用
WO2023216046A1 (fr) * 2022-05-09 2023-11-16 中国科学院遗传与发育生物学研究所 Gène pour réguler et contrôler le nombre de branches de soja, et son utilisation
WO2025178902A1 (fr) * 2024-02-22 2025-08-28 Pairwise Plants Services, Inc. Procédés et compositions pour améliorer les caractéristiques de rendement de plantes
CN118421648A (zh) * 2024-05-14 2024-08-02 三明市农业科学研究院 水稻裂颖基因OsSG2、相关InDel连锁标记及其应用

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