EP1409696A2 - Identification et caracterisation de genes vegetaux - Google Patents

Identification et caracterisation de genes vegetaux

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Publication number
EP1409696A2
EP1409696A2 EP02758266A EP02758266A EP1409696A2 EP 1409696 A2 EP1409696 A2 EP 1409696A2 EP 02758266 A EP02758266 A EP 02758266A EP 02758266 A EP02758266 A EP 02758266A EP 1409696 A2 EP1409696 A2 EP 1409696A2
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Prior art keywords
polypeptide
nucleic acid
activity
length polypeptide
full
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP02758266A
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German (de)
English (en)
Inventor
Allen Torrey Mesa Research Instit. Inc. SESSIONS
Steven BRIGGS
Bret COOPER
Stephen A. Sygenta Biotechnology Inc GOFF
Todd Sygenta Biotechnology Inc MOUGHAMER
Jane GLAZEBROOK
Fumiaki University of Minnesota KATAGIRI
Joel Diversa KREPS
Nicolas University of Toronto PROVART
Darrell Syngenta Biotechnology Inc RICKE
Tong Syngenta Biotechnology Inc ZHU
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Syngenta Participations AG
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Syngenta Participations AG
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Publication of EP1409696A2 publication Critical patent/EP1409696A2/fr
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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    • 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
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    • 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
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    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • 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
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    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
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    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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    • 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
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    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention is in the area of plant biotechnology.
  • the invention relates to nucleic acid molecules obtainable from the rice genome that encode protein products that are involved in the development and timing of flower formation in plants and which can be used to modulate flower architecture and flowering time.
  • inflorescence meristems which give rise to flower meristems. From the flower meristems floral organs develop such as sepals and petals.
  • Flower formation is the result of a series of distinct developmental steps including floral induction, the formation of flower primordia and the production of flower organs.
  • floral induction may lead to the transformation of a vegetative shoot meristem into an inflorescens meristem, which then gives rise to a flower meristems.
  • Flower meristems produce floral organ primordia, which develop individually into sepals, petals, stamens or carpels.
  • the ABC model of pattern formation offers a developmental understanding of the flower parts in both monocotyledonous and dicotyledonous plants. It states that genes 'A' and 'B' and C C interact with one another to specify different floral identities. Sepals, petals, stamens and carpels are usually arranged in three, four or five concentric whorls. The sepals arise below the petals, and the stamens arise below the carpels. Sepals and petals are sterile appendages that are attached under the fertile floral parts(stamens and carpels).
  • 'A' gene is Apetalal (API)
  • 'B' genes are Apetala3 (AP3) and Pistillata (PI)
  • ⁇ C gene is Agamous (AG).
  • the Leafy (LFY) gene in Arabidopsis has been shown to regulate the expression of 'A' gene, API, and ⁇ C gene, AG (Busch M., et al., Science 285:585 (1999) and Wagner D., et al., Science 285: 582 (1999)). While LFY ⁇ Unusual Floral Organ (UFO) control the expression of B gene, AP3 (Wilkinson M., et al., Plant Cell 1995 7:1485 (1995)).
  • the homologue of the Arabidopsis LEAFY gene is FLORICAULA (Coen, et al., Cell, 63:1311, 1990) and that of the APETALA1 gene is SQUAMOSA (Huijser, et al., EMBO J., 11 : 1239, 1992).
  • the latter pair contains MADS box domains.
  • Flowering plants exhibit one of two types of inflorescence architecture: indeterminate, in which the inflorescence grows indefinitely, or determinate, in which a terminal flower is produced.
  • TFL serves to restrict the expression of the meristem identity genes to floral meristems, where they promote the patterned expression of floral organ identity genes AP2, AP3, PI and AG, which are also affected by the regulatory genes ANT, UFO, and SUP [194, 195].
  • the present invention provides and relates to nucleotide sequences encoding at least one polypeptide involved in the development and/or timing of flower formation and/or whole plant architecture, as well as the polypeptides encoded thereby, or antigene sequences thereof, which have numerous applications using techniques that are known to those skilled in the art of molecular biology, biotechnology, biochemistry, genetics, physiology or plant pathology. These techniques include the use of nucleotide molecules as hybridization probes, for chromosome and gene mapping, in PCR technologies, in the production of sense or antisense nucleic acids.
  • the present mvention provides the ability to modulate development and/or timing of flower formation and/or whole plant architecture in plants, by modulating gene expression (e.g. over-expressing, under-expressing or knocking out) one or more genes involved in or regulating these processes or their gene products, in a host cell, preferably in a plant cell, in vitro or in planta.
  • gene expression e.g. over-expressing, under-expressing or knocking out
  • Expression vectors comprising at least one nucleotide sequence encoding a polypeptide the activity of which is involved in development and/or timing of flower formation and/or whole plant architecture or the regulation thereof, or its antigene, operably linked to at least one suitable promoter and/or regulatory sequence can be used to study the role of polypeptides encoded by said sequences, for example by transforming a host cell with said expression vector and measuring the effects of overexpression , underexpression or complete knock-out of thses sequences.
  • a host cell transformed with at least one expression vector comprising nucleotide sequences encoding a polypeptide the activity of which is involved in the development and or timing or flower formation in plants and/or whole plant architecture, or the regulation thereof operably linked to suitable promoters and/or regulatory sequences can be useful to produce a polypeptide having a defined polypeptide profile that results in a plant showing a modified architecture or timing of flower development.
  • the present invention further provides, in a collective embodiment, a transformed plant host cell, or one obtained through breeding, capable of over-expressing, under- expressing, or having a knock out of a flower development or flower time gene or a gene regulating these processes and/or its gene products.
  • a plant cell transformed with at least one expression vector comprising nucleotide sequences involved in the development and/or timing of flower formation in plants and/or whole plant architecture, operably linked to suitable promoters and or regulatory sequences, wherein the plant host cell can be used to regenerate plant tissue or an entire plant, or seed therefrom, in which the effects of expression, including overexpression or underexpression or knock-out, of the introduced sequence or sequences can be measured in vitro or in planta.
  • the present invention provides nucleotide sequences including regions of nucleotide sequence encoding polypeptides having homology to at least one functional protein domain (FPD).
  • FPD functional protein domain
  • Embodiments of the invention further provide polypeptides including regions of amino acid sequence having homology to an FPD.
  • the polypeptide may represent a paralogous sequence or paralog, or may represent a variant allele of a gene encoding the FPD.
  • polypeptides may represent orthologous sequences, or orthologs, of the FPD.
  • nucleic acid molecules disclosed herein or respresentative parts thereof can be used in hybridization-based assays for detecting and identifying nucleic acid molecules that encode protein products that are involved in the development and or timing of flower formation in plants and/or whole plant architecture other than rice, but especially in plants belonging to the cereal group.
  • Embodiments of the present invention provide a unique oligonucleotide having a sequence identical to or complementary to a region of a polynucleotide sequence encoding at least a portion of a homologue of a protein according to the invention representatives of which are identified by the SEQ ID NOs provided in the Sequence Listing and/or an FPD thereof, the oligonucleotide being identified by the methods disclosed herein.
  • the unique oligonucleotide has a length of between 12 and 250 nucleotide bases.
  • Embodiments of the present invention also provide a nucleotide microarray comprising the unique oligonucleotide having a sequence identical to or complementary to a region of polynucleotide sequence encoding at least a portion of a homologue of a protein according to the invention representatives of which are identified by the SEQ ID NOs provided in the Sequence Listing and/or an FPD thereof.
  • the microarray includes a plurality of different, unique oligonucleotides, the sequences corresponding to a plurality of homologues of a protein according to the mvention representatives of which are identified by the SEQ ID NOs provided in the Sequence Listing and/or an FPD thereof.
  • the microarray contains at least about 96 different unique oligonucleotides, wherein each of the 96 different unique oligonucleotides has a sequence that is identical, complementary, or has substantial similarity to a segment of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 65 and 123 - 145.
  • Embodiments of the present invention also provide a kit for detecting the presence of a polynucleotide, the kit containing a first nucleotide probe which can hybridize with a region of a nucleotide sequence including the nucleotide sequences of SEQ ID NOs: 1 to SEQ ID NO: 65 and SEQ ID NOs: 123 - 145, a fragment or a variant thereof, and a complementary sequence thereto, the kit further containing at least one additional component such as, for example: a second nucleotide probe, a buffer, an enzyme, a label, a molecular weight standard, a reaction chamber, and a micropipette tip.
  • a second nucleotide probe such as, for example: a second nucleotide probe, a buffer, an enzyme, a label, a molecular weight standard, a reaction chamber, and a micropipette tip.
  • Embodiments of the present invention further provide a kit for detecting the presence of a polypeptide, the kit containing a first probe which can hybridize with a region of a polypeptide including the amino acid sequences of SEQ ID NOs: 2 to SEQ ID NOs: 66 and SEQ ID NOs: 124 - 146, a fragment or a variant thereof, and optionally, the kit further containing at least one additional component such as, for example: a probe, a buffer, an enzyme, a label, a molecular weight standard, a reaction chamber, and a micropipette tip.
  • Probes useful in kit embodiments include antibodies, affinity tags, protein A, protein G, or protein-binding substances including chromatographic media.
  • An additional aspect provides a method for selecting plants, for example cereals, having an altered heading time or whole plant architecture comprising obtaining nucleic acid molecules from the plants to be selected; contacting the nucleic acid molecules with one or more probes that selectively hybridize under stringent or highly stringent conditions to a nucleic acid sequence selected from the group consistmg of SEQ ID NOs. 1-66, and 124 - 146; detecting the hybridization of the one or more probes to the nucleic acid sequences wherein the presence of the hybridization indicates the presence of a gene associated with altered heading time or whole plant architecture; and selecting plants on the basis of the presence or absence of such hybridization.
  • marker-assisted selection is accomplished in rice.
  • marker assisted selection is accomplished in wheat using one or more probes which selectively hybridize under stringent or highly stringent conditions to sequences selected from the group consisting of SEQ ID NOs: 81 - 105.
  • marker assisted selection is accomplished in maize or corn using one or more probes which selectively hybridize under stringent or highly stringent conditions to sequences selected from the group consisting of SEQ ID NOs: 106 - 122.
  • marker assisted selection is accomplished in banana using one or more probes which selectively hybridize under stringent or highly stringent conditions to sequences selected from the group consisting of SEQ ID NOs: 67 - 80.
  • marker-assisted selection can be accomplished using a probe or probes to a single sequence or multiple sequences. If multiple sequences are used they can be used simultaneously or sequentially.
  • a computer readable medium containing one or more of the nucleotide sequences of the mvention is provided as well as methods of use for the computer readable medium.
  • This medium allows a nucleotide sequence corresponding to at least one of the sequences selected from the group of SEQ ID NOs: 1-66, and 123 - 146, and 67 - 122 provided in the Sequence Listing (open reading frames or fragments thereof), to be used as a reference sequence to search against a database.
  • This medium also allows for computer-based manipulation of a nucleotide sequence corresponding to at least one of the sequences selected from the group consistmg of SEQ ID NOs: 1-66, and 123 - 146, and 67 - 122 provided in the Sequence Listing.
  • a further aspect provides a computer readable medium having stored thereon computer executable instructions for performing a method comprising receiving data on nucleotide sequence expression in a test plant of at least one nucleic acid molecule having at least 70%, at least 80%, at least 90% or at least 95%, sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: SEQ ID NOs. 1-66, and 123 - 146; and 67 - 122 and comparing expression data from said test plant to expression data for the same nucleotide sequence or sequences in a plant during grain filling.
  • Odd numbered SEQ ID NOs: 1 - 65 are representing a first sub-group (sub-group I) of polynucleotides comprising nucleotide sequences of rice encoding polypeptides involved in flower timing in plants and/or whole plant architecture or the regulation thereof.
  • Even numbered SEQ ID NOs: 2 - 66 are polypeptides involved in flower timing in plants and/or whole plant architecture or the regulation thereof encoded by the immediately preceding nucleotide sequence e.g., SEQ ID NO: 2 is the protein encoded by the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:4 is the protein encoded by the nucleotide sequence of SEQ ID NO:3, etc..
  • SEQ ID NOs: 67 - 80 are banana sequences which show homology to the rice sequences provided in SEQ ID NOs: 1 -65.
  • SEQ ID NOs: 81 - 105 are wheat sequences which show homology to the rice sequences provided in SEQ ID NOs: 1 -65.
  • SEQ ID NOs: 106 - 122 are maize sequences which show homology to the rice sequences provided in SEQ ID NOs: 1 -65.
  • Odd numbered SEQ ID NOs: 123 - 145 are representing a second sub-group (subgroup II) of polynucleotides comprising nucleotide sequences that have homologies between 80% and 99.9% to the nucleotide sequences of sub-group I and possible variants or familiy members of rice sequences provided in SEQ ID NOs: 1-65.
  • the correlation between the sequences in sub-groups I and II is illustrated in Tables 1 through 3.
  • Even numbered SEQ ID NOs: 124 - 146 are protein sequences encoded by the immediately preceding nucleotide sequence.
  • genes are used broadly to refer to any segment of nucleic acid with or without its native regulatory sequences such a promoters and polyadenylation sites, signal sequences, etc, associated with a biological function.
  • genes include coding sequences and/or the regulatory sequences required for their expression.
  • gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
  • Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins.
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • a chimeric gene refers to any gene that contains 1) DNA sequences, including regulatory and coding sequences, that are not found together in nature, or 2) sequences encoding parts of proteins not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature.
  • transgene refers to a gene that has been introduced into the genome by transformation and is stably maintained.
  • Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes.
  • endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “foreign” gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.
  • oligonucleotide corresponding to a nucleotide sequence of the invention, e.g., for use in probing or amplification reactions, may be about 30 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21 or 24, or any number between 9 and 30).
  • primers are upwards of 14 nucleotides in length.
  • primers 16 to 24 nucleotides in length may be preferred.
  • probing can be done with entire restriction fragments of the gene disclosed herein which may be 100's or even lOOO's of nucleotides in length.
  • protein protein
  • peptide and “polypeptide” are used interchangeably herein.
  • a "partial-length polypeptide” refers to any part of a given full-length polypeptide that has substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full- length polypeptide.
  • the nucleotide sequences of the invention can be introduced into any plant.
  • the genes to be introduced can be conveniently used in expression cassettes for introduction and expression in any plant of interest.
  • Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest.
  • Preferred promoters include constitutive, tissue-specific, developmental-specific, inducible and/or viral promoters.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti- plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al., 1991; Proudfoot, 1991; Sanfacon et al., 1991; Mogen et al., 1990; Munroe et al., 1990; Ballas et al., 1989; Joshi et al., 1987.
  • Coding sequence refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an "uninterrupted coding sequence", i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions.
  • An "intron” is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.
  • open reading frame and “ORF” refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
  • initiation codon and “termination codon” refer to a unit of three adjacent nucleotides
  • RNA transcript refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence.
  • primary transcript When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.
  • Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell.
  • cDNA refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA.
  • regulatory sequences each refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. As is noted above, the term “suitable regulatory sequences” is not limited to promoters. "5' non-coding sequence” refers to a nucleotide sequence located 5' (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency (Turner et al., 1995).
  • 3' non-coding sequence refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • the use of different 3' non-coding sequences is exemplified by Ingelbrecht et al., 1989.
  • translation leader sequence refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon.
  • the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
  • Signal peptide refers to the amino terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into the secretory pathway.
  • signal sequence refers to a nucleotide sequence that encodes the signal peptide.
  • Promoter refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
  • Promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression.
  • Promoter also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an "enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.
  • the "initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.
  • promoter elements particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters.”
  • minimal or core promoters In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.
  • a “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.
  • Constant expression refers to expression using a constitutive or regulated promoter.
  • Consditional and regulated expression refer to expression controlled by a regulated promoter.
  • Constant promoter refers to a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant.
  • ORF open reading frame
  • Each of the transcription-activating elements do not exhibit an absolute tissue-specificity, but mediate transcriptional activation in most plant parts at a level of >1% of the level reached in the part of the plant in which transcription is most active.
  • Regular promoter refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and includes both tissue-specific and inducible promoters. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in plant cells are constantly being discovered, numerous examples may be found in the compilation by Okamuro et al. (1989).
  • Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysome-inducible systems.
  • tissue-specific promoter refers to regulated promoters that are not expressed in all plant cells but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence.
  • “Inducible promoter” refers to those regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, or a pathogen.
  • “Operably-linked” refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other.
  • a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
  • “Expression” refers to the transcription and/or translation of an endogenous gene, ORF or portion thereof, or a transgene in plants.
  • expression may refer to the transcription of the antisense DNA only.
  • expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA.
  • Expression may also refer to the production of protein.
  • Specific expression is the expression of gene products which is limited to one or a few plant tissues (spatial limitation) and/or to one or a few plant developmental stages (temporal limitation). It is acknowledged that hardly a true specificity exists: promoters seem to be preferably switch on in some tissues, while in other tissues there can be no or only little activity. This phenomenon is known as leaky expression. However, with specific expression in this invention is meant preferable expression in one or a few plant tissues.
  • the "expression pattern" of a promoter is the pattern of expression levels which shows where in the plant and in what developmental stage transcription is initiated by said promoter. Expression patterns of a set of promoters are said to be complementary when the expression pattern of one promoter shows little overlap with the expression pattern of the other promoter.
  • the level of expression of a promoter can be determined by measuring the 'steady state' concentration of a standard transcribed reporter mRNA. This measurement is indirect since the concentration of the reporter mRNA is dependent not only on its synthesis rate, but also on the rate with which the mRNA is degraded. Therefore, the steady state level is the product of synthesis rates and degradation rates.
  • the rate of degradation can however be considered to proceed at a fixed rate when the transcribed sequences are identical, and thus this value can serve as a measure of synthesis rates.
  • promoters are compared in this way techniques available to those skilled in the art are hybridization SI -RNAse analysis, northern blots and competitive RT-PCR. This list of techniques in no way represents all available techniques, but rather describes commonly used procedures used to analyze transcription activity and expression levels of mRNA.
  • GUS ⁇ -glucuronidase
  • CAT chloramphenicol acetyl transferase
  • GFP green fluorescent protein
  • Detection systems can readily be created or are available which are based on, e.g., immunochemical, enzymatic, fluorescent detection and quantification. Protein levels can be determined in plant tissue extracts or in intact tissue using in situ analysis of protein expression.
  • “Overexpression” refers to the level of expression in transgenic cells or organisms that exceeds levels of expression in normal or untransformed (nontransgenic) cells or organisms.
  • Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of protein from an endogenous gene or a transgene.
  • Gene silencing refers to homology-dependent suppression of viral genes, transgenes, or endogenous nuclear genes. Gene silencing may be transcriptional, when the suppression is due to decreased transcription of the affected genes, or post-transcriptional, when the suppression is due to increased turnover (degradation) of RNA species homologous to the affected genes (English et al., 1996). Gene silencing includes virus-induced gene silencing (Ruiz et al. 1998).
  • heterologous DNA sequence each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.
  • the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
  • a "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
  • “Homologous to” in the context of nucleotide sequence identity refers to the similarity between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of two protein molecules.
  • “homology” and “homologous” refer to an evaluation of the similarity between two sequences based on measurements of sequence identity adjusted for variables including gaps, insertions, frame shifts, conservative substitutions, and sequencing errors, as described below.
  • Two nucleotide sequences or polypeptides are the 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.
  • complementary to is used herein to mean that the sequence can form a Watson-Crick base pair with a reference polynucleotide sequence.
  • Complementary sequences can include nucleotides, such as inosine, that neither disrupt Watson-Crick base pairing nor contribute to the pairing.
  • a "reverse complement" of a sequence corresponds to the complementary sequence, but in the opposite orientation of bases from 5' to 3', or to the complement of the primary sequence, if the primary sequence is in a reverse orientation of bases from 5' to 3'.
  • BLAST Basic Local Alignment Search Tool
  • BLASTN compares a nucleotide query sequence against a nucleotide sequence database
  • BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database
  • TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands).
  • TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • the BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.
  • High-scoring segment pairs are preferably identified (aligned) by means of a scoring matrix selected from the many scoring matrices known in the art.
  • the scoring matrix used is the BLOSUM 62 matrix (Gonnet et al, Science 256:1443 (1992); Henikoff and Henikoff, Proteins 17:49 (1993)).
  • the PAM or PAM250 matrices may also be used (Schwartz and Dayhoff, In Atlas of Protein Sequence and Structure, Dayhoff, ed., Natl. Biomed. Res. Found., pp. 353-358 (1978)).
  • the BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user- specified percent homology.
  • a user-specified threshold of significance such as a user- specified percent homology.
  • the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (Karlin and Altschul (1990) supra). "Percentage of sequence identity" can be determined from alignments performed using algorithms known in the art.
  • Alignment of nucleotide or polypeptide sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (Add APL Math 2:482 (1981)), by the homology alignment algorithm of Needleman and Wunsch (J Mol Biol 48:443 (1970)), by the search for similarity method of Pearson and Lipman (Proc Natl Acad Sci USA 85:2444 (1988)), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group), or by inspection. When two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. In a preferred embodiment, percenty identity is determined using the GAP program for global alignment using default parameters, using the version of GAP found in the GCG package (Wisconsin Package Version 10.1, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin).
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window may include additions or deletions, including for example gaps or overhangs, as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the term "substantially similar”, when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure as the polypeptide encoded by the reference nucleotide sequence.
  • the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence.
  • substantially similar refers to nucleotide sequences having at least 50% sequence identity, preferably at least 60%, 70%, 80% or 85%, more preferably at least 90% or 95%, and even more preferably, at least 96%, 97% or 99% sequence identity compared to a reference sequence containing nucleotide sequences of Table I, that encode a protein having at least 50% identity, more preferably at least 85% identity, yet still more preferably at least 90% identity to a region of sequence of a BIOPATH protein and/or an FPD, wherein the protein sequence comparisons are conducted using GAP analysis as described below.
  • substantially similar preferably also refers to nucleotide sequences having at least 50% identity, more preferably at least 80% identity, still more preferably 95% identity, yet still more preferably at least 99% identity, to a region of nucleotide sequence encoding a BIOPATH protein and/or an FPD, wherein the nucleotide sequence comparisons are conducted using GAP analysis as described below.
  • the term “substantially similar” is specifically intended to include nucleotide sequences wherein the sequence has been modified to optimize expression in particular cells.
  • a polynucleotide including a nucleotide sequence "substantially similar" to the reference nucleotide sequence preferably hybridizes to a polynucleotide including the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M Na
  • substantially similar when used herein with respect to a protein or polypeptide, means a protein or polypeptide corresponding to a reference protein, wherein the protein has substantially the same structure and function as the reference protein, where only changes in amino acids sequence that do not materially affect the polypeptide function occur.
  • the percentage of identity between the substantially similar and the reference protein or amino acid sequence desirably is preferably at least 30%, more preferably at least 40%, 50%, 60%, 70%, 80%, 85%, or 90%, still more preferably at least 95% , still more preferably at least 99% with every individual number falling within this range of at least 30% to at least 99% also being part of the invention, using default GAP analysis parameters with the University of Wisconsin GCG (version 10), SEQ WEB application of GAP, based on the algorithm of Needleman and Wunsch (1970), supra.
  • polypeptide of the present invention refers to an amino acid sequence encoded by a DNA molecule including a nucleotide sequence substantially similar to an Nucleotide sequence according to the invention.
  • Homologs of BIOPATH protein and/or FPDs include amino acid sequences that are at least 30%) identical to BIOPATH protein and/or FPD sequences found in searchable databases, as measured using the parameters described above.
  • Target gene refers to a gene on the replicon that expresses the desired target coding sequence, functional RNA, or protein. The target gene is not essential for replicon replication.
  • target genes may comprise native non-viral genes inserted into a non-native organism, or chimeric genes, and will be under the control of suitable regulatory sequences.
  • the regulatory sequences in the target gene may come from any source, including the virus.
  • Target genes may include coding sequences that are either heterologous or homologous to the genes of a particular plant to be transformed. However, target genes do not include native viral genes.
  • Typical target genes include, but are not limited to genes encoding a structural protein, a seed storage protein, a protein that conveys herbicide resistance, and a protein that conveys insect resistance. Proteins encoded by target genes are known as "foreign proteins". The expression of a target gene in a plant will typically produce an altered plant trait.
  • altered plant trait means any phenotypic or genotypic change in a transgenic plant relative to the wild-type or non-transgenic plant host.
  • Chrosomally-integrated refers to the integration of a foreign gene or DNA construct into the host DNA by covalent bonds. Where genes are not “chromosomally integrated” they may be “transiently expressed.” Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of an autonomously replicating plasmid or expression cassette, for example, or as part of another biological system such as a virus.
  • transgenic refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance.
  • Host cells containing the transformed nucleic acid fragments are referred to as "transgenic” cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".
  • transgenic organisms Examples of methods of transformation of plants and plant cells include Agrobacterium-mediated transformation (De Blaere et al., 1987) and particle bombardment technology (Klein et al. 1987; U.S. Patent No. 4,945,050).
  • Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan (see, for example, Fromm et al., 1990).
  • Transformed refers to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome generally known in the art and are disclosed in Sambrook et al., 1989. See also Innis et al., 1995 and Gelfand, 1995; and Innis and Gelfand, 1999.
  • Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene- specific primers, vector-specific primers, partially mismatched primers, and the like.
  • “transformed,” “transformant,” and “transgenic” plants or calli have been through the transformation process and contain a foreign gene integrated into their chromosome.
  • the term "untransformed” refers to normal plants that have not been through the transformation process.
  • Transiently transformed refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium-mediated transformation or biolistic bombardment), but not selected for stable maintenance.
  • “Stably transformed” refers to cells that have been selected and regenerated on a selection media following transformation.
  • Transient expression refers to expression in cells in which a virus or a transgene is introduced by viral infection or by such methods as Agrobacterium-mediated transformation, electroporation, or biolistic bombardment, but not selected for its stable maintenance.
  • Genetically stable and “heritable” refer to chromosomally-integrated genetic elements that are stably maintained in the plant and stably inherited by progeny through successive generations.
  • Primary transformant and “TO generation” refer to transgenic plants that are of the same genetic generation as the tissue which is initially transformed (i.e., not having gone through meiosis and fertilization since transformation).
  • Secondary transformants and the “TI, T2, T3, etc. generations” refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants.
  • Wild-type refers to a virus or organism found in nature without any known mutation.
  • Gene refers to the complete genetic material of an organism.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985; Rossolini et al. 1994).
  • a "nucleic acid fragment" is a fraction of a given nucleic acid molecule.
  • nucleotide sequence refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • an "isolated” or “purified” DNA molecule or an “isolated” or “purified” polypeptide is a DNA molecule or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
  • an "isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • a protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%>, 20%, 10%), 5%, (by dry weight) of contaminating protein.
  • culture medium represents less than about 30%>, 20%, 10%>, or 5% (by dry weight) of chemical precursors or non-protein of interest chemicals.
  • nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant (variant) forms. Such variants will continue to possess the desired activity, i.e., either promoter activity or the activity of the product encoded by the open reading frame of the non-variant nucleotide sequence.
  • variants are intended substantially similar sequences.
  • variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
  • variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein.
  • nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 91%, to 98%o and 99%> nucleotide sequence identity to the native (wild type or endogenous) nucleotide sequence.
  • Consatively modified variations of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein.
  • nucleic acid variations are “silent variations” which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted.
  • each codon in a nucleic acid except ATG, which is ordinarily the only codon for methionine
  • each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • the nucleic acid molecules of the invention can be "optimized" for enhanced expression in plants of interest.
  • nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used. Variant nucleotide sequences and proteins also encompass sequences and protein derived from a mutagenic and recombinogenic procedure such as DNA shuffling.
  • libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo.
  • Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, 1994; Stemmer, 1994; Crameri et al., 1997; Moore et al, 1997; Zhang et al., 1997; Crameri et al., 1998; and U.S. Patent Nos. 5,605,793 and 5,837,458.
  • variant polypeptide is intended a polypeptide derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variants may result from, for example, genetic polymo ⁇ hism or from human manipulation. Methods for such manipulations are generally known in the art.
  • polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, 1985; Kunkel et al., 1987; U. S. Patent No. 4,873,192; Walker and Gaastra, 1983 and the references cited therein.
  • Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978). Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred.
  • pression cassette means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.
  • Vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast or fungal cells).
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell.
  • a host cell such as a microbial, e.g. bacterial, or plant cell.
  • the vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • Codoning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance.
  • a "transgenic plant” is a plant having one or more plant cells that contain an expression vector.
  • Plant tissue includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture such as single cells, protoplast, embryos, and callus tissue.
  • the plant tissue may be in plants or in organ, tissue or cell culture.
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity.
  • Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics,
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • Gapped BLAST in BLAST 2.0
  • PSI-BLAST in BLAST 2.0
  • the default parameters of the respective programs e.g. BLASTN for nucleotide sequences, BLASTX for proteins
  • W wordlength
  • E expectation
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). See htt ://www.ncbi .n 1 .nih . gov. Alignment may also be performed manually by inspection.
  • comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein is preferably made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program.
  • equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program.
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which 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 the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
  • Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%>, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%), 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • nucleotide sequences can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
  • Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, more preferably at least 80%, 90%, and most preferably at least 95%.
  • Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • stringent conditions encompass temperatures in the range of about 1°C to about 20°C, depending upon the desired degree of stringency as otherwise qualified herein.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970).
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Bod(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
  • T m can be approximated from the equation of Meinkoth and Wahl, 1984; T ra 81.5°C + 16.6 (log M) +0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity.
  • the T m can be decreased 10°C.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point I for the specific sequence and its complement at a defined ionic strength and pH.
  • severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal melting point I;
  • moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point I;
  • low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point I.
  • hybridization and wash compositions those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T of less than 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used.
  • An extensive guide to the hybridization of nucleic acids is found in Tijssen, 1993.
  • highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point T m for the specific sequence at a defined ionic strength and pH.
  • An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2X SSC wash at 65°C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer).
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides is IX SSC at 45°C for 15 minutes.
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4-6X SSC at 40°C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C and at least about 60°C for long robes (e.g., >50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a signal to noise ratio of 2X (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50%> formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0. IX SSC at 60 to 65°C.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C.
  • a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodec
  • DNA shuffling is a method to introduce mutations or rea ⁇ angements, preferably randomly, in a DNA molecule or to generate exchanges of DNA sequences between two or more DNA molecules, preferably randomly.
  • the DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-naturally occurring DNA molecule derived from at least one template DNA molecule.
  • the shuffled DNA preferably encodes a variant polypeptide modified with respect to the polypeptide encoded by the template DNA, and may have an altered biological activity with respect to the polypeptide encoded by the template DNA.
  • Recombinant DNA molecule is a combination of DNA sequences that are joined together using recombinant DNA technology and procedures used to join together DNA sequences as described, for example, in Sambrook et al., 1989.
  • plant refers to any plant, particularly to seed plant, and "plant cell” is a structural and physiological unit of the plant, which comprises a cell wall but may also refer to a protoplast.
  • the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, or a plant organ.
  • Signal increase is an increase that is larger than the margin of error inherent in the measurement technique, preferably an increase by about 2-fold or greater.
  • “Significantly less” means that the decrease is larger than the margin of error inherent in the measurement technique, preferably a decrease by about 2-fold or greater.
  • the transition between vegetative growth and flowering (reproduction) represents a major developmental shift in the plant life cycle.
  • Identification and modulation of flowering time genes can enable the timing of flowering and whole plant architecture to be altered. Altering the timing of flowering has important agricultural implications.
  • the period to produce plant seeds and fruits can be extended when crop plants flower earlier in the growing season. This can enhance the development of plant fruits and seeds by increasing the growing period, or increasing crop yield with multiple crops per season.
  • Rice for example, requires more than six months to grow and mature in some agricultural areas. Early flowering can provide two rice crops per year. Also, early flowering is a helpful adaptation to some cold climates (Johansen, U. et al., Science 5490: 344 (2000)). When crop plants flower later in the growing season, plant crops such as spinach and lettuce tend to bolt. The delay allows them to dedicate less energy for reproduction and more energy towards vegetative growth. Larger and greener leaves and stems are commercially valuable.
  • FLC flowering locus C
  • plants over- expressing the FLC gene or relatives took substantially longer to flower or did not flower at all, and showed large (i.e. 10-fold) increases in biomass.
  • Planting in Arabidopsis is initiated by flowering time genes that activate floral meristem identity genes leading to the patterned expression of floral organ identity genes.
  • the rice genome contains clear single copy homologs of the Arabidopsis flowering time genes GI, CO, LD, and FCA (e.g. SEQ ID NOs: 33, 17, 49, 27).
  • the LUMINIDEPENDENS (LD) gene is demonostrated (Plant Cell 1994 Jan;6(l):75- 83)) to be involved in the timing of flowering in Arabidopsis. Mutations in this gene render Arabidopsis late flowering and appear to affect light perception. The late-flowering phenotype of the Id mutation is partially suppressed by vernalization. Genomic and cDNA clones of the LD gene were characterized. The predicted amino acid sequence of the LD protein contains 953 residues and includes two putative bipartite nuclear localization signals and a glutamine- rich region.
  • the CO gene from Arabidopsis is isolated, and two zinc fingers that show a similar spacing of cysteines, but little direct homology, to members of the GATA1 family were identified in the amino acid sequence, co mutations were shown to affect amino acids that are conserved in both fingers.
  • Some transgenic plants containing extra copies of CO flowered earlier than wild type, suggesting that CO activity is limiting on flowering time.
  • Double mutants were constructed containing co and mutations affecting gibberellic acid responses, meristem identity, or phytochrome function, and their phenotypes suggested a model for the role of CO in promoting flowering (Cell 80, 847-857 (1995)).
  • the rice genome contains a family of nucleotide sequences that encode homologs (SEQ ID NOs: 18; 34; and 50) of the Arabidopsis CO flowering time protein.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity and abundance of which can alter flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NO: 18; 34;and 50, or a partial- length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least 80%, even more preferably at least 90%> to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity and abundance of which can alter flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99%o amino acid sequence identity to the polypeptide of SEQ ID NO: 18; 34;and 50, with any individual number within this range of between 70% and 99% also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%>, more preferably at least 80%>, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity and abundance of which can alter flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NO: 18; 34;and 50, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%o, more preferably at least 80%), even more preferably at least 90% to 95%> the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NO: 17; 33;and 49, or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90%> to 95% the activity of the full-length polypeptide.; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO: 17; 33;and 49 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • FCA The Arabidopsis FCA genes has been cloned and shown (Cell 89, 737-745 (1997)) to encode a protein containing two RNA-binding domains and a WW protein interaction domain. This suggests that FCA functions in the posttranscriptional regulation of transcripts involved in the flowering process.
  • the FCA transcript is alternatively spliced with only one form encoding the entire FCA protein. Plants carrying the FCA gene fused to the strong constitutive 35S promoter flowered earlier, and the ratio and abundance of the different FCA transcripts were altered.
  • FCA appears to be a component of a posttranscriptional cascade involved in the control of flowering time.
  • the rice genome encodes at least two homologs of the Arabidopsis FCA protein which are given in SEQ ID NOs: 14, 28 and 52, respectively.
  • One of these homologs is encoded by a nucleotide sequence that maps to rice QTL Hd-3, which controls heading date in rice.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which functions in the posttranscriptional regulation of transcripts involved in the flowering process and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NO: 14, 28 and 52, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90% to 95%> the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which functions in the posttranscriptional regulation of transcripts involved in the flowering process and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99%> amino acid sequence identity to the polypeptide of SEQ ID NO: 14, 28 and 52, with any individual number within this range of between 70% and 99% also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which functions in the posttranscriptional regulation of transcripts involved in the flowering process and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NO: 14, 28 and 52 or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NO: 13, 27 and 51, or a part thereof which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90%) to 95% the activity of the full-length polypeptide..; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO: 13, 27 and 51, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • the process of vernalization (the acceleration of flowering by a cold period of 3-8 weeks at 4°-8°C) is an important characteristic in many plants.
  • the FRIGIDA (FRI) gene ensures that flowering is delayed until after winter so that the plant flowers in the favorable conditions of spring.
  • the Arabidopsis FRIGIDA gene was isolated (Mol Gen Genet 1994 Jan;242(l):81-9; Science 290, 344-347 (2000)) and can now be used to alter the vernalization requirements of crop plants to benefit farmers.
  • the FRI genes were isolated from different lines of Arabidopsis and their DNA sequences compared. It was found that the genes isolated from the summer Arabidopsis lines all had parts of the gene sequence missing — the genes were in fact damaged.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which can modulate the vernalization requirements and flowering time of plants and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 6 and 8, or a partial-length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90%) to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which can modulate the vernalization requirements and flowering time of plants and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to the polypeptide of SEQ ID NOs: 6 and 8, with any individual number within this range of between 70% and 99% also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least 80%>, even more preferably at least 90%> to 95% the activity of the full- length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which can modulate the vernalization requirements and flowering time of plants and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 6 and 8, or a partial- length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NOs: 5 and 7, or a part thereof, which still encodes , a partial-length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%, more preferably at least 80%), even more preferably at least 90%> to 95% the activity of the full-length polypeptide.; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NOs: 5 and 7 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • Another gene that can potentially used to alter the vernalization requirements of crop plants is the rice homolog of the Arabidopsis VRN1 gene, which is involved in the vernalization response of Arabidopsis plants.
  • vrnl and vrn2 mutations did not affect the acclimation response as judged by expression of cold-induced transcripts and freezing tolerance assays
  • vrnl-1 affected the short-day vernalization response of Landsberg erecta and reduced the vernalization response of other late-flowering Arabidopsis mutants.
  • the acceleration of flowering by GA3 was not affected by vrnl-1.
  • the VRN 1 locus was mapped to chromosome 3.
  • the rice genome contains at least two nucleotide sequence which show homology to the Arabidopsis VRN1 gene (SEQ ID NOs: 3, 55).
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which can modulate the vernalization requirements of plants and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 4 and 56, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%), even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which can modulate the vernalization requirements of plants and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%>, and 99% amino acid sequence identity to the polypeptide of SEQ ID NOs: 4 and 56, with any individual number within this range of between 70% and 99% also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which can modulate the vernalization requirements of plants and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 4 and 56, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NOs: 3 and 55, or a part thereof, which still encodes , a partial-length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide.; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NOs: 3 and 55 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • the FTITFL gene family in Arabidopsis encodes proteins with homology to RAF kinase inhibitor protein (Science 286, 1962 (1999) )originally described in humans and contains 6 members. Orthologs from other plants have been shown to be mvolved in major aspects of whole plant architecture (Pnueli, L., et al., Development, 1998. 125(11): p. 1979-89 ;Bradley, D., et al., Nature, 1996. 379(6568): p. 791-7).
  • the overall aerial architecture of flowering plants depends on a group of meristematic cells in the shoot apex. It was demonstrated (Development 125, 1609-1615 (1998) that the Arabidopsis TERMINAL FLOWER 1 gene has a unified effect on the rate of progression of the shoot apex through different developmental phases. In transgenic Arabidopsis plants which ectopically express TERMINAL FLOWER 1 , both the vegetative and reproductive phases are greatly extended. As a consequence, these plants exhibit dramatic changes in their overall morphology, producing an enlarged vegetative rosette of leaves, followed by a highly branched inflorescence which eventually forms normal flowers. Activity of the floral meristem identity genes LEAFY and APETALA 1 is not directly inhibited by TERMINAL FLOWER 1 , but their upregulation is markedly delayed compared to wild-type controls.
  • the rice genome encodes for at least 17 members of this gene family, one member is most similar to TFL (SEQ ID NO 19), and nine are more similar to FT (including, for example, SEQ ID NOs: 11, 15, 25, 31, 37 and 61 ).
  • At least two of the rice genes (SEQ ID NOs: 37 and 61) were shown to map to rice QTL Hd-3, which controls rice heading date.
  • SEQ ID NOs: 37 and 61 which map to another rice QTL that controls heading date, Hd-6, also show homology to the Arabidopsis TFL gene.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulating flowering time, and whole plant architecture by modulating plant branching patterns, and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NO: 20, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90% to 95%> the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulating flowering time, and whole plant architecture by modulating plant branching patterns, and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%>, and 99% amino acid sequence identity to the polypeptide of SEQ ID NO: 20, with any individual number within this range of between 70% and 99%> also being part of the invention, or a partial -length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%), even more preferably at least 90%> to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulating flowering time, and whole plant architecture by modulating plant branching patterns, and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NO: 20, or a partial-length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%>, more preferably at least 80%>, even more preferably at least 90%) to 95%> the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NO: 19, or a part thereof, which still encodes a partial- length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90% to 95%> the activity of the full-length polypeptide.; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO: 19 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • FT activation tagging mutagenesis in whole plants
  • Ectopic expression of FT leads to early flowering regardless of daylength, and causes formation of a terminal flower.
  • Loss-of-function EMS-induced mutant alleles ft-1, 2 and 3 have been described previously as late flowering (Korneef et al, 1991).
  • FT protein is 50% identical to TFL. Missense mutations in TFL cause early flowering and formation of a terminal flower, whereas overexpression of TFL dramatically extends vegetative stage. So it is remarkable that gain-of-function and loss- of-function phenotypes of FT and TFL mutants are completely opposite.
  • FT is acting in parallel with LEAFY, probably by regulating competence of the apical meristem to respond to LEAFY activity, and TFL protein acts as antagonist of FT in promoting flowering.
  • Temporal regulation of FT expression together with the phenotype of mutants suggest that FT is one of the master genes necessary and sufficient for commitment to flowering in response to day length.
  • the rice genome encodes for at least 17 members of this FT/TFL gene family, nine of which are most similar to the Arabidopsis FT gene (see, for exampel; SEQ ID NOs: 12, 16, 26, 32, 38 and 62).
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in flowering time and capable of inducing early flowering and terminal flower formation, and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 12, 16, 26, 32, 38 and 62, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90%> to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in flowering time and capable of inducing early flowering and terminal flower formation, and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to the polypeptide of SEQ ID NOs: 12, 16, 26, 32, 38 and 62, with any individual number within this range of between 70% and 99% also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least 80%), even more preferably at least 90% to 95%> the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in flowering time and capable of inducing early flowering and terminal flower formation, and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 12, 16, 26, 32, 38 and 62, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90%> to 95% the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NOs: 11, 15, 25, 31, 37 and 61, or a part thereof, which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90%> to 95% the activity of the full-length polypeptide.; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NOs: 11, 15, 25, 31, 37 and 61 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • SOC1 or AGL20 FLOWERING LOCUS T(FT) gene and also a second gene, called SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1 or AGL20) are early target genes of CO and are required for CO to promote flowering.
  • SOC1 or AGL20 is an Arabidopsis MADS box transcritpion factor which is at the intersection of 2 distinct flowering pathways (the "long day” and the “constitutive” pathways) and is likely to be a central regulator of flowering in Arabidopsis.
  • TheSOCi or AGL20 and the FT genes are also regulated by a second flowering- time pathway that acts independently of CO.
  • early target genes of CO define common components of distinct flowering-time pathways.
  • the rice genome encodes for several members of this SOC1/AGL20 gene family, 4 of which (SEQ ID NOs: 9, 47, 57, and 59) are most similar to the Arabidopsis SOC1 (AGL20) gene.
  • SEQ ID NOs: 9, 47, 57, and 59 are most similar to the Arabidopsis SOC1 (AGL20) gene.
  • At lest one of the nucleotide sequences encoding a homolog of the Arabidopsis AGL20 protein (SEQ ID NO: 60) was shown to map to rice QTL Hd-6, controlling heading date in rice by photoperiod response.
  • SEQ ID NO: 1 which was shown to map to rice QTL HD- 3, has high homology to the Arabidopsis SOC1 (AGL20) gene.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time, and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 2, 10, 48, 58, and 60, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90% to 95%> the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to the polypeptide of SEQ ID NOs: 2, 10, 48, 58, and 60, with any individual number within this range of between 70% and 99% also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide..
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 2, 10, 48, 58, and 60, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90% to 95%> the activity of the full- length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NOs: 1, 9, 47, 57, and 59, or a part thereof, which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%), even more preferably at least 90%> to 95%> the activity of the full- length polypeptide.; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NOs: 1, 9, 47, 57, and 59, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • MADS -box genes encode putative transcription factors that have been shown to play important regulatory roles in plants, yeast and animals. In plants, MADS-box genes regulate key events in flower development, such as the specification of flower-meristem and flower-organ identity, as well as the differentiation of each flower organ. In Arabidopsis, more than two dozen MADS-box genes have been cloned.
  • CAULIFLOWER (CAL) MADS-box genes have overlapping functions that specify flower- meristem identity, cal single mutant plants are indistinguishable from wild-type plants, however, mutations in the CAL gene dramatically enhance the phenotype of apl single mutants (Bowman, J.L., et al, Development 119, 721-743 (1993). The functional redundancy observed between the CAL and API genes has been attributed to their closely related sequences and expression patterns, suggesting that they regulate many of the same targets.
  • Homologs of CAL a MADS domain gene, could be identified among the large rice MADS domain gene family including those given in SEQ ID NOs: 1, 47 and 57. At least one of the rice homologs (SEQ ID NO: 1) was shown to map to rice QTL Hd-3, which controls rice heading date. Also SEQ ID NO: 59, which maps to another rice controlling rice heading date, QTL Hd-6, shows homology to the CAL gene.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in specifying flower-meristem identity and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 2, 48, 58, and 60, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90%> to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in specifying flower-meristem identity and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%>, and 99%> amino acid sequence identity to the polypeptide of SEQ ID NOs: 2, 48, 58, and 60, with any individual number within this range of between 70% and 99% also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90%> to 95%> the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in specifying flower-meristem identity and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 2, 48, 58, and 60, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%>, even more preferably at least 90% to 95%> the activity of the full- length polypeptide.4858
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NOs: 1, 47, 57, and 59, or a part thereof, which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least
  • the phenotype caused by mutations that affect the timing of flowering in Arabidopsis thaliana has been most extensively analyzed in the Landsberg erecta (Ler) genetic background.
  • Ler the late-flowering phenotype of FRIGIDA and mutations in LUMINIDEPENDENS is suppressed by the Ler allele of FLC.
  • FLC the Columbia ecotype
  • Homologs of FLC, a MADS domain gene could be identified among the large rice MADS domain gene family including those given in SEQ ID NOs: 9; 29, 47; and 57.
  • SEQ ID NOs: 1 and 59 which were shown to to map to rice QTL Hd-3 and Hd- 6, respectively, which controls heading date in rice, have high homology to the Arabidopsis FLC gene.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is capable of modulating flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 2, 10; 30, 48; 58 and 60, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is capable of modulating flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to the polypeptide of SEQ ID NOs: 2, 10; 30, 48; 58 and 60, with any individual number within this range of between 70% and 99%) also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90%) to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is capable of modulating flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 2, 10; 30, 48; 58 and 60, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%>, even more preferably at least 90% to 95%> the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NOs: 1 , 9; 29, 47; 57 and 59, or a part thereof, which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%), even more preferably at least 90%> to 95%> the activity of the full-length polypeptide.; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in SEQ ID NOs: 1, 9; 29, 47; 57 and 59, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • the rice genome encodes for at least 1 members of this gene family (SEQ IDNO: 21).
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in flowering time and capable of inducing early flowering and terminal flower formation, and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NO:22, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90%> to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in flowering time and capable of inducing early flowering and terminal flower formation, and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 10%, and 99% amino acid sequence identity to the polypeptide of SEQ ID NO: 22, with any individual number within this range of between 70% and 99%> also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full- length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in flowering time and capable of inducing early flowering and terminal flower formation, and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NO: 22, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%>, even more preferably at least 90% to 95%> the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NO: 21, or a part thereof, which still encodes a partial- length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95%> the activity of the full-length polypeptide., b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in SEQ ID NO: 21 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • the dominant late flowering Arabidopsis mutant Ihy ( late elongated hypocotyl (Ihy)) was shown to disrupte circadian clock regulation of gene expression and leaf movements and caused flowering to occur independently of photoperiod.
  • LHY was shown to encode a MYB DNA-binding protein.
  • the LHY mRNA showed a circadian pattern of expression with a peak around dawn but in the mutant was expressed constantly at high levels.
  • Increased LHY expression from a transgene caused the endogenous gene to be expressed at a constant level, suggesting that LHY ' was part of a feedback circuit that regulated its own expression.
  • constant expression of LHY disrupts several distinct circadian rhythms in Arabidopsis, and LHY m&y be closely associated with the central oscillator of the circadian clock (Schaffer et al, Cell 93, 1219-1229 (1998)).
  • the rice genome encodes for at least 2 members of this gene family (SEQ ID Nos: 43 and 45).
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 44 and 46, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99%> amino acid sequence identity to the polypeptide of SEQ ID NOs: 44 and 46, with any individual number within this range of between 10% and 99%> also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 44 and 46, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90%> to 95 %> the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NOs: 43 and 45, or a part thereof, which still encodes a partial-length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90%> to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in SEQ ID NOs: 43 and 45 or the complement thereof; e) complementary to (a), (b) or (c); and .
  • FPA is a gene that regulates flowering time in Arabidopsis via a pathway that is independent of daylength (the autonomous pathway). Mutations in FPA result in extremely delayed flowering. FPA was identified by means of positional cloning. The predicted FPA protein contains three RNA recognition motifs in the N-terminal region. FPA is expressed most strongly in developing tissues, similar to the expression of FCA and LUMINIDEPENDENS, two components of the autonomous pathway discussed herein previously. Overexpression of FPA in Arabidopsis causes early flowering in noninductive short days and creates plants that exhibit a more day-neutral flowering behavior. (Schomburg et al, The Plant Cell, Vol. 13, 1427-1436, June 2001)
  • the rice genome contains at least two homologs of the Arabidopsis FPA gene, which map to rice QTLs (Hd-3 and Hd-6, respectively) that are regulate heading time in rice.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 14 and 52, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least 80%>, even more preferably at least 90%> to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to the polypeptide of SEQ ID NOs: 14 and 52, with any individual number within this range of between 70% and 99%> also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%o, more preferably at least 80%>, even more preferably at least 90%) to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 14 and 52, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90%> to 95% the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NOs: 13 and 51, or a part thereof, which still encodes a partial-length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90% to 95%> the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in SEQ ID NOs: 13 and 51 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • FVE FVE
  • WD40 repeat protein encodes a WD40 repeat protein with similarity to proteins involved in transcription repression complexes and FY, which interacts with this WW domain in FCA.
  • the rice genome contains at least two nucleotide sequence which shows homology to either FVE or FY, or to both.
  • FVE and FY map to rice QTLs Hd-3 and Hd-6, respectivley, which regulates heading time in rice.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 24 and 36, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90%> to 95%> the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%>, and 99%o amino acid sequence identity to the polypeptide of SEQ ID NOs: 24 and 36, with any individual number within this range of between 10% and 99% also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least 80%>, even more preferably at least 90%) to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 24 and 36, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%), even more preferably at least 90% to 95%> the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NOs: 23 and 35, or a part thereof, which still encodes a partial -length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90% to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in SEQ ID NOs: 23 and 35 or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • FWA was identified by loss-of-function mutations in normally flowering revertants of thefwa mutant, and it encodes a homeodomain-containing transcription factor.
  • the DNA sequence of wild-type and fwa mutant alleles was identical in the genomic region of FWA.
  • the transition to flowering in Arabidopsis thaliana is delayed in fwa mutant plants.
  • the FWA gene is ectopically expressed in fwa mutants and silenced in mature wild-type plants. This silencing is associated with extensive methylation of two direct repeats in the 5' region of the gene. (Soppe et al, Molecular Cell, Vol. 6, 791-802, October, 2000
  • the rice genome contains at least 5 nucleotide sequences that show homology to the the Arabidopsis FWA gene.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 40, 42, 54, 64 and 66, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%o, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to the polypeptide of SEQ ID NOs: 40, 42, 54, 64 and 66, with any individual number within this range of between 70%) and 99% also being part of the invention, or a partial -length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90%) to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide the activity of which is involved in regulation of flowering time and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NOs: 40, 42, 54, 64 and 66, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%>, even more preferably at least 90%> to 95%> the activity of the fttll- length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NOs: 39, 41, 53, 63 and 65, or a part thereof, which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%), even more preferably at least 90%> to 95% the activity of the full-length polypeptide; b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid comprising 50 to 200 or more consecutive nucleotides of nucleotides given in SEQ ID NOs: 39, 41, 53, 63 and 65, or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • the ability to align regions of heterologous cereal chromosomes to the rice genome provides the opportunity to connect mapped cereal trait genes (quantitative trait loci or QTL) to the rice genome, and assign candidate genes to the traits that these genes influence.
  • mapped cereal trait genes quantitative trait loci or QTL
  • Mapped cereal QTL can be placed on the rice genome map. Positioning of molecular markers within the genome will further facilitate mapping and identification of trait-controlling genes.
  • a comparison of mapped rice QTL controlling flowering time with the positions of rice flowering time gene homologs make it possible to identify and/or confirm functions for many of these rice genes.
  • One such set of QTL from rice is known to control heading of rice. Heading date is a critical trait for adaptation to different cultivation areas and cropping seasons and therefore a major obejective in rice breeding programs. Heading in rice is basically determined by two factors, duration of the basic vegetative growth (BVG) and photoperiod-sensitivity (PS). Several genes are known to be involved in controlling these two factors . Yano et al (Theoretical and Applied Genetics (1997) 95: 1025-1032) reported five
  • Hdl-Hd5 which control rice heading date in an F2 population from a cross between a japonica variety, Nipponbare, and an indica variety, Kasalath. Scan.analysis revealed that two QTL, Hd-1 and Hd-2, had large effects, while three others, Hd-3, Hd-4 and Hd-5, were found to cause minor effects.
  • the nearly isogenic line with QTL (QTL- NIL) that carries the chromosomal segment from Kasalath for the Hd6 region in Nipponbare's genetic background was developed by marker-assisted selection.
  • QTL-NIL QTL- NIL
  • the QTL-NIL for Hd6 prominently increased days to heading under a 13.5-hr day length compared with the recurrent parent, Nipponbare, suggesting that Hd6 controls photoperiod sensitivity.
  • QTL analysis of the F 2 population derived from a cross between the QTL-NILs revealed existence of an epistatic interaction between Hd2, which is one of the photoperiod sensitivity genes detected in a previous analysis, and Hd6.
  • the day-length treatment tests of these QTL-NILs including the line introgressingbothHi2 and Hd6, also indicated an epistatic interaction for photoperiod sensitivity between them.
  • the present invention provides nucleotide sequences as given in the Sequence Listing with the SEQ ID NOs being identified in column C of table 2 corresponding to genes which are located between markers C844 and S1205S on rice chromosome 7 that flank the Hd-2 QTL controlling flowering time in rice (see TAG 95:1025-1032 (1997)) and these are candidate genes controlling this QTL.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide involved in control of flowering time in rice and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in the Sequence Listing with the SEQ ID NOs of the corresponding nucleotide sequences being identified in column C of table 2; or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%>, even more preferably at least 90%> to 95%> the activity of the full- length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is involved in control of flowering time in rice and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to the polypeptide given in the Sequence Listing with the SEQ ID NOs of the corresponding nucleotide sequences being identified in column C of table 2, with any individual number within this range of between 70% and 99%o also being part of the invention, or a partial-length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%>, more preferably at least 80%>, even more preferably at least 90%) to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is involved in control of flowering time in rice nd, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide as given in the Sequence Listing with the SEQ ID NOs of the corresponding nucleotide sequences being identified in column C of table 2; or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90%> to 95% the activity of the full- length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in the Sequence Listing with the SEQ ID NOs being identified in column C of table 2 or a part thereof, which still encodes a partial -length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90%> to 95%> the activity of the full-length polypeptide, b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid as given in the Sequence Listing with the SEQ ID NOs being identified in column C of table 2 comprising 50 to 200 or more consecutive nucleotides or the complement thereof; e) complementary to (a), (b) or (c); and f) which is the reverse complement of (a), (b) or (c).
  • the invention further provides nucleotide sequences as given in the Sequence Listing with the SEQ ID NOs being identified in column D of table 2 corresponding to genes which are located between markers Rl 952 and S 1520 on rice chromosome 6 that flank the Hd-3 QTL controlling flowering time in rice (see TAG 95 : 1025- 1032 ( 1997)) and these are candidate genes controlling this QTL.
  • SEQ ID NOs: 41, 13, 23, 37 and 61 are homologous to known Arabidopsis genes involved in control of flowering time as discussed hereinbefore.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide involved in control of flowering time in rice and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in the Sequence Listing with the SEQ ID NOs of the corresponding nucleic acid molecules being identified in column D of table 2, but especially a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 42, 14, 24, 38 and 62; or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%),-more preferably at least 80%>, even more preferably at least 90%> to 95%> the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is involved in control of flowering time in rice and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to the polypeptide as given in the Sequence Listing with the SEQ ID NOs of the corresponding nucleic acid molecules being identified in column D of table 2, but especially to a polypeptide as given in SEQ ID NOs: 42, 14, 24, 38 and 62, with any individual number within this range of between 70% and 99%> also being part of the invention; or a partial-length polypeptide having substantially the same activity as the full- length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is involved in control of flowering time in rice nd, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide as given in the Sequence Listing with the SEQ ID NOs of the corresponding nucleic acid molecules being identified in column D of table 2, but especially a polypeptide of SEQ ID NOs: 42, 14, 24, 38 and 62; or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90%> to 95%> the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in the Sequence Listing with the SEQ ID NOs being identified in column D of table 2; but especially in SEQ ID NOs: 41, 13, 23, 37 and 61 or a part thereof, which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least 80%>, even more preferably at least 90% to 95% the activity of the full-length polypeptide, b) having substantial similarity to (a); c) capable of hybridizing to (a) or the complement thereof; d) capable of hybridizing to a nucleic acid molecule as given in the Sequence Listing with the SEQ ID NOs being identified in column D of table 2, but especially in SEQ ID NOs: 41, 13, 23, 37 and 61 and , comprising 50 to
  • the invention also provides nucleotide sequences as given in the Sequence Listing with the SEQ ID NOs being identified in column E of table 2 corresponding to genes located between markers R2462 and R2966 on rice chromosome 3 that flank the Hd-6 QTL controlling flowering time in rice (see Genetics 154:885-891 (2000)) and these are candidate genes controlling this QTL.
  • SEQ ID NOs: 35, 51, and 59 are homologous to known Arabidopsis genes involved in control of flowering time as discussed hereinbefore.
  • the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide involved in control of flowering time in rice and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in the Sequence Listing with the SEQ ID NOs of the co ⁇ esponding nucleic acid molecule being identified in column E of table 2, but especially to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 36, 52, and 60; or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90%> to 95%> the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is involved in control of flowering time in rice and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to the polypeptide as given in the Sequence Listing with the SEQ ID NOs of the co ⁇ esponding nucleic acid molecule being identified in column E of table 2, but especially to a polypeptide as given in SEQ ID NOs: 36, 52, and 60, with any individual number within this range of between 70%> and 99% also being part of the invention; or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is involved in control of flowering time in rice nd, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide as given in the Sequence Listing with the SEQ ID NOs of the corresponding nucleic acid molecule being identified in column E of table 2, but especially a polypeptide of SEQ ID NOs: 36, 52, and 60; or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%>, more preferably at least 80%, even more preferably at least 90%o to 95%> the activity of the full-length polypeptide.
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence a) as given in the Sequence Listing with the SEQ ID NOs being identified in column E of table 2, but especially in SEQ ID NOs: 35, 51, and 59 or a part thereof, which still encodes a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least 80%>, even more preferably at least 90%> to
  • a further embodiment of the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide involved in control of flowering time in rice and, optionally, a regulatory region, which induces transcription of the coding region, which nucleic acid molecule is substantially similar to a nucleic acid encoding a polypeptide as given in SEQ ID NOs: 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 54, 56, 58, 62, 64, and 66, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%>, even more preferably at least 90%> to 95%> the activity of the full- length polypeptide.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is involved in control of flowering time in rice and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is substantially similar, and preferably has at least between 70%, and 99% amino acid sequence identity to the polypeptide as given in SEQ ID NOs: 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 54, 56, 58, 62, 64, and 66, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%, more preferably at least 80%, even more preferably at least 90% to 95% the activity of the full-length polypeptide, with any individual number within this range of between 70%) and 99% also being part of the invention.
  • the invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide which is involved in control of flowering time in rice and, optionally, a regulatory region, which induces transcription of the coding region, which polypeptide is immunologically reactive with antibodies raised against a polypeptide as given SEQ ID NOs: 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 28, 30, 32, 34, 38, 40, 42, 44, 46, 48, 50, 54, 56, 58, 62, 64, and 66, or a partial-length polypeptide having substantially the same activity as the full-length polypeptide, e.g., at least 50%), more preferably at least 80%>, even more preferably at least 90% to 95% the activity of the full-length polypeptide
  • the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence g) as given in SEQ ID NOs 3, 5, 7, 9, 11, 15, 17, 376478, 21, 25, 27, 29, 31,
  • a further subset of nucleic acid molecules according to the invention comprises at lest one polynucleotide which contains one or more Simple Sequence Repeats (SSRs) in the coding region.
  • SSRs Simple Sequence Repeats
  • Microsatellites are tandem repeats of short units of DNA that occur with high frequency throughout the genomes of many organisms. SSRs are more or less evenly distributed over the plant genome with a frequency of 1 SSR every 9 Kb on the average. SSRs are present in coding as well as non-coding regions. Their length is dependent on the number of repetitions of the simple di-, tri-, or tetranucleotide. Thus, the length of an SSR may vary from species to species or from gene to gene. Microsatellite loci have a high degree of variability that is due to a high rate of mutations that alter microsatellite length. The primary mutational mechanism leading to changes in microsatellite length is polymerase template slippage.
  • microsatellite loci During replication of a repetitive region, DNA strands may dissociate, and reassociate incorrectly. Renewed replication in this misaligned state leads to insertion or deletion of repeat units, thus altering allele length.
  • the abundance and high level of allelic variation at microsatellite loci in the genomes of many organisms has made them popular genetic markers.
  • Genetic distance measures based on microsatellites can be used to answer questions concerning population structure and divergence.
  • This size change can by captured by PCR with primers flanking the SSRs.
  • the SSR becomes a marker to distinguish any two individual species with different copy number of that particular SSR.
  • SSRs are abundant, highly variable, and easy to assay. Using a number of SSR markers to genotype a segregating population, as described herein, one can generate a genetic map, map a trait of interest, and clone the underlying gene by map-based cloning.
  • the invention provides an isolated nucleic acid molecule including a repeat region, the repeat region having at least about 3 consecutive dinucleotide, trinucleotide, or tetranucleotide repeat units, the nucleic acid further including either a first flanking region of at least about 4 nucleotides at a first end of the repeat region, or a second flanking region of at least about 4 nucleotides at a second end of the repeat region, or both a first and a second flanking region.
  • the SSR can be either some or all of the repeat units, with one or both of the flanking regions.
  • the flanking region can be as little as one base adjacent to the repeat units, and can be typically several bases in length.
  • the size of the flanking region is preferably in a range of between about 4 to 250 nucleotides, with each individual number of nucleotides within this range being also part of the invention, or can even be longer than that.
  • Preferred is a flanking region where the nucleotide sequence is between about 5 to about 9, preferably between about 10 to about 30, and more preferably between about 31 to 50 nucleotides in length.
  • the nucleic acid molecule according to the invention can be adapted for a use such as, for example, use as a chromosomal marker to identify the location of a corresponding or complementary nucleic acid on a native or artificial chromosome; use as a marker for RFLP analysis; use as a marker for quantitative trait linked breeding; use as a marker for marker- assisted breeding; use as a bait sequence in a two-hybrid system to identify sequence encoding polypeptides interacting with the polypeptide encoded by the bait sequence; use as a diagnostic tool for genotyping or identifying an individual or population of individuals; use for genetic analysis to identify boundaries of genes or exons; and the like.
  • the invention further provides a method of modifying the frequency of a gene in a plant population, including the steps of: identifying an SSR within a coding region of a gene; screening a plurality of plants using the SSR as a marker to determine the presence or absence of the gene in an individual plant; selecting at least one individual plant for breeding based on the presence or absence of the gene; and breeding at least one plant thus selected to produce a population of plants having a modified frequency of the gene.
  • a method of plant breeding to select for or against a trait of interest including the steps of: identifying the trait of interest; identifying at least one SSR that can be used as a marker for the trait; screening at least one plant for the presence of the at least one SSR; selecting at least one plant based on presence or absence of the at least one SSR; breeding at least one plant thus selected to produce a population of plants having a modified frequency of the at least one SSR; and screening at least one plant of the population for the presence or absence of the trait.
  • the trait of interest can be, for example, disease resistance, drought resistance, weather resistance, early ripening, pest resistance, salt resistance, cold tolerance, yield, flavor, nutritional value, texture, ease of harvesting, ease of processing, and the like.
  • the invention provides a method of mapping a genome of a plant species, including screening nucleic acid sequences from the plant species to identify an SSR therein, the SSR having a first and a second flanking region; and identifying a first position in the genome, the first position corresponding to the SSR, the first position being relative to a second position in the genome.
  • the second position can correspond to a location on a known natural or artificial chromosome, such as a location of an SSR, or a location of a gene.
  • the second position can also correspond to a map location of a trait.
  • the invention also provides a genetic map of a plant, the map including map positions, wherein the map positions can include positions of a plurality of SSRs, such as at least about 100 SSRs; at least about 500 SSRs; at least about 1000 SSRs; or more.
  • the plurality of SSRs can include at least about 10 SSRs, each SSR having a repeat region and at least a first or a second flanking region, or both a first and a second flanking region.
  • the nucleotide sequences according to the invention can also be used to generate markers, including single-sequence repeats (SSRs) and microsatellite markers for QTL to assist marker-assisted breeding.
  • SSRs single-sequence repeats
  • the nucleotide sequences of the invention can further be used to identify QTL and isolate alleles as described by Li et al. in a study of QTL involved in resistance to a pathogen of rice. (Li et al, Mol Gen Genet 261 :58 (1999)).
  • the nucleotide sequences of the invention can also be used to isolate alleles from the corresponding QTL(s) of wild relatives.
  • Nucleotides of the present invention can also be used for nucleic acid analysis in a variety of ways.
  • One prefe ⁇ ed use involves microbead technology and capture probe hybridization. This technology is described in copending U.S. Patent Application No. 09/565,214, entitled “Novel Assay for Nucleic Acid Analysis,” which was filed on May 4, 2000, and which is hereby incorporated by reference in its entirety.
  • the present invention is directed to a nucleic acid molecule comprising a nucleotide sequence isolated or obtained from any plant which encodes a polypeptide having at least 70%> amino acid sequence identity to a polypeptide encoded by a gene comprising any one of SEQ ID NOs provided in the Sequence Listing.
  • orthologs may be identified or isolated from the genome of any desired organism, preferably from another plant, according to well known techniques based on their sequence similarity to the Oryza nucleic acid sequences, e.g., hybridization, PCR or computer generated sequence comparisons. For example, all or a portion of a particular Oryza nucleic acid sequence is used as a probe that selectively hybridizes to other gene sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen source organism.
  • genomic and cDNA libraries may be prepared from any cell or tissue of an organism.
  • Such techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g., Sambrook et al., 1989) and amplification by PCR using oligonucleotide primers preferably co ⁇ esponding to sequence domains conserved among related polypeptide or subsequences of the nucleotide sequences provided herein (see, e.g., Innis et al., 1990). These methods are particularly well suited to the isolation of gene sequences from organisms closely related to the organism from which the probe sequence is derived.
  • oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art.
  • 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 organism.
  • the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32 P, or any other detectable marker.
  • probes for hybridization can be made by labeling synthetic oligonucleotides based on the sequence of the invention.
  • sequences that hybridize to the sequences disclosed herein will have at least 40% to 50%, about 60% to 70% and even about 80% 85%, 90%, 95% to 98% or more identity with the disclosed sequences. That is, the sequence similarity of sequences may range, sharing at least about 40% to 50%, about 60%> to 70%>, and even about 80%, 85%, 90%, 95%o to 98% sequence similarity, with each individual number within the ranges given above also being part of the invention.
  • nucleic acid molecules of the invention can also be identified by, for example, a search of known databases for genes encoding polypeptides having. a specified amino acid sequence identity or DNA having a specified nucleotide sequence identity. Methods of alignment of sequences for comparison are well known in the art and are described hereinabove.
  • the present invention further provides a composition, an expression cassette or a recombinant vector containing the nucleic acid molecule of the invention, and host cells comprising the expression cassette or vector, e.g., comprising a plasmid.
  • the present invention provides an expression cassette or a recombinant vector comprising a promoter as disclosed herein linked to a nucleic acid segment which, when present in a plant, plant cell or plant tissue, results in transcription of the linked nucleic acid segment.
  • the invention also provides an expression cassette or a recombinant vector comprising a plant nucleotide sequence comprising an open reading frame of the invention such as, for example, those given in the SEQ ID NOs provided in the Sequence Listing which, when present in a plant, plant cell or plant tissue, results in expression of the protein product encoded by the open reading frame.
  • the invention provides isolated polypeptides encoded by any one of the open reading frames given in the SEQ ID NOs provided in the Sequence Listing, a fragment thereof which encodes a polypeptide which has substantially the same activity as the corresponding polypeptide encoded by an ORF listed in the SEQ ID NOs provided in the Sequences Listing, or the orthologs thereof.
  • Virtually any DNA composition may be used for delivery to recipient plant cells, e.g., monocotyledonous cells, to ultimately produce fertile transgenic plants in accordance with the present invention.
  • DNA segments or fragments in the form of vectors and plasmids, or linear DNA segments or fragments, in some instances containing only the DNA element to be expressed in the plant, and the like may be employed.
  • the construction of vectors which may be employed in conjunction with the present invention will be known to those of skill of the art in light of the present disclosure (see, e.g., Sambrook et al., 1989; Gelvin et al, 1990).
  • the invention provides an expression cassette or vector containing an isolated nucleic acid molecule having a nucleotide sequence that directs transscription of a linked nucleic acid segment of interest in a cell, which nucleotide sequence is from a gene which encodes a polypeptide having at least 70% identity to an Oryz polypeptide encoded by a gene having one of the promoters listed in the SEQ ID NOs provided in the Sequence Listing, and which nucleotide sequence is optionally operably linked to other suitable regulatory sequences, e.g., a transcription terminator sequence, operator, repressor binding site, transcription factor binding site and/or an enhancer.
  • suitable regulatory sequences e.g., a transcription terminator sequence, operator, repressor binding site, transcription factor binding site and/or an enhancer.
  • nucleic acid segment of interest can, for example, code for a ribosomal RNA, an antisense RNA or any other type of RNA that is not translated into protein.
  • the nucleic acid segment of interest is translated into a protein product.
  • the nucleotide sequence which directs transcription and/or the nucleic acid segment may be of homologous or heterologous origin with respect to the plant to be transformed.
  • a recombinant DNA molecule useful for introduction into plant cells includes that which has been derived or isolated from any source, that may be subsequently characterized as to structure, size and/or function, chemically altered, and later introduced into plants.
  • nucleotide sequence or segment of interest "derived" from a source would be a nucleotide sequence or segment that is identified as a useful fragment within a given organism, and which is then chemically synthesized in essentially pure form.
  • An example of such a nucleotide sequence or segment of interest "isolated” from a source would be nucleotide sequence or segment that is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering.
  • Such a nucleotide sequence or segment is commonly referred to as "recombinant.”
  • a useful nucleotide sequence, segment or fragment of interest includes completely synthetic DNA, semi-synthetic DNA, DNA isolated from biological sources, and DNA derived from introduced RNA.
  • the introduced DNA is not originally resident in the plant genotype which is the recipient of the DNA, but it is within the scope of the invention to isolate a gene from a given plant genotype, and to subsequently introduce multiple copies of the gene into the same genotype, e.g., to enhance production of a given gene product as given in the SEQ ID NOs provided in the Sequence Listing.
  • the introduced recombinant DNA molecule includes but is not limited to, DNA from plant genes, and non-plant genes such as those from bacteria, yeasts, animals or viruses.
  • the introduced DNA can include modified genes, portions of genes, or chimeric genes, including genes from the same or different genotype.
  • the term "chimeric gene” or “chimeric DNA” is defined as a gene or DNA sequence or segment comprising at least two DNA sequences or segments from species which do not combine DNA under natural conditions, or which DNA sequences or segments are positioned or linked in a manner which does not normally occur in the native genome of untransformed plant.
  • the introduced recombinant DNA molecule used for transformation herein may be circular or linear, double-stranded or single-stranded.
  • the DNA is in the form of chimeric DNA, such as plasmid DNA, that can also contain coding regions flanked by regulatory sequences which promote the expression of the recombinant DNA present in the resultant plant.
  • the introduced recombinant DNA molecule will be relatively small, i.e., less than about 30 kb to minimize any susceptibility to physical, chemical, or enzymatic degradation which is known to increase as the size of the nucleotide molecule increases.
  • the number of proteins, RNA transcripts or mixtures thereof which is introduced into the plant genome is preferably preselected and defined, e.g., from one to about 5-10 such products of the introduced DNA may be formed.
  • This expression cassette or vector may be contained in a host cell.
  • the expression cassette or vector may augment the genome of a transformed plant or may be maintained extrachromosomally.
  • the expression cassette may be operatively linked to a structural gene, the open reading frame thereof, or a portion thereof.
  • the expression cassette may further comprise a Ti plasmid and be contained in an Agrobacterium tumefaciens cell; it may be carried on a microparticle, wherein the microparticle is suitable for ballistic transformation of a plant cell; or it may be contained in a plant cell or protoplast.
  • the expression cassette or vector can be contained in a transformed plant or cells thereof, and the plant may be a dicot or a monocot. In particular, the plant may be a cereal plant.
  • Overexpression can be achieved by insertion of one or more than one extra copy of the selected gene. It is, however, not unknown for plants or their progeny, originally transformed with one or more than one extra copy of a nucleotide sequence, to exhibit the effects of underexpression as well as overexpression.
  • underexpression there are two principle methods which are commonly referred to in the art as “antisense downregulation” and “sense downregulation” (sense downregulation is also referred to as “cosuppression”).
  • antisense downregulation and “sense downregulation” (sense downregulation is also referred to as “cosuppression”).
  • gene silencing is also referred to as "gene silencing". Both of these methods lead to an inhibition of expression of the target gene.
  • the invention hence also provides sense and anti-sense nucleic acid molecules corresponding to the open reading frames identified in the SEQ ID NOs provided in the Sequence Listing as well as their orthologs. .
  • genes and open reading frames according to the present invention which are substantially similar to a nucleotide sequence encoding a polypeptide as identified in the SEQ ID NOs provided in the Sequence Listing including any corresponding anti-sense constructs can be operably linked to any promoter that is functional within the plant host including the promoter sequences according to the invention or mutants thereof.
  • promoters which are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive (Odell et al., 1985), temporally regulated, spatially regulated, tissue-specific, and spatio-temporally regulated. Where expression in specific tissues or organs is desired, tissue-specific promoters may be used. In contrast, where gene expression in response to a stimulus is desired, inducible promoters are the regulatory elements of choice. Where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. Additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in expression constructs of transformation vectors to bring about varying levels of expression of heterologous nucleotide sequences in a transgenic plant.
  • the 3N nontranslated regulatory DNA sequence preferably includes from about 50 to about 1,000, more preferably about 100 to about 1,000, nucleotide base pairs and contains plant transcriptional and translational termination sequences.
  • Appropriate transcriptional terminators and those which are known to function in plants include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3N end of the protease inhibitor I or II genes from potato or tomato, although other 3N elements known to those of skill in the art can also be employed.
  • Preferred 3N elements include those from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan et al., 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato.
  • leader sequences are contemplated to include those which include sequences predicted to direct optimum expression of the attached gene, i.e., to include a preferred consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation.
  • sequences will be known to those of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants will be most preferred.
  • sequences that have been found to enhance gene expression in transgenic plants include intron sequences (e.g., from Adhl, bronzel, actinl, actin 2 (WO 00/760067), or the sucrose synthase intron) and viral leader sequences (e.g., from TMV, MCMV and AMV).
  • viral leader sequences e.g., from TMV, MCMV and AMV.
  • TMV Tobacco Mosaic Virus
  • MCMV Maize Chlorotic Mottle Virus
  • AMV Alfalfa Mosaic Virus
  • Picornavirus leaders for example, EMCV leader (Encephalomyocarditis 5 noncoding region) (Elroy-Stein et al., 1989); Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus); MDMV leader (Maize Dwarf Mosaic Virus); Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak et al., 1991); Untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling et al., 1987; Tobacco mosaic virus leader (TMV), (Gallie et al., 1989; and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel et al., 1991. See also, Della-Cioppa et al., 1987.
  • Adh intron 1 (Callis et al., 1987), sucrose synthase intron (Vasil et al., 1989) or TMV omega element (Gallie, et al., 1989), may further be included where desired.
  • enhancers include elements from the CaMV 35S promoter, octopine synthase genes (Ellis el al., 1987), the rice actin I gene, the maize alcohol dehydrogenase gene (Callis et al., 1987), the maize shrunken I gene (Vasil et al., 1989), TMV Omega element (Gallie et al., 1989) and promoters from non-plant eukaryotes (e.g. yeast; Ma et al., 1988).
  • promoters from non-plant eukaryotes e.g. yeast; Ma et al., 1988.
  • the present invention further provides a method of augmenting a plant genome by contacting plant cells with a nucleic acid molecule of the invention, e.g., one having a nucleotide sequence that directs constitutive, tissue-specific, or tissue-preferential and/or induced transcription of a linked nucleic acid segment encoding a polypeptide that is substantially similar to a polypeptide encoded by the an Oi ⁇ za gene having a sequence according to any one of the SEQ ID NOs provided in the Sequence Listing so as to yield transformed plant cells; and regenerating the transformed plant cells to provide a differentiated transformed plant, wherein the differentiated transformed plant expresses the nucleic acid molecule in the cells of the plant.
  • a nucleic acid molecule of the invention e.g., one having a nucleotide sequence that directs constitutive, tissue-specific, or tissue-preferential and/or induced transcription of a linked nucleic acid segment encoding a polypeptide that is substantially similar to a poly
  • the nucleic acid molecule may be present in the nucleus, chloroplast, mitochondria and/or plastid of the cells of the plant.
  • Plant species may be transformed with the DNA construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art. Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector of the present invention.
  • organogenesis means a process by which shoots and roots are developed sequentially from meristematic centers; the term “embryogenesis,” as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
  • tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and ultilane meristem).
  • existing meristematic tissue e.g., apical meristems, axillary buds, and root meristems
  • induced meristem tissue e.g., cotyledon meristem and ultilane meristem.
  • Plants of the present invention may take a variety of forms.
  • the plants may be chimeras of transformed cells and non-transformed cells; the plants may be clonal transformants (e.g., all cells transformed to contain the expression cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species).
  • the transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, first generation (or TI) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques.
  • a dominant selectable marker (such as npt II) can be associated with the expression cassette to assist in breeding.
  • the present invention provides a transformed (transgenic) plant cell, in planta or ex planta, including a transformed plastid or other organelle, e.g., nucleus, mitochondria or chloroplast.
  • the present invention may be used for transformation of any plant species, including, but not limited to, cells from corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
  • juncea particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculent
  • Duckweed (Lemna, see WO 00/07210) includes members of the family Lemnaceae. There are known four genera and 34 species of duckweed as follows: genus Lemna (L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L.turionifera, L. valdiviana); genus Spirodela (S. intermedia, S. polyrrhiza, S. punctata); genus Woffia (Wa. Angusta, Wa. Arrhiza, Wa.
  • genus Lemna L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscur
  • Lemna species can be classified using the taxonomic scheme described by Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Study. Geobatanischen Institut ETH, founded Rubel, Zurich (1986)). Vegetables within the scope of the invention include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C.
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcher ma), and chrysanthemum.
  • Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata), Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga ultilane); Sitka sp ⁇ ice (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
  • pines such as loblolly pine (Pinus taeda), slash pine (
  • Leguminous plants include beans and peas.
  • Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
  • Legumes include, but are not limited to, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., common bean and lima bean, Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo.
  • Preferred forage and turf grass for use in the methods of the invention include alfalfa, orchard grass, tall fescue, perennial ryegrass, creeping bent grass, and redtop.
  • plants within the scope of the invention include Acacia, aneth, artichoke, arugula, blackberry, canola, cilantro, Clementines, escarole, eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon, lime, mushroom, nut, okra, orange, parsley, persimmon, plantain, pomegranate, poplar, radiata pine, radicchio, Southern pine, sweetgum, tangerine, triticale, vine, yams, apple, pear, quince, cherry, apricot, melon, hemp, buckwheat, grape, raspberry, chenopodium, blueberry, nectarine, peach, plum, strawberry, watermelon, eggplant, pepper, cauliflower, Brassica, e.g., broccoli, cabbage, ultilan sprouts, onion, carrot, leek, beet, broad bean, celery, radish,
  • Pelargonium Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia. Other plants within the scope of the invention are shown in Table 2 (above).
  • transgenic plants of the present invention are crop plants and in particular cereals (for example, com, alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), and even more preferably com, rice and soybean.
  • cereals for example, com, alfalfa, sunflower, rice, Brassica, canola, soybean, barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.
  • the present invention also provides a transgenic plant prepared by this method, a seed from such a plant and progeny plants from such a plant including hybrids and inbreds.
  • Preferred transgenic plants are transgenic maize, soybean, barley, alfalfa, sunflower, canola, soybean, cotton, peanut, sorghum, tobacco, sugarbeet, rice, wheat, rye, turfgrass, millet, sugarcane, tomato, or potato.
  • a transformed (transgenic) plant of the invention includes plants, the genome of which is augmented by a nucleic acid molecule of the invention, or in which the co ⁇ esponding gene has been disrupted, e.g., to result in a loss, a decrease or an alteration, in the function of the product encoded by the gene, which plant may also have increased yields and/or produce a better-quality product than the co ⁇ esponding wild-type plant.
  • the nucleic acid molecules of the invention are thus useful for targeted gene disruption, as well as markers and probes.
  • the invention also provides a method of plant breeding, e.g., to prepare a crossed fertile transgenic plant.
  • the method comprises crossing a fertile transgenic plant comprising a particular nucleic acid molecule of the invention with itself or with a second plant, e.g., one lacking the particular nucleic acid molecule, to prepare the seed of a crossed fertile transgenic plant comprising the particular nucleic acid molecule.
  • the seed is then planted to obtain a crossed fertile transgenic plant.
  • the plant may be a monocot or a dicot.
  • the plant is a cereal plant.
  • the crossed fertile transgenic plant may have the particular nucleic acid molecule inherited through a female parent or through a male parent.
  • the second plant may be an inbred plant.
  • the crossed fertile transgenic may be a hybrid. Also included within the present invention are seeds of any of these crossed fertile transgenic plants. Transformation of plants can be undertaken with a single DNA molecule or multiple
  • DNA molecules i.e., co-transformation
  • co-transformation DNA molecules
  • Numerous transformation vectors are available for plant transformation, and the expression cassettes of this invention can be used in conjunction with any such vectors. The selection of vector will depend upon the prefe ⁇ ed transformation technique and the target species for transformation.
  • a variety of techniques are available and known to those skilled in the art for introduction of constructs into a plant cell host. These techniques generally include transformation with DNA employing tumefaciens or A.
  • rhizogenes as the transforming agent, liposomes, PEG precipitation, electroporation, DNA injection, direct DNA uptake, microprojectile bombardment, particle acceleration, and the like (See, for example, EP 295959 and EP 138341) (see below).
  • cells other than plant cells may be transformed with the expression cassettes of the invention.
  • the general descriptions of plant expression vectors and reporter genes, and Agrobacterium and Agrobacterium-mediated gene transfer, can be found in Gruber et al. (1993).
  • Expression vectors containing genomic or synthetic fragments can be introduced into protoplasts or into intact tissues or isolated cells.
  • expression vectors are introduced into intact tissue.
  • General methods of culturing plant tissues are provided for example by Maki et al., (1993); and by Phillips et al. (1988).
  • expression vectors are introduced into maize or other plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation and the like. More preferably expression vectors are introduced into plant tissues using the microprojectile media delivery with the biolistic device. See, for example, Tomes et al. (1995).
  • the vectors of the invention can not only be used for expression of structural genes but may also be used in exon-trap cloning, or promoter trap procedures to detect differential gene expression in varieties of tissues, (Lindsey et al., 1993; Auch & Reth et al.). It is particularly prefe ⁇ ed to use the binary type vectors of Ti and Ri plasmids of
  • Agrobacterium spp. Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti et al., 1985: Byrne et al., 1987; Sukhapinda et al., 1987; Lorz et al., 1985; Potrykus, 1985; Park et al., 1985: Hiei et al., 1994).
  • the use of T-DNA to transform plant cells has received extensive study and is amply described (EP 120516; Hoekema, 1985; Knauf, et al., 1983; and An et al., 1985).
  • the chimeric genes of the invention can be inserted into binary vectors as described in the examples.
  • transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs (see EP 295959), techniques of electroporation (Fromm et al., 1986) or high velocity ballistic bombardment with metal particles coated with the nucleic acid constructs (Kline et al., 1987, and U.S. Patent No. 4,945,050). Once transformed, the cells can be regenerated by those skilled in the art.
  • rapeseed (De Block et al., 1989), sunflower (Everett et al., 1987), soybean (McCabe et al., 1988; Hinchee et al., 1988; Chee et al., 1989; Christou et al., 1989; EP 301749), rice (Hiei et al., 1994), and com (Gordon Kamm et al., 1990; Fromm et al, 1990).
  • Suitable methods of transforming plant cells include, but are not limited to, microinjection (Crossway et al., 1986), electroporation (Riggs et al., 1986), Agrobacterium-mediated transformation (Hinchee et al., 1988), direct gene transfer (Paszkowski et al., 1984), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wis. And BioRad, Hercules, Calif, (see, for example, Sanford et al., U.S. Pat. No.
  • a nucleotide sequence of the present invention is directly transformed into the plastid genome.
  • Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et al., 1994.
  • the basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation).
  • the 1 to 1.5 kb flanking regions facilitate orthologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome.
  • point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al., 1990; Staub et al., 1992). This resulted in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub et al., 1993).
  • Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3N-adenyltransferase (Svab et al., 1993).
  • selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the invention. Typically, approximately 15-20 cell division cycles following transformation are required to reach a homoplastidic state.
  • Plastid expression in which genes are inserted by orthologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10%> of the total soluble plant protein.
  • a nucleotide sequence of the present invention is inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleotide sequence.
  • Agrobacterium tumefaciens cells containing a vector comprising an expression cassette of the present invention, wherein the vector comprises a Ti plasmid are useful in methods of making transformed plants. Plant cells are infected with an Agrobacterium tumefaciens as described above to produce a transformed plant cell, and then a plant is regenerated from the transformed plant cell. Numerous Agrobacterium vector systems useful in carrying out the present invention are known. For example, vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, 1984).
  • the expression cassettes of the present invention may be inserted into either of the binary vectors pCIB200 and pCIB2001 for use with Agrobacterium.
  • These vector cassettes for Agrobacterium-mediated transformation wear constructed in the following manner.
  • PTJS75kan was created by Narl digestion of pTJS75 (Schmidhauser & Helinski, 1985) allowing excision of the tetracycline-resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTII (Messing & Vierra, 1982; Bevan et al., 1983; McBride et al., 1990).
  • Xhol linkers were ligated to the EcoRV fragment of pCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al., 1987), and the Xhol- digested fragment was cloned into Sail-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19).
  • PCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bglll, Xbal, and Sail.
  • the plasmid pCIB2001 is a derivative of pCIB200 which was created by the insertion into the polylinker of additional restriction sites.
  • Unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sail, Mlul, Bell, Avrll, Apal, Hpal, and Stul.
  • PCIB2001 in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-mediated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the OriT and OriV functions also from RK2.
  • the pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
  • An additional vector useful for Agrobacterium-mediated transformation is the binary vector pCIB 10, which contains a gene encoding kanamycin resistance for selection in plants, T-DNA right and left border sequences and incorporates sequences from the wide host- range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al., 1987. Various derivatives of pCIBlO have been constructed which inco ⁇ orate the gene for hygromycin B phosphotransferase described by Gritz et al., 1983.
  • hygromycin only pCIB743
  • hygromycin and kanamycin pCIB715, pCIB717
  • Methods using either a form of direct gene transfer or Agrobacterium-mediated transfer usually, but not necessarily, are undertaken with a selectable marker which may provide resistance to an antibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide (e.g., phosphinothricin).
  • antibiotic e.g., kanamycin, hygromycin or methotrexate
  • phosphinothricin e.g., phosphinothricin
  • selection markers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics (Messing & Vierra, 1982; Bevan et al., 1983), the bar gene which confers resistance to the herbicide phosphinothricin (White et al., 1990, Spencer et al., 1990), the hph gene which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al., 1983).
  • pCIB3064 One such vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is pCIB3064.
  • This vector is based on the plasmid pCIB246, which comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278, herein incorporated by reference.
  • One gene useful for conferring resistance to phosphinothricin is the bar gene from Streptomyces viridochromogenes (Thompson et al., 1987). This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
  • An additional transformation vector is pSOG35 which utilizes the E. coli gene dihydrofolate reductase (DHFR) as a selectable marker conferring resistance to methotrexate.
  • PCR was used to amplify the 35S promoter (about 800 bp), intron 6 from the maize Adhl gene (about 550 bp) and 18 bp of the GUS untranslated leader sequence from pSOGlO. A 250 bp fragment encoding the E.
  • coli dihydrofolate reductase type II gene was also amplified by PCR and these two PCR fragments were assembled with a Sacl-Pstl fragment from pBI221 (Clontech) which comprised the pUC 19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generated pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus check (MCMV) generated the vector pSOG35. pSOG19 and pSOG35 carry the pUC-derived gene for ampicillin resistance and have Hindlll, Sphl, Pstl and EcoRI sites available for the cloning of foreign sequences.
  • MCMV Maize Chlorotic Mottle Virus check
  • Transgenic plant cells are then placed in an appropriate selective medium for selection of transgenic cells which are then grown to callus.
  • Shoots are grown from callus and plantlets generated from the shoot by growing in rooting medium.
  • the various constmcts normally will be joined to a marker for selection in plant cells.
  • the marker may be resistance to a biocide (particularly an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide, or the like).
  • the particular marker used will allow for selection of transformed cells as compared to cells lacking the DNA which has been introduced.
  • Components of DNA constmcts including transcription cassettes of this invention may be prepared from sequences which are native (endogenous) or foreign (exogenous) to the host.
  • foreign it is meant that the sequence is not found in the wild-type host into which the constmct is introduced.
  • Heterologous constmcts will contain at least one region which is not native to the gene from which the transcription-initiation-region is derived.
  • Such assays include, for example, "molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, in situ hybridization and nucleic acid-based amplification methods such as PCR or RT-PCR; "biochemical” assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as seed assays; and also, by analyzing the phenotype of the whole regenerated plant, e.g., for disease or pest resistance.
  • “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, in situ hybridization and nucleic acid-based amplification methods such as PCR or RT-PCR
  • biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by en
  • DNA may be isolated from cell lines or any plant parts to determine the presence of the preselected nucleic acid segment through the use of techniques well known to those skilled in the art. Note that intact sequences will not always be present, presumably due to rea ⁇ angement or deletion of sequences in the cell.
  • nucleic acid elements introduced through the methods of this invention may be determined by polymerase chain reaction (PCR). Using this technique discreet fragments of nucleic acid are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a preselected nucleic acid segment is present in a stable transformant, but does not prove integration of the introduced preselected nucleic acid segment into the host cell genome. In addition, it is not possible using PCR techniques to determine whether transformants have exogenous genes introduced into different sites in the genome, i.e., whether transformants are of independent origin. It is contemplated that using PCR techniques it would be possible to clone fragments of the host genomic DNA adjacent to an introduced preselected DNA segment.
  • PCR polymerase chain reaction
  • Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced preselected DNA segments in high molecular weight DNA, i.e., confirm that the introduced preselected DNA segment has been integrated into the host cell genome.
  • the technique of Southern hybridization provides information that is obtained using PCR, e.g., the presence of a preselected DNA segment, but also demonstrates integration into the genome and characterizes each individual transformant.
  • RNA may only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues.
  • PCR techniques may also be used for detection and quantitation of RNA produced from introduced preselected DNA segments. In this application of PCR it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR techniques amplify the DNA. In most instances PCR techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA.
  • RNA species can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northem blotting and will only demonstrate the presence or absence of an RNA species. While Southern blotting and PCR may be used to detect the preselected DNA segment in question, they do not provide infonnation as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced preselected DNA segments or evaluating the phenotypic changes brought about by their expression. Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins.
  • Unique physical- chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography.
  • electrophoretic procedures such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography.
  • the unique stmctures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.
  • Assay procedures may also be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed.
  • bioassays Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, mo ⁇ hology, or physiological properties of the plant. Mo ⁇ hological changes may include greater stature or thicker stalks. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays.
  • the present invention also provides a method to identify a nucleotide sequence that encodes a protein product that is involved in the development and/or timing of flower formation in a respective plant by contacting a probe of plant nucleic acid, e.g., cRNA from rice, isolated from various tissues of a plant, with a plurality of isolated nucleic acid samples on one or more, i.e., a plurality of, solid substrates so as to form a complex between at least a portion of the probe and a nucleic acid sample(s) having sequences that are structurally related to the sequences in the probe.
  • Each sample comprises one or a plurality of oligonucleotides corresponding to at least a portion of a plant gene.
  • RNAs are expressed in the particular tissue of the plant.
  • the probe and or samples may be nucleic acid from a dicot or from a monocot.
  • compositions of the invention include plant nucleic acid molecules, and the amino acid sequences for the polypeptides or partial-length polypeptides encoded by the nucleic acid molecule which comprises an open reading frame. These sequences can be employed to alter expression of a particular gene corresponding to the open reading frame by decreasing or eliminating expression of that plant gene or by overexpressing a particular gene product.
  • Methods of this embodiment of the invention include stably transforming a plant with the nucleic acid molecule which includes an open reading frame operably linked to a promoter capable of driving expression of that open reading frame (sense or antisense) in a plant cell.
  • portion or “fragment”, as it relates to a nucleic acid molecule which comprises an open reading frame or a fragment thereof encoding a partial-length polypeptide having the activity of the full length polypeptide, is meant a sequence having at least 80 nucleotides, more preferably at least 150 nucleotides, and still more preferably at least 400 nucleotides.
  • a "portion" or “fragment” means at least 9, preferably 12, more preferably 15, even more preferably at least 20, consecutive nucleotides, e.g., probes and primers (oligonucleotides), corresponding to the nucleotide sequence of the nucleic acid molecules of the invention such as those identified in the SEQ ID NOs provided in the Sequence Listing.
  • the method comprises introducing to a plant, plant cell, or plant tissue an expression cassette comprising a promoter linked to an open reading frame so as to yield a transformed differentiated plant, transformed cell or transformed tissue. Transformed cells or tissue can be regenerated to provide a transformed differentiated plant.
  • the transformed differentiated plant or cells thereof preferably expresses the open reading frame in an amount that alters the amount of the gene product in the plant or cells thereof, which product is encoded by the open reading frame.
  • the present invention also provides a transformed plant prepared by the method, progeny and seed thereof The invention further includes a nucleotide sequence which is complementary to one
  • test sequence which hybridizes under stringent conditions with a nucleic acid molecule of the invention as well as RNA which is transcribed from the nucleic acid molecule.
  • test sequence which hybridizes under stringent conditions with a nucleic acid molecule of the invention as well as RNA which is transcribed from the nucleic acid molecule.
  • either a denatured test or nucleic acid molecule of the invention is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of, e.g., between 55 and 70°C, in double strength citrate buffered saline (SC) containing 0.1 % SDS followed by rinsing of the support at the same temperature but with a buffer having a reduced SC concentration.
  • SC citrate buffered saline
  • SC citrate buffered saline
  • a buffer having a reduced SC concentration buffers are typically single strength SC containing 0.1 % SDS, half strength SC containing 0.1 % SDS and one-tenth strength SC containing 0.1 % SDS.
  • Polynucleotides derived from nucleotide sequences of the present invention having any of the nucleotide sequences selected from the group consisting of of SEQ ID NOs: 1-66, and 123 - 146, and 67 - 122 provided in the Sequence Listing are useful to detect the presence in a test sample of at least one copy of a nucleotide sequence containing the same or substantially the same sequence, or a fragment, complement, or variant thereof.
  • the sequence of the probes and/or primers of the instant invention need not be identical to those provided in the Sequence Listing or the complements thereof. Some variation in probe or primer sequence and/or length can allow additional family members to be detected, as well as orthologous genes and more taxonomically distant related sequences.
  • probes and/or primers of the invention can include additional nucleotides that serve as a label for detecting duplexes, for isolation of duplexed polynucleotides, or for cloning pu ⁇ oses.
  • Preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides containing a contiguous span of between at least 12 to at least 1000 nucleotides of any of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 -66, and 123 - 146, and 67 - 122 provided in the Sequence Listing or the complements thereof, with each individual number of nucleotides within this range also being part of the invention.
  • Prefe ⁇ ed are isolated, purified, or recombinant polynucleotides containing a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 750, or 1000 nucleotides of any of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-66, and 123 - 146, and 67 - 122 provided in the Sequence Listing or the complements thereof.
  • the appropriate length for primers and probes will vary depending on the application. For use as PCR primers, probes are 12-40 nucleotides, preferably 18-30 nucleotides long.
  • probes are 50 to 500 nucleotides, preferably 100-250 nucleotides long.
  • probes as long as several kilobases can be used.
  • the appropriate length for primers and probes under a particular set of assay conditions may be empirically determined by one of skill in the art.
  • the primers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al. (Meth Enzymol 68: 90 (1979)), the diethylphosphoramidite method, the triester method of Matteucci et al. (J Am Chem Soc 103: 3185 (1981)), or according to Urdea et al. (Proc Natl Acad 80: 7461 (1981)), the solid support method described in EP 0 707 592, or using commercially available automated oligonucleotide synthesizers.
  • a method such as the phosphodiester method of Narang et al. (Meth Enzymol 68: 90 (1979)), the diethylphosphoramidite method, the triester method of Matteucci et al. (J Am Chem Soc 103: 3185 (1981)), or according to Urde
  • Detection probes are generally nucleotide sequences or uncharged nucleotide analogs such as, for example peptide nucleotides which are disclosed in International Patent
  • the probe may have to be rendered "non-extendable" such that additional dNTPs cannot be added to the probe.
  • Analogs are usually non- extendable, and nucleotide probes can be rendered non-extendable by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3' end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group.
  • the 3' hydroxyl group simply can be cleaved, replaced or modified so as to render the probe non-extendable.
  • Any of the polynucleotides of the present invention can be labeled, if desired, by inco ⁇ orating a label detectable by specfroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive substances ( P, S, H, I), fluorescent dyes (5-bromodesoxyuridine, fluorescein, acetylaminofluorene, digoxigenin) or biotin.
  • polynucleotides are labeled at their 3' and 5' ends.
  • probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as described in EP 0 225 807.
  • a label can also be used to capture the primer so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support.
  • a capture label is attached to the primers or probes and can be a specific binding member that forms a binding pair with the solid's phase reagent's specific binding member, for example biotin and streptavidin. Therefore depending upon the type of label carried by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, primers or probes provided herein, may, themselves, serve as the capture label.
  • a solid phase reagent's binding member is a nucleotide sequence
  • it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase.
  • a polynucleotide probe itself serves as the binding member
  • the probe will contain a sequence or "tail" that is not complementary to the target.
  • a polynucleotide primer itself serves as the capture label
  • at least a portion of the primer will be free to hybridize with a nucleotide on a solid phase. DNA labeling techniques are well known in the art.
  • Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others.
  • the solid support is not critical and can be selected by one skilled in the art.
  • latex particles, microparticles, magnetic or nonmagnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples.
  • a solid support refers to any material that is insoluble, or can be made insoluble by a subsequent reaction.
  • the solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
  • the solid phase can retain an additional receptor that has the ability to attract and immobilize the capture reagent.
  • the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
  • the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction.
  • the receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay.
  • the solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes and other configurations known to those of ordinary skill in the art.
  • polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support.
  • polynucleotides other than those of the invention may be attached to the same solid support as one or more polynucleotides of the invention.
  • the polynucleotides of the invention that are expressed or repressed in response to environmental stimuli such as, for example, stress or treatment with chemicals or pathogens or at different developmental stages can be identified by employing an anay of nucleic acid samples, e.g., each sample having a plurality of oligonucleotides, and each plurality co ⁇ esponding to a different plant gene, on a solid substrate, e.g., a DNA chip, and probes co ⁇ esponding to nucleic acid expressed in, for example, one or more plant tissues and/or at one or more developmental stages, e.g., probes corresponding to nucleic acid expressed in seed of a plant relative to control nucleic acid from sources other than seed.
  • an anay of nucleic acid samples e.g., each sample having a plurality of oligonucleotides, and each plurality co ⁇ esponding to a different plant gene
  • a solid substrate e.g., a DNA
  • genes that are upregulated or downregulated in the majority of tissues at a majority of developmental stages, or upregulated or downregulated in one tissue such as in seed can be systematically identified.
  • the probes may also correspond to nucleic acid expressed in respone to a defined treatment such as, for example, a treatment with a variety of plant hormones or the exposure to specific environmental conditions involving, for example, an abiotic stress or exposure to light.
  • labeled rice cRNA probes were hybridized to the rice DNA array, expression levels were determined by laser scanning and then rice genes were identified that had a particular expression pattern.
  • the rice oligonucleotide probe anay consists of probes from over 18,000 unique rice genes, which covers approximately 40-50% of the genome. This genome array permits a broader, more complete and less biased analysis of gene expression.
  • the invention also deals with a method for detecting the presence of a polynucleotide including a nucleotide sequence which is substantially similar to a nucleotide sequence given in SEQ ID NOs: 1 to SEQ ID NO: 66, or a fragment or a variant thereof, or a complementary sequence thereto, in a sample, the method including the following steps of: (a) bringing into contact a nucleotide probe or a plurality of nucleotide probes which can hybridize with a polynucleotide having a nucleotide sequence which is substantially similar to a nucleotide sequence given in SEQ ID NOs: 1 to SEQ ID NO: 66, a fragment or a variant thereof, or a complementary sequence thereto and the sample to be assayed.
  • the invention further concerns a kit for detecting the presence of a polynucleotide including a nucleotide sequence which is substantially similar to a nucleotide sequence given in SEQ ID NOs: 1 to SEQ ID NO: 66, a fragment or a variant thereof, or a complementary sequence thereto, in a sample, the kit including a nucleotide probe or a plurality of nucleotide probes which can hybridize with a nucleotide sequence included in a polynucleotide, which nucleotide sequence is substantially similar to a nucleotide sequence given in of SEQ ID NOs: 1 to SEQ ID NO: 66, a fragment or a variant thereof, or a complementary sequence thereto and, optionally, the reagents necessary for performing the hybridization reaction.
  • the nucleotide probe or the plurality of nucleotide probes are labeled with a detectable molecule.
  • the nucleotide probe or the plurality of nucleotide probes has been immobilized on a substrate.
  • the isolated polynucleotides of the invention can be used to create various types of genetic and physical maps of the genome of rice or other plants. Such maps are used to devise positional cloning strategies for isolating novel genes from the mapped crop species.
  • the sequences of the present invention are also useful for chromosome mapping, chromosome identification, tagging of genes involved in regulating flowering time, and whole plant architecture.
  • the isolated polynucleotides of the invention can further be used as probes for identifying polymo ⁇ hisms associated with phenotypes of interest such as, for example, early or delayed flowering time. Briefly, total DNA is isolated from an individual or isogenic line, cleaved with one or more restriction enzymes, separated according to mass, transfened to a solid support, and hybridized with a probe molecule according to the invention. The pattern of fragments hybridizing to a probe molecule is compared for DNA from different individuals or lines, where differences in fragment size signals a polymo ⁇ hism associated with a particular nucleotide sequence according to the present invention. After identification of polymo ⁇ hic sequences, linkage studies can be conducted.
  • linkage studies can be conducted by using the individuals showing polymo ⁇ hisms as parents in crossing programs. Recombinants, F 2 progeny recombinants or recombinant inbreds, can then be analyzed using the same restriction enzyme/hybridization procedure.
  • the order of DNA polymo ⁇ hisms along the chromosomes can be infened based on the frequency with which they are inherited together versus inherited independently. The closer together two polymo ⁇ hisms occur in a chromosome, the higher the probability that they are inherited together.
  • polymo ⁇ hisms and associated marker sequences are sufficiently numerous to produce a genetic map of sufficiently high resolution to locate one or more loci of interest.
  • the nucleotide sequences of the present invention can also be used for simple sequence repeat identification, also known as single sequence repeat, (SSR) mapping.
  • SSR mapping in rice has been described by Miyao et al. (DNA Res 3:233 (1996)) and Yang et al. (Mol Gen Genet 245: 187 (1994)), and in maize by Ahn et al. (Mol Gen Genet 241 :483 (1993)).
  • SSR mapping can be achieved using various methods. In one instance, polymo ⁇ hisms are identified when sequence specific probes flanking an SSR contained within an sequence of the invention are made and used in polymerase chain reaction (PCR) assays with template DNA from two or more individuals or, in plants, near isogenic lines.
  • PCR polymerase chain reaction
  • a change in the number of tandem repeats between the SSR-flanking sequence produces differently sized fragments (U.S. Patent No. 5,766,847).
  • polymo ⁇ hisms can be identified by using the PCR fragment produced from the SSR-flanking sequence specific primer reaction as a probe against Southern blots representing different individuals (Refseth et al., Electrophoresis 18:1519 (1997)).
  • Rice SSRs were used to map a molecular marker closely linked to a nuclear restorer gene for fertility in rice as described by Akagi et al. (Genome 39:205 (1996)).
  • the nucleotide sequences of the present invention can be used to identify and develop a variety of microsatellite markers, including the SSRs described above, as genetic markers for comparative analysis and mapping of genomes.
  • the nucleotide sequences of the present invention can be used in a variation of the SSR technique known as inter-SSR (ISSR), which uses microsatellite oligonucleotides as primers to amplify genomic segments different from the repeat region itself (Zietkiewicz et al., Genomics 20:176 (1994)).
  • ISSR inter-SSR
  • ISSR employs oligonucleotides based on a simple sequence repeat anchored or not at their 5'- or 3 '-end by two to four arbitrarily chosen nucleotides, which triggers site-specific annealing and initiates PCR amplification of genomic segments which are flanked by inversely orientated and closely spaced repeat sequences.
  • microsatellite markers derived from the nucleotide sequences disclosed in the Sequence Listing, or substantially similar sequences or allelic variants thereof, may be used to detect the appearance or disappearance of markers indicating genomic instability as described by Leroy et al. (Electron.
  • Microsatellite markers derived from nucleotide sequences as provided in the Sequence Listing will be useful for detecting genomic alterations such as the change observed by Leroy et al. (Electron. J Biotechnol, 3(2), supra (2000)) which appeared to be the consequence of microsatellite instability at the primer binding site or modification of the region between the microsatellites, and illustrated somaclonal variation leading to genomic instability. Consequently, the nucleotide sequences of the present invention are useful for detecting genomic alterations involved in somaclonal variation, which is an important source of new phenotypes.
  • SSRs single-sequence repeats
  • microsatellite markers for QTLs involved in heading time and/or whole plant architecture
  • the nucleotide sequences of the invention can be used to identify QTLs involved in in heading time and/or whole plant architecture, and isolate alleles as described by Li et al. in a study of QTLs involved in resistance to a pathogen of rice. (Li et al, Mol Gen Genet 261 :58 (1999)).
  • the nucleotide sequences of the invention can also be used to isolate alleles from the conesponding QTL(s) of wild relatives. Transgenic plants having various combinations of QTL alleles can then be created and the effects of the combinations measured.
  • nucleotide sequences of the invention can be used to help create physical maps of the genome of maize, Arabidopsis and related species.
  • nucleotide sequences of the invention have been ordered on a genetic map, as described above, then the nucleotide sequences of the invention can be used as probes to discover which clones in large libraries of plant DNA fragments in YACs, PACs, etc. contain the same nucleotide sequences of the invention or similar sequences, thereby facilitating the assignment of the large DNA fragments to chromosomal positions. Subsequently, the large BACs, YACs, etc.
  • nucleotide sequences of the invention themselves may provide the means of joining cloned sequences into a contig, and are useful for constmcting physical maps.
  • the nucleotide sequences of the present invention may be useful in mapping and characterizing the genomes of other cereals.
  • Rice has been proposed as a model for cereal genome analysis (Havukkala, Curr Opin Genet Devel 6:711 (1996)), based largely on its smaller genome size and higher gene density, combined with the considerable conserved gene order among cereal genomes (Ahn et al, Mol Gen Genet 241:483 (1993)).
  • the cereals demonstrate both general conservation of gene order (synteny) and considerable sequence homology among various cereal gene families.
  • nucleotide sequences according to the invention can also be used to physically characterize homologous chromosomes in other cereals, as described by Sarma et al. (Genome 43:191 (2000)), and their use can be extended to non-cereal monocots such as sugarcane, grasses, and lilies.
  • the nucleotide sequences of the present invention can be used to obtain molecular markers for mapping and, potentially, for positional cloning.
  • Kilian et al. described the use of probes from the rice genomic region of interest to isolate a saturating number of polymo ⁇ hic markers in barley, which were shown to map to syntenic regions in rice and barley, suggesting that the nucleotide sequences of the invention derived from the rice genome would be useful in positional cloning of syntenic genes of interest from other cereal species that are involved in regulating flowering time, and whole plant architecture.
  • Rice marker technology utilizing the nucleotide sequences of the present invention can also be used to identify QTL alleles for modified heading time from a wild relative of cultivated rice, for example as described by Xiao, et al. (Genetics 150:899 (1998)). Wild relatives of domesticated plants represent untapped pools of genetic resources for abiotic and biotic stress resistance, apomixis and other breeding strategies, plant architecture, determinants of yield, secondary metabolites, and other valuable traits. In rice, Xiao et al. (supra) used molecular markers to introduce an average of approximately 5%> of the genome of a wild relative, and the resulting plants were scored for phenotypes such as plant height, panicle length and 1000-grain weight.
  • nucleotide sequences of the invention such as those provided in the Sequence Listing can be employed as molecular markers to identify QTL alleles regulating heading time in plants from a wild relative, by which these valuable traits can be introgressed from wild relatives using methods including, but not limited to, that described by Xiao et al. ((1998) supra). Accordingly, the nucleotide sequences of the invention can be employed in a variety of molecular marker technologies for yield improvement.
  • any individual (or line) can be genotyped. Genotyping a large number of DNA polymo ⁇ hisms such as single nucleotide polymo ⁇ hisms (SNPs), in breeding lines makes it possible to find associations between certain polymo ⁇ hisms or groups of polymo ⁇ hisms, and certain phenotypes. In addition to sequence polymo ⁇ hisms, length polymo ⁇ hisms such as triplet repeats are studied to find associations between polymo ⁇ hism and phenotype. Genotypes can be used for the identification of particular cultivars, varieties, lines, ecotypes, and genetically modified plants or can serve as tools for subsequent genetic studies of complex traits involving multiple phenotypes.
  • SNPs single nucleotide polymo ⁇ hisms
  • the patent publication WO95/35505 and U.S. Patent Nos. 5,445,943 and 5,410,270 describe scanning multiple alleles of a plurality of loci using hybridization to anays of oligonucleotides.
  • the nucleotide sequences of the invention are suitable for use in genotyping techniques useful for each of the types of mapping discussed above.
  • the nucleotide sequences of the invention are useful for identifying and isolating a least one unique stretch of protein-encoding nucleotide sequence.
  • the nucleotide sequences of the invention are compared with other coding sequences having sequence similarity with the sequences provided in the Sequence Listing, using a program such as BLAST.
  • nucleotide sequences of the invention permits the identification of one or more unique stretches of coding sequences encoding proteins that are involved in regulating flowering time, and whole plant architecture and that are not identical to the conesponding coding sequence being screened.
  • a unique stretch of coding sequence of about 25 base pairs (bp) long is identified, more preferably 25 bp, or even more preferably 22 bp, or 20 bp, or yet even more preferably 18 bp or 16 bp or 14 bp.
  • a plurality of nucleotide sequences is screened to identify unique coding sequences accroding to the invention.
  • one or more unique coding sequences accroding to the invention can be applied to a chip as part of an anay, or used in a non-chip anay system.
  • a plurality of unique coding sequences accroding to the invention is used in a screening anay.
  • one or more unique coding sequences accroding to the invention can be used as immobilized or as probes in solution.
  • one or more unique coding sequences accroding to the invention can be used as primers for PCR.
  • one or more unique coding sequences accroding to the invention can be used as organism-specific primers for PCR in a solution containing DNA from a plurality of sources.
  • unique stretches of nucleotide sequences according to the invention are identified that are preferably about 30 bp, more preferably 50 bp or 75 bp, yet more preferably 100 bp, 150 bp, 200 bp, 250, 500 bp, 750 bp, or 1000 bp.
  • the length of an unique coding sequence may be chosen by one of skill in the art depending on its intended use and on the characteristics of the nucleotide sequence being used.
  • unique coding sequences accroding to the invention may be used as probes to screen libraries to find homologs, orthologs, or paralogs.
  • unique coding sequences accroding to the invention may be used as probes to screen genomic DNA or cDNA to find homologs, orthologs, or paralogs.
  • unique coding sequences accroding to the invention may be used to study gene evolution and genome evolution.
  • Probes and primers of the invention can be used to identify and/or isolate polynucleotides related to the nucleotide sequences according to the invention provided in the Sequence Listing, or allelic variants of the nucleotide sequences according to the invention.
  • related polynucleotides have similar sequences or encode polypeptides with similar biological activity, but are found at other loci within an organism, or are found in other organisms.
  • Identification and isolation of related sequences can provide important tools for functional genomics, to study the evolution of genomes, and to predict gene and protein function, interaction, and regulation.
  • Related sequences including paralogs and orthologs are particularly important in identifying Clusters of Orthologous Groups (COGs) of proteins, which aids protein function prediction and the functional and phylogenetic annotation of newly sequenced genomes.
  • COGs Clusters of Orthologous Groups
  • Hybridization of the nucleotide sequences according to the invention to nucleotides obtained from other organisms can be used to identify and isolate paralogous sequences, or paralogs, which are additional members of gene families.
  • paralogous sequence and “paralog” as used herein encompass both full-length genes and regions and fragments thereof. Paralogs may be located in the same or a different region of the genome in which the sequence used as a probe is located. Paralogs generally have a high sequence identity with the probe sequence or the gene from which the probe was prepared; however, paralogs may have overall sequence identity with a probe sequence as low as 20 to 30% and still be recognizable as members of the same gene family with similar functions, as reported by Takata et al.
  • RAD51B paralogs (Mol Cell Biol 20:6476 (2000)).
  • sequence similarity among paralogs of a gene family is often concentrated into one or a few portions of the sequence, notably in a portion encoding a protein or RNA that has an enzymatic or stmctural function.
  • the degree of identity in the amino acid sequence of the domain that defines the gene family can be as low as 20%>, and is preferably at least 50%, more preferably 75%, even more preferably 80 to 95%, most preferably 85 to 99%.
  • Paralogs may differ in their expression profiles, indicating that they may act at different time, a different place, or at a different developmental stage, even when their function appears to be similar.
  • paralogs encode polypeptides that are "remodeled" during plant evolution, for example to create new forms of oxidized carotenoids in tomato as described by Bouvier et al. (Eur J Biochem 267:6346 (2000)).
  • paralogs may be isolated by hybridizing an nucleotide sequence according to the invention probe to a Southern blot containing the appropriate genomic DNA or cDNA of the organism. To search for paralogs within a species, low stringency hybridization is usually performed, that this will depend on size, distribution and degree of sequence divergence of domains that define the gene family. Given the resulting hybridization data, one or ordinary skill in the art could distinguish and isolate the correct DNA fragments by size, restriction sites and stated hybridization conditions from a gel or from a library. Alternately, paralogs may be isolated by large-scale sequencing followed by BLAST analysis of sequences to identify putative paralogs, as described by Ospina-Giraldo et al.
  • paralogs may be isolated using reverse-transcriptase polymerase chain reaction (RT-PCR) using primers to conserved regions of sequence. Paralogs may be cloned using standard techniques described below to screen libraries using at least one Nucleotide sequence according to the invention as a probe.
  • An orthologous sequence, or orthologous gene, or ortholog has a high degree of sequence similarity to a known sequence or gene of interest, with the similarity often occuning along the entire length of the coding portion of the gene.
  • the terms "orthologous sequence” and “ortholog” as used herein encompass both full-length genes and regions and fragments thereof.
  • ortholog often encodes a gene product that performs a similar function in the organism. Functions for orthologous genes are expected to be the same as or very similar to that of the gene from which the probe was prepared.
  • the degree of identity is a function of evolutionary separation and, in closely related species, the degree of sequence identity can be 98 to 100%.
  • Orthologous sequences sometimes have significantly lower levels of sequence identity, for example as described by Shoes et al. (Plant Cell 12:1345 (2000)) where orthologs of sucrose transporters from Arabidopsis, tomato, and potato had 47%> similarity to the previously characterized sucrose transporter.
  • the amino acid sequence of a protein encoded by an orthologous gene can be less than 50% identical, but tends to be at least 50%, or at least 70% or at least 80% identical, more preferably at least 90%, most preferably at least 95%> identical to the amino acid sequence of the reference protein.
  • probes are hybridized to nucleotides from a species of interest under low stringency conditions and blots are then washed under conditions of increasing stringency. It is preferable that the wash stringency be such that sequences that are 85 to 100%) identical will hybridize. More preferably, sequences 90 to 100%> identical will hybridize and most preferably only sequences greater than 95%> identical will hybridize.
  • the low stringency condition is preferably one where sequences containing as much as 40-45%) mismatches will be able to hybridize. This condition is established by T m - 40°C to T m - 48°C (see below).
  • T m - 40°C to T m - 48°C (see below).
  • amino acid sequences that are identical can be encoded by DNA sequences as little as 67% identical.
  • orthologous sequences may be isolated by hybridizing a nucleotide sequence according to the invention probe to a Southern blot containing the appropriate genomic DNA or cDNA of a different organism.
  • orthologs may be isolated by large-scale sequencing followed by BLAST analysis of sequences to identify putative orthologous sequences and full-length orthologs.
  • orthologs may be isolated using reverse-transcriptase polymerase chain reaction (RT-PCR) using primers to conserved regions of sequence in an Nucleotide sequence according to the invention.
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • Orthologs and/or orthologous sequences may be cloned using standard techniques described below to screen libraries using at least one nucleotide sequence according to the invention as a probe.
  • Probes for Southern blotting to distinguish individual restriction fragments can range in size from 15 to 20 nucleotides to several thousand nucleotides. More preferably, the probe is 100 to 1000 nucleotides long for identifying members of a gene family when it is found that repetitive sequences would complicate the hybridization. For identifying an entire conesponding gene in another species, the probe is more preferably the length of the gene, typically 2000 to 10,000 nucleotides, but probes 50-1,000 nucleotides long might be used. Some genes, however, might require probes up to 15,000 nucleotides long or overlapping probes constituting the full-length sequence to span their lengths.
  • the probe derived from nucleotide sequences according to the invention of the present invention is homogeneous, having a single sequence.
  • a probe designed to represent or identify members of a gene family having diverse sequences can be generated using PCR to amplify genomic DNA or RNA templates using primers derived from nucleotide sequences according to the invention that include sequences that define the gene family.
  • the probe for Southern blotting most preferably would be the genomic copy of the probe gene. This allows all elements of the gene to be identified in the other species.
  • the next most preferable probe is a cDNA spanning the entire coding sequence (for example, as indicated by CDS coordinates in the Sequence Listing) which allows the entire mRNA-coding portion of the gene to be identified; in this case it is possible that some introns in the gene might be missed.
  • Probes for Southern blotting can easily be generated from Nucleotide sequences according to the invention by making primers having the sequence at the ends of the Nucleotide sequence according to the invention and using rice (Oryza sativa) genomic DNA as a template.
  • primers including the conserved sequence can be used for PCR with genomic DNA from a species of interest to obtain a probe.
  • that portion of the Nucleotide sequence according to the invention can be used to make primers and, with appropriate template DNA, used to make a probe to identify genes containing the domain.
  • the PCR products can be resolved, for example by gel electrophoresis, and cloned and/or sequenced. In this manner, the variants of the domain among members of a gene family, both within and across species, can be examined.
  • the nucleotide sequences according to the invention can be used to isolate the conesponding DNA from the same organism or other organisms. Either cDNA or genomic DNA can be isolated. Libraries of genomic DNA, or lambda, cosmid, BAC or YAC, or other large insert genomic library from the plant of interest can be constmcted using standard molecular biology techniques as described in detail by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) and by Ausubel et al. (Current Protocols in Molecular Biology, Greene Publishing (1992)) with updates).
  • recombinant lambda clones are plated out on appropriate bacterial medium using an appropriate E. coli host strain.
  • the resulting plaques are lifted from the plates using nylon or nitrocellulose filters.
  • the plaque lifts are processed through denaturation, neutralization, and washing treatments following the standard protocols outlined by Ausubel et al. ((1992) supra).
  • the plaque lifts are hybridized to either radioactively labeled or non-radioactively labeled AC DNA at room temperature for about 16 hours, usually in the presence of 50% formamide and 5X SSC (sodium chloride and sodium citrate) buffer and blocking reagents.
  • formamide and 5X SSC sodium chloride and sodium citrate
  • the plaque lifts are then washed at 42°C with 1% sodium dodecyl sulfate (SDS) and at a particular concentration of SSC.
  • SSC concentration used is dependent upon the stringency at which hybridization occuned in the initial Southern blot analysis performed. For example, if a fragment hybridized under medium stringency such as T m - 20°C, then this condition is maintained or preferably adjusted to a less stringent condition such as T m - 30°C, to wash the plaque lifts.
  • Positive clones showing hybridization to the probe are detected by exposure to X-ray films or chromogen formation or any other suitable detection method, and subsequently isolated for purification using the same general protocol outlined above.
  • restriction analysis can be conducted to nanow the region conesponding to the gene of interest. Restriction analysis and succeeding subcloning steps can be done using procedures described by, for example, Sambrook et al. ((1989) supra).
  • the procedures outlined for the lambda library are essentially similar except the YAC clones are harbored in bacterial colonies. The YAC clones are plated out at reasonable density on nitrocellulose or nylon filters supported by appropriate bacterial medium in petri plates. Following the growth of the bacterial clones, the filters are processed through the denaturation, neutralization, and washing steps following the procedures of Ausubel et al. ((1992) supra). The same hybridization procedures for lambda library screening are followed.
  • the library can be constmcted in a lambda vector appropriate for cloning cDNA such as ⁇ gtl 1.
  • the cDNA library can be made in a plasmid vector.
  • cDNA for cloning can be prepared by any of the methods known in the art, but is preferably prepared as described above.
  • a cDNA library will include a high proportion of full-length clones.
  • Sequence analysis and mapping of related sequences can be used in phylogenetic analyses of the evolution of the sequences in question, including the determination of gene duplication and reanangements.
  • expression studies of related sequences can be used to further understand the evolutionary history and function of the paralogs and orthologs, and to suggest future uses for the sequences.
  • Nucleotide sequences according to the invention encompassing regulatory regions can be used to identify coordinately expressed genes by using the regulatory region of the portion of the Nucleotide sequence according to the invention as a probe.
  • the Nucleotide sequences according to the invention can also be used as probes to assay plants of different species for those phenotypes.
  • the practitioner will preferably adjust the amount of target DNA of each species so that, as nearly as is practical, the same number of genome equivalents is present for each species examined. This prevents faint signals from species having large genomes, and thus small numbers of genome equivalents per mass of DNA, from enoneously being inte ⁇ reted as absence of the conesponding gene in the genome.
  • Nucleotide sequences according to the invention can be applied to substrates for use in anay applications such as, but not limited to, assays of global gene expression, including to measure expression profiles of organisms under specific conditions of development, growth conditions, or for diagnostic or forensic methods.
  • anay applications such as, but not limited to, assays of global gene expression, including to measure expression profiles of organisms under specific conditions of development, growth conditions, or for diagnostic or forensic methods.
  • Anay applications are cmcial to all aspects of functional genomics, as anays permit the easy, rapid, and reproducible detection of the presence and amount of large numbers of gene transcripts to generate large data sets for complex analyses of the function of genes.
  • a substrate including a plurality of Nucleotide sequences according to the invention, or oligonucleotide primers or probes derived from Nucleotide sequences according to the invention of the invention may be used for detecting targeted sequences in a polynucleotide including any of SEQ ID NOs: 1 to SEQ ID NO: 66 for amplifying targeted sequences in a polynucleotide including any of SEQ ID NOs: 1 to SEQ ID NO: 66 or for detecting mutations in the coding or in the non-coding sequences of polynucleotide including any of SEQ ID NO: 1 to SEQ ID NO:66.
  • any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support.
  • the polynucleotides of the invention may be attached in an ordered anay wherein each polynucleotide is attached to a distinct region of the solid support that does not overlap with the attachment site of any other polynucleotide.
  • such an ordered anay of polynucleotides is designed to be "addressable" where the distinct locations are recorded and can be accessed as part of an assay procedure.
  • Addressable polynucleotide anays typically include a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these "addressable" anays particularly useful in hybridization assays. Any addressable anay technology known in the art can be employed with the polynucleotides of the invention.
  • VLSIPSTM Very Large Scale Immobilized Polymer Synthesis
  • VLSIPSTM technologies are provided in US Patents 5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, which describe methods for forming oligonucleotide anays through techniques such as light-directed synthesis techniques.
  • Further presentation strategies aimed at providing anays of nucleotides immobilized on solid supports were developed to order and display the oligonucleotide anays on the chips in an attempt to maximize hybridization patterns and sequence information as disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.
  • an oligonucleotide probe matrix may advantageously be used to detect mutations occuning in a polynucleotide including any of SEQ ID NOs: 1 to SEQ ID NO: 66 and preferably in the regulatory region of these sequences.
  • probes are specifically designed to have a nucleotide sequence allowing their hybridization to the genes that carry known mutations (either by deletion, insertion or substitution of one or several nucleotides).
  • known mutations it is meant, mutations of a polynucleotide including any of SEQ ID NOs: 1 to SEQ ID NO: 66 that have been identified using techniques known in the art.
  • Another technique that is used to detect mutations in a polynucleotide including any of SEQ ID NOs: 1 to SEQ ID NO: 66 is the use of a high-density DNA anay, where single base mutations are encompassed by this technique.
  • Each oligonucleotide probe constituting a unit element of the high density DNA anay is designed to match a specific subsequence of the genomic DNA or cDNA of interest.
  • an anay containing oligonucleotides complementary to subsequences of the target gene sequence is used to determine the identity of the target sequence with the "wild-type" nucleotide sequence, measure its amount, and detect differences between the target sequence and the reference wild-type nucleotide sequence.
  • a 4L tiled anay is implemented using a set of four probes (A, C, G, T), preferably 15-nucleotide oligomers.
  • A, C, G, T preferably 15-nucleotide oligomers.
  • the perfect complement will hybridize more strongly than mismatched probes. Consequently, a nucleotide target of length L is scanned for mutations with a tiled anay containing 4L probes; the whole probe set containing all the possible mutations in the known wild reference sequence.
  • the hybridization signals of the 15-mer probe set tiled anay are perturbed by a single base change in the target sequence. As a consequence, there is a characteristic loss of signal or a "footprint" for the probes flanking a mutation position. This technique was described by Chee et al (Science 274:610 (1996)).
  • Another embodiment provides an anay containing any of a variety of types of oligonucleotides, either alone or in a mixture of oligonucleotides types on an anay, including for example, non-unique oligonucleotides, oligonucleotides that are identical to or complementary to a portion of a nucleotide sequence in a public database, oligonucleotides that do not conespond to a BIOPATH protein and/or an FPD, degenerate oligonucleotides, or random oligonucleotides alone or in a mixture.
  • the invention concerns an anay of nucleotide molecules including one or more nucleotide sequences according to the invention useful as probes and primers.
  • the invention concerns an anay of nucleotides including at least two polynucleotides described above, or fragments or variants thereof, as probes and primers.
  • a computer readable medium containing one or more of the nucleotide sequences of the invention as well as methods of use for the computer readable medium are provided.
  • This medium allows a nucleotide sequence conesponding to at least one of SEQ ID NOs provided in the Sequence Listing (open reading frames or fragments thereof), to be used as a reference sequence to search against a database.
  • This medium also allows for computer-based manipulation of a nucleotide sequence conesponding to at least one of SEQ ID NOs provided in the Sequence Listing.
  • Genomic DNA is isolated from nuclei of Oryza sativa L. ssp japonica cv Nipponbare and then sheared to produce fragments of approximately 500 bp.
  • seeds are germinated on cheese cloth immersed in water and grown for 4-6 weeks under greenhouse conditions. After plants reached a height of approximately 5-8 inches, the upper parts of the green leaves are harvested and wrapped in aluminum foil at 4°C overnight. Leaf material is then stored at -80°C or directly used for extraction of nuclei.
  • Intact nuclei are isolated by homogenization (in a blender for fresh material or by grinding with mortar and pestle for frozen material) in a buffer containing 10 mM Trizma base, 80 mM KCI, 10 mM EDTA, 1 mM spermidine, 1 mM spermine, 0.5 M sucrose, 0.5% Triton-X-100, 0.15% ⁇ -mercaptoethanol pH 9.5.
  • the homogenate is filtered and nuclei recovered by gentle centrifugation using a fixed-angle rotor at 1 ,800 g at 4 C for 20 minutes.
  • the pellet recovered after centrifugation is gently resuspended with the assistance of a small paint b sh soaked in ice cold wash buffer and wash buffer added.
  • Particulate matter remaining in the suspension is removed by filtering the resuspended nuclei into a 50 ml centrifuge tube through two layers of miracloth by gravity and centrifuging the filtrate at 57 g (500 ⁇ m), 4 C for 2 minutes to remove intact cells and tissue residues.
  • the supernatant fluid is transfened into a fresh centrifuge tube and nuclei are pelleted by centrifugation at 1,800 g, 4 C for 15 minutes in a swinging bucket centrifuge.
  • DNA is isolated from the nuclear preparation by phenol/chloroform extraction, as in Sambrook et al (supra).
  • Isolated total genomic DNA is physically sheared (Hydroshear) to generate for generating random DNA fragments, and fragments of approximately 500 bp are recovered.
  • DNA is eluted and the ends filled in using T DNA polymerase, Klenow fragments, and dNTPs.
  • Double-stranded DNA is linkered and cloned into a Novartis proprietary medium-copy vector derived from pSCl 01.
  • Vector inserts are amplified by PCR and sequenced using the MegaBACE sequencing system (Molecular Dynamics, Amersham). The amplification reaction is diluted before use and is not purified using an exonuclease/alkaline phosphatase procedure. Sequencing reactions are performed using DYEnamic ET Terminator Kit.
  • the reactions contained approximately 50 ng of amplicon, DYEnamic ET Terminator premix, and 5 pmol of -40 M13 forward primer.
  • the sequencing reaction is amplified for 30 cycles, and reaction products are concentrated and purified using ethanol precipitation.
  • the sample is electrokinetically injected into the capillary at 3 kV for 45 sec and separated via electrophoresis at 9 kV for 120 min. 1.2 Isolation and sequencing ofcDNA fragments
  • RNA is purified from rice plant tissue using standard total RNA purification methods.
  • PolyA+ RNA is isolated from the total RNA using the Qiagen Oligotex mRNA purification system (Qiagen, Valencia, CA), and cDNA is generated using cDNA synthesis reagents from Life Technologies (RockviUe, MD).
  • First strand cDNA synthesis is catalyzed by reverse transcriptase using oligo dT primers with a Notl restriction site.
  • Second strand synthesis is catalyzed by DNA polymerase.
  • An oligonucleotide linker with a Sail restriction endonuclease site is attached to the 5' end of the cDNAs using DNA ligase.
  • the cDNAs are digested with Notl and Sail restriction endonucleases and inserted into an E. eo/z " -replicating plasmid harboring a selectable marker.
  • E. coli is transfected with the recombinant plasmids and grown on selectable media. E. coli colonies are individually picked off the selectable media and placed into storage plates.
  • Sequencing the rice cDNA library The DNA sequence of the cDNA cloned into the plasmid purified from an E. coli colony is determined using standard dideoxy sequencing methods. Oligonucleotide primers respectively conesponding to plasmid DNA regions upstream of the 5' end of the cDNA insert (Forward reaction) and downstream of the 3' end of the cDNA insert (Reverse reaction) are used in the dideoxy sequencing reactions. If the DNA sequence determined as a result of the Forward and Reverse reactions from the cDNA overlapped, the two sequences could be merged into a contig using computerized analysis software (Consed, University of Washington,Seattle), to assemble a full-length sequence of the cDNA.
  • Computerized analysis software Consed, University of Washington,Seattle
  • the DNA sequence of the separating region is determined using one of two dideoxy sequencing methods.
  • a primer specifically conesponding to the 3' end of the DNA sequence determined from the Forward reaction is used in a second dedeoxy sequencing reaction. The primer walking procedure is repeated until the DNA sequence that separated the original Forward and Reverse is resolved and a contig could be assembled.
  • the clone harboring the cDNA is subjected to transposon in vitro insertion dideoxysequencing (Epicentre, Madison, WI).
  • the insertion process is random and the result is multiple DNA sequence coverage over the targeted cDNA, where the sequences thus obtained are assembled into a contig.
  • Candidate transcript translations are examined for known Prosite motifs and full-length Pfam domains as evidence of accurate predictions. Additional candidate transcripts are identified from plant and fungal mRNA (BLASTN and TBLASTX) and protein (TBLASTN) searches against the rice genome. Frameshifts caused by InDels and gaps between assembled sequence contigs are considered during the analysis, and software is adjusted to compensate for these potential problems. The percent coverage of each candidate transcript is summed across all overlapping similarities as the percent "evidence" for each candidate transcript. The results of gene prediction and homology searches are combined into an integrated dataset. For overlapping candidate transcripts, the transcript with the highest evidence is selected.
  • Table 1 provides provides a subset of rice genes that show homology to known Arabidopsis flowering time genes and further identifies those Arabidopsis genes. Further provided are SEQ ID NOs of banana, wheat and maize representing nucleotide sequences that are homologous to the rice sequences show in column 1.
  • the ability to align regions of heterologous cereal chromosomes to the rice genome provides the opportunity to connect mapped cereal trait genes (quantitative trait loci or QTL) to the rice genome, and assign candidate genes to the traits that these genes influence.
  • mapped cereal trait genes quantitative trait loci or QTL
  • Mapped cereal QTL can be placed on the rice genome map. Positioning of molecular markers within the genome will further facilitate mapping and identification of trait-controlling genes.
  • Heading date is a critical trait for adaptation to different cultivation areas and cropping seasons and therefore a major obejective in rice breeding programs. Heading in rice is basically determined by two factors, duration of the basic vegetative growth (BVG) and photoperiod-sensitivity (PS). Several genes are known to be involved in controlling these two factors.
  • BVG basic vegetative growth
  • PS photoperiod-sensitivity
  • Hdl-Hd7 Seven QTL, designated Hdl-Hd7, are known to control rice heading date (Yano et al (Theoretical and Applied Genetics (1997) 95: 1025-1032); Yamamoto et al (Genetics (February 2000) 154: 885-891)).
  • Tables 2 and 3 provides a first set of rice genes that can be found in QTL Hd-3 (Table 2) and Hd-6 (Table 3) and also show homology to known Arabidopsis flowering time genes.
  • Table 2 provides a subset of rice genes from the Hd-3 QTL that show homology to known Arabidopsis flowering time genes and further identifies those Arabidopsis genes. Further provided are SEQ ID NOs of banana, wheat and maize representing nucleotide sequences that are homologous to the rice sequences show in column 1.
  • Table 3 provides a subset of rice genes from the Hd-6 QTL that show homology to known Arabidopsis flowering time genes and further identifies those Arabidopsis genes. Further provided are SEQ ID NOs of banana, wheat and maize representing nucleotide sequences that are homologous to the rice sequences show in column 1.
  • Sequence homology can be helpful in identifying possible functions of many genes.
  • reverse genetics the process of identifying the function of a gene by obtaining and studying the phenotype of an individual containing a mutation in that gene, is another approach to identify the function of a gene.
  • T-DNA left border sequences from individual plants are amplified using a modified thermal asymmetric interlaced-polymerase chain reaction (TAIL-PCR) protocol (Liu et al., (1995) . Plant J. 8, 457-463).
  • TAIL-PCR modified thermal asymmetric interlaced-polymerase chain reaction
  • Left border TAIL-PCR products are sequenced and assembled into a database that associates sequence tags with each of the approximately 100,000 plants in the mutant collection. Screening the collection for insertions in genes of interest involves a simple gene name or sequence BLAST query of the insertion site flanking sequence database, and search results point to individual lines. Insertions are confirmed using PCR.
  • the Arabidopsis T-DNA GARLIC insertion collection is used to investigate the roles of certain genes in the timing of flowering and whole plant architecture.
  • Target genes are chosen using a variety of criteria, including public reports of mutant phenotypes, RNA profiling experiments, and sequence similarity to genes implicated in the timing of flowering and whole plant architecture.
  • Plant lines with insertions in genes of interest are then identified. Each T-DNA insertion line is represented by a seed lot collected from a plant that is hemizygous for a particular T-DNA insertion. Plants homozygous for insertions of interest are identified using a PCR assay. The seed produced by these plants is homozygous for the T- DNA insertion mutation of interest.
  • Homozygous mutant plants are tested for altered flowering time or whole plant architecture.
  • the genes interrapted in these mutants contribute to the observed phenotype.
  • the genes interrupted in these mutants interfere with the normal timing and development of the plant flower.
  • Rice orthologs of the Arabidopsis genes affecting flowering time and flower architecture in plants are identified by similarity searching of a rice database using the Double- Affine Smith-Waterman algorithm (BLASP with e values better than ⁇ ] °).
  • Genomic DNA Plant genomic DNA samples can be isolated from frozen tissues, according to one of the three procedures, e.g., standard procedures described by Ausubel et al. (1995), a quick leaf prep described by Klimyuk et al. (1993), or using FTA paper (Life
  • a piece of leaf is excised from the plant, placed on top of the FTA paper and covered with a small piece of parafilm that serves as a banier material to prevent contamination of the crashing device.
  • a crashing device is used to mash the tissue into the FTA paper.
  • the FTA paper is air dried for an hour.
  • Two mm punches are removed from the specimen area on the FTA paper using a 2 mm Hanis Micro PunchTM and placed into PCR tubes.
  • Candidate cDNA A candidate cDNA is amplified from total RNA isolated from rice tissue after reverse transcription using primers designed against the computationally predicted cDNA. Primers designed based on the genomic sequence can be used to PCR amplify the full-length cDNA (start to stop codon) from first strand cDNA prepared from rice cultivar Nipponbare tissue.
  • the Qiagen RNeasy kit (Qiagen, Hilden, Germany) is used for extraction of total RNA.
  • the Superscript II kit (Invitrogen, Carlsbad, USA) is used for the reverse transcription reaction. PCR amplification of the candidate cDNA is carried out using the reverse primer sequence located at the translation start of the candidate gene in 5' - 3' direction. This is performed with high-fidelity Taq polymerase (Invitrogen, Carlsbad, USA).
  • PCR fragment is then cloned into pCR2.1-TOPO (Invitrogen) or the pGEM-T easy vector (Promega Co ⁇ oration, Madison, Wis., USA) per the manufacturer's instructions, and several individual clones are subjected to sequencing analysis.
  • pCR2.1-TOPO Invitrogen
  • pGEM-T easy vector Promega Co ⁇ oration, Madison, Wis., USA
  • DNA sequencing DNA preps for 2-4 independent clones are miniprepped following the manufacturer's instructions (Qiagen). DNA is subjected to sequencing analysis using the BigDyeTM Terminator Kit according to manufacturer's instructions (ABI) .
  • Sequencing makes use of primers designed to both strands of the predicted gene of interest.
  • DNA sequencing is performed using standard dye-terminator sequencing procedures and automated sequencers (models 373 and 377; Applied Biosystems, Foster City, CA). All sequencing data are analyzed and assembled using the Phred Phrap/Consed software package
  • the consensus sequence from the sequencing analysis is then to be validated as being intact and the correct gene in several ways.
  • the coding region is checked for being full length
  • a plant complementation assay can be used for the functional characterization of the flowering time genes according to the invention.
  • Rice and Arabidopsis putative orthologue pairs are identified using BLAST comparisons, TFASTXY comparisons, and Double-Affine Smith- Waterman similarity searches.
  • Constmcts containing a rice cDNA or genomic clone inserted between the promoter and terminator of the Arabidopsis orthologue are generated using overlap PCR (Gene 77, 61- 68 (1989)) and GATEWAY cloning (Life Technologies Invitrogen).
  • overlap PCR Gene 77, 61- 68 (1989)
  • GATEWAY cloning Life Technologies Invitrogen
  • rice cDNA clones are prefened to rice genomic clones.
  • a three stage PCR strategy is used to make these constmcts.
  • primers are used to PCR amplify: (i) 2Kb upstream of the translation start site of the Arabidopsis orthologue, (ii) the coding region or cDNA of the rice orthologue, and (iii) the 500 bp immediately downstream of the Arabidopsis orthogue's translation stop site.
  • Primers are designed to inco ⁇ orate onto their 5' ends at least 16 bases of the 3' end of the adjacent fragment, except in the case of the most distal primers which flank the gene constmct (the forward primer of the promoter and the reverse primer of the terminator).
  • the forward primer of the promoters contains on their 5' ends partial AttBl sites, and the reverse primer of the terminators contains on their 5' ends partial AttB2 sites, for Gateway cloning.
  • overlap PCR is used to join either the promoter and the coding region, or the coding region and the terminator.
  • the promoter-coding region product can be joined to the terminator or the coding region-terminator product can be joined to the promoter, using overlap PCR and amplification with fulll Att site-containing primers, to link all three fragments, and put full Att sites at the constmct termini.
  • the fused three-fragment piece flanked by Gateway cloning sites are introduced into the LTI donor vector pDONR201 using the BP clonase reaction, for confirmation by sequencing. Confirmed sequenced constmcts are introduced into a binary vector containing Gateway cloning sites, using the LR clonase reaction such as, for example, pAS200.
  • the pAS200 vector was created by inserting the Gateway cloning cassette RfA into the
  • pNOV3510 was created by ligation of inverted pNOV2114 VSl binary into pNOV3507, a vector containing a PTX5' Arab Protox promoter driving the PPO gene with the Nos terminator.
  • pNOV2114 was created by insertion of virGN54D (Pazour et al. 1992, J . Bacteriol.
  • pHiNK085 was created by deleting the 35S:PMI cassette and M13 ori in pVictorHiNK.
  • pPVictorHiNK was created by modifying the T-DNA of pVictor (described in WO 97/04112) to delete M13 derived sequences and to improve its cloning versatility by introducing the BIGLINK polylinker.
  • the sequence of the pVictor HiiMK vector is disclosed in SEQ ID NO: 5 in WO 00/6837, which is incorporated herein by reference.
  • the pVictorHiNK vector contains the following constituents that are of functional importance:
  • the origin of replication (ORI) functional in Agrobacterium is derived from the Pseudomonas aeruginosa plasmid pVSl (Itoh et al. 1984. Plasmid 11 : 206-220; Itoh and Haas, 1985. Gene 36: 27-36).
  • the pVSl ORI is only functional in Agrobacterium and can be mobilised by the helper plasmid pRK2013 from E.coli into A. tumefaciens by means of a triparental mating procedure (Ditta et al, 1980. Proc. Natl. Acad. Sci USA 77: 7347-7351).
  • the ColEl origin of replication functional in E. coli is derived from pUC19 (Yannisch-Penon et al, 1985. Gene 33: 103-119).
  • the bacterial resistance to spectinomycin and streptomycin encoded by a 0.93 kb fragment from transposon Tn7 functions as selectable marker for maintenance of the vector in E. coli and Agrobacterium .
  • the gene is fused to the tac promoter for efficient bacterial expression (Amman et al, 1983. Gene 25: 167-178).
  • T-DNA border fragments of 1.9 kb and 0.9 kb that comprise the 24 bp border repeats have been derived from the Ti-plasmid of the nopaline type Agrobacterium tumefaciens strains pTiT37 (Yadav et al, 1982. Proc. Natl. Acad. Sci. USA. 79: 6322-6326).
  • the plasmid is introduced into Agrobacterium tumefaciens GV3101pMP90 by electroporation.
  • the positive bacterial transformants are selected on LB medium containing 50 ⁇ g/ ⁇ l kanamycin and 25 ⁇ g/ ⁇ l gentamycin. Plants are transformed by standard methodology (e.g., by dipping flowers into a solution containing the Agrobacterium) except that 0.02% Silwet -77 (Lehle Seeds, Round Rock, TX) is added to the bacterial suspension and the vacuum step omitted. Five hundred (500) mg of seeds are planted per 2 ft 2 flat of soil and , and progeny seeds are selected for transformants using PPO selection.
  • Primary transformants are analyzed for complementation. Primary transformants are genotyped for the Arabidopsis mutation and presence of the transgene. When possible, >50 mutants harboring the transgene should be phenotyped to observe variation due to transgene copy number and expression.
  • overexpression are designed to overexpress the protein of interest and are of two general types, biolistic and binary, depending on the plant transformation method to be used.
  • biolistic transformation For biolistic transformation (biolistic vectors), the requirements are as follows: 1. a backbone with a bacterial selectable marker (typically, an antibiotic resistance gene) and origin of replication functional in Escherichia coli (E. coli; eg. ColEl), and 2. a plant-specific portion consisting of: a. a gene expression cassette consisting of a promoter (eg. ZmUBIint MOD), the gene of interest (typically, a full-length cDNA) and a transcriptional terminator (eg. Agrobacterium tumefaciens nos terminator); b. a plant selectable marker cassette, consisting of a promoter (eg. rice Actl D-BV MOD), selectable marker gene (eg. phosphomannose isomerase, PMI) and transcriptional terminator (eg. CaMV terminator).
  • a promoter eg. ZmUBIint MOD
  • the gene of interest typically, a full-length cDNA
  • Vectors designed for transformation by Agrobacterium tumefaciens consist of:
  • A. tumefaciens right and left border sequences which mediate transfer of the DNA flanked by these two sequences to the plant.
  • Knock out vectors designed for reducing or abolishing expression of a single gene or of a family or related genes are also of two general types conesponding to the methodology used to downregulate gene expression: antisense or double-stranded RNA interference (dsRNAi).
  • dsRNAi double-stranded RNA interference
  • a full-length or partial gene fragment (typically, a portion of the cDNA) can be used in the same vectors described for full-length expression, as part of the gene expression cassette.
  • the coding region of the gene or gene fragment will be in the opposite orientation relative to the promoter; thus, mRNA will be made from the non-coding (antisense) strand in planta.
  • dsRNAi vectors For dsRNAi vectors, a partial gene fragment (typically, 300 to 500 basepairs long) is used in the gene expression cassette, and is expressed in both the sense and antisense orientations, separated by a spacer region (typically, a plant intron, eg. the OsSHl intron 1, or a selectable marker, eg. confening kanamycin resistance).
  • a spacer region typically, a plant intron, eg. the OsSHl intron 1, or a selectable marker, eg. confening kanamycin resistance.
  • Vectors of this type are designed to form a double- stranded mRNA stem, resulting from the basepairing of the two complementary gene fragments in planta.
  • Biolistic or binary vectors designed for overexpression or knockout can vary in a number of different ways, including eg. the selectable markers used in plant and bacteria, the transcriptional terminators used in the gene expression and plant selectable marker cassettes, and the methodologies used for cloning in gene or gene fragments of interest (typically, conventional restriction enzyme-mediated or Gateway recombinase-based cloning).
  • An important variant is the nature of the gene expression cassette promoter driving expression of the gene or gene fragment of interest in most tissues of the plants (constitutive, eg. ZmUBIint MOD), in specific plant tissues (eg. maize ADP-gpp for endosperm-specific expression), or in an inducible fashion (eg. GAL4bsBzl for estradiol-inducible expression in lines constitutively expressing the cognate transcriptional activator for this promoter).
  • Example 8 Insertion of a "candidate gene" involved in flower timing into a Plant
  • a validated rice cDNA clone in pCR2.1-TOPO or the pGEM-T easy vector is subcloned using conventional restriction enzyme-based cloning into a vector, downstream of the maize ubiquitin promoter and intron, and upstream of the Agrobacterium tumefaciens nos 3' end transcriptional terminator.
  • the resultant gene expression cassette (promoter, "candidate gene” and terminator) is further subcloned, using conventional restriction enzyme-based cloning, into the pNOV2117 binary vector (Negrotto et al (2000) Plant Cell Reports 19, 798-803; plasmid pNOVl 17 discosed in this article conesponds to pNOV2117 described herein), generating pNOVCAND.
  • the pNOVCAND binary vector is designed for transformation and over-expression of the "candidate gene" in monocots.
  • pNOV2117 contains the T-DNA portion flanked by the right and left border sequences, and including the PositechTM (Syngenta) plant selectable marker (WO 94/20627) and the "candidate gene” gene expression cassette.
  • the PositechTM plant selectable marker confers resistance to mannose and in this instance consists of the maize ubiquitin promoter driving expression of the PMI (phosphomannose isomerase) gene, followed by the cauliflower mosaic viras transcriptional terminator.
  • Plasmid pNOV21 17 is introduced into Agrobacterium tumefaciens LBA4404 (pAL4404; pSBl) by electroporation.
  • Plasmid pAL4404 is a disarmed helper plasmid (Ooms et al (1982) Plasmid 7, 15-29).
  • Plasmid pSBl is a plasmid with a wide host range that contains a region of homology to pNOV2117 and a 15.2 kb Kpnl fragment from the vimlence region of pTiBo542 (Ishida et al (1996) Nat Biotechnol 14, 745-750).
  • Plasmid pNOV2117 into Agrobacterium strain LBA4404 results in a co-integration of pNOV2117 and pSBl .
  • plasmid pCIB7613 which contains the hygromycin phosphotransferase (hpt) gene (Gritz and Davies, Gene 25, 179-188, 1983) as a selectable marker, may be employed for transformation.
  • Plasmid pCIB7613 (see WO 98/06860, inco ⁇ orated herein by reference in its entirety) is selected for rice transformation.
  • pCIB7613 the transcription of the nucleic acid sequence coding hygromycin-phosphotransferase (HYG gene) is driven by the com ubiquitin promoter (ZmUbi) and enhanced by com ubiquitin intron 1.
  • the 3 'polyadenylation signal is provided by NOS 3' nontranslated region.
  • plasmids include pNADII002 (GAL4-ER-VP16) which contains the yeast GAL4 DNA Binding domain (Keegan et al., Science, 231 :699 (1986)), the mammalian estrogen receptor ligand binding domain (Greene et al., Science. 231 : 1150 (1986)) and the transcriptional activation domain of the HSV VP16 protein (Triezenberg et al.,1988).
  • yeast GAL4 DNA Binding domain yeast GAL4 DNA Binding domain
  • the mammalian estrogen receptor ligand binding domain Greene et al., Science. 231 : 1150 (1986)
  • the transcriptional activation domain of the HSV VP16 protein Triezenberg et al.,1988).
  • Both hpt and GAL4-ER-VP16 are constitutively expressed using the maize Ubiquitin promoter, and pSGCDLl (GAL4BS Bzl Luciferase), which carries the firefly luciferase reporter gene under control of a minimal maize Bronze 1 (Bzl) promoter with 10 upstream synthetic GAL4 binding sites. All constructs use termination signals from the nopaline synthase gene.
  • Agrobacterium is grown on YPC solid plates for 2-3 days prior to experiment initiation. Agrobacterial colonies are suspended in liquid MS media to an OD of 0.2 at ⁇ 600nm. Acetosyringone is added to the agrobacterial suspension to a concentration of 200 ⁇ M and agro is induced for 30min. Three- week-old calli which are induced from the scutellum of mature seeds in the N6 medium (Chu, C.C. et al., Sci, Sin., 18, 659-668(1975)) are incubated in the agrobacterium solution in a 100 x 25 petri plate for 30 minutes with occasional shaking. The solution is then removed with a pipet and the callus transfered to a MSAs medium which is overlayed with sterile filter paper.
  • Co-Cultivation is continued for 2 days in the dark at 22°C.
  • Colonies are tranfered to MS20SorbKinTim regeneration media in plates for 2 weeks in light.
  • Small plantlets are transfe ⁇ ed to MS20SorbKinTim regeneration media in GA7 containers. When they reach the lid, they are transfered to soil in the greenhouse.
  • transgenic T 0 plants Expression of the "candidate gene” in transgenic T 0 plants is analyzed. Additional rice cultivars, such as but not limited to, Nipponbare, Taipei 309 and Fuzisaka 2 are also transformed and assayed for expression of the "candidate gene” product and enhanced protein expression.
  • pNOVCAND is transformed into immature maize embryos. Transformation of immature maize embryos is performed essentially as described in Negrotto et al., (2000) Plant Cell Reports 19: 798-803. For this example, all media constituents are as described in Negrotto et al., supra. However, various media constituents described in the literature may be substituted.
  • the genes used for transformation are cloned into a vector suitable for maize transformation as described in Example 17.
  • Vectors used contain the phosphomannose isomerase (PMI) gene (Negrotto et al. (2000) Plant Cell Reports 19: 798-803).
  • PMI phosphomannose isomerase
  • Agrobacterium strain LBA4404 (pSBl) containing the plant transformation plasmid is grown on YEP (yeast extract (5 g/L), peptone (lOg/L), NaCl (5g/L),15g/l agar, pH 6.8) solid medium for 2 to 4 days at 28°C.
  • YEP yeast extract
  • peptone lOg/L
  • NaCl NaCl
  • 15g/l agar, pH 6.8 solid medium for 2 to 4 days at 28°C.
  • Approximately 0.8X 10 9 Agrobacteria are suspended in LS- inf media supplemented with 100 ⁇ M acetosyringone (As) (Negrotto et al, (2000) Plant Cell Rep 19: 798-803). Bacteria are pre-induced in this medium for 30-60 minutes.
  • Immature embryos from A188 or other suitable maize genotypes are excised from 8 - 12 day old ears into liquid LS-inf + 100 ⁇ M As. Embryos are rinsed once with fresh infection medium. Agrobacterium solution is then added and embryos are vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos are then transfened scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between 20 and 25 embryos per petri plate are transfened to LSDc medium supplemented with cefotaxime (250 mg/1) and silver nitrate (1.6 mg/1) and cultured in the dark for 28°C for 10 days.
  • Immature embryos producing embryogenic callus are transfened to LSD1M0.5S medium.
  • the cultures are selected on this medium for 6 weeks with a subculture step at 3 weeks.
  • Surviving calli are transfened either to LSD1M0.5S medium to be bulked-up or to Regl medium.
  • Green tissues are then transfened to Reg2 medium without growth regulators and incubated for 1 -2 weeks.
  • Plantlets are transfened to Magenta GA-7 boxes (Magenta Co ⁇ , Chicago 111.) containing Reg3 medium and grown in the light. Plants that are PCR positive for the candidate gene cassette are transfened to soil and grown in the greenhouse.
  • Example 10 Method of modifying the gene frequency
  • the invention further provides a method of modifying the frequency of a gene in a plant population, including the steps of: identifying an SSR within a coding region of a gene; screening a plurality of plants using the SSR as a marker to determine the presence or absence of the gene in an individual plant; selecting at least one individual plant for breeding based on the presence or absence of the gene; and breeding at least one plant thus selected to produce a population of plants having a modified frequency of the gene.
  • the identification of the SSR within the coding region of a gene can be accomplished based on sequence similarity between the nucleic acid molecules of the invention and the region within the gene of interest flanking the SSR.
  • Example 11 Chromosomal Markers to Identify the Location of a Nucleic Acid Sequence
  • the sequences of the present invention can also be used for SSR mapping.
  • SSR mapping in rice has been described by Miyao et al. (DNA Res 3:233 (1996)) and Yang et al. (Mol Gen Genet 245:187 (1994)), and in maize by Ahn et al. (Mol Gen Genet 241:483 (1993)).
  • SSR mapping can be achieved using various methods. In one instance, polymo ⁇ hisms are identified when sequence specific probes flanking an SSR contained within a sequence are made and used in polymerase chain reaction (PCR) assays with template DNA from two or more individuals or, in plants, near isogenic lines.
  • PCR polymerase chain reaction
  • a change in the number of tandem repeats between the SSR-flanking sequence produces differently sized fragments (U.S. Patent No. 5,766,847).
  • polymo ⁇ hisms can be identified by using the PCR fragment produced from the SSR-flanking sequence specific primer reaction as a probe against Southern blots representing different individuals (Refseth et al, Electrophoresis 18: 1519 (1997)).
  • Rice SSRs can be used to map a molecular marker closely linked to functional gene, as described by Akagi et al. (Genome 39:205 (1996)).
  • sequences of the present invention can be used to identify and develop a variety of microsatellite markers, including the SSRs described above, as genetic markers for comparative analysis and mapping of genomes.
  • Many of the polynucleotides listed in Tables 1 to 4 contain at least 3 consecutive di-, tri- or tetranucleotide repeat units in their coding region that can potentially be developed into SSR markers.
  • Trinucleotide motifs that can be commonly found in the coding regions of said polynucleotides and easily identified by screening the polynucleotides sequences for said motifs are, for example: CGG; GCC, CGC, GGC, etc (see Table 4 for further details).
  • primers can be designed which are complementary to the region flanking the repeat unit and used in any of the methods described below. Sequences of the present invention can also be used in a variation of the SSR technique known as inter-SSR (ISSR), which uses microsatellite oligonucleotides as primers to amplify genomic segments different from the repeat region itself (Zietkiewicz et al, Genomics 20:176 (1994)).
  • ISSR inter-SSR
  • ISSR employs oligonucleotides based on a simple sequence repeat anchored or not at their 5'- or 3 '-end by two to four arbitrarily chosen nucleotides, which triggers site-specific annealing and initiates PCR amplification of genomic segments which are flanked by inversely orientated and closely spaced repeat sequences.
  • microsatellite markers as disclosed herein, or substantially similar sequences or allelic variants thereof, may be used to detect the appearance or disappearance of markers indicating genomic instability as described by Leroy et al. (Electron.
  • Microsatellite markers are useful for detecting genomic alterations such as the change observed by Leroy et al. (Electron. J Biotechnol, 3(2), supra (2000)) which appeared to be the consequence of microsatellite instability at the primer binding site or modification of the region between the microsatellites, and illustrated somaclonal variation leading to genomic instability. Consequently, sequences of the present invention are useful for detecting genomic alterations involved in somaclonal variation, which is an important source of new phenotypes.
  • QTLs Quantitative Trait Loci
  • Many important crop traits are quantitative traits and result from the combined interactions of several genes. These genes reside at different loci in the genome, often on different chromosomes, and generally exhibit multiple alleles at each locus.
  • Developing markers, tools, and methods to identify and isolate the QTLs involved in a trait enables marker-assisted breeding to enhance desirable traits or suppress undesirable traits.
  • the sequences disclosed herein can be used as markers for QTLs to assist marker-assisted breeding.
  • the sequences of the invention can be used to identify QTLs and isolate alleles as described by Li et al.
  • sequences of the invention can also be used to isolate alleles from the conesponding QTL(s) of wild relatives. Transgenic plants having various combinations of QTL alleles can then be created and the effects of the combinations measured. Once an ideal allele combination has been identified, crop improvement can be accomplished either through biotechnological means or by directed conventional breeding programs. (Flowers et al, J Exp Bot 51 :99 (2000); Tanksley and McCouch, Science 277: 1063 (1997)).
  • Example 13 Marker-Assisted Breeding Markers or genes associated with specific desirable or undesirable traits are known and used in marker assisted breeding programs. It is particularly beneficial to be able to screen large numbers of markers and large numbers of candidate parental plants or progeny plants.
  • the methods of the invention allow high volume, multiplex screening for numerous markers from numerous individuals simultaneously. Markers or genes associated with specific desirable or undesirable traits are known and used in marker assisted breeding programs. It is particularly beneficial to be able to screen large numbers of markers and large numbers of candidate parental plants or progeny plants.
  • the methods of the invention allow high volume, multiplex screening for numerous markers from numerous individuals simultaneously.
  • a multiplex assay is designed providing SSRs specific to each of the markers of interest. The SSRs are linked to different classes of beads.
  • RNA is extracted from root tissue of 1000 different individual plants and hybridized in parallel reactions with the different classes of beads. Each class of beads is analyzed for each sample using a microfluidics analyzer. For the classes of beads conesponding to qualitative traits, qualitative measures of presence or absence of the target gene are recorded. For the classes of beads conesponding to quantitative traits, quantitative measures of gene activity are recorded. Individuals showing activity of all of the qualitative genes and highest expression levels of the quantitative traits are selected for further breeding steps. In procedures wherein no individuals have desirable results for all the measured genes, individuals having the most desirable, and fewest undesirable, results are selected for ftirther breeding steps. In either case, progeny are screened to further select for homozygotes with high quantitative levels of expression of the quantitative traits.
  • Table 4 This table identifies the start and end points and the nucleotide sequence of trinucleotide repeat units in the coding sequence of selected flowering time genes
  • Salivary glue protein SGS-3 precursor (Dentin matrix precursor protein-3) (DMP-3) [Contains:
  • DPP distal phosphophoryn
  • Zinc finger protein constans- PROTEIN APETALA3 like 14
  • RNA-binding protein 10 Description: FLOWERING LOCUS T (RNA binding motif protein 10) protein (DXS8237E) 50
  • GLABRA2 Homeobox-leucine zipper protein protein ATHB-10) (HD-ZIP protein
  • ATHB-10) (HD-ZIP protein 60 ATHB-10) ATHB-10)
  • Agamous-like MADS box GLABRA2 (Homeobox-leucine zipper protein AGL6 protein 70 ATHB- 10) (HD-ZIP protein

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Abstract

La présente invention concerne des molécules d'acides nucléiques pouvant être obtenues à partir du génome du riz, codant pour des produits protéiques impliqués dans le développement et le rythme de la formation de fleurs dans des végétaux. Ces molécules d'acides nucléiques peuvent être utilisées pour moduler le développement des fleurs, la structure et le rythme de floraison.
EP02758266A 2001-06-22 2002-06-24 Identification et caracterisation de genes vegetaux Withdrawn EP1409696A2 (fr)

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WO2004022755A2 (fr) * 2002-09-05 2004-03-18 Genesis Research And Development Corporation Limited Compositions isolees a partir de graminees fourrageres et procedes d'utilisation de ces compositions
US7538260B2 (en) 2002-09-05 2009-05-26 Jeroen Demmer Compositions isolated from forage grasses and methods for their use
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WO2006026287A2 (fr) * 2004-08-25 2006-03-09 The Samuel Roberts Noble Foundation, Inc. Plantes a floraison retardee
JP2008052371A (ja) 2006-08-22 2008-03-06 Fujitsu Ltd アウトバンド認証を伴うネットワークシステム
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CN104928284A (zh) * 2015-06-30 2015-09-23 华南农业大学 大豆春化基因GmVRN1及其克隆方法和应用
CN105671054B (zh) * 2016-03-03 2019-04-26 中国农业科学院作物科学研究所 水稻开花调节基因OsWRKY104在调节植物光周期和开花时间中的应用
CN106995853B (zh) * 2017-05-16 2020-08-25 浙江海洋大学 筛选与少根紫萍叶生长相关的分子标记的方法
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